<%BANNER%>

Biological Denitrification of High Nitrate Industrial Streams

Permanent Link: http://ufdc.ufl.edu/UFE0022265/00001

Material Information

Title: Biological Denitrification of High Nitrate Industrial Streams
Physical Description: 1 online resource (64 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: biological, denitrification, treatment, water
Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The purpose of this project was to develop a biological denitrification system for high nitrate industrial streams. High nitrate samples obtained from an industrial source and synthetic nitrate solution prepared using nitric acid and DI water were used in the experiments. The goal was to obtain a denitrification rate of 2 mg NO3-N/L/min. Attached growth systems and continuous stirred tank reactors were used for the study. The reactors were kept in anoxic conditions in order to maintain denitrification conditions. Effects of micronutrient deficiency, volatile contaminants high nitrate concentration, and sodium on denitrification rates were studied. From the study, it was concluded that the industrial stream was treatable; however, volatile contaminants present in the feed solution must be aerated out and metals must be removed by ion exchange prior to denitrification. It was also concluded that micronutrients should be added directly to the reactor in order to avoid its precipitation in the feed and deficiency in the reactor. Studies comparing sodium and potassium neutralized nitrate solutions did not show any significant differences in denitrification rates. Thus, it was concluded that sodium did not affect the denitrification process considerably. In the suspended growth system, denitrification rate of 4 mg NO3-N/L/min was achieved with synthetic nitrate solution as the feed, and a denitrification rate of 2 mg NO3-N/L/min was achieved using industrial nitrate solution. An assay was also developed in order to test the biotreatability of high nitrate solutions. In the assay procedure, a batch test is conducted using the test sample along with a synthetic nitrate solution containing the same NO3-N concentration. The absorbance values and denitrification rates of the test sample and synthetic nitrate solution are obtained after 4, 8, and 24 hours. YNO and ?maxNO of the sample and synthetic nitrate solution are obtained from the absorbance and NO3-N measurements. The biotreatability index, BI, of the test sample is obtained by taking the ratio of ?maxNO of the sample to ?maxNO of a synthetic nitrate solution containing the same nitrate concentration. A BI value of 1 means that the test sample can be successfully denitrified, and values considerably less than 1 show that the denitrification will be very difficult.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Svoronos, Spyros.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022265:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022265/00001

Material Information

Title: Biological Denitrification of High Nitrate Industrial Streams
Physical Description: 1 online resource (64 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: biological, denitrification, treatment, water
Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The purpose of this project was to develop a biological denitrification system for high nitrate industrial streams. High nitrate samples obtained from an industrial source and synthetic nitrate solution prepared using nitric acid and DI water were used in the experiments. The goal was to obtain a denitrification rate of 2 mg NO3-N/L/min. Attached growth systems and continuous stirred tank reactors were used for the study. The reactors were kept in anoxic conditions in order to maintain denitrification conditions. Effects of micronutrient deficiency, volatile contaminants high nitrate concentration, and sodium on denitrification rates were studied. From the study, it was concluded that the industrial stream was treatable; however, volatile contaminants present in the feed solution must be aerated out and metals must be removed by ion exchange prior to denitrification. It was also concluded that micronutrients should be added directly to the reactor in order to avoid its precipitation in the feed and deficiency in the reactor. Studies comparing sodium and potassium neutralized nitrate solutions did not show any significant differences in denitrification rates. Thus, it was concluded that sodium did not affect the denitrification process considerably. In the suspended growth system, denitrification rate of 4 mg NO3-N/L/min was achieved with synthetic nitrate solution as the feed, and a denitrification rate of 2 mg NO3-N/L/min was achieved using industrial nitrate solution. An assay was also developed in order to test the biotreatability of high nitrate solutions. In the assay procedure, a batch test is conducted using the test sample along with a synthetic nitrate solution containing the same NO3-N concentration. The absorbance values and denitrification rates of the test sample and synthetic nitrate solution are obtained after 4, 8, and 24 hours. YNO and ?maxNO of the sample and synthetic nitrate solution are obtained from the absorbance and NO3-N measurements. The biotreatability index, BI, of the test sample is obtained by taking the ratio of ?maxNO of the sample to ?maxNO of a synthetic nitrate solution containing the same nitrate concentration. A BI value of 1 means that the test sample can be successfully denitrified, and values considerably less than 1 show that the denitrification will be very difficult.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Svoronos, Spyros.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022265:00001


This item has the following downloads:


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101203_AAAAAE INGEST_TIME 2010-12-03T06:25:58Z PACKAGE UFE0022265_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 97085 DFID F20101203_AAACQK ORIGIN DEPOSITOR PATH peter_s_Page_19.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
5334ecbcac6de0680fcd5d5118927325
SHA-1
745a2bed09caad6871998efc8885ba85c90b9a41
401101 F20101203_AAACLN peter_s_Page_59.jp2
226c4cbe832df7c1d6bcce44dd691123
1f334f8cf3cb35aaded30050a0dc6aa8e49b4890
593752 F20101203_AAACVH peter_s_Page_47.jp2
11e40ac116ee5f3bd75a1db7d0e5e8b0
b6b5cac2032309184060f0f816495f659fde8f03
53674 F20101203_AAACQL peter_s_Page_43.pro
bc77016f4b69b07dd76dce92c2b54bf7
a8f4e344be52ad22060e7af340f85dff136b81ff
93719 F20101203_AAACLO peter_s_Page_18.jpg
eddedfaf5ed80e2c6d09786a47bd598c
e29028868a0ced8016b80f258d3c9a6fb57e2427
1051986 F20101203_AAACVI peter_s_Page_48.jp2
5e67ce3fe043e00037c71ff7383de39f
3ef7477bf86b56ff6c43dbec1f6438ff02e20823
5267 F20101203_AAACGR peter_s_Page_59thm.jpg
be4734acd03e59bbe8838db422ce210e
a2edae46eff621029a5c9b1fb79b54f9dd5d1698
92431 F20101203_AAACQM peter_s_Page_46.jpg
a3004c5e342ee6c26c7b929231b93f99
ccba8f6de082ade13886216c33f8f9c46b9d67d4
951 F20101203_AAACLP peter_s_Page_51.txt
f9436a196b6dc5f4016d8d21f8346986
c4b59fabb474702a15ea612d9db163b040977051
527231 F20101203_AAACVJ peter_s_Page_51.jp2
676876a1ae92f94f8212817c9b3e71f9
1dc2b3d70df34219273cf27ee69678117628d735
70527 F20101203_AAACGS peter_s_Page_08.pro
ee0d3aa5ad7eb176cd4097d481f86b41
27a72f9a2ac4f2c78e97997178ee1b9cd456b612
1051961 F20101203_AAACQN peter_s_Page_54.jp2
199eaeb1d004e4b0865d8c4ebb5c6fa5
d5d791533b0bb9d4d8b695ab893f07182fd2d9e6
107080 F20101203_AAACLQ peter_s_Page_13.jpg
938f3616c8990b436f15589f25969aa8
ce4519fa7be0de3f7a93c0e98522077a1ab6f737
F20101203_AAACVK peter_s_Page_52.jp2
b059297c292110e9db8b2b970373e58d
47e6e1f4835c5db584650f240ca1f38e73630387
94420 F20101203_AAACQO peter_s_Page_31.jpg
0f6c5e32c2698428b3ed86092ddb7819
e2b96053115442d074d3d8a20385c28c969700ca
884150 F20101203_AAACVL peter_s_Page_58.jp2
1420a066df38f2570cf138d24a8b2ddc
672c0daa1610f6cf08c51502da08ebe7469be195
2199 F20101203_AAACGT peter_s_Page_32.txt
ec312b8299d07bbf8fcfbd0758f1d37f
825cf9ad870ceb557d3099075aad9f778eaf6a68
1815 F20101203_AAACQP peter_s_Page_27.txt
52f9428253425001f025880df617ff13
641b13b61d6a2bf8c89a5c7b91c6d5c45355df9d
7085 F20101203_AAACLR peter_s_Page_16thm.jpg
b4175800d67c2e82f0bec573194647a8
1d9fd31293011b9a0c1ffe5d3c2a87074ba5eae4
25271604 F20101203_AAACVM peter_s_Page_02.tif
a7df848e846cddc092ab282418a2a926
ced6bd5655f4b8f30ea557e7a0ed5b5a32c8fe58
94022 F20101203_AAACGU peter_s_Page_22.jpg
2aad01278334bd463a66dd359c217602
6e7c3601cbb062869ff970d63614682390bc3ab7
F20101203_AAACQQ peter_s_Page_11.tif
e997685102f341f782b00829c3ab797e
9909ee3f4bc5beb9d17724a9853a39ac9ff2d7ce
46231 F20101203_AAACLS peter_s_Page_29.pro
91d7a4da08637840967106c7f93e432e
03c12639a4f0ebc6e92e6baf7ba9a64d1cd25f2d
F20101203_AAACVN peter_s_Page_05.tif
eca355f3da6c3079c5a23fc87ab65e30
ef4ead3390d3f1c0c144390e3becebdc721aa1fc
35340 F20101203_AAACGV peter_s_Page_41.pro
59333bb7b99155ba7b00abaf716046f3
d8e544ff7e56b72227b045156b3147a34ddd1b93
96559 F20101203_AAACQR peter_s_Page_35.jpg
9314639fee16302985550c5f205b07af
0b08cb4d29596a4d2798f46f7d9a019609004d2b
65464 F20101203_AAACLT peter_s_Page_09.jpg
f90bdd62f227c8ce5f8e3f9f3b76cab4
55c684a806605d8d8e53df4c739f1aebcc14b4ba
F20101203_AAACVO peter_s_Page_06.tif
0f6d35de7f90962b86bbda56f538920e
673b0171386e86dd11317f5ac5c74841cd711a4f
8889 F20101203_AAACGW peter_s_Page_55thm.jpg
1ace565a5ae366a37986376293bf84ae
8d59e2140f29452044e2238024b0ff72653555ff
F20101203_AAACQS peter_s_Page_49.tif
ce05097195f844655ff817b84ab884ab
664250fe7fbe98d5c8c7c923d9c9cf8b09b4910b
F20101203_AAACLU peter_s_Page_28.tif
f922d37c5a7436c411e073fe5b760ca2
186c4f7862f8f63638f3c603f34bf7245a6330cd
F20101203_AAACVP peter_s_Page_07.tif
3a5b20301df4fe6cf79148484bcfb6b4
50b03eb671f1d51e137df9f3dc006150f4eb9a25
F20101203_AAACGX peter_s_Page_35.tif
55ceaacd1c9a341890b0bd8c553c86c6
12529dd48b284ca84d06e3ef53becaee4ceb9620
50670 F20101203_AAACQT peter_s_Page_51.jpg
6666775d2e9b22fc4e10328c8bd94408
5dafff1fc942661a1d3423c3f54076ce5a8a33d5
8178 F20101203_AAACLV peter_s_Page_58thm.jpg
246cff13c1de5b01196ddbac520ede59
7d53a9403d90cd0c555cd993ca38f7dfbd063b4c
F20101203_AAACVQ peter_s_Page_13.tif
7cda9c83c6bde451b30f8e8d3f850da9
101440ffed2356321a374c58b3a7be3fc23df2c4
F20101203_AAACGY peter_s_Page_53.jp2
bf11f9ccb270ccb6136b3ceda08e2e52
ed76e13a230f872b9c4f87ea9e1d43fa0cd1c5f3
1766 F20101203_AAACQU peter_s_Page_03.pro
c81d6a2a7c1785be78eac5ae5fd435d3
680f2fe165f84a52e89f64eef13850285d52c029
53427 F20101203_AAACLW peter_s_Page_24.pro
26a0cfba7eeb94a173637a6e2231504b
bbaeb23ec425e097031d9ed91bb96516f1ec3046
F20101203_AAACVR peter_s_Page_19.tif
fdfee0490f88755ea1e8e5be93387841
59e79979d5edc61c7f99d46cbb696ff9189d9713
27164 F20101203_AAACGZ peter_s_Page_05.QC.jpg
75b8f25edc2951c8ef2439acfeb6074b
422ec4a2e245c9e08ff9364a065b8af27e6296ed
2111 F20101203_AAACQV peter_s_Page_24.txt
a2ca997fb877346ebdecbe0c5839a3bd
44f361db75a4e148b54bbaf68c36f0c2b4e84aeb
732809 F20101203_AAACLX peter_s_Page_37.jp2
dcfa0b78d8d64f9a9ef2f775cdbfc2e1
29659ff6b8bfa536ce67eb611b2fb8770d883760
F20101203_AAACVS peter_s_Page_40.tif
2b64ffd34949dd7c97ff6234dc780a76
fb7a6d19bcaa1aa88e62310083cf5cd45c25e38c
F20101203_AAACJA peter_s_Page_20.tif
34eb7393f866df9da6ec7461d051839b
776fc795ed4875605624542c9075211fec733fd4
1051949 F20101203_AAACLY peter_s_Page_29.jp2
c4f8669c6f6bb3cb1ee47e35d4776e86
2cab6a0f9175d343f305524df2fe11daf1044079
F20101203_AAACVT peter_s_Page_43.tif
610253544cd863ca224b1cffbcd81913
0f009fb3f519bca2a1e75595f1bd89e405fb4d58
32216 F20101203_AAACQW peter_s_Page_62.QC.jpg
d7e63b73208119fd5a31678257a12e6b
8bcc8b77d194abc04509e3073afc76e61a235291
52844 F20101203_AAACJB peter_s_Page_25.pro
b2308f0e463e06b3b8649f3f10184981
152582e405827334dae0c8707d5ffdc02604a0e0
4787 F20101203_AAACLZ peter_s_Page_21thm.jpg
b6b4f2c00b7d5d0c2b550c0fe87d564f
faee237fd97c0b34ee00dde00d42d1e168c5d925
F20101203_AAACVU peter_s_Page_50.tif
85136a5b2f77191e0e724c75319b57e2
e86e032732960a2bef4cfeaf74616cf1ab385f21
427 F20101203_AAACQX peter_s_Page_01.txt
957a2effeeaccee0aa0611c7492b0d0f
ef61bb29a6f15e37259a00e110963b9f4cadb1e6
35992 F20101203_AAACJC peter_s_Page_20.QC.jpg
ae77de3a68e92935307c4b83947b5625
195785accc11f2d6dbc4ee9d220b7df37016641d
F20101203_AAACVV peter_s_Page_53.tif
11678426ea5b7099f42c9ccb24e7f796
56a7778962744de73ddba55ff69ca12a884d0808
F20101203_AAACQY peter_s_Page_54.tif
5ba663888ee9cc6d6a074f845fabe41f
2a9bd5df7601d32467f19f159aaeb08d2eb89171
7449 F20101203_AAACJD peter_s_Page_08thm.jpg
782efaeef61eb49cbec94c5ea2a6b198
a900f108a48ffaba7f8d2c3a89fc8b53c89f9c5a
F20101203_AAACVW peter_s_Page_59.tif
2191c0a038bfc7d7152afeb8aabdc522
7a7ab3c7eb1b03d8e96e1d50423236ff8dd7f893
34316 F20101203_AAACOA peter_s_Page_04.jpg
9695a8ce7293a002aa8018c2206eab47
cf1ef19cd350af28962cbe810480816188a9e77c
9280 F20101203_AAACQZ peter_s_Page_27thm.jpg
64d194e5a9f6148d80faed452f48f781
d34a5efc22260b8dde88b2d0b76db6187f4954e5
F20101203_AAACJE peter_s_Page_03.tif
68895ff040cffe2d3604c26fd8adac03
e57952d4decad6be08ac421b509d73038cbb2826
F20101203_AAACVX peter_s_Page_62.tif
ed0b71c3b07057c761c7c4ab6a772ced
9d01895fe0c22a218f451a7b4b00ddee1a460b45
39683 F20101203_AAACOB peter_s_Page_42.pro
81f75359779e912936864e0ef11c0073
bb941ccd17878b30f4e9ae641305333391ce202b
2011 F20101203_AAACJF peter_s_Page_62.txt
af3463b19bb3a48c56ab9dd87feb9ab5
2df5b3535a8a69f3dff1bd4b4f0b43450e19d750
37487 F20101203_AAACVY peter_s_Page_09.pro
4ba699c5f2db0fdea019064a3aa6b1d8
d189cb87958f52c5eba99294fb2516e56a72d091
843 F20101203_AAACOC peter_s_Page_07.txt
f7ed91055b52dd7334476d9a19663bd0
c8cadf86e64c5fa8d118dd07493e66033fe875bd
37984 F20101203_AAACJG peter_s_Page_32.QC.jpg
b10be89a5dc5c6bf823ee0276bf7e50f
aa4432ba8460824dc784f13ea58a0d5762a111a8
39567 F20101203_AAACTA peter_s_Page_58.pro
6911f2b1832d4624f1f3462f9ae65468
25f777d0177c41948812e4ddab4cebcaf083182b
53718 F20101203_AAACVZ peter_s_Page_20.pro
e2aeeb0a618139f46bd2f90502d4a256
c4e31d3d842f86f90a9dab8c905a577db2d136b8
109634 F20101203_AAACOD peter_s_Page_17.jpg
ed09bcd435d55b90f5a83df0a9b4b71e
dfadb468b46c92bc9b3082c0d3917ca1d1ae8672
7232 F20101203_AAACJH peter_s_Page_10thm.jpg
d1d627bd22f479d38e75f8c518a6c3e7
7df25addebdfed8dcdd1a4c4cc9ff3dd97adf26b
20869 F20101203_AAACTB peter_s_Page_34.pro
69a5fcfd95f7f0275586f1288223910a
304ad55dd6ed3de2d40c4195d335716b0ddfdf56
980943 F20101203_AAACOE peter_s_Page_35.jp2
0216e2f1101d7b757669b8bd80b02c03
a85ad5a635d5dca25aff1415a076f104880396ce
F20101203_AAACJI peter_s_Page_29.tif
bca227a030c449fc0748337f4b1cd990
6315b9a16cbeaa380bdd22fb0c1a91102b0b511c
8321 F20101203_AAACTC peter_s_Page_40thm.jpg
6e79e0d95d8f4c338368e1aac1886f44
2e8daf688af4acb7ff6900ab6283c2ab8f49d46b
1959 F20101203_AAACOF peter_s_Page_29.txt
e4e91618da7452983b084e2ebf6e90d9
4bfc1930ab94a6376b98ec91d0465bdf4403535a
7674 F20101203_AAACYA peter_s_Page_41thm.jpg
ac8e9359e847b1969305d8f1333eb5aa
72d971afeef2400fc260ac6906557b52b89b5763
5744 F20101203_AAACJJ peter_s_Page_61thm.jpg
d1a83ccf14efb2b2b850a9dc766cf665
7109a56dcc2e3fcb86a079d81b2a181d839f05f9
49162 F20101203_AAACTD peter_s_Page_62.pro
b66bf2b546300981ed6cb5bdd16bf21f
7dae627880739c3e655868ffd9dd6208d7785a0e
36126 F20101203_AAACOG peter_s_Page_48.QC.jpg
3b876f7b26b249f9c551beca1eee4968
62e16828869a1cb329f6cf59978669b9070c1b71
8246 F20101203_AAACYB peter_s_Page_46thm.jpg
3249647b1b7865b8b862044e53456541
07aa9214dc05a48ef0f749a1350b5f23ebb27324
45626 F20101203_AAACJK peter_s_Page_03.jp2
77539c33fc6b386473e3f352da005bbc
e1056c7d0985789e1d82f11aef92e9832f1aec3a
43913 F20101203_AAACTE peter_s_Page_16.pro
cb5601a2643f414bcf5dd7000f92350d
a76172716672b20a3fd4b09d5289f5917ed6b558
110963 F20101203_AAACOH peter_s_Page_43.jpg
fe2bd9dd404bc69be6f434e6c44bae5f
c949e979e5d2a0d4502e68dce19bccb54d7a353a
F20101203_AAACJL peter_s_Page_64.tif
6d5272406433dbcaefbecd634ab5cc03
a9526ceeb717bc03a443f59e7102ab5bf359ae36
51144 F20101203_AAACTF peter_s_Page_54.pro
60cdbfc30a88363523b2fc20a2119c4a
8474f463e1ea8ad0aec21c0c6dcc6a6c6cba5e9f
2101 F20101203_AAACOI peter_s_Page_50.txt
34b3b71529e8cebcfd5cc25ed7d93f86
1242b46f0c68a18762ca1cded48f446c7c376d59
1900 F20101203_AAACJM peter_s_Page_10.txt
ade6379c47e8166e1f2e0dc24796843d
0e34338d6b036b8626498876ffec4ea7922cd734
33220 F20101203_AAACTG peter_s_Page_52.QC.jpg
d0ea3e5de91cf333ce91e150381bcb5c
15226e0340f3cfd85090e73b2cc993b916e88a67
4823 F20101203_AAACOJ peter_s_Page_11thm.jpg
1e7a51c9a3ae4333f06025bc3b175df2
b392124f6ea4e4139daf9454b1cb5d37152c711d
56113 F20101203_AAACJN peter_s_Page_32.pro
aac2bf4f832ab0978fcf4eca60084f27
500f13f2f13562b17d8906430b74982ad2c2aa67
F20101203_AAACTH peter_s_Page_61.tif
d2232ac3aead5bdc027053badb35dfe2
2d02b2d4d7583a1be1e905aa907286d37f1c6d98
6200 F20101203_AAACOK peter_s_Page_34thm.jpg
7f1d0e1960829485d1926c9cf5fcb45e
7267a40ab83369a118c4ed207abd92c7129d41ef
1051966 F20101203_AAACJO peter_s_Page_23.jp2
020b41c31d1b13a2ce48901d8819a7e5
16adc5fcc7626655c49469e0b6f1e3ff632c1074
F20101203_AAACTI peter_s_Page_48.tif
0c27f3487c2f38d37febc5e856e666d7
fe6edddb41a3c2a3b6c45e4698d9a77d83677cb2
43350 F20101203_AAACOL peter_s_Page_10.pro
ae59820711690e6d22d2a5a768ee66ac
5c03349d53d867f8bd8341366d0473011576a3f9
48378 F20101203_AAACTJ peter_s_Page_18.pro
8743933afe45f190350b9be1e397dfc9
f3783234960aa28c858335fd8efe132bb77f829b
20172 F20101203_AAACOM peter_s_Page_07.pro
f1c57c0d70a68ca95b914a6369a31b6b
07d4a96c282a3d7c5fa05021572f7c6255af11d5
92099 F20101203_AAACTK peter_s_Page_16.jp2
7ed27a9a31900bbc70e9123eaeebfbd6
10776c0ea8fffb34f2708bc0dd31d2329ad96ee7
37523 F20101203_AAACON peter_s_Page_45.QC.jpg
635cd3f14d30e04d7090d7582a2fe978
1cccc5150b2be3febf153d178e76c0b304cd1d41
F20101203_AAACJP peter_s_Page_24.tif
eb4e660ab830b4e404f6d5031e37816d
d133a77eb893ca59b625251f019fa96af09a0d03
F20101203_AAACTL peter_s_Page_23.tif
60d15bcf02c37f4595500b5b6737d69d
afbf8e7749f43f6b11309934fb57d934ad75b204
6600 F20101203_AAACOO peter_s_Page_05thm.jpg
395b4675ed6b724b13c6b2d30c4182ff
48a68496aa5dc89ddb06b3da42e7e00c491daf0c
20761 F20101203_AAACJQ peter_s_Page_64.jpg
88fddc7338e3ee2a8243aca462e036b6
5a464ff3466419b91a231c2224ceba550f129f4d
31376 F20101203_AAACTM peter_s_Page_06.pro
7227d0bb8da30faa29705311cab3ffa1
0faaed37203938ed84cedcc67d85df521e2c3325
2987 F20101203_AAACOP peter_s_Page_06thm.jpg
75ad242267d42684a86b004635adee86
94161052508a6b6825da2e0851264084c5af7278
1789 F20101203_AAACJR peter_s_Page_39.txt
016430a54411c035bb674dbd6a7891db
446ca866d908b1f50eee537a2059c43bf05953de
2051 F20101203_AAACTN peter_s_Page_23.txt
16daead2004723bb763f75030492aa0e
3f10b97fa07ccb027a4555cf1ea55992fa6874d9
56436 F20101203_AAACOQ peter_s_Page_11.jpg
f55ed93117a1ebd3879355fb6922f5ef
6f7a7bac3062f31ee19636f2002e768934942642
2083 F20101203_AAACJS peter_s_Page_13.txt
208102a3d55ac4c86dafb97b599807e0
401d9af5a1115e0a40e614c780db978f52923ffb
F20101203_AAACTO peter_s_Page_44.tif
7593c78cafd215530db1f7347535512a
e13ac20d8492b3adf185ce245987755f7bc3c3cb
109598 F20101203_AAACOR peter_s_Page_20.jpg
234526e5181fafc25556f66f2dd2681a
42bbe27dbc77776e11bb8b3faca5bcd6dae7698e
780 F20101203_AAACJT peter_s_Page_02.pro
9384b1016a25a739eb1afe073a17e551
b65ac802f314fbfe19c2beed4a85f084cf47c6dd
49660 F20101203_AAACTP peter_s_Page_14.pro
797aae7ef6da658964d4f5abaf224f06
c75c8cffb14d5bbf87807a32f15ee7c3aa0d0934
F20101203_AAACOS peter_s_Page_36.tif
6e113d6e1e34fca89b9bec394f8df0c0
8644356f24e46b7328c3c68629db9341d487cfcb
F20101203_AAACJU peter_s_Page_47.tif
fde541bfc1f913569678b38fbb650c6e
fef4f3b22d0fe9df19038cb40d507a4bf97a3f43
8755 F20101203_AAACTQ peter_s_Page_22thm.jpg
e1a8c7f5f7e3946a934c81dc68bdc012
4a3398cf42aa7141e3ce85974dd7c4d73e3bdd86
31389 F20101203_AAACOT peter_s_Page_46.QC.jpg
33e447ea3ef20b376b1dbab7795ffdb3
77d211f1060848a2309f0a8a948186449e4ad41d
1051983 F20101203_AAACJV peter_s_Page_62.jp2
fc013c61321822c20cd33e2ab7e6f31e
1305b37e6d8cd06b836ae5493385f5835398bc2c
6514 F20101203_AAACTR peter_s_Page_36thm.jpg
c3489c4f228cd3d783a42b017a493820
2d9f00c0385a4f52cb569547b645e8df384be2d5
14700 F20101203_AAACJW peter_s_Page_04.pro
71b778efa8c8af0aaed7b443d31df18e
bd8baeca77c486b9ea326afde38f7e46d6cb12c4
25041 F20101203_AAACTS peter_s_Page_38.QC.jpg
f5ccef8aaefeef3c1561412eb1bedd30
d76fb3c599c82467bb4adc4a5b1ab2e2aaee108c
112883 F20101203_AAACOU peter_s_Page_45.jpg
ddf34eb428746e916ab4851b6b26c543
1804dfe099f3bde97a2ebc1a99c70846dc0ceaf0
40065 F20101203_AAACJX peter_s_Page_35.pro
1521d4c35490b82fe7da8ebc2d498dc2
a0f0a42dfd04af4b18e56c0566dc5b34a6878271
66865 F20101203_AAACTT peter_s_Page_61.jpg
b6b19980c25a24b293dfea54e7cc90df
5422a8e7dc402b4ef3a584eece4a3bc08c530778
101652 F20101203_AAACOV peter_s_Page_29.jpg
c389d631a4b320bfa812a19f8c811f8b
569a79cef339afac44986f673e8ededd23a8e283
13984 F20101203_AAACHA peter_s_Page_60.pro
540d0761103b20de4174e59b7a65be33
c406d8eb05933ebd1e884aada02f23204b63ea15
2110 F20101203_AAACJY peter_s_Page_20.txt
3295698920aef64f59e94cc37a7334e0
37ac0d3c961a42aae9fdb0a8edbd2cec25790fcc
100294 F20101203_AAACTU peter_s_Page_28.jpg
d50d273b58425d0422ecb1fc5ef33d70
02c60f4f5c0fb1af336f1ec041c41d819f911cac
29859 F20101203_AAACOW peter_s_Page_39.QC.jpg
11e033407c42cd0c1a98108ed910e4b8
becb3a7582a4bac3e81110f3c6c0fda32cb9cf12
22097 F20101203_AAACHB peter_s_Page_47.QC.jpg
24306a56cc2aa9337a72bfd4903c6047
a59cfe9911939e4b1152137b2c82f6462318246e
F20101203_AAACJZ peter_s_Page_08.tif
362639ea3934cf45cd870889726b5299
0ed4692e47be2ec992e414154250301ccd62a3e1
F20101203_AAACTV peter_s_Page_60.tif
336f7ea1d509612dbf8af79462f8be1a
8687a94011f9b63a71c0ecc7b1797919454d2597
1047278 F20101203_AAACOX peter_s_Page_19.jp2
ffb16693a43853bfc21859eaa32428e1
019a401f927af48d8d608abfe55bd6688a16176e
F20101203_AAACHC peter_s_Page_33.tif
b2a0186a2987d8cd5c711d6d2797453b
717bd4b7344094e6f479274b7b5e47c2bd5cea03
8654 F20101203_AAACTW peter_s_Page_52thm.jpg
4e3f317ead64dafcc666f8315f7da296
84ce3d00d125ec40146971a0d4f135eadfa72a7f
108915 F20101203_AAACMA peter_s_Page_25.jpg
0233f8ad3d3838995628fc084afbd43e
573ae824858f7ce3c720c271e62cd78b909cb888
345921 F20101203_AAACOY peter_s_Page_04.jp2
f33320abeec15638e7a9a8b7ddc43f6f
a58f1fd3b28a9394fc3807904243899ca70d7473
27503 F20101203_AAACHD peter_s_Page_37.QC.jpg
2522c8c5dc31c11c0a51aabe7af964e5
319a924b4c413a9d009244a3925b9b9e28a37874
56621 F20101203_AAACTX peter_s_Page_21.jpg
ecd140971f9ae1b9623c156db55f1705
12762d63717a0954ae2525b60008b7ffc4db0bf6
85095 F20101203_AAACMB peter_s_Page_49.jpg
06b7224da6e642f7769cdfbffc83122c
65bdeb19554db71cb9887e0c6e4799e34a6d5589
36608 F20101203_AAACOZ peter_s_Page_31.pro
1927d723cda72a7da5ffce0c065b8b79
79b088c8c63a7ced35331c896286c756721a47ab
816 F20101203_AAACHE peter_s_Page_59.txt
877e02bc7e9169fbf065c3164b7cdad3
30f4a9b4bd1cc0f20651cf81beccf127858ab224
8625 F20101203_AAACTY peter_s_Page_25thm.jpg
3c9f39c690ceb93c88492699d1fde479
4086af743dfc88acc9925c0ccb7db45c8faed496
21922 F20101203_AAACMC peter_s_Page_56.QC.jpg
e5ff8f4de32665da063d89f6e4438401
62a4274285e02456da014bbedc7c179cdfda37f6
828 F20101203_AAACHF peter_s_Page_60.txt
7f52626a9960dd68638c7e3804201f1f
da106631fed389f93f9f8acca93870941da6ed38
89012 F20101203_AAACMD peter_s_Page_63.jpg
693088d50840508edb7e8e0bc0b7f63a
290c14098abe21e30aeff64f610d509ac595a6c5
1488 F20101203_AAACHG peter_s_Page_09.txt
e4375024f0a50951492e116e815f8efa
58c054d0913102ca95d243dcb0d5d75691815ca6
1079 F20101203_AAACRA peter_s_Page_34.txt
fc98b5b2deccdfaf92ad554f64763354
c35e3cfba4b756a7a58b1debe6825a377d2a87e2
F20101203_AAACME peter_s_Page_01.tif
13060b9c7749e2bbfc30a6b5f4392cea
9fe106bec5ecb5e77e23f6b049aebc878b490ef9
1051973 F20101203_AAACHH peter_s_Page_17.jp2
813d6c2a58e9c48a5ec358587bfa1c32
05b641cac14d87ee2d2769dcbd428693ea9e95d4
F20101203_AAACRB peter_s_Page_38.tif
df16084297a260075c1d4548edf1e377
d5395130e4648e06f08a6e5ffef6a0aab9625583
54027 F20101203_AAACTZ peter_s_Page_12.pro
775f7bc17fdea23d6baa2cc0e86643d9
02a1be316bc85a3c2c3cc36b7a54ed275e9c9c8e
12559 F20101203_AAACMF peter_s_Page_57.pro
bd062680747593a7fc2c290e85e8c956
21f2f63d117ed2446f7a68ec1638fcbf7ee702a3
26159 F20101203_AAACHI peter_s_Page_56.pro
cbf5a0845bc8d0700d76c9f4991c0a51
85be274a166bf01ac38999e3899ad33e83383610
53786 F20101203_AAACRC peter_s_Page_52.pro
52b42ba269966d322e2a11c98a58d168
823b4faade509376dffc7b1d4ecd9f773ad8e58a
9068 F20101203_AAACMG peter_s_Page_29thm.jpg
78eb18989e4afb8b19df958efcf157c2
4935c1026002d8005c784ff5ce9b51d31494ab13
51837 F20101203_AAACWA peter_s_Page_23.pro
b7d444e6dd82ab602878b6188e3d5712
51ea106bc6cf2e1800638e0ac7e108a5eb978800
F20101203_AAACHJ peter_s_Page_37.tif
7d030f8695f6107a12dd822af67a7275
ff3d1baceee54bb4fed0995bb2cf7985a7d1083f
30093 F20101203_AAACRD peter_s_Page_22.QC.jpg
69c514b6754348f5a968425d2ae9e44c
e72b663b5b29a292173647831f18cdc77b862509
596628 F20101203_AAACMH peter_s_Page_07.jp2
d8aeb8861ad2e226b95e32791fb0f388
744af2b8a00a04b4db153418ff85e486194c8e79
9065 F20101203_AAACWB peter_s_Page_30.pro
eaab74d913055a29d84c3110e275c59c
ac79bfb7fac7096c29ea7ddd12c4a18a66a2831d
1051982 F20101203_AAACHK peter_s_Page_50.jp2
d3fec4082935a67ede8be73d8e4768ed
6b5c43cdde03ed645028d69ebfd927361811a641
F20101203_AAACRE peter_s_Page_25.tif
08c5942233e25a08a37e1930aeeae104
ba878d051eee120dce1460a8235b6bf94bd2ff57
F20101203_AAACMI peter_s_Page_17.tif
b8a65db4574695f35bffbb69c41d146f
d684b03112e9916515909c073740b96b7832204a
42188 F20101203_AAACWC peter_s_Page_39.pro
ba9ef2c5915a3b85a2457b51efe54a87
62784b743a18b4c4232d9b58fe9d907d4024bad5
52198 F20101203_AAACHL peter_s_Page_53.pro
b9abc4b0ecefc6d258596674d77b0a2d
0de2f7075ec1ae2040de07bd844598c50a1ce639
17952 F20101203_AAACRF peter_s_Page_57.QC.jpg
e67e85755c6e57b458f10a40deafec48
ecd6d0e3162e43e654eff9b6d0edd8fe538aca0a
25864 F20101203_AAACWD peter_s_Page_44.pro
f67a67819de97cae1b3e39578e029e4a
9c2e0879293c909c3b78ab181a9adcfd2720d257
597957 F20101203_AAACMJ peter_s_Page_34.jp2
836b10d20c256f88cc40229fe99b0701
f4c124ca771c9917a5da6fecfec5bf77344d8df8
45864 F20101203_AAACHM peter_s_Page_28.pro
f1a2d010e2b0a375877f6f52b4a0352f
8078515dbc91b6023cdb0f85e668fce412a1ff84
1051950 F20101203_AAACRG peter_s_Page_63.jp2
9c34153a47db7583c230d0b66b2d5bdf
2b42b85163220b36e17d3c6b96935519a7bc460e
46460 F20101203_AAACWE peter_s_Page_46.pro
f6edee3c182ac4440d60adce32fa81c9
c017c39126c0f1c4d53ffb1ccef5a12ea2a42989
1997 F20101203_AAACMK peter_s_Page_01thm.jpg
8d80b5d8a2b356bc830a293a614d7ff5
0a4126f0bff74f993317d30ee06f651d92abda3e
F20101203_AAACRH peter_s_Page_46.tif
4735c5c0a25654333f559134f7e1273e
d39784dce538fe7b8627ec59a2ad28473b5ec291
33166 F20101203_AAACWF peter_s_Page_49.pro
4255caf6ed8b2466c520a5094e93497e
2e2d0ee99a9eca5eefc559bac886cf07edc124b3
959054 F20101203_AAACML peter_s_Page_39.jp2
894318f4bfc54c8cd0983b646622b84a
e3d04dba1a0f1e7a88e34cdd331d5e51edfa6016
26394 F20101203_AAACHN peter_s_Page_63.QC.jpg
31499ff64885f1c43e0bfebd6b105e6b
7772e45345c0458219b47e905543423c93771dba
2462 F20101203_AAACRI peter_s_Page_43.txt
e21094717d4daa06178f636b7d9fc863
4e6bc84def0d52ed84c6c31bdfeeeeabc30e5d69
23820 F20101203_AAACWG peter_s_Page_51.pro
57cb0388ddfe617cb3c4fb0cba892731
585e42ce61331426d3562ec75144a9db54d10ed6
737250 F20101203_AAACHO peter_s_Page_36.jp2
b0c4a5e6337c6fdf3a3b7f7b2029bc60
126c82ed8f05a9cb5e7cc8e0ea9a2f3616275a55
F20101203_AAACRJ peter_s_Page_55.tif
2c194e078853e8b58b5909abda005a9c
9795df649c4fe298d0fe531f2deb340d6b665237
35387 F20101203_AAACMM peter_s_Page_25.QC.jpg
a492359852ff106ce69884750029b33f
53574dd2bb9fee982cb712d405139e17bccd46f1
54609 F20101203_AAACWH peter_s_Page_55.pro
35f6ad793f104552d8b3fd5127dff4bc
5f4846063404edb951c6181856ecc7641c790c14
9153 F20101203_AAACHP peter_s_Page_43thm.jpg
0d641829667970bef07d51686f931653
78708eda22dbd5eb561d98e9e060d2c4f4a34b5f
1920 F20101203_AAACRK peter_s_Page_42.txt
6d2105739066131b089e8096d2d7fe97
f38cab056a1843f0e7d93690fc5f75c8c35fdab3
5146 F20101203_AAACMN peter_s_Page_60thm.jpg
7b646f9885b920e017e79e4e94645fde
c58c97d1ed4223f5ca45bde2066afc00b0afbe70
13564 F20101203_AAACWI peter_s_Page_59.pro
52faac8a3ba3931a9ba031fa8ebf8c8f
5d420078333aa314b183933d6bab0255ec9096f8
100236 F20101203_AAACHQ peter_s_Page_27.jpg
163122daa7ac3c2d45b07e37c89674bc
21824ccb9fafc1f9e7ab202ee9f38dd69ffabb29
F20101203_AAACRL peter_s_Page_52.tif
5ea37754cbe1c0f5c7aaac693db29087
104c7f362a6c410ec0e40cfffcd971cfd550c9c8
608592 F20101203_AAACMO peter_s_Page_56.jp2
75b85a3bcee4937053fbbe91984324f9
d48b5269820ba69787d62cf86969ba406bd51b8d
41870 F20101203_AAACWJ peter_s_Page_63.pro
6b2acb4cd3325cc7864b6e665d6a95db
da481af4de267f0a4c576d70920f8a70d11a7a9f
8786 F20101203_AAACHR peter_s_Page_32thm.jpg
f61a37c7ca27e08db89bcb554dcf2259
0098f1416dbb2511afd3be9bcec0b31d4d29cf89
37403 F20101203_AAACRM peter_s_Page_38.pro
f14472b6464f2a2ad975124cd7bace78
60440cf81d79a7018b592e7793b2318e4a276d5c
7870 F20101203_AAACMP peter_s_Page_18thm.jpg
6322f1a8c75a281224989c1373843ba7
c0125a5ce26f8adbe0a823f21c7d10188d1859b4
126 F20101203_AAACWK peter_s_Page_03.txt
50f561e75a028240719c5f630f280ec3
14ed0c0b7838adeba67ccfd2605310b857a6ef22
27151 F20101203_AAACHS peter_s_Page_11.pro
795e58f9b6d18dd651c2633aefbaa6e7
1ade9f018e2ac487bece3fe9fda365b081433bc6
505 F20101203_AAACRN peter_s_Page_02thm.jpg
c17fb58d4b4bc838b99292b6d7de84c1
73e7945137ba338feaad8e1da904243fc372f3c1
46389 F20101203_AAACMQ peter_s_Page_26.pro
9c5555c90970165d8ebd8661d32c2d07
7a3c4b69e66ecbb2aae8d30cdc56f052a3cb9177
624 F20101203_AAACWL peter_s_Page_04.txt
1318b4c67e9313d32121da195fc335a8
4a781df5615634f5fc0e81a722dc947a91305e19
F20101203_AAACHT peter_s_Page_56.tif
4b4cd0f528d14ff5a872e0451eacabdf
128799b9d4f74a31f771905274855bc763d71bf2
1097 F20101203_AAACRO peter_s_Page_02.QC.jpg
4e39b15b2fc9fa99bdfb04f12e17c9cf
e8eecc63ef203c725541d9b04882d24b09afdd97
8133 F20101203_AAACMR peter_s_Page_19thm.jpg
dc58a06247c9d3232bc95727d5105d9b
abb2ab2ef68e2cfaa2d18c8bae7088247b053374
1994 F20101203_AAACWM peter_s_Page_14.txt
ecfee8ecbb4ec961aebc74bef38f4e91
7fc7b902845e8f50d855c2218be1fe2bba21333c
90673 F20101203_AAACHU peter_s_Page_10.jpg
445bfe086bc7a47685a8139618dd6304
6995674ad12d36fb2434cd2d02cd1eb73d49e691
F20101203_AAACRP peter_s_Page_45.tif
0a235c2b79c26da00ee6b7c624a011ec
8f2e5e1785cda9111619762b55a43a33eeef8c9b
1858 F20101203_AAACWN peter_s_Page_16.txt
3d4104589d4fdb93abb20df9bb7714f2
92ed93424516b1b6bb80a1f5043b59f7cea0000b
53131 F20101203_AAACHV peter_s_Page_57.jpg
6a69d19706ab229e18fb2e91523e7126
284b56a3d58cca68fd5a4e9e64d89c26a6c8665c
32947 F20101203_AAACRQ peter_s_Page_15.QC.jpg
60795699f122fe41dece7556a1487929
95bf93e10c80fae5cc825597f080f50c09842331
9101 F20101203_AAACMS peter_s_Page_24thm.jpg
98d53d1959e17d5927a341d51bc31df4
bd0bec08ccaa107e9c551e4abfda3de0e5866852
1119 F20101203_AAACWO peter_s_Page_21.txt
ca7361e5276f9c86cd8e81d68b038ea0
95a882a362567bb675608b639d7cb82d3c510b50
51558 F20101203_AAACHW peter_s_Page_15.pro
ffe9c1da49da49ab5c5b30f746c1a2ec
8803a42d52ba72d41636812d61764ac2487b53a0
88151 F20101203_AAACRR peter_s_Page_16.jpg
40b7fcf34030defe15f2ae883cc3051a
ee31df8c2512045cfb3ba0e1b6c871985898c8ef
108473 F20101203_AAACMT peter_s_Page_55.jpg
3b742e0133f0c9bca5cab48e6898c86b
48e738ea2bab2c305d546b7514b1c407dc7b29a6
2112 F20101203_AAACWP peter_s_Page_25.txt
007febafd51dbe8048fd98b78f717f13
f87c5ffa23d708dadd1d51127cf9994f5eb60eea
91808 F20101203_AAACHX peter_s_Page_58.jpg
53b7d3bf5faeea63849b30b31cbc0157
f7b836114e29e512e42e66fac72ad8d4604ec319
F20101203_AAACRS peter_s_Page_58.tif
dbd31bc30ea5ebd7f580e0ecb23b5c92
c1093e228fb4be5399803c6b501f9db629635063
8596 F20101203_AAACMU peter_s_Page_31thm.jpg
55050c6b55f4827ad92a1f7ca1ada67b
d18200ccf80e3bcf6d31e179b13a73a9efd51e1d
2342 F20101203_AAACWQ peter_s_Page_28.txt
50571ca08bcef38981fde00f9a60cde5
755f099ae15003d3d0c24110b0c212cc87d58d7a
27156 F20101203_AAACHY peter_s_Page_10.QC.jpg
e730ed008c7063cb90f36dc66036d8e5
621a8f1f2732f222dffe0ad742e86aeaf898a262
8871 F20101203_AAACRT peter_s_Page_50thm.jpg
f25be46da8cef36dadaeb985fbf8acbc
94f0fa426b2126599691919d640d4dc82216ebf6
16512 F20101203_AAACMV peter_s_Page_51.QC.jpg
2a0a5d6dc8ac516fd2ebf431bd8fb6d6
bc1284d3cc62977bf63ded321580820fd3dc7c20
1594 F20101203_AAACWR peter_s_Page_38.txt
087c011ba64ec1593033495240d64d0c
e3006d26e3b4c2f5a857425a7a0a8231871988eb
2240 F20101203_AAACHZ peter_s_Page_17.txt
ca9df3fc38bd608ec213254242b35022
eb917476493150b2cdbd779ef98ef5fca5031ea8
34147 F20101203_AAACRU peter_s_Page_17.QC.jpg
b73bcf1dd341868dabeb1c6b4411b252
8e5e807657875270020b0742bb75a0c9875b7e5a
8651 F20101203_AAACMW peter_s_Page_48thm.jpg
4847997418d69c7480b6e4f94fe1379d
307f2c37341321350a664eb7c7b483ca443b3460
2235 F20101203_AAACWS peter_s_Page_46.txt
a1f4b6793a9725b7fce38f184ab31122
32e21b92f7275d8a5a03ed4212d69123b6c511d3
708 F20101203_AAACRV peter_s_Page_03thm.jpg
11f5e22613de3b05a0774a8d58cfd272
1d31b1d1bfaab3dbff9d7216cf44b0276ff84a40
121622 F20101203_AAACMX peter_s_Page_05.jpg
1f732fe3825d8211aeeac5d8d2fa4bda
2730b6f7de56735b60827026e10ee7396d2881a8
1253 F20101203_AAACWT peter_s_Page_47.txt
d0236b0bddabb844f9adb879fe81f8ef
92071ad89b7e631ffee4c707331d75d6140a985e
2217 F20101203_AAACRW peter_s_Page_12.txt
e972742e124c1f0b829ccbf785381bf3
1bd86675d808e56e0187f45c0d1941c6cddef457
11164 F20101203_AAACKA peter_s_Page_04.QC.jpg
be5da78dbecc5bcd3b09e95b80b5bb3d
a8ea12a5b8dea746a30b2194b5ec2252431c1359
1323 F20101203_AAACMY peter_s_Page_56.txt
1a59717cd933585c39af9e68850935d3
bfafa339eb82716b31d65571ccf3ff1f978ecbb6
2279 F20101203_AAACWU peter_s_Page_52.txt
fd22eaba2387c5dbae9b4d951f473c10
cd482fcbc732c634dbfdf549d8e660cbde52b73d
47685 F20101203_AAACKB peter_s_Page_19.pro
84f0798d9c58bef72108ed19e0171bd6
4acf72e16f18d931aa79983f86934cf89a3a2c18
1053954 F20101203_AAACMZ peter_s_Page_16.tif
03e61e9c75593d2fd4897b239dd63e1f
81f86d75b63c5779b903b4e16e54a7f587e0e015
F20101203_AAACWV peter_s_Page_53.txt
6e2362d7672a11d3a84b65871707878f
05708e3ede15537e76763776b49d812f40431caf
100921 F20101203_AAACRX peter_s_Page_14.jpg
fa6846822f3737a12e567c5f7c1d7f87
6894ec82d57153f55ba1acb2f2f00a5789aee4cd
8505 F20101203_AAACKC peter_s_Page_17thm.jpg
54d30fcb751a5db92b1671059bde76ae
8f5d994ac6b19a0192f6e55cee436f2e1edc7aed
1691 F20101203_AAACWW peter_s_Page_58.txt
95943645dfa2be02bdda9c733f948e04
c4638cabccf80cc66ba81b429efca1866b51dcdc
F20101203_AAACPA peter_s_Page_05.jp2
8b5fba437ae1ba6c6436a30634728765
1121eab06af63376949686b187775bb1305d4420
7921 F20101203_AAACRY peter_s_Page_64.pro
cb5ad2936379f3a09c6eddfd707865e8
a945da5676c4864f4ec89743bd5058468b7791fd
F20101203_AAACKD peter_s_Page_12.tif
a80c16760f8f7152d978468cf2190244
7fb62dc5940af144b60ad8450a4720960e06bda3
353 F20101203_AAACWX peter_s_Page_64.txt
777993806e68445269d75927c11b39bd
3d8b484ac8d78e44b6b08e2fe70ad85d99d45b93
1051947 F20101203_AAACPB peter_s_Page_27.jp2
0d7e5085da3a11424243f5c3e73d1cf7
19e0be360a2ba726666d6e0fbae939d1e04a2a5e
84338 F20101203_AAACRZ peter_s_Page_38.jpg
a0091563385da2a8d3a6ed5a05d5d5c3
e26916f9bb102ec151180e91daf8937b7da0b3c5
388705 F20101203_AAACKE peter_s_Page_60.jp2
8c01b9f808b21ffb6d91f5c039b315d6
230717be574a108a5279f1babf297e520f379c4e
22074 F20101203_AAACWY peter_s_Page_61.QC.jpg
5250e6eacb18215ca25ea59a58a874d6
5cce0fcf3e02e13f19192ed0041844d1756b06af
F20101203_AAACPC peter_s_Page_51.tif
cca434ac63194e39eedf83c496a36bf8
a6e4561d8b680f04f42187b71fcfb85a8ba418a3
1728 F20101203_AAACKF peter_s_Page_49.txt
a403460bbe58c6086af745a3a2e6ceea
554358e91124cd75269dd0600bb1cf72a937dfeb
30386 F20101203_AAACWZ peter_s_Page_58.QC.jpg
2a922a87bd6265166b80f147b30ab936
ad6104468a0c8e0975868ff4a4f97fd34b6ac8c5
6911 F20101203_AAACPD peter_s_Page_47thm.jpg
22f54f677b94a2e992e92011395b60cb
fb7c7324b0c97d5203da4273747a4f7f0b0c9973
1643 F20101203_AAACKG peter_s_Page_63.txt
4fed70a7a29fbefab1500249bccd4db1
d3c6150da43507d5418cbed5bd5e1200dd97cb01
435496 F20101203_AAACUA peter_s_Page_57.jp2
0d59fd5caea8342666edc95fb6a173c2
f1d64fc9fceff6c2c20f68408678ed666f46d01d
F20101203_AAACPE peter_s_Page_39.tif
2298427dccd5476c9bf8bc8fc1cbbd26
329aa94a12a17c28cd00955cf9dd0b3aee57808f
F20101203_AAACKH peter_s_Page_32.tif
08a5e7186c8949223526e43e1180b55f
6cabc33ce3e985d33a87955efbecf4a1b61eedfb
9100 F20101203_AAACUB peter_s_Page_28thm.jpg
46ddfe70e13a182fe7fbd2dee2619e22
f2f99174d946fde0807456c846cf91ff2143cdd2
3897 F20101203_AAACPF peter_s_Page_05.txt
da1514ed9ed8884e8c722899610d42a8
7f4a65c2db3203bbc7c242017aba774d5b5bd463
34126 F20101203_AAACKI peter_s_Page_28.QC.jpg
c28b4245f046ecc231a40cfafba25829
b9e6c6e464a9bd63d534b009af0dea8ea3d274de
1515 F20101203_AAACUC peter_s_Page_41.txt
c7fa766086b25fd2e2a5245a1721c559
9c36c2eb9ca1a0953ee7f8c088bed9109cceb514
1051939 F20101203_AAACPG peter_s_Page_55.jp2
c2464d9e85d901438db6fa99f4065f52
1f65998357713dc05a5b79497286834c222d1e90
52067 F20101203_AAACKJ peter_s_Page_33.pro
1c29b65110ef13fed9e8fd44dd4796ca
2fb5474c02bca4674fe1117efcfa38d3355395c4
369 F20101203_AAACUD peter_s_Page_30.txt
1eca5c6a1ede40aa847849558d20f2cb
5488a1b5daab5a9c9f0898f2bce90ed0d49146a1
33123 F20101203_AAACPH peter_s_Page_54.QC.jpg
6e370ebc715e8c514a6bc6b643c5544f
38308eb350d0e933b09fdc4561300814d88d5a34
35507 F20101203_AAACKK peter_s_Page_43.QC.jpg
f40871f6c9f723d69395ba5bddc52497
5116a69e001004225dcaa81210f9aa7b275e79d1
75547 F20101203_AAACUE UFE0022265_00001.mets FULL
c4b93438e70e372cce5f4ddce312cf8a
a815b890b257f3b6bd4d8248d7aaf7ae1d903de0
1911 F20101203_AAACKL peter_s_Page_19.txt
c1ccdc81582a8a1d4668c786c690ceda
7164eee8a92203e52d65783558098b7db8796947
7872 F20101203_AAACPI peter_s_Page_62thm.jpg
9afa35860043821d2985a5e7e5cf728d
e00592dc99fe1799cdba5254472ec0464b255c91
27178 F20101203_AAACKM peter_s_Page_21.pro
8a8a80980f0290397128fa87b20385ea
75a53fe73a09231afbb28a3b5f13dea83cd55f7e
1051984 F20101203_AAACPJ peter_s_Page_32.jp2
38327629e4bc184c77a022a809a69dce
404959a967977d6ddb7b034471195069ee72c555
21716 F20101203_AAACKN peter_s_Page_34.QC.jpg
f322042f1f3cc06398301e25319d2e26
c6b7f1ea2ddc21a27649fd540ae517792ac31c3a
44378 F20101203_AAACUH peter_s_Page_06.jpg
d227d06afcc7096b752179515780ba50
dde75f32897b783328ba4def09dc8ff0dd0ba399
94936 F20101203_AAACPK peter_s_Page_05.pro
1c6d3df492a5d7c38cd0e36de6d36e43
2f5eb3af22d25e8d8469780e49a3ca06f373d872
28614 F20101203_AAACKO peter_s_Page_37.pro
b21a6dfa99a064c855dbd8a1c7900d20
ce65802f8a5d97d8f33040ac0cbc211657cb81ab
34876 F20101203_AAACUI peter_s_Page_07.jpg
8f2b53501107006a35fb3bee18ddf1b1
bc0de5e0e0688cfc80f52c0d5cab1e76173267db
F20101203_AAACPL peter_s_Page_34.tif
e1ace4d7b2dc91d2c1473f8d5b38e23a
cc38d4f0a0af644910879431f271aec153e0d3ae
2169 F20101203_AAACKP peter_s_Page_54.txt
7ec10218282288c2c0a83d43da656618
dc434d7a3191b8a449eedc9445390d3e94d129a3
105624 F20101203_AAACUJ peter_s_Page_23.jpg
5dfee0d6ec4f585f92bcdcbe3c7e1894
0111ef63ea9faffa4d2a8eb2630124c49a4e7f3a
2034 F20101203_AAACPM peter_s_Page_15.txt
db3e55413439f9f07d58951b4fb65069
6851add199ad8d8ea839cf59681b0a92ce5d168c
21282 F20101203_AAACUK peter_s_Page_30.jpg
c6061ca6af566d3b327d5fd7fc2d9f3a
825336d11cfe9d6b16ced594a6f0e4a7f9448703
1081 F20101203_AAACPN peter_s_Page_11.txt
052c064ebef4b3542e7e050b4a6c31d1
238858c41421d9aea90f6b40b533eac260041dae
7934 F20101203_AAACKQ peter_s_Page_33thm.jpg
1e80d9fc8d0248057b471862056e7af9
a520a1c4ac52563f5662ec35ebe39e1e340f3820
112150 F20101203_AAACUL peter_s_Page_32.jpg
ec9b0585a874b67299fc7717570dccaf
e6cda013b4c2ef42b5e9129ed2fbd544372df9e0
28260 F20101203_AAACPO peter_s_Page_16.QC.jpg
4bcf1afd2702defbe6b1d1c5e8e6047a
bbd5e2830988ab670a02c662a6ec53cea9018a3a
99945 F20101203_AAACKR peter_s_Page_62.jpg
56a0b1eab44e2ab715d5a61021b35f68
346ee02a8f567c77e34acf92aa30a18e2aaf9370
59825 F20101203_AAACUM peter_s_Page_34.jpg
98bb0257e520f22e0811eeec78c2efe4
942e18dbc88ee9054e387490bd7694330301affe
1051976 F20101203_AAACPP peter_s_Page_12.jp2
c7e45c60f3f23409b752e94b90385f08
25cdb4f6373f161ab64ab5d07a050ec5548605b0
1051955 F20101203_AAACKS peter_s_Page_33.jp2
ee2492e515ae4777abab23b88bf8365d
d1b1156d5a41dafe285041fca207267436e5a775
73823 F20101203_AAACUN peter_s_Page_37.jpg
bb7658a99140d79c3ce3224966032228
9c7fe8dda267afc9552ba328ea5aef0329613f64
882822 F20101203_AAACPQ peter_s_Page_42.jp2
d8c063fa3e1aebb10d89122cfa64e863
ad54c42504912b1b07f18ead9431ce0bcba7694a
55769 F20101203_AAACKT peter_s_Page_17.pro
d8602928e3b1e7d717ad2bfd87939cfc
916cd51b72ac704de4f72675b411deab0a393de3
95369 F20101203_AAACUO peter_s_Page_39.jpg
5710d687606a1e460b63d16a58a9d053
71615f71ff91cf5ee229494042ae4eac19ca48d2
F20101203_AAACPR peter_s_Page_25.jp2
ec61eec64efe5a5b1be02172fff70e2c
92f8f8f68ad646204d6670a7727d6d0ff2cee9df
F20101203_AAACKU peter_s_Page_27.tif
8382566d6e0d5aef4a0fd8d161c398c4
3ca02e8dd00b85780f2ec34ac34112373a8a829b
86468 F20101203_AAACUP peter_s_Page_41.jpg
277b7824a8b7c8b5592b3feb191b4456
a4d79da0af9fdabc0beaccb6d65b8f33ca8beca8
2830 F20101203_AAACPS peter_s_Page_08.txt
dd0be8efa486602737cf51f9e2f08d26
27ee6565b9b1b94c0f78b546a76398c440e3e26e
30411 F20101203_AAACKV peter_s_Page_40.QC.jpg
78e655b65c00d6a03d86571e71552fd5
d6fc6a6d0f8bd5707d1abd4a4184c6674b112e42
87403 F20101203_AAACUQ peter_s_Page_42.jpg
fb946f0287b37b9773fe451a5277a3e7
720855dbddd9220a8422eaf6842f857e50d9a4e9
871402 F20101203_AAACPT peter_s_Page_41.jp2
3d93f8593808c14725a31465ac713096
d849a30fb63d20bbee928c42c926cd40f5ebd45f
1414 F20101203_AAACKW peter_s_Page_37.txt
e7b0ac0e5444aa1149e7d93c1a019812
2943ffff288665d594caf56a6a1abc68eeca1c15
63846 F20101203_AAACUR peter_s_Page_47.jpg
20973186a3f2c0152895bbe9f3d2d208
883657761ad623ebf646a7d8c7aa616ec0aecdb3
2065 F20101203_AAACPU peter_s_Page_40.txt
d1d72f9484cd51d2313808fa0c293e46
7a453b0957b0aab7484b8db0349f9ac255e59ad9
20421 F20101203_AAACKX peter_s_Page_47.pro
20997b0baff393b807aa15fad4d169a8
581fa78b3a7a217542bbd1e88a3f4d2c04148eed
99131 F20101203_AAACUS peter_s_Page_54.jpg
7e2b552124accf61dccb34e7ea0d851d
b2365096e71f3b73131f57886f91e1f56b8fb94a
F20101203_AAACKY peter_s_Page_63.tif
d3b80161eccb95406d159778ff370441
df6f8c5a82855d18b035c6bfff9761480a67d037
63007 F20101203_AAACUT peter_s_Page_56.jpg
d9f152cfd28311d78b7816e3f6f83ca7
ca413f3b72d9feeeec497e908569711c342737a1
53565 F20101203_AAACPV peter_s_Page_48.pro
33a7e8c82c49304fbce333b248135dcb
80be95d5cbb4fe946db677425596f28bd0e17cd5
6665 F20101203_AAACIA peter_s_Page_63thm.jpg
a28117303795fd8c615e2f1aca30f092
5dcf2f2063b1d7af7f40577cb87721ffb59f11a2
616040 F20101203_AAACKZ peter_s_Page_21.jp2
7bafe4cad356eba2d7b30f9daef8b2cd
3b62f165414770dec3ede920ef49ea6ef1488ee5
218613 F20101203_AAACUU peter_s_Page_01.jp2
835dbcfa2a282188b5ea97932dfa8ed2
4d89bbaf157105f352d119756e1328147266908e
F20101203_AAACPW peter_s_Page_21.tif
457804fe85725f151d60b7eb4d5a8e77
46986c1f399383ad36a41384faae6e056d786c56
F20101203_AAACIB peter_s_Page_18.tif
c74a9b700ad0497acc66704bc2db1551
ac4482e7aa66b9dbcb42579ea22681bd667c4f2d
F20101203_AAACUV peter_s_Page_09.jp2
981f38dd2cf54477f7cb23759e73e590
c81c98021f78138f97ce6cdeb4b9ddfad8f62efe
30894 F20101203_AAACPX peter_s_Page_31.QC.jpg
e36d04914622f03c4ca545ee750892ec
787b56967b0228c139c1002a3299fcba36ef3e5e
8800 F20101203_AAACIC peter_s_Page_45thm.jpg
493574aea07d3257e74974812a198866
ae912dcbec1da4b35c72d975e95887c8c03f62d9
971971 F20101203_AAACUW peter_s_Page_10.jp2
e054ebd56251e2500fdc6153c1308748
fa1ac7a595858b6892b8b4d2e0b3e1bf3d09edde
7747 F20101203_AAACNA peter_s_Page_01.QC.jpg
5aa8138877d86e749aa4b3d83e7ae9fc
f6f2c13d515ecd512b39e58519d936baf7fde1e7
22974 F20101203_AAACPY peter_s_Page_02.jp2
8a7827de20093c09d9805fff64a48f04
40f6f211472de358730ca6afeb564905a24bf0a5
7251 F20101203_AAACID peter_s_Page_30.QC.jpg
641c38a09305fe4af6af23ec4e607c85
d84f835edfbff6a9c195bb4ee4a4e3e90f6416ba
597879 F20101203_AAACUX peter_s_Page_11.jp2
6b69f8f7356e349eb1e2eaa2ccba6e50
0600f0732ae21fc8d9331d2527c237fccad53371
8689 F20101203_AAACNB peter_s_Page_49thm.jpg
25d4d61171aae785002096484af56a4a
108c9cab3fb00a0f79c9b5a329e504871d96f78c
F20101203_AAACPZ peter_s_Page_42.tif
f3efa6f8121ee7fce8af0773a355295f
17b58068604a98bee59835408f7c34082e7b5254
75827 F20101203_AAACIE peter_s_Page_44.jpg
f8a60ca3c1fe6bd61c89b107d819a36c
98afe122221140fa26c5e2cc7a24d2e904a9e873
1051970 F20101203_AAACUY peter_s_Page_13.jp2
305fc74fac39ad8701fba402e6ab5a16
c7af16ddd699cf1b677c7f34c34a0bc90ff305c8
F20101203_AAACNC peter_s_Page_41.tif
4010d33891f552f9a030e091cb2198bb
60de52eb830a591627cd9a7c737f0b6af06acb99
28134 F20101203_AAACIF peter_s_Page_41.QC.jpg
b8dd742534eee3ca96c0f2d9a6743732
e10e99f2989ed8390c7fd193c371ee4fbff04abc
195555 F20101203_AAACSA peter_s_Page_64.jp2
e4040dc4b4b87bdb0a559b254b821403
cffcefa5af35b1a069bfd33bda5e1b9aa00c9ff6
1003855 F20101203_AAACUZ peter_s_Page_18.jp2
500789590db9852065c7638bc96462cb
c878bf531816d975f22191093ae758e3f63bbf10
90 F20101203_AAACND peter_s_Page_02.txt
ea3f505250761c09c348c088bace22fe
3607acf8ad62d5936722aef7f445fa5e9b6fc725
73275 F20101203_AAACIG peter_s_Page_36.jpg
619c3c20db7363a8f298e3757f761a2c
105ea61b1456fef7d0e188f6b15384a5a2aedb04
52924 F20101203_AAACSB peter_s_Page_13.pro
039f1cf30b79aa78f2ab43fccf9677ab
8c976d8f5eb13c0c68f1ea8bbe8dbaf6b76724f0
6193 F20101203_AAACNE peter_s_Page_64.QC.jpg
17fecbc7bb51842bcd6808c6d1fbab23
7e9885b6e7f71eb53f647d19687375c1a5f77477
3630 F20101203_AAACIH peter_s_Page_02.jpg
92d22a6d59f6f5495d0a50af9933be8d
7c783b26cf7a88cc416c69e6b064c7c932f9cfc4
8586 F20101203_AAACSC peter_s_Page_12thm.jpg
d465b0e9bc944b6a846d6c341e17815e
e099673c07276b547b258029aac78d1c29cf6740
F20101203_AAACNF peter_s_Page_30.tif
348e996fb4332057c55530fd31369ede
22169f0b9566237e2b7db42bd0f28a0e1c02160a
33181 F20101203_AAACII peter_s_Page_61.pro
a5e87d429e504447f94c5caf755d3c16
caccb7ef922a1cda055e72cdf5e030dc1f9d37d2
8218 F20101203_AAACXA peter_s_Page_44thm.jpg
9bd61f50601e94bd18318fbf23bd44aa
6bca2d9df5bdff3c358e162b0b040f0e9f0e146a
F20101203_AAACSD peter_s_Page_14.tif
04d620ec8ce72590d9296b03ee995974
8a38e6d472d70222b1f84cc7da8a58eef837c3ed
55904 F20101203_AAACNG peter_s_Page_45.pro
729364aaa44ef272adb931b2501f9f4c
01cfe6a4c13974decdc910d7c4609250af79aab6
16659 F20101203_AAACIJ peter_s_Page_59.QC.jpg
225912e4f0775ae9786818c715a3d805
07e61717c9d63691d1fa7377d325b0d0a743b5b8
7778 F20101203_AAACXB peter_s_Page_37thm.jpg
28ddfbf3b2997e2387531f1fea0766f0
2d520141e4d786e61d2ee1c5f294722cf50b0d29
F20101203_AAACNH peter_s_Page_45.jp2
798edebd17ae412679e14ea12fae7254
0e9e182c7f30c95b7334353fd695202309a91cc1
35722 F20101203_AAACIK peter_s_Page_50.QC.jpg
f208673221ecdfb298356f2287e00a73
e561d5029ab87d40734cc375650e49c72beae066
8001 F20101203_AAACSE peter_s_Page_39thm.jpg
69c0b4860f7469185c7bb48c92e00268
6d46a372688e47bc645eb95038f9f66d86b26040
30447 F20101203_AAACXC peter_s_Page_35.QC.jpg
b7e01450b7f866615610e5bbeae50d5d
0890001d8bbb09d28aaf4fbdc5a2a6e2e9b5c522
763802 F20101203_AAACNI peter_s_Page_06.jp2
9d4dc7501c70fe581ccef34fa552cf65
d515faea3bd6b3b8b827e7815be7125f6f432e71
653 F20101203_AAACIL peter_s_Page_57.txt
b52d37c40b42c6bfd5ad02e00ea596a0
12709768311b6df7fe050e3acd8d98ddb5e49fbc
98349 F20101203_AAACSF peter_s_Page_33.jpg
4e00a1a37f6bdc4890425f22620ff803
b7db56028d09b0845f2edaf6fa6657b5ced3624e
8322 F20101203_AAACXD peter_s_Page_53thm.jpg
8dd102cfef6b147873fd6a04df52b898
50c93a5e9016c99b275db2faf73cc87fde0f2add
2903 F20101203_AAACNJ peter_s_Page_07thm.jpg
45b15ff936952a1965b57d46c62f9e82
a4f7d497fad289f23cd038b2c2e1fe9455d7df99
4697 F20101203_AAACIM peter_s_Page_09thm.jpg
866cfa5dd73afa0893aecf86e0459b4f
cc93ffec31e8abf0f058969c6dc9bf80c37227cf
102296 F20101203_AAACSG peter_s_Page_53.jpg
55b55a0ec03889c12b2cf6a055f015a8
3fcccb705f6a6f9fda1d8371f9be89691618d0e8
29184 F20101203_AAACXE peter_s_Page_42.QC.jpg
f235f63e6f21d31c12071886da0df1c8
680701499f5f4cb883fea27d026d380bd5d2141d
F20101203_AAACNK peter_s_Page_15.tif
1956dea70eec6af393e60de4a228fed7
6cdecd0c068e2ae9675572b19aca8f608c232830
F20101203_AAACIN peter_s_Page_04.tif
8ecf1a700a5074869925ced8a810b5f7
73f0d9e01c5a52e462b8a461ff1aa3df444d83f9
2208 F20101203_AAACSH peter_s_Page_45.txt
5e0e4de462221b1858417ecc26a853e6
67670f058922c139e16751f40f11ff6a91ff3aec
10593 F20101203_AAACXF peter_s_Page_07.QC.jpg
438c12d81a354b9e94b7705751754c5a
7c29eacffe6083f01a94eaa786c36f7855918660
34386 F20101203_AAACNL peter_s_Page_13.QC.jpg
75116135c169cb1407579a1a191062b5
11a10f3d078f5015ef689b6cdf5d7aa7460e3827
1765 F20101203_AAACSI peter_s_Page_35.txt
ab73f4ba7b8606f839b3505040e66b5a
c3308786a11f38ca26b714a2f247cacebebbf421
15979 F20101203_AAACXG peter_s_Page_60.QC.jpg
7c83686e0420c6eb5220179286170e6b
238a692ccab48824f793b8cb56c9fdfd3cd0c24a
2047 F20101203_AAACNM peter_s_Page_36.txt
b00d58a9e00650aedb38e7af4493cbd5
f07a0f0785d8b52137f17a4c48f4f70db896acf7
F20101203_AAACIO peter_s_Page_08.jp2
c3f38f6577c91b1ffec50d694f8c266e
ccc1a1aa5a22e2f3e483c82982a2fe42caad2acc
F20101203_AAACSJ peter_s_Page_09.tif
5659830d439af217d5f5be224df41a0a
9216e145bb0f847f3ea3906d0487396f84fc7e2e
8805 F20101203_AAACXH peter_s_Page_35thm.jpg
70a7df6b6cfeb342d6632dd8ce668eae
a243aed28e0e45b2ce3dd21d73989b7f7fe7d732
22907 F20101203_AAACNN peter_s_Page_01.jpg
02a7752e578bf0d3b8f9aa946f45bd8a
3fa5c57384eb560a0ee49781639f7d81bebdcfc2
F20101203_AAACIP peter_s_Page_22.tif
126d943e34dc6d2877114ad60d410b9f
c5b90f11482e2a353e5a6f32065d9528f1f462f1
1051945 F20101203_AAACSK peter_s_Page_22.jp2
5837b7766c5aca5041616c85e1f09710
7f755eb243f61593459ec37bcd524a508b55ae0f
4251 F20101203_AAACXI peter_s_Page_51thm.jpg
63ef94cd9790a333427fb70288d3fd32
54666bf93b5a6e8ae7014cabbbb367f2673d799c
7426 F20101203_AAACNO peter_s_Page_01.pro
56f265e391e0d80d40e6bb0474cdb0a9
8ee6d27d767156aab040247e18e087d2ab40c04f
F20101203_AAACIQ peter_s_Page_15.jp2
467b310d5991f257e5be8cea6205d56e
5632194cb296486f1794762726a87b82e3248f70
695580 F20101203_AAACSL peter_s_Page_61.jp2
2890638134fb3ecb8ea62fc217606b74
d1df4de2355f457dd355efbe4c848e0b01fa0ede
9357 F20101203_AAACXJ peter_s_Page_26thm.jpg
641af8330afbd60499db734b0b537276
fd069eee408bea2fadaebdeae556ec118fb8f58d
2267 F20101203_AAACNP peter_s_Page_26.txt
24b0a1876432b6550294903c0353a256
ac900221506a7a23437fcf38ef470f175157ad27
32241 F20101203_AAACIR peter_s_Page_14.QC.jpg
8e6df5ada74feeb70288d54c4b92cbaf
a0d64a84c29d2afba9befb01ebe3bac2fcad4a51
1772 F20101203_AAACSM peter_s_Page_64thm.jpg
269c44ff9a46cfada92e15b4f02cd687
d6e4cb168a5b02f19583f8e4496211d3906a3965
34970 F20101203_AAACXK peter_s_Page_26.QC.jpg
0b35f9d0e96aae3c776f26f0d460f3a9
a293d91956ac93923bfa0b217955cc639dc4cf69
7692 F20101203_AAACNQ peter_s_Page_42thm.jpg
d7eadb6e414419a60645ccec88702abc
6a801f78ac56c9c9e6af46a0086b5745b3df572c
108706 F20101203_AAACIS peter_s_Page_52.jpg
9ba4df707c6b52d37e9b4ec5d06a946f
0fd11b1b26bda3c5a9593c71fb3f2bc1a005c358
106055 F20101203_AAACSN peter_s_Page_26.jpg
fa0ffea4a5e7204cc10bd2f7ee852085
1654771e2466107ea74cea930d9f9fe987ef7f5a
5256 F20101203_AAACXL peter_s_Page_57thm.jpg
6d5d2ccbfb52b44b1f899000580e9395
ecf8fde7f478e76fc117f71d4f6e665b5a4ecf00
F20101203_AAACNR peter_s_Page_26.tif
5d1a03e24ab0fefe7179b85d0654d486
d98e7c13db49dcbfe73f1b3377530dc16b5b2c95
1776 F20101203_AAACIT peter_s_Page_31.txt
dc656bb7342f1fe324229fa47ff320c6
6852e9747ac7a593418793950507807718710604
F20101203_AAACSO peter_s_Page_20thm.jpg
3dfd6d66f3de644617a4b8f890fe6eb9
bab4b94e3a50be511a7c57b5a010fbcded6f273d
9072 F20101203_AAACXM peter_s_Page_23thm.jpg
b74c5950374b46183c5941be287f41c7
82d0b3e3e870ca4a25eef2fd87f3aaa814f8ea25
899602 F20101203_AAACNS peter_s_Page_49.jp2
2c61e7e5602f95c8d498d4f5b00fb4fc
99049a0fba0533521fd3d3a70bd827483164aafe
F20101203_AAACIU peter_s_Page_10.tif
ebff53fc437c659121dc8298a883d741
96675fe380eb40d9d85b7bf280ef326302e7f3ae
42339 F20101203_AAACSP peter_s_Page_36.pro
c4f6a6df8932e77c78156fc8fb28948d
be9014725da67aeaaf6bee3d227e0d2bba97562e
34747 F20101203_AAACXN peter_s_Page_23.QC.jpg
5e7e2e86f6a610db2d6a7ceaa8f2d7df
c8d41d9a01d79935f5e98e29f040c7f1596b0421
1310 F20101203_AAACIV peter_s_Page_22.txt
91b9af2877270070d070702a870afe7f
96d261cb003d42d63944417e7f924cf3cc919242
108654 F20101203_AAACSQ peter_s_Page_48.jpg
e8c710510fb9fb40c38c6097eeae5b94
661efbbce3a286032c75abb970941f50d3ce6a1c
97552 F20101203_AAACXO UFE0022265_00001.xml
9be0fc3387569ceaac1e09198d493db9
96e8c1689cdea689f39a3e1c0b677f3da20edfb8
32047 F20101203_AAACNT peter_s_Page_19.QC.jpg
f47af01614070f5710e715e8871f5f0b
94442cf90733e99633c016ba4df51e9bb30cd44d
2025 F20101203_AAACIW peter_s_Page_30thm.jpg
96415e3c9ee52fd85261adc0bf8199fa
fc30d3dfe52ecc46f492198b782a19bb67e72209
19101 F20101203_AAACSR peter_s_Page_21.QC.jpg
85520bae1f0b8e88864f91fa69f13643
3543c414eec831613a45297424101b366f1ac4dd
2800 F20101203_AAACXP peter_s_Page_04thm.jpg
990ab3c064a576c83ff752e478d1939e
1576a17ace823178652d30c6edc07aa8d396d1cd
48299 F20101203_AAACNU peter_s_Page_60.jpg
2e1ce7a1af62470cc93fa344f6516c55
80f78b6d8d3ce70cfa0722eb728e07269695dd32
2431 F20101203_AAACIX peter_s_Page_33.txt
32ba708f6f4565aafe7879d5d25acc29
38fb3a7c3e0308b5acfcfb0577ec40c72b992ae1
108909 F20101203_AAACSS peter_s_Page_24.jpg
b2a147acb13466c632b46aea2f431fa9
d6050eb94d064e9a15c7741a831533369185b63d
31698 F20101203_AAACXQ peter_s_Page_08.QC.jpg
d4441978442cfb37db5bc053e40fe9da
64e7eec612ea3302d8915ebdff14f1370ea79be6
47191 F20101203_AAACNV peter_s_Page_59.jpg
8207c1e51c018475391ae52f467252da
9b2309d5477c8e64252a5ad6eb92756e39d7f93e
2153 F20101203_AAACIY peter_s_Page_55.txt
d418aff3c405b3d99ea769e154168103
ca1620a427a87bf5f46007c04461b373cf8b88da
1667 F20101203_AAACST peter_s_Page_03.QC.jpg
a7d0780f417444e54c6e9381bfbd74c2
97d207d4db669b049a6aee7a6dd53a5a609caf1d
18467 F20101203_AAACXR peter_s_Page_11.QC.jpg
ff834451353912274a28e81316692878
d203d4232599296664bf630bcb62403174198575
6084 F20101203_AAACNW peter_s_Page_03.jpg
06f37b34afb160d73ed02d5fd9e0512e
1ae596500316b36aabc9f3b96581f611ab74de78
1374 F20101203_AAACIZ peter_s_Page_06.txt
3632efa4c217fcd7d7a168c765c41d04
288a9354cbbf8dea820a1eadd2ad98cd7a663e93
8605 F20101203_AAACSU peter_s_Page_13thm.jpg
8609cc9b1413e2d1cc76e0e1b38896fa
d237781ae1c353a00b87c5622bd8cf1f89c3c87a
8210 F20101203_AAACXS peter_s_Page_14thm.jpg
9218c7646ea17b0f7f68117cfc2992ff
46d56041316340fa4a37d7467780f3fae339563c
27395 F20101203_AAACNX peter_s_Page_49.QC.jpg
08f2354f8396c3ea6c9927cb742f37ee
3291cd597ca6a43eb3e5a1793d4565a188002e01
210902 F20101203_AAACSV peter_s_Page_30.jp2
46b79df2cdf2e929f86c68162ecda56a
ca4993762f202e15a734d8edd34b426a80751a9f
8290 F20101203_AAACXT peter_s_Page_15thm.jpg
10a23405bb6bace36e8e1169f0152d9c
b61444008deaca66188e0e3f9583c065884ecaac
36053 F20101203_AAACNY peter_s_Page_24.QC.jpg
47623fcd7d2da8f2e4f248f3880547e9
9ee61827fb3849af5fb316045c89cbddec15347c
25966 F20101203_AAACSW peter_s_Page_44.QC.jpg
0f9fd9a5087ae2369738ccbb3d020608
ce7b0ba4e2ed5ed4ebc67e89fff4bface1eb7ae1
35659 F20101203_AAACLA peter_s_Page_12.QC.jpg
7ea4c462f44288732a7c6cfdc6cefe95
ac4fba204f293476b5d2611e1df58808ec22af1d
30286 F20101203_AAACXU peter_s_Page_18.QC.jpg
1e9b18e22560441b6715d29b83fbdd25
ba826d5efdbd53473fe41df833788990017d6464
110601 F20101203_AAACNZ peter_s_Page_08.jpg
0360f4c82a227dfbf51e7adf7ee3d1cf
fa4925af5f77b18befa7ec19c18acdf8e8a31a5c
10493 F20101203_AAACSX peter_s_Page_06.QC.jpg
416673b881e034903df62c15144e3d68
9c5ae5773401c76e287023bb7ce1573d3effb569
1023099 F20101203_AAACLB peter_s_Page_31.jp2
512384d08953e61a8de734a006be38d2
9c31515a7a362155d15f7c182ecb86d2e2096b06
32907 F20101203_AAACXV peter_s_Page_27.QC.jpg
26e5efafbf548703e607029f63a81c8c
2d8d6f0cf85972f282863ba42b50e597806183ae
309270 F20101203_AAACLC peter_s.pdf
9713883704a40da61f03b8cfc118ea7a
e9d8ff64d857ff202edfc6d2e30c4d5d46727951
34614 F20101203_AAACXW peter_s_Page_29.QC.jpg
c081131b552a61ec19419d51ab7bfcdc
2d4069a16ab5e96012b413bebc0c1f5cfb9265d0
6453 F20101203_AAACQA peter_s_Page_56thm.jpg
bf9c6bad763d9a9b9a359d3d3f49388d
ea098dd03f75d299ab045319c212b32add21ed09
90998 F20101203_AAACSY peter_s_Page_40.jpg
b1d936e9c9f87f870f3dfeda59f158e5
4fae3e5a13f92da4a36624dd1a1d89bdd2ec67d4
1547 F20101203_AAACLD peter_s_Page_44.txt
01a9851d122891da3aa54ed51c02e776
875c65fc5297d31061ae2a151f90c7ac41442238
32336 F20101203_AAACXX peter_s_Page_33.QC.jpg
fe3f85e3e221ad225afd1b690171740b
786e87a0a957e763b435bae52557d1c2c3850821
45267 F20101203_AAACQB peter_s_Page_40.pro
87649941df16ea001f8de4aa6bb6dc9b
3863e7b5048471e4148eaf02a2908ebf747250ad
39447 F20101203_AAACSZ peter_s_Page_27.pro
b882266145024768677f52b60a6ed0aa
e4f59b049192ec9e04790c05998c7d0950834b4e
105498 F20101203_AAACLE peter_s_Page_50.jpg
6aa4888c0cc32106abad317d98a867fb
3327e8e2f395e9c5813c19fd61eb20a52b0c2b44
24378 F20101203_AAACXY peter_s_Page_36.QC.jpg
3182c0a339bb9a9a1f11c2a3088c7af3
9c47b5bbc0c7c2e9401e99e23255c302bbf0ab2d
2032 F20101203_AAACQC peter_s_Page_18.txt
26c46c369ccf42d58607af5f27dc0464
42994ce6faa74df9173e643f7cf599dc0eb76861
2108 F20101203_AAACLF peter_s_Page_48.txt
995cff9aeea113c61922ad359a950e11
34ea2fa64e91458ef8d657519dbffdad21788df9
6282 F20101203_AAACXZ peter_s_Page_38thm.jpg
46ca4fb6d2fa4567af92f8411f434d4e
13263b0885c09661e43ee3dcc598cfce11cf7067
F20101203_AAACQD peter_s_Page_57.tif
3e91f847a37d37625bb1b0ff3067593f
7d137654d3a1a6e88dd222ff030d6ce151d9dcfa
1051958 F20101203_AAACLG peter_s_Page_20.jp2
c3c92936d0881a36fe5b8f131dd62ef9
2884bcc721708f20b0f0e57f25d0abf87c8bcd8d
1051942 F20101203_AAACVA peter_s_Page_26.jp2
9a25afe5f05c9ade6f53a9198e1b2163
a627dc1a4e67d923ae0c653a6f4558d2a6e0e783
35767 F20101203_AAACQE peter_s_Page_55.QC.jpg
e0647d4ff2ff63e37926b8f07f63c9d8
edb7717f997dbfdf83ed6fdc0cf9443aba09e037
8490 F20101203_AAACLH peter_s_Page_54thm.jpg
5c29f52e189bd8077da9bce0d475d93f
8fd4f359e8ee785690fdbc351ca9ba1fb525688e
1051941 F20101203_AAACVB peter_s_Page_28.jp2
fc51d72114124bab27c2670c65c082be
e908ee5ee6eccd76d4ca765a2843a64e2c3a3e1c
1455 F20101203_AAACQF peter_s_Page_61.txt
2d797febdff80c5a2d2ae24159015bf7
e5248e7e9a7d100f3461b7ecaeb9a2ddf1c92d6d
F20101203_AAACLI peter_s_Page_14.jp2
69e033b77dacfaf1e97a9b6528aeba28
92e4a267e8548ff4ac3ea0a0183949ed63c09876
856708 F20101203_AAACVC peter_s_Page_38.jp2
210fc629912d63faab035de3169dc729
b5097273283c706ba2358108ef69724f2a1e96d8
109564 F20101203_AAACQG peter_s_Page_12.jpg
75d24d143776361971822e2c91d40a5c
a0c4f7aa579fc13719c98bf252923338bd24fd45
18257 F20101203_AAACLJ peter_s_Page_09.QC.jpg
535bf9447077944c5ebf3908e1f3b4a9
886684528da05753d1a306c7d3fb2e0f6e5fdbeb
943180 F20101203_AAACVD peter_s_Page_40.jp2
398faec8e5c4203aa2e763b3f2f5c1c3
028746025e94de4b9eafd683766c7a61cae98220
102752 F20101203_AAACQH peter_s_Page_15.jpg
f9fa1bf17e55380d5a99f8d2d77a2f11
5ad30ac354fdd70f131cf69a70b763ce00ad1355
25421 F20101203_AAACLK peter_s_Page_22.pro
f878d10197ff7b2609a561b78a59dcbd
f78f59d3fb05d8b557fb4e36d58ecfbb19a86ded
1051974 F20101203_AAACVE peter_s_Page_43.jp2
dfb6cecdd5685734b1e99391763400c7
781fe81729ea4b7e1a9fc710bd85ca3d9d22cff3
1051948 F20101203_AAACQI peter_s_Page_24.jp2
be6154b3b623b9479bbbc37b8b827e1c
e8effef1459dde84bc20d762abb707af691d988e
53064 F20101203_AAACLL peter_s_Page_50.pro
50ef44164d82a2deddd1134a5a304421
90883864783e8a745b6a3b6e16c6efad60933755
765772 F20101203_AAACVF peter_s_Page_44.jp2
c600b9a684a0606d7cfa9d6c9147df60
2f700f172161de812fbae2cce2a6e0672910a1e4
30756 F20101203_AAACQJ peter_s_Page_53.QC.jpg
5bf609986f52ca9d457e6b88a47bfba2
02f0ba9d4b8c1f4d439da17724348c22b050be0a
F20101203_AAACLM peter_s_Page_31.tif
d269d4af67bc95af6e4f89d2eee2dc6c
1c6e0d02ce378ac974cb70548d9742886311a820
962442 F20101203_AAACVG peter_s_Page_46.jp2
16298b65669d03c06a975e63646ffa55
6e3d02a2922d05b0e374d0f742e419425b45affd







BIOLOGICAL DENITRIFICATION OF HIGH NITRATE INDUSTRIAL STREAMS


By

SHERIN PETER



















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008



































2008 Sherin Peter



































To my parents, Peter Korath and Jessy Peter, and to Marun









ACKNOWLEDGMENTS

I would like to gratefully acknowledge my advisor, Dr. Spyros Svoronos, for his

guidance and support throughout my graduate studies. I would also like to thank my committee

member, Dr. Ben Koopman, for his valuable advice through the course of the experiments. I

acknowledge Kiranmai Durvasula, for her guidance and advice in experimental work. I am also

grateful to Darrick, for providing industrial samples and materials used in the experiments.

Finally, I thank my parents for their endless support and advice throughout my studies.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IS T O F T A B L E S ................................................................................. 7

LIST OF FIGURES .................................. .. ..... ..... ................. .8

A B S T R A C T ......... ....................... ............................................................ 10

CHAPTER

1 INTRODUCTION ............... ................. ........... .............................. 12

Biochemical Operations in Wastewater Treatment.....................................................12
B biological D enitrification ......................................................................... ........................ 14
K inetics of D enitrification .................................. ....................................................... 17
Bioreactor Configuration for Denitrification .................... ............ .............. 20
Objective of the Proj ect ............................ .......... .................. ... 21

2 DENTRIFICATION OF HIGH NITRATE INDUSTRIAL STREAMS USING
G R O W TH B IO R E A C TO R S ........................................ .......... .................. ........................22

M materials and M methods ...................................... .. .......... ....... ...... 22
Setting up Bios2 ....................................... ...............22
Feed Preparation and Sampling for Bios2......................... ...............23
S ettin g u p B io s3 ..................... ...........................................................2 4
Feed Preparation and Sampling for Bios3 ............................................ ............... 25
Results and Discussion ..................................... ................. ........ ..... 25
B io s2 R e su lts ..................................................................................................2 5
B io s3 R e su lts ........................................................................................................2 7

3 DENITRIFICATION OF HIGH NITRATE INDUSTRIAL STREAMS USING
SUSPENDED GROWTH BIOREACTORS ..... .................... ...............31

M materials and M ethods ................... ...... .................. .. ..................................... ..... ............. 1
Setting up Suspended Growth Bioreactor ...... ................. ...............31
Sam pling and D ata C collection .............................................................. .....................32
Feed Preparation ................. .. .. ...... ....... ..........33
R e su lts an d D iscu ssio n ..................................................................................................... 3 4
C S T R 1 R esu lts ................................... ...... ............ .. .. ......... ...... ............3 4
A analysis of C STR 1 R results .......... .................................................... .......... ........... 35
Developing Equations ............................... ....... ........ ............... 36
A applying C ST R 1 R results ....................................................................... ................... 37
C ST R 2 R results ........................................................................... 39
C ST R 3 R results ........................................................................... 4 5









C S T R 4 R e su lts .......................................................................................................... 4 8
R starting C STR 4 w ith Sam ple A ........................................................ .....................50

4 ASSAY ON THE BIOLOGICAL TREATABILITY OF INDUSTRIAL STREAMS..........52

D ev elop in g E qu action s............................................................. ................................. .. 53
D evelopm ent of the A ssay .................................................................................. ...... 54
Results and Discussion ..................................... ................. ........ ..... 56
C onclu sions.......... ..........................................................6 1

5 CONCLUSIONS AND FUTURE WORK.................................... ................................... 62

L IS T O F R E F E R E N C E S .................................................................................... .....................63

B IO G R A PH IC A L SK E T C H .............................................................................. .....................64







































6









LIST OF TABLES


Table page

1-1 Oxidation-reduction reactions for denitrification ....................................................... 17

3-1 Contents of the micronutrient solution (Vishniac and Santer, 1957...............................33

3-2 Concentration after the first time period for CSTR1 ................... ........................ 38

3-3 Concentration after the second time period for CSTR1.......................... ................... 38

3-4 Concentration after the third time period for CSTR1 ................. ............... ............... 38

4-1 B iotreatability assay results ....................................................................... ..................6 1









LIST OF FIGURES

Figure page

1-1 D enitrification reaction s............................................................................ ................... 16

2-1 Attached growth bioreactor .......................................... .................... ............... 22

2-2A Bios2 denitrification and feed flow rate vs. time................................... ...... ...............26

2-2B Bios2 pH and nitrate concentrations vs. tim e ........................................ .....................27

2-3A Bios3 denitrification and feed flow rate vs. time.................................... ............... 28

2-3B Bios3 pH and nitrate concentration vs. time................................ ...............29

3-1 Continuous stirred tank reactor......... .................................... ................. ............... 31

3-2 CSTR1 nitrate reduction and feed flow rate vs. time............................................. 34

3-3 CSTR1 pH and nitrate concentrations vs. time....................................... ............... 35

3-4 Time periods used for CSTR1 data analysis................................ ........................ 37

3-5 Dilution rate of CSTR1 with respect to time.............. ...........................................39

3-6 CSTR2 denitrification and feed flow rate vs. time, part I............................................40

3-7 Ratio of C(t)/Cf vs. time for component accumulation in CSTR2...............................41

3-8 Plot of dilution rate vs. time for CSTR2................................... .....................42

3-9 Ratio of C(t)/C(o) vs. time for component deficiency in CSTR2.............................. 42

3-10 CSTR2 denitrification and feed flow rate vs. time, part II. .........................................43

3-11 CSTR2 pH and nitrate concentrations vs. time.....................................44

3-12 C STR 2 absorbance vs. tim e................................ ......................................... ................44

3-13 CSTR3 denitrification and feed flow rate vs. time .................................... .................46

3-14 CSTR3 pH and nitrate concentration vs. time....................................... ............... 47

3-15 C ST R 3 absorbance vs. tim e.......................... ........................................... ....................47

3-16 CSTR4 denitrification and feed flow rate vs. time. ........................... ................................ 49

3-17 CSTR4 pH and nitrate concentrations vs. time.....................................49









3-18 CSTR4 absorbance vs. tim e....... .. .......... .. ......... .............................. ............... 49

4-1 Absorbance vs. nitrate concentration, aerated sample .................................................56

4-2 Absorbance vs. nitrate concentration, non-aerated sample.................................... 56

4-3 Absorbance vs. nitrate concentration, synthetic nitrate solution ....................................57

4-4 ln(P-YNoSno(o)) vs. YNo*time, aerated sample ........................................... ............... 57

4-5 ln( P-YNoSno(o)) vs. YNo*time, non-aerated sample.................... ........................57

4-6 ln(P-YNoSno(o)) vs. YNo*time, synthetic nitrate solution............... ......................... 58

4-7 A bsorbance vs. SNO for sam ple A ........................................................... ............... 59

4-8 ln(P-YNoSNo(0)) vs. YNO t for sample A .................. .......... .................... 59

4-9 Absorbance vs. SNO for aerated sample C....................................... ....................... 59

4-10 ln(P-YNoSNo(0)) vs. YNO t for aerated sample C .......................... ............... 60

4-11 Absorbance vs. SNO for aerated sample D .............................................. ............... 60

4-12 In(p-YNoSNo(O)) vs. YNO t for aerated sample D.....................................................60









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

BIOLOGICAL DENITRIFICATION OF HIGH NITRATE INDUSTRIAL STREAMS

By

Sherin Peter

May 2008
Chair: Spyros Svoronos
Major: Chemical Engineering

The purpose of this project was to develop a biological denitrification system for high

nitrate industrial streams. High nitrate samples obtained from an industrial source and synthetic

nitrate solution prepared using nitric acid and DI water were used in the experiments. The goal

was to obtain a denitrification rate of 2 mg NO3-N/L/min. Attached growth systems and

continuous stirred tank reactors were used for the study. The reactors were kept in anoxic

conditions in order to maintain denitrification conditions. Effects of micronutrient deficiency,

volatile contaminants high nitrate concentration, and sodium on denitrification rates were

studied.

From the study, it was concluded that the industrial stream was treatable; however, volatile

contaminants present in the feed solution must be aerated out and metals must be removed by ion

exchange prior to denitrification. It was also concluded that micronutrients should be added

directly to the reactor in order to avoid its precipitation in the feed and deficiency in the reactor.

Studies comparing sodium and potassium neutralized nitrate solutions did not show any

significant differences in denitrification rates. Thus, it was concluded that sodium did not affect

the denitrification process considerably. In the suspended growth system, denitrification rate of









4 mg NO3-N/L/min was achieved with synthetic nitrate solution as the feed, and a denitrification

rate of 2 mg NO3-N/L/min was achieved using industrial nitrate solution.

An assay was also developed in order to test the biotreatability of high nitrate solutions. In

the assay procedure, a batch test is conducted using the test sample along with a synthetic nitrate

solution containing the same N03-N concentration. The absorbance values and denitrification

rates of the test sample and synthetic nitrate solution are obtained after 4, 8, and 24 hours. YNO

and lImaxNo of the sample and synthetic nitrate solution are obtained from the absorbance and

NO3-N measurements. The biotreatability index, BI, of the test sample is obtained by taking the

ratio of [,maxNO of the sample to [,maxNO of a synthetic nitrate solution containing the same nitrate

concentration. A BI value of 1 means that the test sample can be successfully denitrified, and

values considerably less than 1 show that the denitrification will be very difficult.









CHAPTER 1
INTRODUCTION

Biochemical Operations in Wastewater Treatment

Biochemical operations are used widely in wastewater treatment systems to pollutants

that can cause harm to aquatic environments after discharge (Tchobanoglous, 2003). Pollutants

in aquatic systems can cause low dissolved oxygen concentrations, eutrophication, and increased

toxicity due to organic chemicals. Pollutants in wastewater may be classified by their physical

characteristics, chemical characteristics, by their susceptibility to alteration by microorganisms,

by their origin, and by their effects (Tchobanoglous, 2003). Many of these classifications may

overlap for various components. The goal of wastewater treatment is to remove these pollutants

in an efficient and economical manner by utilizing various unit operations. The unit operations

can be divided into physical, chemical, and biochemical operations. Physical operations are

operated based on laws of physics, chemical operations are operated by utilizing various

chemical reactions to remove toxins, and biochemical operations use enzymatic catalysis of

living microorganisms in order to treat the wastewater (Tchobanoglous, 2003).

Most wastewater containing biodegradable constituents can be treated biologically with

proper control. The primary objective in using biological treatment in industrial wastewater is to

reduce the concentrations of organic and inorganic compounds, as well as remove any nutrients

present, such as nitrate or phosphorus (Tchobanoglous, 2003). Sometimes pretreatment is

needed prior to biological treatment since industrial streams may contain components that are

toxic to bacteria. In biochemical processes, soluble pollutants in the wastewater are converted to

an inert form, such as CO2 or N2, or into new microbial biomass. Generally after the treatment,

excess biomass is removed from treated effluent by a physical operation, such as settling tanks.

Microorganisms may also entrap any insoluble organic matter present in the waste stream after









preliminary treatment, and it is removed through physical operation that follows. Thus, the

effluent from the physical operation is relatively clean, and generally no additional treatment is

required (Tchobanoglous, 2003).

Biochemical operations in wastewater treatment may be classified according to the

biochemical transformation, the biochemical environment, and the bioreactor configuration

(Grady, 1999). Biochemical transformations include removal of soluble organic matter,

stabilization of insoluble organic matter, and conversion of soluble inorganic matter. Removal

of soluble organic matter occurs when the microorganisms use the organic matter as carbon and

energy source. Part of the carbon is converted to carbon dioxide during enzymatic reactions, and

the rest is used to produce more biomass. This process can be carried out in aerobic conditions

and anaerobic conditions (Grady, 1999). Stabilization of insoluble organic matter occurs when

the particulates are entrapped within the biomass, and is converted to stable end products.

Conversion of soluble inorganic matter is utilized in biological nutrient removal processes,

generally to reduce phosphorus and nitrogen concentrations in wastewater. Phosphates in

wastewater are converted ultimately to orthophosphates through microbial activity, and are then

taken up by specialized bacteria that store large quantities of phosphates in granules within the

cell (Grady, 1999). Nitrogen can be present as ammonium or nitrate in wastewater. Ammonium

is converted to nitrate in aerobic environments, and nitrate is converted to inert nitrogen gas in

anaerobic environments.

Biochemical environment where microbial activity takes place is classified mainly

according to the terminal electron acceptor during energy production. Three main types of

electron acceptors are oxygen, inorganic compounds, and organic compounds (Grady, 1999).

When dissolved oxygen is present in high concentration, it becomes the primary electron









acceptor, and the environment is aerobic. When nitrate or nitrite is present in high

concentrations compared to DO levels, the environment is anoxic, and nitrate or nitrite serves as

the terminal electron acceptor. Sometimes organic compounds, carbon dioxide, and sulfate may

act as the terminal electron acceptor, in which the environment is anaerobic (Grady, 1999).

Wide ranges of microbial species thrive in aerobic environments, while anoxic and anaerobic

processes are carried out by a limited number of organisms.

Biological Denitrification

Denitrification is used widely in wastewater treatment plants in order to treat nitrate and

ammonia, in combination with nitrification. Nitrate concentrations in effluent streams are highly

regulated by EPA. Since nitrate can act as a fertilizer, aquatic weeds, grasses and algae can grow

excessively. This can lead to eutrophication and depleted oxygen levels in receiving waters,

causing fish kills (Kanow, 1993). Therefore, unless the water is properly diluted or sufficient

flow of water is employed to prevent accumulation, high nitrate concentrations can cause

ecological imbalance. Nitrate concentrations in contaminated water have also been linked to

outbreaks of methemoglobinemia in infants, and various health problems in animals (Barber,

2000).

The standard for total nitrate concentration in effluent water is < 10 mg/L, or less than the

concentration that exceeds nutrient criteria (US EPA, 2005). Various methods exist for nitrate

reduction in wastewater, including air stripping, break point chlorination, ion-exchange systems,

fluidized bed systems, expanded bed systems, and biological denitrification (Kanow, 1993).

Most of the chemical methods used for denitrification can be costly, and the chemicals involved

may release toxic compounds to the environment (Lee, 2006). Biological denitrification is









comparatively stable and reliable, and can have higher potential removal efficiency and easy

process control (Barber, 2000).

In domestic wastewater plants, biological denitrification is coupled with nitrification.

Nitrification converts ammonia to nitrate, and in denitrification, nitrate is converted ultimately to

nitrogen gas. Two modes of nitrate removal can occur in effluent streams: assimilating and

dissimilating nitrate reduction (Tchobanoglous, 2003). In assimilating nitrate reduction, nitrate

is converted to ammonia for cell synthesis. This usually occurs when low NH4+-N is available,

and the process does not depend on dissolved oxygen concentrations. In dissimilating nitrate

reduction, the nitrate reduction is associated with respiratory electron chain, where nitrate or

nitrite ions are used as terminal electron acceptor for the oxidation of electron donors

(Tchobanoglous, 2003). The bacteria that are used for denitrification are facultative aerobic;

they are able to switch from aerobic mode to anoxic mode (Grady, 1999). In aerobic conditions,

bacteria use oxygen as the terminal electron acceptor. In the presence of nitrate in anoxic

environments, nitrate is used as the terminal electron acceptor. In the absence of dissolved

oxygen, nitrate reductase enzyme in the electron transport chain is induced, which leads to the

transfer of electrons to nitrate. Presence of oxygen can suppress the activity of the enzyme

(Tchobanoglous, 2003). Therefore, low oxygen concentrations and high nitrate concentrations

are preferable for high denitrification rates.

Heterotrophic bacteria such as paracoccus denitrificans, thiobacillus denitrificans, and

other pseudomonas are usually used for denitrification processes (Tchobanoglous, 2003). The

mechanism for biological denitrification is given in Figure 1-1 (Wasser, 2002). The nitrate

content in the waste stream is converted to an inert form. The enzymes involved in the









denitrification process are nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous

oxide reductase (Grady, 1999).


Nitrate reductase
I. N03------ ------------------ NO2
Nitrite reductase
II. NO2- ------------------------------4 NO
Nitric oxide reductase
III. NO ------------------------------ ----- N20
Nitrous oxide reductase
IV. N20 ------------------------------------N2


Figure 1-1. Denitrification reactions

Heterotrophic bacteria use organic carbon as the energy source for metabolism. The

amount of carbon in the stream is measured using chemical oxygen demand. Chemical oxygen

demand measures electrons available in an organic compound. The COD test is based on the

fact that organic compounds can be fully oxidized into carbon dioxide and water with a strong

oxidizing agent. The COD is expressed as the amount of oxygen required to accept the electrons

from the organic compound after complete oxidation (Grady, 1999). Three sources of carbon

may be used in denitrification: COD in the influent wastewater, COD produced during

endogenous decay, or COD from an exogenous source (Tchobanoglous, 2003). Generally,

methanol or acetate is used when exogenous carbon source is needed. Along with carbon, other

nutrients are also required for bacterial metabolism and growth, including phosphorus, sulfur,

potassium, calcium, iron, sodium, chlorine, and magnesium (Tchobanoglous, 2003). Domestic

waters often contain necessary nutrients; for industrial wastewaters, nutrients are added

externally. The energy source used for growth is called the substrate due to the extensive role of

enzymes in microbial metabolism.









Kinetics of Denitrification

Bacterial metabolism can be described as a series of oxidation-reduction reactions. The

substrate is consumed during growth, and additional cells are produced by binary fission, asexual

mode, or by budding (Tchobanoglous, 2003). In binary fission, the cell becomes two organisms.

The time required for binary fission is called generation time, which can range from less than 20

minutes to several days. In water treatment systems, the generation of new cells is limited by

availability of substrate and nutrients. The generalized equation for microbial growth is given in

Equation 1-1 (Tchobanoglous, 2003):

Carbon source + energy source + electron acceptor + nutrients ------- biomass +
CO2 + reduced acceptor + end products (1-1)

For a balanced growth equation, carbon utilization and biomass growth are coupled.

Therefore, the growth yield, Y, is expressed as units of biomass produced per unit of substrate

removed. When the reactions are coupled, the rate of substrate utilization for synthesis and the

rate of biomass growth are proportional, with Y as the proportionality constant. The actual

amount of biomass formed always less than Y, since a portion of energy is used for maintenance

purposes. This yield is referred to as the observed yield (Yobs) (Grady, 1999).

Bacterial growth in anoxic conditions, as in denitrification, with nitrate as the nitrogen

source and terminal electron acceptor, and acetate as the carbon source, follows the oxidation

reduction reactions as given in Table 1-1 (Grady, 1999). The general formula used for biomass

is CsH702N.

Table 1-1. Oxidation-reduction reactions for denitrification
Reaction for bacterial cell synthesis with nitrate 1/28 CsH702N + 11/28 H20 = 1/28 NO3s +
as the nitrogen source: 5/28 CO2 + 29/28 H+ + e-
Reaction for electron acceptor (with nitrate as 1/10 N2 + 3/5 H20 = 1/5 N03- + 6/5 H+ + e
the terminal electron acceptor):
Reaction for electron donor (with acetate as the 1/8 CH3COO- + 3/8 H20 = 1/8 CO2 + 1/8
carbon source): HCO3- + H+ + e









The overall molar-based equation for bacterial growth can be obtained by using the

Equations 1-2a and 1-2b (Grady, 1999).

R = Rd fe*Ra fs*R (1-2a)

fs+ fe = 1 (1-2b)

Rd is the equation for electron donor, Ra is the equation for electron acceptor, Re is the

equation for bacterial cell synthesis, fs is the fraction of transferred electrons used for cell

synthesis, and fe is the fraction of electrons used for energy. The negative terms are inverted

when used in the equation. The fraction of electrons used for synthesis can be obtained from the

growth yield. The carbon for synthesis is shifted to biomass and the carbon used for energy

converted to carbon dioxide during the oxidation-reduction reactions. When industrial carbon

source is used, the formula for the electron donor can be estimated by analyzing the elemental

compositions. Also, when nitrate is the predominant nitrogen form, the nitrogen must be

reduced from +V state to -III state before it can be used as the nitrogen source for cell synthesis

(Grady, 1999).

The equation for microbial growth is often written also with COD basis in terms of true

growth yield. The general formula is given in Equation 1-3,

Ss + (-(1-YH)) SN -----------> YHXBH (1-3)

where Ss is amount of substrate COD, SN is the amount of terminal electron acceptor in COD

units, YH is the growth yield of active heterotrophic bacteria, and XBH is the active heterotrophic

biomass in COD units (Grady, 1999).

Bacteria grow exponentially in favorable conditions. When there is a limiting nutrient

present, the specific growth rate coefficient of the bacteria, [t, will depend on the concentration

of the nutrient. The limiting nutrient can be the carbon source, nutrients, or other factor needed









by the organisms for growth. The growth rate coefficient increases initially with increasing

substrate concentration, and asymptotically approaches a maximum called the maximum specific

growth rate, _tmax (Grady, 1999). A general equation used to characterize bacterial growth is

the Monod equation (Equation 1-4). This equation is developed strictly on empirical basis

(Grady, 1999).

t = tmax (Ss/(Ks + Ss)) (1-4)

In the Monod equation, Ss is the substrate concentration and Ks is the half-saturation

coefficient. Ks is the substrate concentration where the specific growth rate of the bacteria is

equal to half of the maximum growth rate. It can used to predict how fast pmax is obtained.

The Monod equation can be extended to consider the effects of various substrates when multiple

limiting nutrients are present, as shown in Equations 1-5 and 1-6 (Grady, 1999). Equation 1-5 is

used when both growth limiting nutrients are required for biomass growth and they can influence

the specific growth rate at the same time. Then the specific growth rate is calculated by taking

the product of the Monod terms for the two nutrients. Equation 1-6 is used when both nutrients

are required for biomass growth, but only one nutrient will limit the growth at a given time.

Then the specific growth rate is obtained by taking the lowest value obtained from separate

single-substrate models.

[i = max (Ssi/(Ksi + Ssi)) (Ss2/(Ks2 + Ss2)) (1-5)

[ = min (pmax (Ssi/(Ksi + Ssi))), (pmax(Ss2/(Ks2 + Ss2))) (1-6)

The pH range of the system should also be considered during the denitrification. The pH

of the system is generally increased during denitrification since OHf ions are produced. The

optimal pH range for the bacterial culture can be determined using various batch tests, and the









bacterial growth can be recorded. Generally, mild alkaline conditions are preferred for

denitrification (Tchobanoglous, 2003).

Bioreactor Configuration for Denitrification

Two general anoxic models are used for denitrification processes: suspended growth

systems and attached growth systems (Grady, 1999). In attached growth systems, microbes are

grown attached to a packing material, generally rocks, sand, or other synthetic materials. The

wastewater flows upward or downward, coming into contact with the attached biofilm, and the

treated water flows out of the system. In anoxic systems, the packing material can be completely

submerged, with gas space above the biofilm liquid layer (Grady, 1999). Excess biomass that

may exit with the effluent can be separated using a clarifier and can be removed for further

processing.

In suspended growth systems, microbes are kept in a suspended state by appropriate

mixing. Suspended growth systems can be operated in batch mode or continuous mode. In

batch systems feed is added into the reactor with suspended biomass and is allowed to react to

completion. Reaction conditions and growth environment change with time. In continuous

systems feed solution is continuous pumped into the reactor and an effluent is pumped out at the

same flow rate. The concentrations of components in effluent are the same as the concentrations

of components in the reactor. One example of a continuous suspended system is the continuous

stirred tank reactor (CSTR). In a CSTR, the liquid volume is kept constant and sufficient mixing

is added to keep the conditions uniform throughout the reactor. A physical operation, such as

sedimentation, may be implemented in order to separate biomass from the effluent. The

overflow from the sedimentation basin will have very small concentration of biomass. The

underflow will have very high concentration of biomass, most of which is recycled to the

bioreactor. A portion of the concentrated slurry is wasted, and is further treated before









discharging it to the environment. In several operations, flexibility is implemented by placing

several CSTRs in a series. The conditions in various stages of the system can vary, thus

allowing for completion of various transformations. Recycle may also be employed at desired

stage of the system or through the entire chain (Grady, 1999).

Objective of the Project

The objective of this study was to develop a feasible denitrification process for treating

an industrial nitrate stream. Attached growth bioreactors and suspended growth bioreactors were

used for this study. Initial inoculum for the reactors were obtained from the denitrification

basins at University of Florida Water Reclamation Facility. The optimal pH for denitrification

was determined to be between 8 and 9.5 in previous studies. Since industrial nitrate stream does

not contain enough COD to support denitrification, potassium acetate was used as an exogenous

carbon source. The goal of the project was to obtain a denitrification rate of 2 mg N03-N/L/day.









CHAPTER 2
DENTRIFICATION OF HIGH NITRATE INDUSTRIAL STREAMS USING GROWTH
BIOREACTORS

Initial attempts to denitrify the high nitrate industrial stream were performed in attached

growth bioreactors, Bios2 and Bios3. The submerged attached growth bioreactors allow for

short hydraulic residence times with high solids retention times, and low solids waste after

denitrification. A packed bed bioreactor with upward flow and a recycle stream was used to

conduct the experiments. Rock media obtained from Adventus was used to pack the reactor,

which provided high surface area for the attached growth of bacterial cells. The reactor was kept

anoxic in order to allow for proper denitrification.

Materials and Methods

Setting up Bios2

A 3.78 L HDPE bottle was used to construct Bios2. The set up of the bioreactor is shown

in Figure 2-1.
Effluent tubing Recycle pump








Effluent
tank










Feed pump Bioreactor Inlet orts
Figure 2-1. Attached growth bioreactor









The bioreactor contained eight feed inlet ports at the bottom and an outlet port at the top.

This allowed for the feed to enter from all sides of the bioreactor, which allowed for better

contact with the denitrifying bacteria inside. An outlet port was connected back to the feed

stream as the recycle, and another outlet port was used to collect the overflow into a receiving

bottle. A sample port was also constructed along the recycle outlet tubing. The feed stream

was pumped from the feed tank and was allowed to mix with the recycle. This allowed for the

regulation of pH of the stream entering the bioreactor. Feed and recycle streams flowed through

peristaltic pumps, and pump flow dials were used to monitor the flow rates. Feed and recycle

pumps were standardized and calibration curves were used to obtain the flow rates with respect

to pump dials.

The mixed stream flowed through the eight inlet ports, and was allowed contact with the

bacteria as it flows upward and out the bioreactor. The top portion of the bioreactor was used as

the gas separator. Tubing was attached to the lid of the bioreactor to allow the gas to escape.

Cole Parmer L/S 13 tubing was used for all the connections in the bioreactor. The bioreactor

was filled with stone media from Adventus and 1.5 L of inoculum from UF WRF. The working

volume of the bioreactor was 3 L.

Feed Preparation and Sampling for Bios2

Acetate was added as the carbon source for the industrial stream that was fed to the

reactor in the form of sodium acetate. The stream was pre-neutralized in order to increase the

pH, and had a concentration of approximately 1300 mg/L N03-N. The carbon source for Bios2

feed was added in order to obtain 8:1 COD-NO3-N ratio. In order to obtain an 8:1 COD-NO3-N

ratio, 10400 mg COD/L was required in the feed stream. The initial COD was assumed to be

6000 mg/L, and thus 4.4 g/L of COD was needed in the feed solution. Thus 6.445 g/L of acetate









was added to the feed solution. In order to increase the denitrification rate of the bioreactor,

approximately 0.01 g/L of yeast extract and 0.3 ml/L molybdenum (as 1.1 g/L molybdic acid)

were also added to the feed stream, providing nutrients to the bacterial culture.

The initial feed flow for Bios2 was turned on to 0.57 mL/min and the recycle was turned

on to 6.67 mL/min. The recycle flow to feed flow ratio was 11.57. The inlet pH of the feed

solution was maintained at 5. Concentrated hydrochloric acid was added to the feed tank in

increments of 10 mL if the feed stream was measured to be high.

During sampling, it was noticed that the sample port was in fact allowing a mixture of the

feed stream and recycle stream to flow through the valve, and thus the nitrate concentrations

measured through the valve were not a true representation of the NO3-N of the effluent stream of

the bioreactor. Thus the sample port was moved to the recycle outlet, and sample was obtained

by disconnecting the recycle tubing. This allowed for measuring the pH and N03-N of both inlet

stream and outlet stream of the bioreactor. A small volume of the sample was discarded initially

each time in order to flush out the tubing and the sample port valve. The pH and NO3-N tests

were conducted three times a day, and the flow rates were adjusted accordingly. N03-N was

measured using HACH NitraVer test kits and measured using a spectrophotometer. In general,

the sample was diluted to 20 times in order to obtain the nitrate concentration.

Setting up Bios3

The main purpose of Bios3 was to compare the effects of sodium and potassium on

denitrification. The construction and set-up for Bios3 was identical to that of Bios2. The reactor

was filled with rock media from Adventus and 1.1 L of inoculum from UF WRF was added.

The working volume of the reactor was 3 L. Eight inlet ports were still used for feed flow, along

with a recycle outlet, gas outlet, and a sample port for the inlet stream. Computer Controlled









pumps were used for Bios3. The pumps were calibrated by measuring the effluent accumulated

in the receiving bottle for a certain period of time.

Feed Preparation and Sampling for Bios3

Bios3 was initially started with sodium hydroxide neutralized industrial stream (sample

A)-the same feed as Bios2. The feed was then switched to potassium hydroxide neutralized

industrial stream (sample C). Potassium acetate was used as the carbon source for the feed. The

nitrate-nitrogen concentration of sample C was 2100 mg/L, which was much higher than Bios2

feed. 15 g/L of potassium acetate was added to the feed in order to ensure excess carbon, along

with 0.024 g/L potassium phosphate as a source for phosphorus, 0.01 g/L yeast extract, and 3 mL

of 1.1 g molybdenum in 1000 mL molybdic acid. The feed pH was maintained at 5, and 10 mL

increments of hydrochloric acid were added to the feed if high pH was observed. Nitrate and pH

measurements of the effluent were taken three times daily. Nitrate-nitrogen measurements were

taken using HACH NitraVer Test 'N Tubes and a spectrophotometer. A small amount of the

effluent was flushed out of the tubing before samples were taken. Two samples were collected

for each measurement, one corresponding to the effluent stream collected from the recycle outlet,

the other sample corresponding to the inlet stream collected from the sample port valve. The

bioreactor was started with a feed flow of 0.5 mL/min and a recycle flow rate of 2 mL/min.

Results and Discussion

Bios2 Results

As shown in Figure 2-2A, Bios2 initially had high denitrification rates; however, the

denitrification rate began to decrease eventually. A large amount of gas bubbles were noticed in

the reactor throughout its operation. The reactor was agitated periodically to release the gas

bubbles. It was noticed that when the reactor was agitated, the denitrification rates improved

temporarily. However, the nitrate concentration in the reactor increased again. High










denitrification rate was also observed after the addition of micronutrients to the feed and to the

reactor. 40 drops of micronutrients were added to the feed and 5 drops were added directly to

the reactor. The denitrification briefly increased after the addition of micronutrients; however,

the results did not stay positive for long. The highest flow rate achieved by Bios2 was 0.8

mL/min, with a denitrification rate of 95.96%. This corresponded to a denitrification rate of

0.354 mg NO3-N/L/day. Thus, bioreactor failed to denitrify the industrial stream at the desirable

rate, which was 2 mg NO3-N/L/min.


Nitrate reduction and feed rate vs. time

100 P 1.0

90
$6o. .
80 0.8
t. iC

70 0
C
t 60 60 0.6




30
E








6/7Y 6/17 6/27 7/7 7/17 7/27 8/6
New Bios2 Feed pH agitated
Me Bleia stated
Begin decrease ime (Days)Feed w/ K-Acetate % Nitrate Reduction Feed Flow


Figure 2-2A. Bios2 denitrification and feed flow rate vs. time. As shown in the figure, a slight
increase in denitrification was observed when the media was agitated. However, it still
continued to perform poorly. An increase in denitrification was also shown immediately after
the addition of micronutrients. Also, as shown in the figure, when the pH of the effluent was
measured to be higher than 9.5, the pH of the feed was decreased by the addition of concentrated
HC1 to the feed tank. Target feed rate for the reactor was 2 mg N03-N/L/min.










Start-Up 2/5/2007 ; Feeding Begins 2/12/2007


10.00
9.00
8.00
7.00
6.00
5.00
4.00 ---- 4 0 44
3.00
2.00
1.00 *
A.00 Vicronutrient
0.00 s 7'added,
t 6/7 6/17 6/7 77 7/17 127 8/6
I media Agitated System Upset
Feed pH decreases Feed w/ K-Acetate begin



Figure 2-2B. Bios2 pH and nitrate concentrations vs. time

One factor that was taken into account after Bios2 failed to denitrify was the effect of

sodium on denitrification. It was hypothesized that sodium contribute significantly to the

osmotic pressure across the cell membrane, which would affect the metabolism and

denitrification of the bacteria. An alternate to using sodium salt for neutralization and as carbon

source was to use potassium salts. Even though potassium ions contribute to the osmotic

pressure also, their effects would be smaller than those of sodium ions. Therefore, a new

bioreactor was started in order to examine if potassium would lead to high denitrification rates of

the industrial stream samples.

Bios3 Results

Bios3 had initial high denitrification rates; however, on the whole, it had the same results as

Bios2, as shown in Figure 2-3A. It was noticed that Bios3 recovered faster from system upsets

than Bios2, possibly due to the presence of potassium instead of sodium. In general, no










significant changes were observed from Bios2 results. An increase in denitrification was

observed after adding micronutrients to the feed solution (4 drops/L) and directly to the reactor

(5 drops). However, the high denitrification rate was temporary. Gas bubbles were also

observed in the walls of the reactor. Agitation of the reactor failed to increase its performance.

The highest flow rate that was achieved by Bios2 was 1.2 mL/min, with an overall denitrification

of 98.7%. This corresponded to a denitrification rate of 0.612 mg NO3-N/L/day. Therefore,

Bios3 also failed to meet the target denitrification rate of 2 mg NO3-N/L/day. The results

showed using potassium salts did not have any significant impact on denitrification.


nitrate reduction and feed rate vs. time

100 1.00

**
90

*
80 0.80

70 > #
**

S60 0.6
i 50 n I *

S40 0.40

30

20 p 0.20

10

0 i 0.00
7/9 f 7/14 7/19 7/24 7/29 8/ 8/8 813 8/18 8/23
Bios3 Begin Micronutrients Flushed out with water
Time (Days) added % Nitrate Reduction Feed Flow


Figure 2-3A. Bios3 denitrification and feed flow rate vs. time. Bios3 results did not vary
significantly from Bios2 results, even though potassium hydroxide was used to neutralize the
feed rather than sodium hydroxide. Notice that an increase in denitrification was also observed
in Bios3 immediately after the addition of micronutrients.









Start-Up 2/5/2007; Feeding Begins 2/12/2007


10

6 o
S ,. U

0 ~ *. M ronutrlents
0 added pd
7/9 7/14 7/19 7/24 7/29 8/3 8/8 8/13 8/18 8/23
N03-N (100 mg/L) pH


Figure 2-3B. Bios3 pH and nitrate concentration vs. time

After Bios2 and Bios3 failed to denitrify industrial nitrate streams properly, several

factors were considered that might have resulted in low performance of the reactors. One

possibility was the presence of metals in high concentrations in the feed, which can affect

bacterial metabolism. A metals analysis of Bios2 feed, Bios3 feed, and Bios3 effluent was

conducted using Inductively Coupled Plasma (ICP). However, very low concentrations of

metals were found in all samples. Another factor was the high nitrate concentration of the

industrial stream. The effects of high nitrate concentration of the feed on denitrification were not

studied. Also, clogging of the tubing could have occurred due to various inlet ports and low flow

rate of the feed and recycle. Clogging in the pores of the rock media could have occurred also,

due to high concentration of biomass, which can lower the surface area for the attached growth

of bacterial growth. Channeling and short-circuiting inside the reactor is also possible, and

proper contact of the feed stream with the bacteria can be diminished. Another factor considered

was the presence of toxic materials in the feed affecting bacterial growth. It was also possible

that toxic substances present in the feed accumulated on the rock media, which can affect the

metabolism of the attached bacteria and cause low denitrification.









At this point, it was concluded that the low denitrification was attributed to some possible

factors related to reactor design and some possible factors related to the feed. In order to

distinguish the causes, a standard suspended growth reactor was started. The suspended growth

reactor used was Continuous Stirred Tank Reactor (CSTR).









CHAPTER 3
DENITRIFICATION OF HIGH NITRATE INDUSTRIAL STREAMS USING SUSPENDED
GROWTH BIOREACTORS

Suspended growth bioreactors consist of reactor vessels containing biomass growing in a

suspended state rather than attached to a media. Continuous stirred tank reactor (CSTR) is a

suspended growth bioreactor where the contents in the reactor are stirred continuously in order to

keep the conditions homogeneous within the reactor and the culture suspended. The stream to be

treated is fed into the reactor, and the treated liquid mixed with biomass is pumped out as the

effluent. The fractions of constituents in the effluent are identical to those inside the reactor.

Many processes have a recycle stream where most of the biomass is recycled into the reactor.

Materials and Methods

Setting up Suspended Growth Bioreactor

The assembly of the continuous stirred tank is given in Figure 3-1.

as inlet port Gas outlet port

Influent tubing Effluent tubing







Feed tank .4
Computer controlled
pump




Figure 3-1. Continuous stirred tank reactor

As shown in Figure 3-1, the continuous stirred tank reactor was constructed using a 4.2 L

flask. The flask was filled 10% with feed solution, and 90% with inoculum from UF WRF

denitrification basin. The reactor was then set in a stir plate with the stirrer inside and was









stirred continuously. A rubber stopper was used to close the reactor and it was secured to the

reactor using parafilm. The rubber stopper had a feed inlet port, an effluent port, a gas inlet port,

and a gas outlet port. A nitrogen tank was setup beside the reactor and nitrogen was bubbled

through the reactor twice daily for 10 minutes to strip off any oxygen that is present. Initially,

the reactor was started up in the batch mode. Samples for the nitrate tests were taken through the

inlet gas port. The reactor was operated in batch mode until the pH and absorbance increased,

and then it was switched to continuous mode. For the continuous mode, a computer controlled

pump was set up near the reactor with the same inlet and outlet flow rates. The inlet tubing was

secured into the feed port in the rubber stopper, and the outlet tubing was attached to the effluent

port. Cole Parmer L/S 13 tubing was used for feed and effluent connections. The effluent

from the reactor was collected and measured at a specific time period in order to calibrate the

pump.

Sampling and Data Collection

NO3-N, pH, and absorbance measurements were taken twice daily from the effluent of

the reactor. In continuous mode, samples were taken from the effluent. The reactor was purged

with nitrogen gas after each sampling and the gas inlet port was then secured firmly in order to

keep the anoxic conditions in the bioreactor. The gas outlet port was closed only slightly in

order to vent out gas formed in the reactor. Nitrate-nitrogen concentrations were measured using

HACH NitraVer Test'N Tubes and a HACH spectrophotometer. In general, the effluent

sample was filtered and diluted to 20 times before nitrate-nitrogen measurements. Absorbance

measurements were representative of the bacterial concentration in the reactor, and were taken

using a spectrometer. The results from nitrate-nitrogen, pH and absorbance measurements were

used for adjusting the flow rates in the reactor. COD measurements were taken periodically

using HACH COD test kits. The samples were filtered and diluted to 20 times. After the









samples were added to the test tubes with the reactive material, they were placed in a COD

reactor for two hours. COD measurements were then taken using a spectrophotometer.

Feed Preparation

The high nitrate industrial stream obtained was pre-neutralized to appropriate pH using

either sodium hydroxide or potassium hydroxide. Potassium acetate was used as the carbon

source for the bacteria. 17.5 g/L of potassium acetate was used in the feed, along with 0.02 g/L

potassium phosphate. A micronutrient solution was prepared using instructions for trace element

solution by Vishniac & Santer. The contents in the solution are given in Table 3-1. The

micronutrient solution was added in the feed initially in the amount of 4 drops/L. Later in the

experiments, micronutrients were added directly to the reactor rather than in the feed.

Table 3-1. Contents of the micronutrient solution (Vishniac and Santer, 1957)
Ingredient Amount (per 500 mL DI water)
Ethylenediaminetetraacetic acid (EDTA) 25.0 g
ZnSO4 11.2 g
CaCl2 2.79 g
MnCl 2.58 g
FeSO4 2.59 g
Mo7024(NH3)6 0.6 g
CuSO4.5H20 0.785 g
CoCl2.6H20 0.81 g
Magnesium sulfate 2.525 g
Molybdic acid 0.55 g

Artificial nitrate solution used in the experiments was prepared using 69.6 weight percent

nitric acid assay and DI water. The solution was prepared by adding 8.6 mL of HNO3 assay per

one liter of DI water to obtain the same N03-N concentration as sample A. The solution was

neutralized by adding approximately 4 g NaOH pellets per liter of solution. The feed solution

also contained 17.5 g/L potassium acetate as the carbon source and 0.024 g/L potassium

phosphate. The nitrate-nitrogen concentration of the feed was approximately 1450 mg/L, which

was comparable to the nitrate-nitrogen concentration of sample A.










Results and Discussion


CSTR1 Results

CSTR1 was started in order to denitrify sample A neutralized with NaOH in the tote.

The reactor was started in batch mode, and was switched to continuous mode at a flow rate of 1

mL/min once the pH and absorbance increased. Micronutrients were added in the feed of the

reactor in the amount of 4 drops/L. The results of the reactor are shown below:


nitrate reduction and feed rate vs. time (Target feed rate: 2 mg NOs-N/Lnmin)


100.00 **
90.00 *
80.00 *
70.00 *
S60.00
S50.00 *-
S40.00
30.00 *
20.00
10.00
0.00 *
8/5 8/15 8/25 9/4 9/14 9/24
time

+ % nitrate reduction feed flow rate


Figure 3-2. CSTR1 nitrate reduction and feed flow rate vs. time


1


0.8


0.6
z

0.4


0.2


0
"o
o











pH and nitrate concentration vs. time


10
9-
8 1000

6--

4 500




1/5 115 8125 9/4 9/14 9/24
-U-pH N03-N (100 mg/L) time


Figure 3-3. CSTR1 pH and nitrate concentrations vs. time

As shown in Figure 3-2, the denitrification rate of CSTR1 was initially high. However, it

started to decrease after 8 days and continued to decrease without possibility of recovery. The

pH began decreasing and nitrate concentration started decreasing with time, as shown in Figure

3-3. The reactor failed to recover even after switching to batch mode.

After CSTR1 failed, possible causes that might have contributed to the low denitrification

of the industrial stream were considered. It was hypothesized that a toxic component in the

sample might be accumulating in the reactor and was not consumed during the metabolism of the

bacteria. Possible candidates were sodium buildup, high nitrate buildup, or accumulation of

some other toxic component in the industrial solution. An analysis was done to predict the

concentration of a toxic compound that might be accumulating in the reactor after a certain time

period.

Analysis of CSTR1 Results

After the hypothesis that the accumulation of a constituent is causing low denitrification,

a modeling analysis was done on CSTR1 results in order to predict the accumulation period of

the constituent. A mass balance was done on the reactor as shown in Equation 3-1.









d/dt (VC)= FCf FC Vr (3-1)

at constant volume,
V dC/dt= FCf FCo Vr (3-2)

dC/dt = (F/V) Cf (F/V) Co r (3-3)

(F/V) =D (3-4)
(D = dilution rate)

dC/dt= DCf -DC- r (3-5)

Developing Equations

When accumulation is considered, the reaction is neglected; then
dC/dt = D Cf D Co (3-6)

Since dilution rate changes with flow rate and thus with time,
dC/dt + D(t) Co = D(t) Cf (3-7)

Using integrating factor, IF = exp(fD(c)) dc
D(t) = aT + Do (3-8)

exp(J(ac + Do) dz = exp(az2/2 + Do') (3-9)

Multiplying by IF gives
exp(ac2/2 + Do') dC/dt + exp(ac2/2 + Do') Co = exp(ac2/2 + Do') Cf (3-10)
t
I d/dt(exp(ar2/2 + Dor) C) = (exp(ar2/2 + Dor) D(t) Cf (3-11)

C exp(ac2/2 + Do') Co = I D(c) Cf exp(ac2/2 + DoT) d' (3-12)

C exp(ac2/2 + Do') Co = I (ar + Do ) Cf exp(ac2/2 + Do') d' (3-13)

C(t)=Co exp -(at2/2 + Dot)+ exp -(at2/2 + Dot) I (ar + Do ) Cf exp(ac2/2 + Do') d' (3-14)

At constant dilution rate, a = 0, then
C(t) = Co exp -(Dot) + Cf exp -(Dot) I (Do ) exp(Do') d' (3-15)

C(t) = Co exp(-Dot) + Cf (1-exp(-Dot)) (3-16)

When dilution rate is not constant, it is assumed to change linearly with time; then
D = ct + Do. Substituting gives
(ac + Do)d' = d (ac2/2 + Do') (3-17)

Cf oP ((a + Do) exp(acc2/2 + Do') d' = Cf oP exp(acc2/2 + Do') d (ar2c/2 + Do') (3-18)











Cf of exp(az2/2 + DoT) d (a21/2 + DoT) = Cf (exp(cz2/2 + DoT) 1) (3-19)

C(t) = Co exp(-(ca2/2 + DoT)) + Cf exp(-(oz2/2 + DoT)) (exp(aoz2/2 + DoT) 1) (3-20)

C(t) = Co exp(-(ca2/2 + DoT)) + Cf (1- exp(-(az2/2 + DoT))) (3-21)

Applying CSTR1 Results

The results were analyzed over three time periods. As shown in Figure 3-4, time period 1

was taken from to to ti, time period 2 was taken from ti to t2, and time period 3 was taken from t2

to t3. It was assumed that time period 1 and time period 2 had constant dilution rate, and time

period 3 had dilution rate that changed linearly with time. At t = to, Co was taken to be zero; thus

it was assumed that no toxins were present in the system initially. The results of the analysis are

shown in Tables 3-2, 3-3, and 3-4.


100.00 ,

90.00 13

80.00 II

70.00 -

60.00

50.00

40.00

30.00 *
.. *4
20.00

10.00

0.00
8/5 8/10 8/15 8/20 8/25
time


3


2.5


2


1.5 |


1


0.5


0


Figure 3-4. Time periods used for CSTR1 data analysis









Table 3-2. Concentration after the first time period for CSTR1
After the first time period:
C(t) = Co exp(-Dot) + Cf (1-exp(-Dot))
Co= 0
Ci(t) = Cf(1- exp(-Dot))
t = 3420 minutes
Do (dilution rate) = F/V = (1 mL/min)/(4000 mL) = 0.00025 min1
Ci(t) = Cf (1- exp((-0.00025/min)(3420 min))
Ci(t) = 0.575 Cf
Thus, after the first time period, 57.5% of the initial toxin concentration will have accumulated

Table 3-3. Concentration after the second time period for CSTR1
After the second time period:
C2(t) = Ci(t) exp(-Dot) + Cf(1-exp(-Dot))
t = 3365 minutes
Do = F/V = (0.5 mL/min)/(4000 mL) = 0.000125 min-
C2(t) = 0.575 Cfexp((-0.000125/min)(3365 min))+Cf (1- exp(-0.000125/min)(3365 min))
C2(t) = 0.721 Cf
Thus, after the second time period, 72.1% of the initial toxin concentration will have
accumulated in the reactor.

Table 3-4. Concentration after the third time period for CSTR1
After the third time period:
Dilution changes linearly with time. Thus D(t) = ct + Do
t = 4500 minutes
a and Do were determined from the plot of dilution rate vs. time, as shown in Figure 3-5
From the plot, a = 5.4E-8
Do = 0.0002
C3(t) = Co exp(- at2/2 + Dot) + Cf (1- exp(- at2/2 + Dot))
Co = C2(t)= 0.721 Cf
exp(-at2/2 + Dot) = exp{(-5.4E-8*(4500 min)2/2)+((0.000241 min1-)(4500 min))} =
0.196
C3(t) = 0.721 Cf (0.196)+ Cf (0.804)
C3(t) = 0.946 Cf
After the third time period, 94.6% Co will have accumulated in the reactor.










dilution rate vs. time


S0.0006
0.0005 5E-08 + 0 0002
0.0004
0.0003
0.0002
o 0.0001
0
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
time (minutes)




Figure 3-5. Dilution rate of CSTR1 with respect to time. From the linear regression, ao was
calculated to be 5E-08 and Do was obtained to be 0.0002.

The analysis showed that after 57 hours, 57.5% of the initial toxic component would have

accumulated in the reactor. The model predicts that at the time of failure (8/15/2007),

approximately 94.6% of the feed concentration of the toxin would have accumulated in the

reactor, which can cause significant impact on the denitrification rates. Therefore, a new reactor

was started using synthetic nitrate solution to investigate whether the accumulating toxin is a

component present in the industrial feed or the sodium ions added to the feed for neutralization.

CSTR2 Results

CSTR2 was started using synthetic nitrate solution prepared using nitric acid and DI

water. Cole Parmer L/S 13 tubing was used to pump the feed into the reactor, and Cole

Parmer L/S 14 tubing was used to pump out the effluent. Once the pH and the absorbance of

the reactor increased, the reactor was switched to continuous mode. Two computer controlled

pumps were placed near the reactor; one pump was used for feed and the second pump was used

for the effluent. Two pumps were required because of the difference in feed and effluent tubing

sizes. Micronutrients were added to the feed in the amount of 4 drops/L. The results of the

reactor are shown in Figure 3-6.










nitrate reduction and feed rate vs. time: Part 1


100.00 T ----- -*---* --*--"* - '-*-----------
** 4
100.00 -
I 80.00 *
60.00
40.00 : 1
20.00
0.00 0
8131 915 9110 9115
time

% nitrate reduction feed flow rate


Figure 3-6. CSTR2 denitrification and feed flow rate vs. time, part I

Initially on continuous mode, the reactor was started at 0.5 mL/min. The reactor was

denitrifying very well at this flow rate. The flow rate of the feed was increased up to 2 mL/min

and the reactor continued to denitrify at high rates, as shown in Figure 3-6. However, the flow

rate was increased without allowing the reactor to reach stable conditions. Increasing the feed

flow required an increase in effluent flow rate as well, and increasing the flow prematurely can

lead to washout conditions in the reactor. Therefore, the feed flow rate was decreased to 1.2

mL/min in order for the reactor to stabilize. The initial positive results seen in the reactor were

brief, however, and the nitrate concentration began to increase again in the reactor. The reactor

was switched to batch mode after very low denitrification rates were observed.

It was noticed that denitrification rate was high in the reactor for approximately 265

hours (11 days). The low denitrification rates and low absorbance values were observed later on.

An analysis was done on the accumulation of toxins in the reactor using Equation 3-21. The

initial concentration of the toxin, Co, is assumed to be zero. The ratio of C(t)/Cf was plotted

against time. The results are shown in Figure 3-7











CSTR2 accumulation of a toxin


1
0.9
0.8 -
0.7
0.6 -
0.5 -* Series1
0 0.4
0.3
0.2
0.1
0
0 1000 2000 3000 4000 5000 6000 7000
time (minutes)


Figure 3-7. Ratio of C(t)/Cf vs. time for component accumulation in CSTR2

As shown in the Figure 3-7, within after a time period of approximately 6400 minutes, the

ratio C(t)/Cf has reached the value of 0.86. Thus, a deterioration of the bioreactor performance

should have been observed by this time. After 188 hours (9/9/2007), the toxic component

concentration would have reached 99% of the feed concentration. However, as shown in Figure

3-6, the reactor continued to perform well for two more days. Thus, accumulation of a toxin was

not causing the low denitrification rates in the reactor. It was then suspected that deficiency of a

key constituent might be causing the low denitrification. Thus, the results were analyzed in

order to predict the time that deficiency of a component might occur.

Analysis was done for the first 6400 minutes of the operation of the reactor, where a linear

increase in flow rate, and thus dilution rate, was observed. The dilution rate was calculated using

the relationship given in Equation 3-8, where D(t) is equal to at + Do. a and Do were obtained

from the plot of dilution rate vs. time, as shown in Figure 3-8.









y = 5.3931E-08x + 1.3776E-04
0.0006 R2 9.6752E-01
0.0005
0.0004
0.0003 -
0.0002
0.0001
0
0 -----------------------------
0 1000 2000 3000 4000 5000 6000 7000

Figure 3-8. Plot of dilution rate vs. time for CSTR2

Another plot of C(t)/Co vs. time was generated in order to quantify the depletion of a

component initially present in the system. Analysis done using the data obtained from the

reactor showed that after approximately 107 hours, the concentration of a component added

initially to the reactor would have reached approximately 13% of the feed concentration. The

results are shown in Figure 3-9.

CSTR2 deficiency of a nutrient

1.5

0.5
0-
0 ------------------------------.
0 1000 2000 3000 4000 5000 6000 7000
time


Figure 3-9. Ratio of C(t)/C(o) vs. time for component deficiency in CSTR2

Since the reactor was performing very well up to this point, it was noted that the ingredient

that is exhausted is needed only in very small amounts for denitrification. Further analysis of the

data showed that at the time of the failure (9/11/2007), the amount of the nutrient will have

reached 0.6% of its initial concentration. One component that was required for bacterial growth

in very small amounts was the micronutrients. It was noted that attached film bioreactors had

started to recover previously when micronutrients were added to the feed and directly to the










reactor. However, the effect was short-lived and the reactors deteriorated again. This led to the

conjecture that micronutrients added to the feed may have precipitated, as suggested by Ben

Koopman. Thus, most of the micronutrients added to the feed of the CSTR would have been

precipitated out. Taking this into consideration, micronutrients were added directly to the reactor

everyday in proportion to the flow rate of the feed. Generally, 1 mL of micronutrients was added

to the reactor when the feed flow rate is 1 mL/min. Magnesium was also added to the feed

solution in the form of magnesium sulfate heptahydrate in order to ensure its availability.

After the micronutrients were added, an increase in the reactor performance as well as

bacterial growth was observed, as shown in Figure 3-6. These results showed that it was indeed

the deficiency of micronutrients that caused low denitrification in the reactor previously. It was

also determined that the addition of micronutrients to the feed can cause precipitation, and thus,

it must be added directly to the reactor. The amount of potassium phosphate added to the feed

was also increased to 0.5 mg/L in order to avoid phosphorus deficiency.


CSTR2 nitrate reduction and feed rate vs. time Part II

100.00
** < 4 t
80.00 ,
2 60.00
c 40.00 2
20.00
0.00 0
8131 9110 9120 9130 10/10 10120 10130 11/9 11119 11 29 12/9 12/19
time feed rate lowered due to limited
carbon availability
% nitrate reduction feed flowrate


Figure 3-10. CSTR2 denitrification and feed flow rate vs. time, part II. As shown in the figure,
a deficiency in carbon occurred during the operation, and the flow rate to the reactor was set at
the minimum for 2 days. The flow rate was increased rapidly after carbon was available to reach
appropriate steady state.











pH and nitrate concentrations vs. time


9
8
1000
7 8
6
S5

4 500
3*
2
1
0 0
9/15 9/23 10/1 10/9 10/17 10/25 11/2 11/10 11/18 11/26 12/4 12/12 12/20

-pH N03-N (100 mg/L)


Figure 3-11. CSTR2 pH and nitrate concentrations vs. time



Absorbance vs. time

4
3.5
3
c 2.5
2
*0
2 1.5 "........ .... ." .. ..... -, "" .** *.,
S1.5
1
0.5 ..
0
9115 9123 10/1 10/9 10/17 10125 11/2 11110 11118 11/26 12/4 12/12 12/20
time

absorbance



Figure 3-12. CSTR2 absorbance vs. time

After the addition of micronutrients, increase in denitrification rate and absorbance was

observed in the reactor. The overall denitrification remained high with increasing flow rates, as

shown in Figure 3-10. The pH of the reactor was in between 9 and 9.5 throughout its operation,









as shown in Figure 3-11. The absorbance decreased generally with an increase in feed flow rate,

as expected since the effluent flow rate was also increased. The absorbance stabilized once the

reactor reached steady state for each flow rate. The flow rate of the reactor was continuously

increased afterwards up to a flow rate of 11.5 mL/min, as shown in Figure 3-10, which was close

to twice the target flow rate, and the highest denitrification rate observed in CSTR2 was 4 mg

N03-N/L-min. From these results, it was confirmed that high nitrate concentration did not affect

the metabolism and denitrification rate of the denitrifying bacteria.

It was furthermore speculated in the beginning that high amount of sodium can lower

denitrification rates since it will lead to high osmotic pressure across the cell membrane. The

effect of sodium was also tested previously using attached growth bioreactors-by operating a

reactor containing sodium hydroxide neutralized industrial stream and another reactor containing

potassium hydroxide neutralized industrial stream simultaneously. However, the results from the

attached growth bioreactors were not reliable since the reason for poor denitrification could have

been micronutrient deficiencies. Therefore, a new CSTR was started alongside CSTR2 in order

to test the effects of sodium on denitrification rate. The new reactor was started using synthetic

nitrate stream using DI water and 69.6% nitric acid assay, and the solution was neutralized using

potassium hydroxide pellets instead of sodium hydroxide pellets. There weren't any noticeable

differences between the results obtained from sodium hydroxide neutralized solution and

potassium hydroxide neutralized solution. Therefore, it was concluded that sodium does not

have any significant effects on denitrification.

CSTR3 Results

After it was confirmed that high nitrate concentration and sodium did not have

significant effect on denitrification rate, another suspended growth reactor, CSTR3, was started

again with the industrial stream as the feed solution. Effluent from CSTR2 was used as the










inoculum for the reactor. The reactor was initially started with artificial nitrate solution

neutralized with potassium hydroxide pellets as the feed. The feed was later switched to ion-

exchanged industrial stream that was pre-neutralized with potassium hydroxide. The feed

solution contained 17.5 g/L potassium acetate as the carbon source, 0.5 g/L potassium phosphate

as the phosphorus source, and 0.1 g/L magnesium sulfate heptahydrate in order to provide

additional magnesium for bacterial metabolism. The results of the reactor are shown in Figures

3-13, 3-14, and 3-15.

The reactor continued to denitrify at a high rate using sample B (ion-exchanged industrial

stream), as shown in Figure 3-13. The highest flow rate obtained using sample B was 0.8

mL/min with an overall denitrification of 99.97%. This corresponded to a denitrification rate of

0.315 mg N03-N/L-min. The reactor was not able to achieve higher flow rates because of the

shortage of available sample B solution.


CSTR3 nitrate reduction and feed rate vs. time

100.00 0 104, W 1
90.00
C 80.00 0.8
0 70.00 E-
60.00 0.6 "E
50.00 ----------------------_---------- -
40.00 0.42
30.00 Z
S 20.00 0.2 .2
10.00
0.00 0
911: 9123 B 1013 101 3 10123 11/2
A time C

% nitrate reduction -feed flow rate


Figure 3-13. CSTR3 denitrification and feed flow rate vs. time A: HNO3-KOH feed started; B:
Switched feed to sample B, C: Switched feed to sample C












nitrate concentration and pH vs. time


-sitched feed to sample C


10

9

8




8 5

4
3

2,#


0 4.. ._
9/13 9/16 9/19 9/22 9/25 9/28 10/1 10/4 10/7 10/1 10/1 10/1 10/1 10/2 12 1 10/2 10/3 11/3
--pH N03-N (100 mg/L) 2 5 8 1



Figure 3-14. CSTR3 pH and nitrate concentration vs. time





absorbance vs. time


1000
.1



8
500





0


/13 9/16 9/19 9/22 9/25 9/28


10/1 10/4 10/7 10/1 10/1 10/1 10/1 10/2 10/2 10/2 10/3 11/3
0 3 6 9 2 5 8 1
time

* absorbance


Figure 3-15. CSTR3 absorbance vs. time


4
3.5
3
2.5
2
1.5
1
0.5
0
9


* *
*. *. .
*

** S.. .***
S. .
.
e
tao~ee eeo e
( eCo e









After sample B was exhausted, the feed was switched to sample C, which was non-ion

exchanged industrial stream neutralized with potassium hydroxide. Changes in performance of

the reactor were not speculated due to this action, since it was hypothesized that it was indeed the

deficiency of micronutrients that caused low denitrification of the previous industrial streams.

However, as shown in Figure 3-13, the denitrification rate started to decrease as soon as the feed

was switched to sample C. Figures 3-14 and 3-15 correspond to the pH and absorbance of the

reactor, respectively. It can be seen clearly that the change in feed led to decrease in pH and

absorbance as well. The reactor was then put on batch mode for three days and the feed was

switched back to synthetic feed neutralized with potassium hydroxide. The reactor failed to

recover from the low denitrification rate, however. This led to the belief that metal ions are

interfering with bacterial growth and denitrification process. It was also noticed that sample C

had a tint of green that was not observed in previous samples.

CSTR4 Results

After CSTR3 failed to denitrify sample C properly, other factors that may cause poor

denitrification were considered. The main concern was the presence of metals in the industrial

stream, which can affect bacterial metabolism and lower denitrification rates. This was further

supported by the results obtained from denitrification of sample C, which was ion-exchanged.

After CSTR3 was discontinued, two new batches of ion-exchanged industrial stream neutralized

with KOH were received (sample D and sample E). CSTR4 was started up using sample D. The

feed also contained 17.5 mg/L potassium acetate, 0.5 mg/L potassium phosphate, and 0.1 mg/L

magnesium sulfate heptahydrate. Effluent from CSTR2 was used as the inoculum for the

reactor. Micronutrients were added to the reactor daily in amounts proportional to the feed flow

rate. The results of the reactor are shown in Figures 3-16, 3-17, and 3-18.













100.00
80.00
60.00
40.00
20.00
0.00


nitrate reduction and feed flow rate vs. time



E

0.5 z
r\ 0
10/ 101 10/ 11 111 111 11/ 111 11/ 11/ 11/ 11/ 12/ 12/ 12/ 12/ 12/ 12/ 12/ 12/
25 28 31 3 6 9 2 15 18 21 24 27 30 3 6 9 12 15 18 21 24

Sample D Sample E Sample A time
synthetic nitrate solution % nitrate reduction feed flow rate
synthetic nitrate solution


Figure 3-16. CSTR4 denitrification and feed flow rate vs. time. Low denitrification rates were
observed for both ion-exchanged samples. High denitrification was observed once the feed was
switched to sample A.


pH and nitrate vs. time


11/3 11/11 11/19 11/27
pH NO3-N(10mg/L)


1300
1200
1100 ,
880 g)_
;8 Ce-

600


188
0


12/13 12/21


Figure 3-17. CSTR4 pH and nitrate concentrations vs. time


Absorbance


0/26


11/11


11/19


11/27


12/13


12/21


* absorbance


Figure 3-18. CSTR4 absorbance vs. time


W1 C
I.5 .2
t ;
UC3


~~ur~~~ e d-,I-~-I, ter-,~~


4
3.5
3
2.5
2
1.5
1
0.5
0
1I


, *, % .<* ** ***@ **C'** **^*, ***4*** ******* *
"-. .

*% *


4111 WL









CSTR4 did not have positive denitrification results initially. The pH and absorbance

decreased steadily as shown in Figures 3-17 and 3-18, and the denitrification was deteriorating

slowly as shown in Figure 3-16. The results were puzzling since the sample was ion-exchanged,

and thus it invalidated the assumption that metal ions may be causing the problem. The reactor

failed to recover after switching to synthetic feed. In order to be certain that the ion-exchange

was effective, an analysis of the samples were done at the industrial site, and it was observed that

one of the samples had high concentration of metal ions that might inhibit the bacterial growth

and lower the denitrification rate. It wasn't positive which of the samples contained the high

metal ion content. It was possible that the low denitrification of the reactor was due to presence

of metal ions in the feed.

It was then decided to restart CSTR4 and observe the denitrification rates of sample E.

Therefore, the reactor was restarted with synthetic feed and inoculum obtained from CSTR2

effluent. Once the reactor was in steady state with a flow rate of 1 mL/min, the feed was

switched to sample E. The denitrification rate was high using this sample; however, the

absorbance started decreasing. This led to the conclusion that metals were not the inhibition

factor in low denitrification rates observed.

Restarting CSTR4 with Sample A

CSTR4 was restarted with sample A since it was suspected that previous low

denitrification rates were caused by deficiency of micronutrients. When sample A was

previously fed into the reactor, micronutrients were added in the feed solution rather than

directly to the reactor. Thus, the reactor was restarted in continuous mode at lmL/min. Effluent

from CSTR2 was used as the inoculum for the reactor.

High denitrification rates were observed throughout the operation of the reactor, and pH

remained steady. The reactor was able to achieve high flow rates with high overall









denitrification. The highest flow rate achieved by the reactor was 5.5 mL/min, with 97.5%

denitrification, as shown in Figure 3-16. This corresponded to a denitrification rate of 1.68 mg

NO3-N/L/day, which was close to the target denitrification rate of 2 mg NO3-N/L/day.

At this point it was concluded that the industrial stream was indeed treatable, but a

component in the stream is acting as an inhibitory factor. Causes for denitrification include

presence of metal ions and presence of volatile contaminants. Thus, a tool was needed to judge

the treatability of the samples. Thus, a batch test was developed in order to test the treatability of

high nitrate streams. It was revealed from the results of the batch tests that volatile contaminants

are indeed present in the industrial stream and they might be the components contributing to the

low denitrification rates and low absorbance values.









CHAPTER 4
ASSAY ON THE BIOLOGICAL TREATABILITY OF INDUSTRIAL STREAMS

A batch test procedure was developed to test the biological treatability of high nitrate water

streams. The assay was inspired by studies on Daphnia by Gasith et. al. Since the parameter to

be tested was denitrification, Daphnia was replaced with denitrifying bacterial culture. The

denitrification results of the test solution are compared with those of the synthetic nitrate

solution. The experiments were mainly conducted on ion-exchanged high nitrate industrial

stream neutralized with potassium hydroxide, and non-ion exchanged industrial stream

neutralized with sodium hydroxide. The batch tests primarily investigated the effects of volatile

contaminants and settled particles on denitrification and bacterial growth. The general procedure

developed to test the treatability of the high nitrate streams is as follows:

1. Grow denitrifying bacteria in a high nitrate stream so that they can be used for the batch
tests.

2. Measure the nitrate-nitrogen concentration and pH of the test solution using HACH
NitraVer Test 'N Tube test kits

3. Prepare synthetic nitrate solution using nitric acid with the same nitrate-nitrogen
concentration as the test solution.

4. Add appropriate chemicals (given as follows) to variable and control solutions in order
to support bacterial growth and metabolism:
a. 17.5 g/L potassium acetate as carbon source
b. 0.5 g/L potassium phosphate as phosphorus source
c. 0.1 g/L magnesium sulfate heptahydrate as magnesium source
d. 0.3 g/L ammonium chloride as the ammonium source

5. Neutralize the test solution and synthetic nitrate solution to pH 8 using sodium hydroxide
pellets

6. Filter the solution using 2 |tm filters.

7. Aerate approximately 200 mL of test solution, and keep the rest non-aerated.

8. Obtain 9 125-mL Erlenmeyer flasks; fill three flasks with 60 mL of aerated test solution,
three flasks with 60 mL of non-aerated test solution, and three flasks with 60 mL of
synthetic nitrate solution.










9. Add 10 mL of bacterial culture and 0.2 mL micronutrient solution-prepared using
instructions for trace element solution by Vishniac & Santer to each flask.

10. Measure the nitrate-nitrogen concentration, pH, and absorbance of each flask. The
nitrate-nitrogen concentration is measured using HACH test kits after filtering the
sample from each flask using 0.45 |tm filters, and diluting the samples to appropriate
concentrations.

11. Secure all flasks with rubber stoppers and place them in an incubator at 37C

12. Measure NO3-N, pH, and absorbance of one aerated flask and one non-aerated flask
three times: first measurement after 3-4 hours, second measurement after 7-9 hours, and
third measurement after 22-24 hours.

13. Obtain ([ max/Y'No)sample / (([ max/'No)synthetic solution from the data obtained in order
to establish the treatability of the high nitrate stream; treatable nitrate solutions will have
a value close to 1.

Developing Equations

Bacterial growth in the test flasks can be quantified since the biomass concentration is

proportional to absorbance values. Biomass growth kinetics is developed assuming the presence

of excess carbon and neglecting decay. The change in absorbance can be characterized by

Monod equation.

dA/dt = maxNo (SNo/(KNo + SNO)) A, nitrate is the limiting component (4-1)

dSNo/dt = maxNO (SNo/(KNo + SNO)) A (4-2)

dSNo/dt = (-l/YNo) (dA/dt) (4-3)

A(t) = Ao + Y'NO (SNO(O) SNo(t)) (4-4)

Thus, a plot of absorbance vs. nitrate concentration will have a slope of -YNO and y-

intercept of Ao. This plot can be obtained from the measurements of absorbance and nitrate

concentration after 0, 4, and 8 hours. It was noted that KNO is typically less than 1, usually for

wastewater streams containing nitrate concentration around 100 mg/L. Since the nitrate

concentration is much higher than 100 mg/Lin the experiments done with this assay, the ratio









(SNO/(KNo + SNO)) is approximated to be equal to 1. Then Equation 4-2 can be rewritten as

shown in Equation 4-5.

dSNo/dt = lmaxNO [Ao + YNo(SNo(O) SNO(t))] (4-6)

(Ao+YNoSNo(o)) = P (4-7)

Sno(On(0t) (dSNO/ P YNOSNO) = lmaxNO of' dt (4-8)

ln(P YNoSNo(t)) = YNO [lmaxNO t + ln(P-YNoSNo(o)) (4-9)

Thus, a plot of ln(P YNoSNo(t)) vs. YNO*t will have a slope of [lmaxNo. Once |JmaxNo of

the test solution is obtained, JmaxNO of the test solution is compared with ,maxNO of the synthetic

nitrate solution. Thus, the ratio BI, given in Equation 4-10, can be considered as a treatability

index. A low BI will indicate that biological denitrification of that sample will be very difficult.

BI=((PJmaxNO) test sample)/((.maxNO) synthetic sample with same N03-N concentration) (4-10)

Development of the Assay

Initially the batch tests were conducted in flasks secured with rubber stoppers with a gas

inlet port and a gas outlet port. The inlet and outlet ports were secured with aluminum foil after

nitrogen gas was bubbled through the flasks. The first batch test was conducted to test the

treatability of the ion-exchanged industrial stream neutralized with potassium hydroxide. This

sample previously had poor denitrification when it was fed to the suspended growth reactor. The

test was conducted alongside sodium hydroxide neutralized industrial stream, which had positive

results after it was fed through the suspended growth reactor. After 24 hours, it was noticed that

all the flasks had high denitrification and absorbance increase. This led to the suspicion that

oxygen had entered the flasks through gas inlet and gas outlet ports, and that the absorbance

increase was mainly due to aerobic growth of the bacteria. When oxygen is present, bacteria can

convert the nitrate to ammonia, which is then used as the nitrogen source for bacterial growth.









In order to limit the presence of oxygen in the flasks, another batch test was conducted

using flasks that were secured tightly with solid rubber stoppers. Prior to securing the flasks,

nitrogen gas was bubbled through the flasks in order to purge out any oxygen. The ion-

exchanged stream neutralized with potassium hydroxide was tested against artificial nitrate

solution prepared using 69.6% HN03 assay. The results were similar to previous test, and high

denitrification and absorbance increase were observed in all flasks. This did not correspond to

the results obtained from suspended growth reactors. Since oxygen was purged out using

nitrogen gas, the possibility of presence of any oxygen in the flasks was discarded.

It was then suspected that volatile contaminants present in the industrial stream might

have caused low denitrification rates in the suspended growth reactor. It was possible that

volatile contaminants may have been removed when nitrogen gas was bubbled through the

flasks. Thus, another batch test was carried out using ion-exchanged sample. The flasks were

again secured with solid rubber stoppers; however, nitrogen gas was not bubbled through the

reactors. At the end of the batch test, it was noticed that little or no denitrification occurred in

the flasks, and no bacterial growth was observed. This supported the hypothesis that volatile

contaminants were causing the low denitrification rates observed previously in the CSTR, and

the high denitrification in the previous batch tests were the result of removal of volatile

contaminants through aeration.

In order to confirm the presence of volatile contaminants in the industrial stream, a batch

test was carried out using aerated and non-aerated ion-exchanged samples. In order to avoid

assimilatory denitrification, 0.3 g/L ammonium chloride was added to the flasks. The flasks

containing aerated samples showed high denitrification and absorbance increase, while the flasks

containing non-aerated samples had low denitrification and no absorbance increase. This










confirmed the theory that volatile contaminants were causing the low denitrification in the

CSTR.

Results and Discussion

The assay was applied to test the treatability of aerated and non-aerated industrial stream

samples neutralized using sodium hydroxide. Inoculum for the batch test was obtained from

CSTR4 effluent. Measurements were taken after 4, 8, and 24 hours. The analyses from batch

test done non ion-exchanged industrial sample are shown in Figures 4-1 through 4-6.


Aerated industrial sample

2.5
S= -87571E- 04. 20 iE-OO
,| 1.5 ^ ^ ^ ^ ^R- = 447E.01
S 1.5 -
0 1
2. 0.5
< 0
0 500 1000 1500 2000 2500


Sno



Figure 4-1. Absorbance vs. nitrate concentration, aerated sample



non aerated industrial sample


1

0.8

0.6

8 0.4

" 0.2

0
1


500


1550 1600 1650 1700 1750 1800 1850 1900


Sno



Figure 4-2. Absorbance vs. nitrate concentration, non-aerated sample


y = -2.0796E-03x 4.0101E+00 **
R- = 7.8896E-01













synthetic nitrate solution


2000


2500


Sno





Figure 4-3. Absorbance vs. nitrate concentration, synthetic nitrate solution


Aerated industrial sample


0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.2


Yno*t




Figure 4-4. ln(3-YNoSno(o)) vs. YNo*time, aerated sample


Figure 4-5. ln( P-YNoSno(o)) vs. YNo*time, non-aerated sample


S= -9 172E-04. 2' 502E-00
R' = 9 94S83E.01


S=2 028,. 0 8511
R- = 0 0


S=5Ii8 21E-01. 1 0 ',i EOC(
R = I:.'""21E-0 1











Synthetic nitrate solution


0.4 -
0.2 = 3.0266x 1.1197
0.2 .
R- = 0.961
C 0
0 -0.2 A 0.1 0.2 0.4 05
o -0.4 -
-0.6
S-0.8
= -1
-1.2
-1.4
Yno*t




Figure 4-6. ln(3-YNoSno(o)) vs. YNo*time, synthetic nitrate solution

Figures 4-1, 4-2, and 4-3 show the plots of absorbance vs. nitrate concentration for

aerated high nitrate industrial stream, non-aerated high nitrate industrial stream, and synthetic

nitrate solution respectively. From the plots, the YNO was obtained to be 8.76E-4 for aerated

stream, 2.08E-3 for non-aerated stream, and 9.19E-4 for synthetic nitrate solution. The plots in

Figures 4-4, 4-5, and 4-6 were used to obtain ,maxNo values for aerated sample, non-aerated

sample, and synthetic nitrate solution respectively. JmaxNo values were obtained to be 2.073 for

aerated sample, 0.569 for non aerated sample, and 3.027 for synthetic nitrate. From [lmaxNO

values, the biotreatability index, BI was calculated. BI for aerated sample was 0.635 and for

non-aerated sample was 0.188. The assay showed that the aerated industrial stream can be

treated biologically. The results were verified by using aerated industrial stream as the feed for a

suspended growth reactor. High denitrification rates and absorbance values were observed. The

assay was done on other industrial samples as well. The results obtained from the industrial

samples are given in Figures 4-7 through 4-12. The BI values are summarized in Table 4-1.











Sample A YNO


0 200 400 600


Sno



Figure 4-7. Absorbance vs. SNO for sample A



Sample A

1



0

-0.5

-1 '

-1.5
Yno*t


800 1000 1200 1400


Figure 4-8. ln(3-YNoSNo(0)) vs. YNO t for sample A




Aerated Sample C YNO


1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
Sno



Figure 4-9. Absorbance vs. SNO for aerated sample C


S.1 13 1E.03- + 1 3 36E
R- 9 5532E-01


+00


, 0RI-:.R. 00OL4
R: 0= 0.42 "'4


= -1 4015E-C5. .5 0?E -01
S= 1 851 TE-.E













aerated Sample C


- 0

C -0.2

S -0.4
ci
g -0.6

-0.8


Yno*t




Figure 4-10. ln(3-YNoSNo(0)) vs. YNO t for aerated sample C





aerated sample D, YNO


Sno




Figure 4-11. Absorbance vs. SNO for aerated sample D


Sample D, aerated


_0 .O-O 4 C0E- 1 8C00

-04
S= 02 'E-01. 1 C001 E+0
-0 CI, h = 9.rI':lEd 01

.1-

-1.2



Figure 4-12. ln(3-YNoSNo(O)) vs. YNO t for aerated sample D


= 1444. 0 5'01
W =CJ


, = -1 4607E.-', 1i33E.01
R: = 8 748-E.01 *









Table 4-1. Biotreatability assay results
Sample A tmaxNO = 0.868
Sample C aerated BI = 0.49
Sample C non-aerated Invalid BI value; bacteria did not grow
Sample D aerated BI = 0.43
Sample D non-aerated Invalid BI value; bacteria did not grow

As shown in Table 4-1, aerated sample C and sample D are treatable biologically. The

non-aerated sample C and non-aerated sample D had negative YNO values, and thus gave invalid

BI values. The absorbance for those samples decreased with nitrate consumption during batch

test.

Conclusions

The objective of the assay is to quantitatively analyze biological treatability of high

nitrate solutions. The biotreatability index, BI is used to determine if the sample can be

denitrified successfully. BI is obtained from the ratio, ([tmaxNO) test sample)/(([lmaxNO) synthetic sample with

same N03-N concentration). YNO values can be obtained from the plots of absorbance vs. SNO observed

during the batch tests, and [tmaxNo is obtained from the plots of ln(3 YNoSNO(O)) vs. YNO*t. If

the value of the ratio is closer to 1, the sample is treatable. A value much lower than 1 shows

that high denitrification will be very difficult to achieve for that sample.









CHAPTER 5
CONCLUSIONS AND FUTURE WORK

The objective of the project was to establish high denitrification rate for an industrial high

nitrate stream using biological treatment. At the end of this study, it was concluded that the

industrial stream was indeed treatable. However, volatile contaminants and metals ions present in

the stream must be removed prior to biological treatment. Volatile contaminants may be

removed through aeration, and metal ions can be removed through an ion exchange process.

During the treatment, micronutrients should be added to the reactor directly rather than in the

feed, since precipitation can occur in the feed. It was also established that neutralization with

sodium did not affect denitrification rates, since the industrial stream sample neutralized with

sodium hydroxide and the stream sample neutralized with potassium hydroxide had same results.

CSTR with sodium hydroxide neutralized synthetic nitrate feed was able to reach a

denitrification rate of 4 mg NO3-N/L/min with a flow rate of 11.5 mL/min and an overall

denitrification of 97%. This denitrification rate was twice the target denitrification rate. CSTR

with the feed of sodium hydroxide neutralized industrial stream was able to reach the target

denitrification rate of 2 mg NO3-N/L/min with a flow rate of 5.5 mL/min and an overall

denitrification of 98%.

The denitrification was performed using potassium acetate as the exogenous carbon

source. Thus, effects of carbon limitation on denitrification were not analyzed. For the next

step, an industrial carbon source may be used for the process. Studies using attached growth

bioreactors can also be conducted with micronutrients added directly to the reactor instead of the

feed. Attached growth bioreactors can be more feasible since sludge concentration in the reactor

effluent will be very low.









LIST OF REFERENCES


Barber W. P. and Stuckey D. C. (2000) Nitrogen removal in a modified anaerobic baffled
reactor (ABR): 2, Denitrification. Water Research 34, 2413-2422.

Gasith A. J., Jop K. M., Dickson, K. L., Parkerton, T. F., and Kaczmarek, S.A. (1988). Protocol
for the identification of toxic fractions in industrial wastewater effluents. Aquatic Toxicology
and Hazard Assessment: 10th Volume, ASTM STP 971. Adams, W.J., Chapman, G.A., and
Landis, W.G. Editors., American Society for Testing and Materials, Philadelphia. pp. 204-215.

Kanow, P.E., 1993. Denitrification Methods United States Patent 5211847.
http://www.freepatentsonline.com/5211847.html March 25, 2008.

Lee S., Maken S., Jang J., Park K., and Park J. (2006). Development of physicochemical
nitrogen removal process for high strength industrial wastewater. Water Research 40, 975-980

Leslie Grady C.P., Daigger, G.T., and Lim, H.C. 1999. Biological Wastewater Treatment 2nd ed.
CRC Press, Boca Raton, USA.

Tchobanoglous, G., Burton F.L., and Stensel, D.H. 2003. Wastewater Engineering: Treatment
and Reuse McGraw-Hill, NY, USA.

US EPA., 2005. Section 62-302.530, Criteriafor Surface Water Quality Classifications
(Florida) http://www.epa.gov/waterscience/standards/wqslibrary/fl/fl 4_62-302t.pdf March 30,
2008

Vishniac S., and Santer M. (1957). The Thiobacilli. Bacteriol Rev 21, 195-213

Wasser I.M., deVries S., Moenne-Loccoz P., Schroder, I., and Kavlin K. D. (2002). Nitric oxide
in biological denitrification: Fe/Cu metalloenzyme and metal complex NOx redox chemistry.
Chemical Reviews (Review) 102, 1201-1234









BIOGRAPHICAL SKETCH

Sherin Peter was born on February 23, 1984, in Muvattupuzha, India. She received her

Bachelor of Science degree in chemical engineering from University of South Florida, Tampa, in

2005. She obtained her MS degree in chemical engineering from University of Florida in 2008.





PAGE 1

1 BIOLOGICAL DENITRIFICATION OF HI GH NITRATE INDUSTRIAL STREAMS By SHERIN PETER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORI DA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Sherin Peter

PAGE 3

3 To my parents, Peter Korath and Jessy Peter, and to Marun

PAGE 4

4 ACKNOWLEDGMENTS I would like to grat efully acknowledge m y advisor, Dr. Spyros Svoronos, for his guidance and support throughout my graduate studies. I would also like to thank my committee member, Dr. Ben Koopman, for his valuable advi ce through the course of the experiments. I acknowledge Kiranmai Durvasula, for her guidance and advice in experimental work. I am also grateful to Darrick, for providing industrial samples and materials used in the experiments. Finally, I thank my parents for their endle ss support and advice throughout my studies.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT...................................................................................................................................10 CHAP TER 1 INTRODUCTION..................................................................................................................12 Biochemical Operations in W astewater Treatment................................................................ 12 Biological Denitrification.......................................................................................................14 Kinetics of Denitrification...............................................................................................17 Bioreactor Configuration for Denitrification ................................................................... 20 Objective of the Project....................................................................................................... ...21 2 DENTRIFICATION OF HIGH NITRA TE INDUST RIAL STREAMS USING GROWTH BIOREACTORS.................................................................................................. 22 Materials and Methods...........................................................................................................22 Setting up Bios2.............................................................................................................. 22 Feed Preparation and Sampling for Bios2....................................................................... 23 Setting up Bios3.............................................................................................................. 24 Feed Preparation and Sampling for Bios3....................................................................... 25 Results and Discussion......................................................................................................... ..25 Bios2 Results...................................................................................................................25 Bios3 Results...................................................................................................................27 3 DENITRIFICATION OF HIGH NITR ATE INDUST RIAL STREAMS USING SUSPENDED GROWTH BIOREACTORS..........................................................................31 Materials and Methods...........................................................................................................31 Setting up Suspended Growth Bioreactor....................................................................... 31 Sampling and Data Collection.........................................................................................32 Feed Preparation.............................................................................................................. 33 Results and Discussion......................................................................................................... ..34 CSTR1 Results................................................................................................................ 34 Analysis of CSTR1 Results.............................................................................................35 Developing Equations..................................................................................................... 36 Applying CSTR1 Results................................................................................................37 CSTR2 Results................................................................................................................ 39 CSTR3 Results................................................................................................................ 45

PAGE 6

6 CSTR4 Results................................................................................................................ 48 Restarting CSTR4 with Sample A...................................................................................50 4 ASSAY ON THE BIOLOGICAL TREATABI LITY OF INDUSTRIAL STREAMS .......... 52 Developing Equations........................................................................................................... ..53 Development of the Assay...................................................................................................... 54 Results and Discussion......................................................................................................... ..56 Conclusions.............................................................................................................................61 5 CONCLUSIONS AND FUTURE WORK ............................................................................. 62 LIST OF REFERENCES...............................................................................................................63 BIOGRAPHICAL SKETCH.........................................................................................................64

PAGE 7

7 LIST OF TABLES Table page 1-1 Oxidation-reduction reactions for denitrification ..............................................................17 3-1 Contents of the micronutrient solution (Vishniac and Santer, 1957 .................................. 33 3-2 Concentration after the first time period for CSTR1......................................................... 38 3-3 Concentration after the s econd tim e period for CSTR1..................................................... 38 3-4 Concentration after the third time period for CSTR1........................................................ 38 4-1 Biotreatability assay results.............................................................................................. .61

PAGE 8

8 LIST OF FIGURES Figure page 1-1 Denitrification reactions.................................................................................................. ...16 2-1 Attached growth bioreactor................................................................................................ 22 2-2A Bios2 denitrification an d feed flow rate vs. tim e.............................................................. 26 2-2B Bios2 pH and nitrate concentrations vs. tim e.................................................................... 27 2-3A Bios3 denitrification and feed flow rate vs. tim e...............................................................28 2-3B Bios3 pH and nitrate concentration vs. time...................................................................... 29 3-1 Continuous stirred tank reactor.......................................................................................... 31 3-2 CSTR1 nitrate reduction a nd feed flow rate vs. tim e......................................................... 34 3-3 CSTR1 pH and nitrate concentrations vs. time.................................................................. 35 3-4 Time periods used for CSTR1 data analysis...................................................................... 37 3-5 Dilution rate of CSTR1 with respect to tim e..................................................................... 39 3-6 CSTR2 denitrificati on and feed flow rate vs. tim e, part I.................................................. 40 3-7 Ratio of C(t)/Cf vs. time for component accumulation in CSTR2..................................... 41 3-8 Plot of dilution rate vs. time for CSTR2........................................................................... 42 3-9 Ratio of C(t)/C(o) vs. time for component deficiency in CSTR2...................................... 42 3-10 CSTR2 denitrificati on and feed flow rate vs. tim e, part II................................................ 43 3-11 CSTR2 pH and nitrate concentrations vs. time.................................................................. 44 3-12 CSTR2 absorbance vs. time............................................................................................... 44 3-13 CSTR3 denitrification and feed flow rate vs. tim e............................................................ 46 3-14 CSTR3 pH and nitrate concentration vs. time................................................................... 47 3-15 CSTR3 absorbance vs. time............................................................................................... 47 3-16 CSTR4 denitrification and feed flow rate vs. tim e............................................................ 49 3-17 CSTR4 pH and nitrate concentrations vs. time.................................................................. 49

PAGE 9

9 3-18 CSTR4 absorbance vs. time............................................................................................... 49 4-1 Absorbance vs. nitrate con centration, aerated sample ....................................................... 56 4-2 Absorbance vs. nitrate con centration, non-aerated sam ple................................................ 56 4-3 Absorbance vs. nitrate concentra tion, synthetic nitrate solution ....................................... 57 4-4 ln( -YNOSno(o)) vs. YNO*time, aerated sample................................................................ 57 4-5 ln( -YNOSno(o)) vs. YNO*time, non-aerated sample........................................................ 57 4-6 ln( -YNOSno(o)) vs. YNO*time, synthetic nitrate solution................................................. 58 4-7 Absorbance vs. SNO for sample A......................................................................................59 4-8 ln( -YNOSNO(0)) vs. YNO t for sample A........................................................................... 59 4-9 Absorbance vs. SNO for aerated sample C.......................................................................... 59 4-10 ln( -YNOSNO(0)) vs. YNO t for aerated sample C............................................................... 60 4-11 Absorbance vs. SNO for aerated sample D......................................................................... 60 4-12 ln( -YNOSNO(0)) vs. YNO t for aerated sample D............................................................... 60

PAGE 10

10 Abstract of Thesis Presente d to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BIOLOGICAL DENITRIFICATION OF HIGH NITRATE INDUSTRIAL STREAMS By Sherin Peter May 2008 Chair: Spyros Svoronos Major: Chemical Engineering The purpose of this project was to develop a biological denitrificat ion system for high nitrate industrial streams. High nitrate samples obtained from an industrial source and synthetic nitrate solution prepared using nitr ic acid and DI water were used in the experiments. The goal was to obtain a denitrific ation rate of 2 mg NO3-N/L/min. Attached growth systems and continuous stirred tank reactors were used for the study. The reactors were kept in anoxic conditions in order to maintain denitrification conditions. Effects of micronutrient deficiency, volatile contaminants high nitr ate concentration, and sodium on denitrification rates were studied. From the study, it was concluded that the industr ial stream was treatable; however, volatile contaminants present in the feed solution must be aerated out and metals must be removed by ion exchange prior to denitrificati on. It was also concluded that micronutrients should be added directly to the reactor in order to avoid its precipitation in the feed and deficiency in the reactor. Studies comparing sodium and potassium neutralized nitrate solutions did not show any significant differences in denitrification rates. Thus, it was concl uded that sodium did not affect the denitrification process considerably. In the suspended growth system, denitrification rate of

PAGE 11

11 4 mg NO3-N/L/min was achieved with synthetic nitrate solution as the feed, and a denitrification rate of 2 mg NO3-N/L/min was achieved using in dustrial nitrate solution. An assay was also developed in order to test the biotreatability of high nitrate solutions. In the assay procedure, a batch test is conducted usi ng the test sample along with a synthetic nitrate solution containing the same NO3-N concentration. The absorbance values and denitrification rates of the test sample and synthetic nitrate solution are obt ained after 4, 8, and 24 hours. YNO and maxNO of the sample and synthetic nitrate solution are obtained from the absorbance and NO3-N measurements. The biotreatab ility index, BI, of the test sa mple is obtained by taking the ratio of maxNO of the sample to maxNO of a synthetic nitrate soluti on containing the same nitrate concentration. A BI value of 1 means that the test sample can be successfully denitrified, and values considerably less than 1 show that the denitrification will be very difficult.

PAGE 12

12 CHAPTER 1 INTRODUCTION Biochemical Operations in Wastewater Treatment Biochem ical operations are used widely in wastewater treatment systems to pollutants that can cause harm to aquatic environments after discharge (Tchobanoglous, 2003). Pollutants in aquatic systems can cause low dissolved oxyge n concentrations, eutrophication, and increased toxicity due to organic chemical s. Pollutants in wastewater ma y be classified by their physical characteristics, chemical characteristics, by th eir susceptibility to alte ration by microorganisms, by their origin, and by their e ffects (Tchobanoglous, 2003). Many of these classifications may overlap for various components. The goal of wast ewater treatment is to remove these pollutants in an efficient and economical manner by utilizi ng various unit operations. The unit operations can be divided into physical, chemical, and biochemical operations. Physical operations are operated based on laws of physics, chemical operations are operated by utilizing various chemical reactions to remove toxins, and bioc hemical operations use enzymatic catalysis of living microorganisms in order to tr eat the wastewater (Tchobanoglous, 2003). Most wastewater containing biodegradable cons tituents can be treated biologically with proper control. The primary objective in using biological treatment in industrial wastewater is to reduce the concentrations of organic and inorga nic compounds, as well as remove any nutrients present, such as nitrate or phosphorus (Tchoba noglous, 2003). Sometimes pretreatment is needed prior to biological treatment since indu strial streams may contain components that are toxic to bacteria. In biochemical processes, soluble pollutants in the wastewater are converted to an inert form, such as CO2 or N2, or into new microbial biomass. Generally after the treatment, excess biomass is removed from treated effluent by a physical operation, su ch as settling tanks. Microorganisms may also entrap any insoluble or ganic matter present in the waste stream after

PAGE 13

13 preliminary treatment, and it is removed through physical operation that follows. Thus, the effluent from the physical operati on is relatively clean, and gene rally no additional treatment is required (Tchobanoglous, 2003). Biochemical operations in wastewater trea tment may be classified according to the biochemical transformation, the biochemical environment, and the bioreactor configuration (Grady, 1999). Biochemical transformations in clude removal of soluble organic matter, stabilization of insoluble organic matter, and co nversion of soluble inorganic matter. Removal of soluble organic matter occurs when the microorganisms use the organic matter as carbon and energy source. Part of the carbon is converted to carbon dioxide during enzymatic reactions, and the rest is used to produce more biomass. This process can be carried out in aerobic conditions and anaerobic conditions (Grady, 1999). Stabiliza tion of insoluble organic matter occurs when the particulates are entrapped within the bioma ss, and is converted to stable end products. Conversion of soluble inorganic ma tter is utilized in biologic al nutrient removal processes, generally to reduce phosphorus a nd nitrogen concentrations in wastewater. Phosphates in wastewater are converted ultimat ely to orthophosphates through microbial activity, and are then taken up by specialized bacteria th at store large quantities of phos phates in granules within the cell (Grady, 1999). Nitrogen can be present as ammonium or nitrat e in wastewater. Ammonium is converted to nitrate in aerobic environments, and nitrate is conve rted to inert nitrogen gas in anaerobic environments. Biochemical environment where microbial activity takes place is classified mainly according to the terminal electron acceptor dur ing energy production. Three main types of electron acceptors are oxygen, inorganic compounds, and organic compounds (Grady, 1999). When dissolved oxygen is present in high con centration, it becomes the primary electron

PAGE 14

14 acceptor, and the environment is aerobic. Wh en nitrate or nitrite is present in high concentrations compared to DO levels, the environment is anoxic, and nitrate or nitrite serves as the terminal electron acceptor. Sometimes or ganic compounds, carbon dioxide, and sulfate may act as the terminal electron acceptor, in which the environment is anaerobic (Grady, 1999). Wide ranges of microbial specie s thrive in aerobic environmen ts, while anoxic and anaerobic processes are carried out by a lim ited number of organisms. Biological Denitrification Denitr ification is used widely in wastewater treatment plants in order to treat nitrate and ammonia, in combination with nitrification. Nitrat e concentrations in effl uent streams are highly regulated by EPA. Since nitrate can act as a fert ilizer, aquatic weeds, grasses and algae can grow excessively. This can lead to eutrophication a nd depleted oxygen levels in receiving waters, causing fish kills (Kanow, 1993). Therefore, unless the water is properly diluted or sufficient flow of water is employed to prevent accumu lation, high nitrate concentrations can cause ecological imbalance. Nitrate conc entrations in contaminated water have also been linked to outbreaks of methemoglobinemia in infants, and various health problems in animals (Barber, 2000). The standard for total nitrate con centration in effluent water is 10 mg/L, or less than the concentration that exceeds nutrient criteria (US EPA, 2005). Various methods exist for nitrate reduction in wastewater, includi ng air stripping, break point chlo rination, ion-exchange systems, fluidized bed systems, expanded bed systems, and biological denitrif ication (Kanow, 1993). Most of the chemical methods used for denitrif ication can be costly, a nd the chemicals involved may release toxic compounds to the environmen t (Lee, 2006). Biologica l denitrification is

PAGE 15

15 comparatively stable and reliable, and can have higher potential removal efficiency and easy process control (Barber, 2000). In domestic wastewater plants, biological denitrifi cation is coupled with nitrification. Nitrification converts ammonia to ni trate, and in denitrification, n itrate is converted ultimately to nitrogen gas. Two modes of nitrate removal can occur in effluent streams: assimilating and dissimilating nitrate reduction (Tchobanoglous, 2003) In assimilating nitr ate reduction, nitrate is converted to ammonia for cell synthesi s. This usually occurs when low NH4 +-N is available, and the process does not depend on dissolved oxyge n concentrations. In dissimilating nitrate reduction, the nitrate reduction is associated with respiratory electron chain, where nitrate or nitrite ions are used as terminal elec tron acceptor for the oxida tion of electron donors (Tchobanoglous, 2003). The bacteria that are used for denitrification are facultative aerobic; they are able to switch from aerobic mode to anoxic mode (Grady, 1999). In aerobic conditions, bacteria use oxygen as the terminal electron accepto r. In the presence of nitrate in anoxic environments, nitrate is used as the terminal electron acceptor. In the absence of dissolved oxygen, nitrate reductase enzyme in the electron transport chain is induced, which leads to the transfer of electrons to nitrat e. Presence of oxygen can suppre ss the activity of the enzyme (Tchobanoglous, 2003). Therefore, low oxygen concen trations and high nitrate concentrations are preferable for high de nitrification rates. Heterotrophic bacteria such as paracoccus denitrificans, thi obacillus denitrificans, and other pseudomonas are usually used for denitr ification processes (Tc hobanoglous, 2003). The mechanism for biological denitrification is gi ven in Figure 1-1 (Wasser, 2002). The nitrate content in the waste stream is converted to an inert form. The enzymes involved in the

PAGE 16

16 denitrification process are nitrate reductase, nitrite reductase, n itric oxide reductase, and nitrous oxide reductase (Grady, 1999). I. NO3 -------------------------------NO2 II. NO2 ------------------------------NO III. NO ------------------------------------N2O IV. N2O --------------------------------------N2 Figure 1-1. Denitrif ication reactions Heterotrophic bacteria use organic carbon as the energy source for metabolism. The amount of carbon in the stream is measured using chemical oxygen demand. Chemical oxygen demand measures electrons available in an or ganic compound. The COD test is based on the fact that organic compounds can be fully oxidized into carbon dioxide and water with a strong oxidizing agent. The COD is expressed as the amount of oxygen required to accept the electrons from the organic compound after complete oxi dation (Grady, 1999). Three sources of carbon may be used in denitrification: COD in the influent wastewater, COD produced during endogenous decay, or COD from an exogenous source (Tchobanoglous, 2003). Generally, methanol or acetate is used when exogenous carb on source is needed. Along with carbon, other nutrients are also required for bacterial me tabolism and growth, incl uding phosphorus, sulfur, potassium, calcium, iron, sodium, chlorine, a nd magnesium (Tchobanoglous, 2003). Domestic waters often contain necessary nutrients; for industrial wastewaters, nutrients are added externally. The energy source used for growth is called the substrate due to the extensive role of enzymes in microbial metabolism. Nitratereductase Nitritereductase Nitricoxidereductase Nitrousoxideredu ctase

PAGE 17

17 Kinetics of Denitrification Bacterial m etabolism can be described as a series of oxidation-re duction reactions. The substrate is consumed during gr owth, and additional cells are pr oduced by binary fission, asexual mode, or by budding (Tchobanoglous, 2003). In binary fission, the cell beco mes two organisms. The time required for binary fission is called gene ration time, which can range from less than 20 minutes to several days. In water treatment sy stems, the generation of new cells is limited by availability of substrate and nutri ents. The generalized equation fo r microbial growth is given in Equation 1-1 (Tchobanoglous, 2003): Carbon source + energy source + elec tron acceptor + nutrients ------biomass + CO2 + reduced acceptor + end products (1-1) For a balanced growth equation, carbon util ization and biomass growth are coupled. Therefore, the growth yield, Y, is expressed as units of biomass produced per unit of substrate removed. When the reactions are coupled, the rate of substrate utilizatio n for synthesis and the rate of biomass growth are proportional, with Y as the proportionality constant. The actual amount of biomass formed always less than Y, si nce a portion of energy is used for maintenance purposes. This yield is referre d to as the observed yield (Yobs) (Grady, 1999). Bacterial growth in anoxic conditions, as in denitrification, with ni trate as the nitrogen source and terminal electron acceptor, and acetate as the carbon source, follows the oxidation reduction reactions as given in Table 1-1 (Grady, 1999). The general formula used for biomass is C5H7O2N. Table 1-1. Oxidation-reduction reactions for denitrification Reaction for bacterial cell synthesis with nitrate as the nitrogen source: 1/28 C5H7O2N + 11/28 H2O = 1/28 NO3 + 5/28 CO2 + 29/28 H+ + eReaction for electron acce ptor (with nitrate as the terminal electron acceptor): 1/10 N2 + 3/5 H2O = 1/5 NO3 + 6/5 H+ + eReaction for electron donor (with acetate as the carbon source): 1/8 CH3COO+ 3/8 H2O = 1/8 CO2 + 1/8 HCO3 + H+ + e

PAGE 18

18 The overall molar-based equation for bacter ial growth can be obtained by using the Equations 1-2a and 1-2b (Grady, 1999). R = Rd fe*Ra fs*Rc (1-2a) fs + fe = 1 (1-2b) Rd is the equation for electron donor, Ra is the equation for electron acceptor, Rc is the equation for bacterial cell synthesis, fs is the fraction of transfe rred electrons used for cell synthesis, and fe is the fraction of electrons used for energy. The negative terms are inverted when used in the equation. The fraction of electr ons used for synthesis can be obtained from the growth yield. The carbon for synthesis is sh ifted to biomass and the carbon used for energy converted to carbon dioxide duri ng the oxidation-reduction reactions. When industrial carbon source is used, the formula for the electron donor can be estimated by analyzing the elemental compositions. Also, when nitrate is the pre dominant nitrogen form, the nitrogen must be reduced from +V state to III state before it can be used as the nitrogen source for cell synthesis (Grady, 1999). The equation for microbial growth is often written also with COD basis in terms of true growth yield. The general formula is given in Equation 1-3, Ss + (-(1-YH)) SN -------------> YHXBH (1-3) where Ss is amount of substrate COD, SN is the amount of termin al electron acceptor in COD units, YH is the growth yield of activ e heterotrophic bacteria, and XBH is the active heterotrophic biomass in COD units (Grady, 1999). Bacteria grow exponentially in favorable cond itions. When there is a limiting nutrient present, the specific growth rate coefficient of the bacteria, will depend on the concentration of the nutrient. The limiting nutrient can be the carbon source, nutrients, or other factor needed

PAGE 19

19 by the organisms for growth. The growth rate coefficient increases initially with increasing substrate concentration, and asymptotically ap proaches a maximum called the maximum specific growth rate, _max (Grady, 1999). A general equation used to characterize bacterial growth is the Monod equation (Equation 1-4). This equati on is developed strictly on empirical basis (Grady, 1999). = _max (Ss/(Ks + Ss)) (1-4) In the Monod equation, Ss is the substrate concentration and Ks is the half-saturation coefficient. Ks is the substrate concentration where the specific growth rate of the bacteria is equal to half of the maximum growth ra te. It can used to predict how fast _max is obtained. The Monod equation can be extended to consider th e effects of various substrates when multiple limiting nutrients are present, as shown in Equa tions 1-5 and 1-6 (Grady, 1999). Equation 1-5 is used when both growth limiting nutrients are required for biomass growth and they can influence the specific growth rate at the same time. Then the specific growth rate is calculated by taking the product of the Monod terms for the two nutrients. Equation 1-6 is used when both nutrients are required for biomass growth, but only one nutrient will limit th e growth at a given time. Then the specific growth rate is obtained by taking the lowest value obtained from separate single-substrate models. = _max (Ss1/(Ks1 + Ss1)) (Ss2/(Ks2 + Ss2)) (1-5) = min ( _max (Ss1/(Ks1 + Ss1))), ( _max(Ss2/(Ks2 + Ss2))) (1-6) The pH range of the system should also be c onsidered during the denitrification. The pH of the system is generally increas ed during denitrif ication since OHions are produced. The optimal pH range for the bacterial culture can be determined using variou s batch tests, and the

PAGE 20

20 bacterial growth can be recorded. Generall y, mild alkaline conditions are preferred for denitrification (Tchobanoglous, 2003). Bioreactor Configuratio n for Denitrif ication Two general anoxic models are used for den itrification processes: suspended growth systems and attached growth systems (Grady, 1999). In attached growth systems, microbes are grown attached to a packing material, generally rocks, sand, or other synthetic materials. The wastewater flows upward or downward, coming into contact with the atta ched biofilm, and the treated water flows out of the system. In anoxic systems, the packing material can be completely submerged, with gas space above the biofilm liquid layer (Grady, 1999). Excess biomass that may exit with the effluent can be separated us ing a clarifier and can be removed for further processing. In suspended growth systems, microbes ar e kept in a suspended state by appropriate mixing. Suspended growth systems can be opera ted in batch mode or continuous mode. In batch systems feed is added into the reactor with suspended biomass and is allowed to react to completion. Reaction conditions and growth envi ronment change with time. In continuous systems feed solution is continuous pumped into the reactor and an effluent is pumped out at the same flow rate. The concentratio ns of components in effluent are the same as the concentrations of components in the reactor. One example of a continuous suspended system is the continuous stirred tank reactor (CSTR). In a CSTR, the liqui d volume is kept constant and sufficient mixing is added to keep the conditions uniform throughout the reactor. A physical operation, such as sedimentation, may be implemented in order to separate biomass from the effluent. The overflow from the sedimentation basin will have very small concentration of biomass. The underflow will have very high concentration of bi omass, most of which is recycled to the bioreactor. A portion of the c oncentrated slurry is wasted, and is further treated before

PAGE 21

21 discharging it to the environment. In several operations, flexibility is implemented by placing several CSTRs in a series. The conditions in various stages of the system can vary, thus allowing for completion of various transformations Recycle may also be employed at desired stage of the system or through the entire chain (Grady, 1999). Objective of the Project The objective of this study was to develop a feasible denitr if ication process for treating an industrial nitrate stream. Attached growth bioreactors and suspended growth bioreactors were used for this study. Initial inoculum for the re actors were obtained from the denitrification basins at University of Florida Water Reclamati on Facility. The optimal pH for denitrification was determined to be between 8 and 9.5 in previo us studies. Since industr ial nitrate stream does not contain enough COD to support denitrification, potassium acetate was used as an exogenous carbon source. The goal of the project was to obtain a denitrification rate of 2 mg NO3-N/L/day.

PAGE 22

22 CHAPTER 2 DENTRIFICATION OF HIGH NITRATE IN DUST RIAL STREAMS USING GROWTH BIOREACTORS Initial attempts to denitrify the high nitrate industr ial stream were performed in attached growth bioreactors, Bios2 and Bios3. The subm erged attached growth bioreactors allow for short hydraulic residence times with high solids retention time s, and low solids waste after denitrification. A packed bed bioreactor with up ward flow and a recycle stream was used to conduct the experiments. Rock media obtained fr om Adventus was used to pack the reactor, which provided high surface area for the attached grow th of bacterial cells. The reactor was kept anoxic in order to allow for proper denitrification. Materials and Methods Setting up Bios2 A 3.78 L HDPE bottle was used to construct Bi os2. The set up of the bioreactor is s hown in Figure 2-1. Figure 2-1. Attached growth bioreactor Inlet ports Bioreactor Effluent tubing Effluent tank Recycle pump Feed pump

PAGE 23

23 The bioreactor contained eight feed inlet port s at the bottom and an outlet port at the top. This allowed for the feed to enter from all si des of the bioreactor, which allowed for better contact with the denitrif ying bacteria inside. An outlet por t was connected back to the feed stream as the recycle, and another outlet port wa s used to collect the overflow into a receiving bottle. A sample port was also constructed along the recycle outlet tubing. The feed stream was pumped from the feed tank and was allowed to mix with the recycle. This allowed for the regulation of pH of the stream entering the biore actor. Feed and recycle streams flowed through peristaltic pumps, and pump flow dials were used to monitor the flow rates. Feed and recycle pumps were standardized and calibration curves were used to obtain the flow rates with respect to pump dials. The mixed stream flowed through the eight in let ports, and was allowed contact with the bacteria as it flows upward and out the bioreactor. The top portion of the bioreactor was used as the gas separator. Tubing was attached to the lid of the bioreactor to al low the gas to escape. Cole Parmer L/S 13 tubing was used for all the c onnections in the bioreactor. The bioreactor was filled with stone media from Adventus and 1.5 L of inoculum from UF WRF. The working volume of the bioreactor was 3 L. Feed Preparation and Sampling for Bios2 Acetate was added as the carbon source for th e industrial stream that was fed to the reactor in the form of sodium acetate. The str eam was pre-neutralized in order to increase the pH, and had a concentration of approximately 1300 mg/L NO3-N. The carbon source for Bios2 feed was added in order to obtain 8:1 COD-NO3-N ratio. In order to obtain an 8:1 COD-NO3-N ratio, 10400 mg COD/L was required in the feed st ream. The initial COD was assumed to be 6000 mg/L, and thus 4.4 g/L of COD was needed in the feed solution. Thus 6.445 g/L of acetate

PAGE 24

24 was added to the feed solution. In order to increase the denitrif ication rate of the bioreactor, approximately 0.01 g/L of yeast extract and 0.3 ml/L molybdenum (as 1.1 g/L molybdic acid) were also added to the feed stream, providi ng nutrients to the ba cterial culture. The initial feed flow for Bios2 was turned on to 0.57 mL/min and the recycle was turned on to 6.67 mL/min. The recycle flow to feed fl ow ratio was 11.57. The inlet pH of the feed solution was maintained at 5. Concentrated hydr ochloric acid was added to the feed tank in increments of 10 mL if the feed st ream was measured to be high. During sampling, it was noticed that the sample port was in fact allowing a mixture of the feed stream and recycle stream to flow through the valve, and thus the nitrate concentrations measured through the valve were no t a true representation of the NO3 -N of the effluent stream of the bioreactor. Thus the sample port was moved to the recycle outlet, and sample was obtained by disconnecting the recycle tubing. This allowed for measuring the pH and NO3-N of both inlet stream and outlet stream of the bioreactor. A small volume of the sample was discarded initially each time in order to flush out the tubing and the sample port valve. The pH and NO3 -N tests were conducted three times a day, and the fl ow rates were adjusted accordingly. NO3-N was measured using HACH NitraVer test kits and measured us ing a spectrophotometer. In general, the sample was diluted to 20 times in order to obtain the nitrate concentration. Setting up Bios3 The m ain purpose of Bios3 was to compare the effects of sodium and potassium on denitrification. The construction and set-up for Bios3 was identical to that of Bios2. The reactor was filled with rock media from Adventus an d 1.1 L of inoculum from UF WRF was added. The working volume of the reactor was 3 L. Eight inlet ports were still us ed for feed flow, along with a recycle outlet, gas outlet, and a sample port for the inle t stream. Computer Controlled

PAGE 25

25 pumps were used for Bios3. The pumps were ca librated by measuring the effluent accumulated in the receiving bottle for a certain period of time. Feed Preparation and Sampling for Bios3 Bios3 was initially started with sodium hydr oxide neutralized industrial stream (sample A)-the same feed as Bios2. The feed was then switched to potassium hydroxide neutralized industrial stream (sample C). Po tassium acetate was used as the ca rbon source for the feed. The nitrate-nitrogen concentration of sample C was 2100 mg/L, which was much higher than Bios2 feed. 15 g/L of potassium acetate was added to the feed in order to ensure excess carbon, along with 0.024 g/L potassium phosphate as a source for phosphorus, 0.01 g/L yeast extract, and 3 mL of 1.1 g molybdenum in 1000 mL mo lybdic acid. The feed pH wa s maintained at 5, and 10 mL increments of hydrochloric acid were added to the feed if high pH was observed. Nitrate and pH measurements of the effluent were taken three ti mes daily. Nitrate-nitrogen measurements were taken using HACH NitraVer Test N Tubes and a spectr ophotometer. A small amount of the effluent was flushed out of the tubing before samples were take n. Two samples were collected for each measurement, one corresponding to the effl uent stream collected from the recycle outlet, the other sample corresponding to the inlet stream collected from the sample port valve. The bioreactor was started with a feed flow of 0.5 mL/min and a recycle flow rate of 2 mL/min. Results and Discussion Bios2 Results As shown in Figure 2-2A, Bios2 initially had high denitrification rates; however, the denitrification rate began to decrease eventually. A large am ount of gas bubbles were noticed in the reactor throughout its operation. The reactor was agitated periodically to release the gas bubbles. It was noticed that when the reactor was agitated, the denitr ification rates improved temporarily. However, the nitrate concentr ation in the reactor increased again. High

PAGE 26

26 denitrification rate was also obs erved after the addition of micronu trients to the feed and to the reactor. 40 drops of micronutrien ts were added to the feed and 5 drops were added directly to the reactor. The denitrification briefly increased after the add ition of micronutrients; however, the results did not stay positive for long. The highest flow rate achieved by Bios2 was 0.8 mL/min, with a denitrification ra te of 95.96%. This corresponded to a denitrification rate of 0.354 mg NO3-N/L/day. Thus, bioreactor failed to deni trify the industrial stream at the desirable rate, which was 2 mg NO3-N/L/min. Nitrate reduction and feed rate vs. time 0 10 20 30 40 50 60 70 80 90 100 6/76/176/277/77/177/278/6 Time (Days)% Nitrate Reduction0.0 0.2 0.4 0.6 0.8 1.0Feed Flow (mg NO3-N/L/min) % Nitrate Reduction Feed Flow New Bios2 Begin Feed pH decreases Media Agitated Feed w/ K-Acetate System Upset Micronutrients added Figure 2-2A. Bios2 denitrification and feed flow rate vs. time. As shown in the figure, a slight increase in denitrification was observed when the media was agitated. However, it still continued to perform poorly. An increase in denitrification wa s also shown immediately after the addition of micronutrients. Also, as shown in the figure, when the pH of the effluent was measured to be higher than 9.5, the pH of the f eed was decreased by the addition of concentrated HCl to the feed tank. Target feed rate for the reactor was 2 mg NO3-N/L/min.

PAGE 27

27 Start-Up 2/5/2007 ; Feeding Begins 2/12/20070.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 6/76/176/277/77/177/278/6 Feed pH decreases Media Agitated Feed w/ K-Acetate begin System Upset Micronutrient s added Figure 2-2B. Bios2 pH and n itrate concentrations vs. time One factor that was taken into account afte r Bios2 failed to denitrify was the effect of sodium on denitrification. It was hypothesized that sodium c ontribute significantly to the osmotic pressure across the cell membrane, which would affect the metabolism and denitrification of the bacteria. An alternate to using sodium salt for neutralization and as carbon source was to use potassium salts. Even t hough potassium ions contribute to the osmotic pressure also, their effects would be smaller than those of sodium ions. Therefore, a new bioreactor was started in order to examine if potassium would lead to high denitrification rates of the industrial stream samples. Bios3 Results Bios3 had initial high denitrificat ion rates; however, on the w hole, it had the same results as Bios2, as shown in Figure 2-3A. It was noticed that Bios3 recovered faster from system upsets than Bios2, possibly due to the presence of potas sium instead of sodium. In general, no

PAGE 28

28 significant changes were observed from Bios2 re sults. An increase in denitrification was observed after adding micronutrients to the feed so lution (4 drops/L) and di rectly to the reactor (5 drops). However, the high denitrificati on rate was temporary. Gas bubbles were also observed in the walls of the reactor Agitation of the reactor failed to increase its performance. The highest flow rate that was achieved by Bios2 was 1.2 mL/min, w ith an overall denitrification of 98.7%. This corresponded to a de nitrification rate of 0.612 mg NO3-N/L/day. Therefore, Bios3 also failed to meet the target denitrification rate of 2 mg NO3-N/L/day. The results showed using potassium salts did not have any significant impact on denitrification. nitrate reduction and feed rate vs. time 0 10 20 30 40 50 60 70 80 90 100 7/97/147/197/247/298/38/88/138/188/23 Time (Days)% Nitrate Reduction0.00 0.20 0.40 0.60 0.80 1.00 % Nitrate Reduction Feed Flow Bios3 Begin System Upset Micronutrients added Flushed out with water Figure 2-3A. Bios3 denitrification and feed flow rate vs. time. Bios3 results did not vary significantly from Bios2 result s, even though potassium hydroxide was used to neutralize the feed rather than sodium hydroxide. Notice that an increase in denitrific ation was also observed in Bios3 immediately after the addition of micronutrients.

PAGE 29

29 Start-Up 2/5/2007 ; Feeding Begins 2/12/2007 0 2 4 6 8 10 7/97/147/197/247/298/38/88/138/188/23 NO3-N (100 mg/L) pH Micronutrients added Figure 2-3B. Bios3 pH and n itrate concentration vs. time After Bios2 and Bios3 failed to denitrify industrial nitrate stre ams properly, several factors were considered that might have result ed in low performance of the reactors. One possibility was the presence of metals in high concentrations in the feed, which can affect bacterial metabolism. A metals analysis of Bios2 feed, Bios3 feed, and Bios3 effluent was conducted using Inductively Coupled Plasma (ICP). However, very low concentrations of metals were found in all samples. Another factor was the high nitrat e concentration of the industrial stream. The effects of high nitrate concentration of the feed on denitrification were not studied. Also, clogging of the tubi ng could have occurred due to various inlet ports and low flow rate of the feed and recycle. Clogging in the po res of the rock media c ould have occurred also, due to high concentration of biomass, which can lower the surface area for the attached growth of bacterial growth. Channeling and short-circuiting inside the reactor is also possible, and proper contact of the feed stream with the bacteria can be diminished. Another factor considered was the presence of toxic materials in the feed a ffecting bacterial growth. It was also possible that toxic substances present in the feed accumu lated on the rock media, which can affect the metabolism of the attached bacteria and cause low de nitrification.

PAGE 30

30 At this point, it was concluded that the low de nitrification was attri buted to some possible factors related to reactor design and some possibl e factors related to th e feed. In order to distinguish the causes, a standard suspended growth reactor was started. The suspended growth reactor used was Continuous Stirred Tank Reactor (CSTR).

PAGE 31

31 CHAPTER 3 DENITRIFICATION OF HIGH NITRATE IN DUST RIAL STREAMS USING SUSPENDED GROWTH BIOREACTORS Suspended growth bioreactors consist of react or vessels containing biomass growing in a suspended state rather than attached to a medi a. Continuous stirred ta nk reactor (CSTR) is a suspended growth bioreactor where the contents in the reactor are stirred c ontinuously in order to keep the conditions homogeneous within the reactor and the culture suspended. The stream to be treated is fed into the reactor, and the treated liquid mixed with biomass is pumped out as the effluent. The fractions of constituents in the effluent are identical to those inside the reactor. Many processes have a recycle stream where most of the biomass is recycled into the reactor. Materials and Methods Setting up Suspended Growth Bioreactor The assembly of the continuous st irred tank is given in Figure 3-1. Figure 3-1. Continuous stirred tank reactor As shown in Figure 3-1, the continuous stirre d tank reactor was constructed using a 4.2 L flask. The flask was filled 10% with feed so lution, and 90% with inoculum from UF WRF denitrification basin. The reactor was then set in a stir plate with the stirrer inside and was Gas outlet port Gas inlet port Effluent tubing Influent tubing Feed tank stirrer Computer controlled pump

PAGE 32

32 stirred continuously. A rubber stopper was used to close the reactor and it was secured to the reactor using parafilm. The rubber stopper had a feed inlet port, an effluent port, a gas inlet port, and a gas outlet port. A nitrogen tank was se tup beside the reactor and nitrogen was bubbled through the reactor twice da ily for 10 minutes to strip off any oxygen that is present. Initially, the reactor was started up in the batch mode. Samples for the nitr ate tests were taken through the inlet gas port. The reactor was operated in ba tch mode until the pH and absorbance increased, and then it was switched to continuous mode. For the continuous mode, a computer controlled pump was set up near the reactor with the same inlet and outlet flow rate s. The inlet tubing was secured into the feed port in th e rubber stopper, and the outlet tubing was attached to the effluent port. Cole Parmer L/S 13 tubing was used for feed and effluent connections. The effluent from the reactor was collected and measured at a specific time period in order to calibrate the pump. Sampling and Data Collection NO3-N, pH, and absorbance measurements were taken twice daily from the effluent of the reactor. In continuous mode, samples were ta ken from the effluent. The reactor was purged with nitrogen gas after each sampling and the gas inlet port was then secured firmly in order to keep the anoxic conditions in the bioreactor. The gas outlet port was closed only slightly in order to vent out gas formed in the reactor. Nitrate-nitrogen concentrations were measured using HACH NitraVer TestN Tubes and a HACH spect rophotometer. In general, the effluent sample was filtered and diluted to 20 times before nitrate-nitrogen measurements. Absorbance measurements were representative of the bacteria l concentration in the reactor, and were taken using a spectrometer. The results from nitratenitrogen, pH and absorbance measurements were used for adjusting the flow rates in the reacto r. COD measurements were taken periodically using HACH COD test kits. The samples were filtered and diluted to 20 times. After the

PAGE 33

33 samples were added to the test tubes with th e reactive material, they were placed in a COD reactor for two hours. COD measurements were then taken using a spectrophotometer. Feed Preparation The high nitrate industrial str eam obtained was pre-neutralized to appropriate pH using either sodium hydroxide or potassium hydroxide. Potassium acetate was used as the carbon source for the bacteria. 17.5 g/L of potassium acetate was used in the feed, along with 0.02 g/L potassium phosphate. A micronutrient solution was prepared using instructions for trace element solution by Vishniac & Santer. The contents in the solu tion are given in Table 3-1. The micronutrient solution was added in the feed initia lly in the amount of 4 drops/L. Later in the experiments, micronutrients were added directly to the reacto r rather than in the feed. Table 3-1. Contents of the micronutri ent solution (Vishniac and Santer, 1957) Ingredient Amount (per 500 mL DI water) Ethylenediaminetetraacetic acid (EDTA) 25.0 g ZnSO4 11.2 g CaCl2 2.79 g MnCl 2.58 g FeSO4 2.59 g Mo7O24(NH3)6 0.6 g CuSO4.5H2O 0.785 g CoCl2.6H2O 0.81 g Magnesium sulfate 2.525 g Molybdic acid 0.55 g Artificial nitrate solution used in the experiments was prepar ed using 69.6 weight percent nitric acid assay and DI water. The so lution was prepared by adding 8.6 mL of HNO3 assay per one liter of DI water to obtain the same NO3-N concentration as sample A. The solution was neutralized by adding approximately 4 g NaOH pelle ts per liter of solution. The feed solution also contained 17.5 g/L potassium acetate as the carbon source and 0.024 g/L potassium phosphate. The nitrate-nitrogen concentration of the feed was approximately 1450 mg/L, which was comparable to the nitrate-nitrogen concentration of sample A.

PAGE 34

34 Results and Discussion CSTR1 Results CSTR1 was started in order to denitrify sam p le A neutralized with NaOH in the tote. The reactor was started in batch mode, and was sw itched to continuous mode at a flow rate of 1 mL/min once the pH and absorbance increased. Micronutrients were added in the feed of the reactor in the amount of 4 dr ops/L. The results of the reactor are shown below: nitrate reduction and feed rate vs. time (Target feed rate: 2 mg NO3-N/L/min)0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 8/58/158/259/49/149/24time% nitrate reductio n 0 0.2 0.4 0.6 0.8 1feed flow rate (mg NO3-N/L/min) % nitrate reduction feed flow rate Figure 3-2. CSTR1 nitrate reducti on and feed flow rate vs. time

PAGE 35

35 pH and nitrate concentration vs. time0 1 2 3 4 5 6 7 8 9 10 8/58/158/259/49/149/24 timepH0 500 1000nitrate concentratio n pH NO3-N (100 mg/L) Figure 3-3. CSTR1 pH and nitr ate concentrations vs. time As shown in Figure 3-2, the deni trification rate of CSTR1 wa s initially high. However, it started to decrease after 8 days and continued to decrease without possibility of recovery. The pH began decreasing and nitrate concentration st arted decreasing with time, as shown in Figure 3-3. The reactor failed to recover ev en after switching to batch mode. After CSTR1 failed, possible causes that might ha ve contributed to the low denitrification of the industrial stream were considered. It was hypothesized that a toxic component in the sample might be accumulating in the reactor and was not consumed during the metabolism of the bacteria. Possible candidates were sodium buildup, high nitrate buildup, or accumulation of some other toxic component in the industrial so lution. An analysis was done to predict the concentration of a toxic compound that might be accumulating in the reactor after a certain time period. Analysis of CSTR1 Results After the hypothesis that the accumulation of a constituent is causing low denitrification, a modeling analysis was done on CSTR1 results in order to predict the accumulation period of the constituent. A mass balance was done on the reactor as shown in Equation 3-1.

PAGE 36

36 d/dt (VC) = FCf FCo V r (3-1) at constant volume, V dC/dt = FCf FCo V r (3-2) dC/dt = (F/V) Cf (F/V) Co r (3-3) (F/V) = D (3-4) (D = dilution rate) dC/dt = D Cf D Co r (3-5) Developing Equations When accumulation is considered, the reaction is neglected; then dC/dt = D Cf D Co (3-6) Since dilution rate changes with fl ow rate and thus with time, dC/dt + D(t) Co = D(t) Cf (3-7) Using integrating factor, IF = exp( D( )) d D( ) = + Do (3-8) exp( ( + Do) d = exp( 2/2 + Do ) (3-9) Multiplying by IF gives exp( 2/2 + Do ) dC/dt + exp( 2/2 + Do ) Co = exp( 2/2 + Do ) Cf (3-10) t d/dt(exp( 2/2 + Do ) C) = (exp( 2/2 + Do ) D(t) Cf (3-11) C exp( 2/2 + Do ) Co = D( ) Cf exp( 2/2 + Do ) d (3-12) C exp( 2/2 + Do ) Co = ( + Do ) Cf exp( 2/2 + Do ) d (3-13) C(t)=Co exp -( t2/2 + Dot)+ exp -( t2/2 + Dot) ( + Do ) Cf exp( 2/2 + Do ) d (3-14) At constant dilution rate, = 0, then C(t) = Co exp -(Dot) + Cf exp -(Dot) (Do ) exp(Do ) d (3-15) C(t) = Co exp(-Dot) + Cf (1-exp(-Dot)) (3-16) When dilution rate is not constant, it is a ssumed to change linearly with time; then D = + Do. Substituting gives ( + Do)d = d ( 2/2 + Do ) (3-17) Cf 0t ( + Do) exp(2/2 + Do ) d = Cf 0t exp( 2/2 + Do ) d ( 2/2 + Do ) (3-18)

PAGE 37

37 Cf 0t exp( 2/2 + Do ) d ( 2/2 + Do ) = Cf (exp( 2/2 + Do ) 1) (3-19) C(t) = Co exp(-( 2/2 + Do )) + Cf exp(-(2/2 + Do )) (exp( 2/2 + Do ) 1) (3-20) C(t) = Co exp(-( 2/2 + Do )) + Cf (1exp(-( 2/2 + Do ))) (3-21) Applying CSTR1 Results The results were analy zed over three time peri ods. As shown in Figure 3-4, time period 1 was taken from to to t1, time period 2 was taken from t1 to t2, and time period 3 was taken from t2 to t3. It was assumed that time period 1 and tim e period 2 had constant dilution rate, and time period 3 had dilution rate that cha nged linearly with time. At t = to, Co was taken to be zero; thus it was assumed that no toxins were present in the system initially. The results of the analysis are shown in Tables 3-2, 3-3, and 3-4. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 8/58/108/158/208/25time% nitrate reductio n 0 0.5 1 1.5 2 2.5 3feed flow rate Figure 3-4. Time periods used for CSTR1 data analysis to t1 t2 t3

PAGE 38

38 Table 3-2. Concentration after the first time period for CSTR1 After the first time period: C(t) = Co exp(-Dot) + Cf (1-exp(-Dot)) Co = 0 C1(t) = Cf (1exp(-Dot)) t = 3420 minutes Do (dilution rate) = F/V = (1 mL/min)/(4000 mL) = 0.00025 min-1 C1(t) = Cf (1exp((-0.00025/min)(3420 min)) C1(t) = 0.575 Cf Thus, after the first time period, 57.5% of the initi al toxin concentration will have accumulated Table 3-3. Concentration after the second time period for CSTR1 After the second time period: C2(t) = C1(t) exp(-Dot) + Cf (1-exp(-Dot)) t = 3365 minutes Do = F/V = (0.5 mL/min)/(4000 mL) = 0.000125 min-1 C2(t) = 0.575 Cf exp((-0.000125/min)(3365 min))+Cf (1exp(-0.000125/min)(3365 min)) C2(t) = 0.721 Cf Thus, after the second time period, 72.1% of th e initial toxin concentration will have accumulated in the reactor. Table 3-4. Concentration after the third time period for CSTR1 After the third time period: Dilution changes linearly with time. Thus D(t) = + Do t = 4500 minutes and Do were determined from the plot of dilution rate vs. time, as shown in Figure 3-5 From the plot, = 5.4E-8 Do = 0.0002 C3(t) = Co exp(t2/2 + Dot) + Cf (1exp(t2/2 + Dot)) Co = C2(t) = 0.721 Cf exp(t2/2 + Dot) = exp{(-5.4E-8*(4500 min)2/2)+((0.000241 min-1)(4500 min))} = 0.196 C3(t) = 0.721 Cf (0.196) + Cf (0.804) C3(t) = 0.946 Cf After the third time period, 94.6% Co w ill have accumulated in the reactor.

PAGE 39

39 dilution rate vs. timey = 5E-08x + 0.0002 R2 = 0.9859 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0500100015002000250030003500400045005000 time (minutes)dilution rate (min^-1) Figure 3-5. Dilution rate of CSTR1 with respect to time. From the linear regression, was calculated to be 5E-08 and Do was obtained to be 0.0002. The analysis showed that af ter 57 hours, 57.5% of the initial toxic component would have accumulated in the reactor. The model predicts that at the time of failure (8/15/2007), approximately 94.6% of the feed concentration of the toxin would have accumulated in the reactor, which can cause significant impact on the de nitrification rates. Therefore, a new reactor was started using synthetic nitr ate solution to investigate whether the accumulating toxin is a component present in the industria l feed or the sodium ions adde d to the feed for neutralization. CSTR2 Results CSTR2 was started using synt hetic nitrate solution prepared using nitric acid and DI water. Cole Parmer L/S 13 tubing was used to pump the feed into the reactor, and Cole Parmer L/S 14 tubing was used to pump out the effluent. Once the pH and the absorbance of the reactor increased, the reacto r was switched to continuous m ode. Two computer controlled pumps were placed near the reactor; one pump wa s used for feed and the second pump was used for the effluent. Two pumps were required becaus e of the difference in feed and effluent tubing sizes. Micronutrients were added to the feed in the amount of 4 drops/L. The results of the reactor are shown in Figure 3-6.

PAGE 40

40 nitrate reduction and feed rate vs. time: Part 1 0.00 20.00 40.00 60.00 80.00 100.00 8/319/59/109/15time 0 2 4 % nitrate reduction feed flow rate Figure 3-6. CSTR2 denitrif ication and feed flow rate vs. time, part I Initially on continuous mode, the reactor wa s started at 0.5 mL/min. The reactor was denitrifying very well at this flow rate. The flow rate of the fe ed was increased up to 2 mL/min and the reactor continued to denitr ify at high rates, as shown in Figure 3-6. However, the flow rate was increased without allowing the reactor to reach stable conditions. Increasing the feed flow required an increase in effl uent flow rate as well, and in creasing the flow prematurely can lead to washout conditions in the reactor. Ther efore, the feed flow ra te was decreased to 1.2 mL/min in order for the reactor to stabilize. Th e initial positive results seen in the reactor were brief, however, and the nitrate concentration began to increase again in the reactor. The reactor was switched to batch mode after very low denitrification rates were observed. It was noticed that denitr ification rate was high in th e reactor for approximately 265 hours (11 days). The low denitrif ication rates and low absorbance va lues were observed later on. An analysis was done on the accumulation of toxins in the reactor using Equation 3-21. The initial concentration of the toxin, Co, is assumed to be zero. The ratio of C(t)/Cf was plotted against time. The results are shown in Figure 3-7

PAGE 41

41 CSTR2 accumulation of a toxin0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 01000200030004000500060007000 time (minutes)C(t)/Cf Series1 Figure 3-7. Ratio of C(t)/Cf vs. time for component accumulation in CSTR2 As shown in the Figure 3-7, within after a time period of approximately 6400 minutes, the ratio C(t)/Cf has reached the value of 0.86. Thus, a deterioration of the bioreactor performance should have been observed by this time. After 188 hours (9/9/2007), the toxic component concentration would have reached 99% of the feed concentration. However, as shown in Figure 3-6, the reactor continued to perform well for tw o more days. Thus, accumulation of a toxin was not causing the low denitrification rates in the reacto r. It was then suspected that deficiency of a key constituent might be causing the low denitrif ication. Thus, the results were analyzed in order to predict the time that defici ency of a component might occur. Analysis was done for the first 6400 minutes of the operation of the reactor, where a linear increase in flow rate, and thus dilution rate, wa s observed. The dilution rate was calculated using the relationship given in Equati on 3-8, where D(t) is equal to t + Do. and Do were obtained from the plot of dilution rate vs time, as shown in Figure 3-8.

PAGE 42

42 y = 5.3931E-08x + 1.3776E-04 R2 = 9.6752E-01 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 01000200030004000500060007000 Figure 3-8. Plot of dilution rate vs. time for CSTR2 Another plot of C(t)/Co vs. time was generate d in order to quantify the depletion of a component initially present in the system. Analysis done using the data obtained from the reactor showed that after approximately 107 ho urs, the concentration of a component added initially to the reactor would have reached appr oximately 13% of the feed concentration. The results are shown in Figure 3-9. CSTR2 deficiency of a nutrient 0 0.5 1 1.5 01000200030004000500060007000 timeC(t)/Co Figure 3-9. Ratio of C(t)/C(o) vs. time for component deficiency in CSTR2 Since the reactor was performing very well up to this point, it was noted that the ingredient that is exhausted is needed only in very small am ounts for denitrification. Further analysis of the data showed that at the time of the failure (9/11/2007), the am ount of the nutrient will have reached 0.6% of its initial concentration. One component that was required for bacterial growth in very small amounts was the micronutrients. It was noted that attached film bioreactors had started to recover previously wh en micronutrients were added to the feed and directly to the

PAGE 43

43 reactor. However, the effect wa s short-lived and the reactors deteri orated again. This led to the conjecture that micronutrients added to the feed may have pr ecipitated, as suggested by Ben Koopman. Thus, most of the mi cronutrients added to the feed of the CSTR would have been precipitated out. Taking th is into consideration, micronutrients were added directly to the reactor everyday in proportion to the flow rate of the feed. Generally, 1 mL of micronutrients was added to the reactor when the feed flow rate is 1 mL/min. Magnesium was also added to the feed solution in the form of magnesium sulfate heptahydrate in order to ensure its availability. After the micronutrients were added, an incr ease in the reactor performance as well as bacterial growth was observed, as shown in Figure 3-6. These results showed that it was indeed the deficiency of micronut rients that caused low denitrification in the reactor previously. It was also determined that the additi on of micronutrients to the feed can cause precipitation, and thus, it must be added directly to the reactor. Th e amount of potassium phosphate added to the feed was also increased to 0.5 mg/L in or der to avoid phosphorus deficiency. CSTR2 nitrate reduction and feed rate vs. time Part II 0.00 20.00 40.00 60.00 80.00 100.00 8/319/109/209/3010/1010/2010/3011/911/1911/2912/912/19 time% nitrate reduction0 2 4feed flow rate (mg NO3-N/L/min) % nitrate reduction feed flow rate feed rate lowered due to limited carbon availab ility Figure 3-10. CSTR2 denitrification and feed flow rate vs. time, part II. As shown in the figure, a deficiency in carbon occurred during the operation, and the flow rate to the reactor was set at the minimum for 2 days. The flow rate was increas ed rapidly after carbon was available to reach appropriate steady state.

PAGE 44

44 pH and nitrate concentrations vs. time0 1 2 3 4 5 6 7 8 9 109/159/2310/110/910/1710/2511/211/1011/1811/2612/412/1212/20timepH0 500 1000nitrate concentratio n pH NO3-N (100 mg/L) Figure 3-11. CSTR2 pH and ni trate concentrations vs. time Absorbance vs. time 0 0.5 1 1.5 2 2.5 3 3.5 49/159/2310/110/910/1710/2511/211/1011/1811/2612/412/1212/20 timeAbsorbance absorbance Figure 3-12. CSTR2 absorbance vs. time After the addition of micronutrients, increase in denitrification rate and absorbance was observed in the reactor. The overa ll denitrification remained high with increasing flow rates, as shown in Figure 3-10. The pH of the reactor was in between 9 and 9.5 throughout its operation,

PAGE 45

45 as shown in Figure 3-11. The ab sorbance decreased generally with an increase in feed flow rate, as expected since the effluent flow rate was also increased. The absorbance stabilized once the reactor reached steady state for e ach flow rate. The flow rate of the reactor was continuously increased afterwards up to a flow rate of 11.5 mL/min, as shown in Figure 3-10, which was close to twice the target flow rate, and the highest denitrification ra te observed in CSTR2 was 4 mg NO3-N/L-min. From these results, it was confirme d that high nitrate concentration did not affect the metabolism and denitrification rate of the denitrifying bacteria. It was furthermore speculated in the begi nning that high amount of sodium can lower denitrification rates since it wi ll lead to high osmotic pressure across the cell membrane. The effect of sodium was also tested previously using attached growth bioreactors-by operating a reactor containing sodium hydroxide neutralized industria l stream and anothe r reactor containing potassium hydroxide neutralized indu strial stream simultaneously. However, the results from the attached growth bioreactors were not reliable since the reason fo r poor denitrification could have been micronutrient deficiencies. Therefore, a new CSTR was started alongside CSTR2 in order to test the effects of sodium on denitrification rate. The new r eactor was started using synthetic nitrate stream using DI water a nd 69.6% nitric acid assay, and th e solution was neutralized using potassium hydroxide pellets instead of sodium h ydroxide pellets. Ther e werent any noticeable differences between the results obtained from sodium hydroxide neutralized solution and potassium hydroxide neutralized solution. Theref ore, it was concluded that sodium does not have any significant eff ects on denitrification. CSTR3 Results After it was confirmed that high nitrat e concentration and sodium did not have significant effect on denitrifica tion rate, another suspended grow th reactor, CSTR3, was started again with the industrial stream as the feed solution. Effluent from CSTR2 was used as the

PAGE 46

46 inoculum for the reactor. The reactor was init ially started with artificial nitrate solution neutralized with potassium hydroxide pellets as the feed. The f eed was later switched to ionexchanged industrial stream that was pre-neut ralized with potassium hydroxide. The feed solution contained 17.5 g/L potassi um acetate as the carbon sour ce, 0.5 g/L potassium phosphate as the phosphorus source, and 0.1 g/L magnesium sulfate heptahydrate in order to provide additional magnesium for bacterial metabolism. Th e results of the reacto r are shown in Figures 3-13, 3-14, and 3-15. The reactor continued to denitr ify at a high rate using samp le B (ion-exchanged industrial stream), as shown in Figure 3-13. The highest flow rate obtained using sample B was 0.8 mL/min with an overall denitrific ation of 99.97%. This corresponded to a denitrification rate of 0.315 mg NO3-N/L-min. The reactor was not able to achieve higher flow rates because of the shortage of available sample B solution. CSTR3 nitrate reduction and feed rate vs. time 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 9/139/2310/310/1310/2311/2time% nitrate reduction0 0.2 0.4 0.6 0.8 1feed flow rate (mg NO3-N/L/min) % nitrate reduction feed flow rate A B C Figure 3-13. CSTR3 denitr ification and feed flow rate vs. time A: HNO3-KOH feed started; B: Switched feed to sample B, C: Switched feed to sample C

PAGE 47

47 nitrate concentration and pH vs. time0 1 2 3 4 5 6 7 8 9 109/139/169/199/229/259/2810/110/410/710/1 0 10/1 3 10/1 6 10/1 9 10/2 2 10/2 5 10/2 8 10/3 1 11/3timepH0 500 1000nitrate concentratio n pH NO3-N (100 mg/L) switched feed to sample C Figure 3-14. CSTR3 pH and ni trate concentration vs. time absorbance vs. time 0 0.5 1 1.5 2 2.5 3 3.5 4 9/139/169/199/229/259/2810/110/410/710/1 0 10/1 3 10/1 6 10/1 9 10/2 2 10/2 5 10/2 8 10/3 1 11/3timeabsorbance absorbance Figure 3-15. CSTR3 absorbance vs. time

PAGE 48

48 After sample B was exhausted, the feed was switched to sample C, which was non-ion exchanged industrial stream neutralized with pot assium hydroxide. Changes in performance of the reactor were not speculated due to this actio n, since it was hypothesized that it was indeed the deficiency of micronutrients that caused low denitrification of the previous industrial streams. However, as shown in Figure 3-13, the denitrificati on rate started to decrea se as soon as the feed was switched to sample C. Figures 3-14 and 315 correspond to the pH and absorbance of the reactor, respectively. It can be seen clearly that the change in feed led to decrease in pH and absorbance as well. The reactor was then put on batch mode for three days and the feed was switched back to synthetic feed neutralized w ith potassium hydroxide. The reactor failed to recover from the low denitrification rate, however. This led to the belief that metal ions are interfering with bacterial growth and denitrificati on process. It was also noticed that sample C had a tint of green that was not observed in previous samples. CSTR4 Results After CSTR3 failed to denitrify sample C properly, other factors that may cause poor denitrification were considered. The main concer n was the presence of metals in the industrial stream, which can affect bacterial metabolism and lower denitrification rates. This was further supported by the results obtained from denitrific ation of sample C, which was ion-exchanged. After CSTR3 was discontinued, two new batches of ion-exchanged industrial stream neutralized with KOH were received (sample D and sample E) CSTR4 was started up using sample D. The feed also contained 17.5 mg/L potassium acet ate, 0.5 mg/L potassium phosphate, and 0.1 mg/L magnesium sulfate heptahydrate. Effluent fr om CSTR2 was used as the inoculum for the reactor. Micronutrients were a dded to the reactor daily in amount s proportional to the feed flow rate. The results of th e reactor are shown in Figures 3-16, 3-17, and 3-18.

PAGE 49

49 nitrate reduction and feed flow rate vs. time 0.00 20.00 40.00 60.00 80.00 100.00 10/ 25 10/ 28 10/ 31 11/ 3 11/ 6 11/ 9 11/ 12 11/ 15 11/ 18 11/ 21 11/ 24 11/ 27 11/ 30 12/ 3 12/ 6 12/ 9 12/ 12 12/ 15 12/ 18 12/ 21 12/ 24 time% nitrate reduction0 0.5 1 1.5 2feed rate (mg NO3N/L/min) % nitrate reduction feed flow rate Sample D Sample E synthetic nitrate solution Sample A Figure 3-16. CSTR4 denitrificati on and feed flow rate vs. time. Low denitrification rates were observed for both ion-exchanged samples. High denitrification was observed once the feed was switched to sample A. pH and nitrate vs. time 0 2 4 6 8 1010/26 11/3 11/11 11/19 11/27 12/5 12/13 12/21timepH0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300nitrate-nitroge n concentration pH NO3-N (100 mg/L) Figure 3-17. CSTR4 pH and ni trate concentrations vs. time Absorbance 0 0.5 1 1.5 2 2.5 3 3.5 4 10/2611/311/1111/1911/2712/512/1312/21 timeAbsorbance absorbance Figure 3-18. CSTR4 absorbance vs. time

PAGE 50

50 CSTR4 did not have positive denitrificati on results initially. The pH and absorbance decreased steadily as shown in Figures 3-17 and 3-18, and the denitrification was deteriorating slowly as shown in Figure 3-16. The results we re puzzling since the sample was ion-exchanged, and thus it invalidated the assumption that meta l ions may be causing the problem. The reactor failed to recover after switching to synthetic feed. In order to be certain that the ion-exchange was effective, an analysis of the samples were done at the industrial site, and it was observed that one of the samples had high concentration of meta l ions that might inhibi t the bacterial growth and lower the denitrification rate It wasnt positive which of the samples contained the high metal ion content. It was possibl e that the low denitrification of the reactor was due to presence of metal ions in the feed. It was then decided to restart CSTR4 and obser ve the denitrification rates of sample E. Therefore, the reactor was rest arted with synthetic feed and inoculum obtained from CSTR2 effluent. Once the reactor was in steady state with a flow rate of 1 mL/min, the feed was switched to sample E. The denitrification rate was high using this sample; however, the absorbance started decreasing. Th is led to the conclusion that metals were not the inhibition factor in low denitrification rates observed. Restarting CSTR4 with Sample A CSTR4 was restarted with sample A since it was suspected that previous low denitrification rates were caused by deficien cy of micronutrients. When sample A was previously fed into the reactor, micronutrients were added in the feed solution rather than directly to the reactor. Thus, the reactor was restarted in conti nuous mode at 1mL/min. Effluent from CSTR2 was used as the inoculum for the reactor. High denitrification rates were observed thr oughout the operation of the reactor, and pH remained steady. The reactor was able to achieve high flow rates with high overall

PAGE 51

51 denitrification. The highest flow rate achie ved by the reactor was 5.5 mL/min, with 97.5% denitrification, as shown in Figure 3-16. This corresponded to a denitrif ication rate of 1.68 mg NO3-N/L/day, which was close to the target denitrification rate of 2 mg NO3-N/L/day. At this point it was conclude d that the industrial stream was indeed treatable, but a component in the stream is acting as an inhibito ry factor. Causes for denitrification include presence of metal ions and presence of volatile contaminants. Thus, a tool was needed to judge the treatability of the samples. Thus, a batch test was developed in order to test the treatability of high nitrate streams. It was reve aled from the results of the batch tests that volatile contaminants are indeed present in the industr ial stream and they might be the components contributing to the low denitrification rates and low absorbance values.

PAGE 52

52 CHAPTER 4 ASSAY ON THE BIOLOGICAL TREATABI L ITY OF INDUSTRIAL STREAMS A batch test procedure was deve loped to test the biological tr eatability of high nitrate water streams. The assay was inspired by studies on Daphnia by Gasith et. al. Since the parameter to be tested was denitrification, Daphnia was replaced with denitrifying bacterial culture. The denitrification results of the te st solution are compared with those of the synthetic nitrate solution. The experiments were mainly conducted on ion-exchanged high nitrate industrial stream neutralized with potassium hydroxide and non-ion exchanged industrial stream neutralized with sodium hydroxide. The batch tests primarily invest igated the eff ects of volatile contaminants and settled particles on denitrificat ion and bacterial growth. The general procedure developed to test the treatability of the high nitrate streams is as follows: 1. Grow denitrifying bacteria in a high nitrate stream so that th ey can be used for the batch tests. 2. Measure the nitrate-nitrogen concentration and pH of the test solution using HACH NitraVer Test N Tube test kits 3. Prepare synthetic nitrate solution using nitr ic acid with the same nitrate-nitrogen concentration as the test solution. 4. Add appropriate chemicals (given as follows) to variable and contro l solutions in order to support bacterial growth and metabolism: a. 17.5 g/L potassium acetate as carbon source b. 0.5 g/L potassium phosphate as phosphorus source c. 0.1 g/L magnesium sulfate heptahydrate as magnesium source d. 0.3 g/L ammonium chloride as the ammonium source 5. Neutralize the test solution a nd synthetic nitrate solution to pH 8 using sodium hydroxide pellets 6. Filter the solution using 2 m filters. 7. Aerate approximately 200 mL of test solution, and keep the rest non-aerated. 8. Obtain 9 125-mL Erlenmeyer flasks; fill three flasks with 60 mL of aerated test solution, three flasks with 60 mL of non-aerated test solution, and three fl asks with 60 mL of synthetic nitrate solution.

PAGE 53

53 9. Add 10 mL of bacterial cu lture and 0.2 mL micronutrien t solution-prepared using instructions for trace element solution by Vishniac & Santer to each flask. 10. Measure the nitrate-nitrogen concentration, pH, and absorbance of each flask. The nitrate-nitrogen concentration is measured using HACH test kits after filtering the sample from each flask using 0.45 m filters, and diluting the samples to appropriate concentrations. 11. Secure all flasks with rubber stoppers and place them in an incubator at 37oC 12. Measure NO3-N, pH, and absorbance of one aera ted flask and one non-aerated flask three times: first measurement after 3-4 hour s, second measurement after 7-9 hours, and third measurement after 22-24 hours. 13. Obtain ( _max/YNO)sample / (( _max/YNO)synthetic solution from the data obtained in order to establish the treatability of the high nitrate stream; treatable nitrate solutions will have a value close to 1. Developing Equations Bacterial growth in the test flasks can be quantified since the bi omass concentration is proportional to absorbance values. Biomass growth kinetics is developed assuming the presence of excess carbon and neglecting decay. The change in absorbance can be characterized by Monod equation. dA/dt = maxNO (SNO/(KNO + SNO)) A, nitrate is the limiting component (4-1) dSNO/dt = maxNO (SNO/(KNO + SNO)) A (4-2) dSNO/dt = (-1/YNO) (dA/dt) (4-3) A(t) = Ao + YNO (SNO(o) SNO(t)) (4-4) Thus, a plot of absorbance vs. nitrate concentration will have a slope of -YNO and yintercept of Ao. This plot can be obtained fr om the measurements of absorbance and nitrate concentration after 0, 4, and 8 hours. It was noted that KNO is typically less than 1, usually for wastewater streams containing nitrate concentration around 100 mg/L. Since the nitrate concentration is much higher th an 100 mg/Lin the experiments done with this assay, the ratio

PAGE 54

54 (SNO/(KNO + SNO)) is approximated to be equal to 1. Then Equation 4-2 can be rewritten as shown in Equation 4-5. dSNO/dt = maxNO [Ao + YNO(SNO(o) SNO(t))] (4-6) (Ao+YNOSNO(o)) = (4-7) Sno(0)Sno(t) (dSNO/ YNOSNO) = maxNO 0t dt (4-8) ln( YNOSNO(t)) = YNO maxNO t + ln( -YNOSNO(o)) (4-9) Thus, a plot of ln( YNOSNO(t)) vs. YNO*t will have a slope of maxNO. Once maxNO of the test solution is obtained, maxNO of the test solution is compared with maxNO of the synthetic nitrate solution. Thus, the ratio BI, given in Equa tion 4-10, can be considered as a treatability index. A low BI will indicate that biological denitrif ication of that sample will be very difficult. BI=(( maxNO) test sample)/((maxNO) synthetic sample with same NO3-N concentration) (4-10) Development of the Assay Initially the batch tests were conducted in fl asks secured with rubbe r stoppers with a gas inlet port and a gas outlet port. The inlet and outlet ports we re secured with aluminum foil after nitrogen gas was bubbled through the flasks. The first batch test was conducted to test the treatability of the ion-exchange d industrial stream neutralized with potassium hydroxide. This sample previously had poor denitrification when it was fed to the suspended growth reactor. The test was conducted alongside sodium hydroxide ne utralized industrial stream, which had positive results after it was fed through the suspended growth reactor. Af ter 24 hours, it was noticed that all the flasks had high de nitrification and absorbance increase. This led to the suspicion that oxygen had entered the flasks through gas inlet and gas outlet ports, and that the absorbance increase was mainly due to aerobic growth of th e bacteria. When oxygen is present, bacteria can convert the nitrate to ammonia, which is then us ed as the nitrogen source for bacterial growth.

PAGE 55

55 In order to limit the presence of oxygen in the flasks, another batch test was conducted using flasks that were secured tightly with soli d rubber stoppers. Prior to securing the flasks, nitrogen gas was bubbled through the flasks in order to purge out any oxygen. The ionexchanged stream neutralized with potassium hydroxide was tested against artificial nitrate solution prepared using 69.6% HNO3 assay. The results were sim ilar to previous test, and high denitrification and absorbance increase were obser ved in all flasks. This did not correspond to the results obtained from suspended growth reactors. Since oxyge n was purged out using nitrogen gas, the possibility of presence of any oxygen in the flasks was discarded. It was then suspected that vol atile contaminants present in the industrial stream might have caused low denitrification rates in the su spended growth reactor. It was possible that volatile contaminants may have been remove d when nitrogen gas was bubbled through the flasks. Thus, another batch test was carried out using ion-exchanged sample. The flasks were again secured with solid rubber stoppers; however, nitrogen ga s was not bubbled through the reactors. At the end of the batc h test, it was noticed th at little or no denitrification occurred in the flasks, and no bacterial growth was observed. This supported the hypothesis that volatile contaminants were causing the low denitrification rates observed previously in the CSTR, and the high denitrification in the previous batch tests were the result of removal of volatile contaminants through aeration. In order to confirm the presen ce of volatile contaminants in the industrial stream, a batch test was carried out using aerated and non-aerated ion-exchanged samples. In order to avoid assimilatory denitrification, 0.3 g/L ammonium ch loride was added to the flasks. The flasks containing aerated samples showed high denitrifi cation and absorbance increase, while the flasks containing non-aerated samples had low denitrif ication and no absorbance increase. This

PAGE 56

56 confirmed the theory that volatile contaminants were causing the low denitrification in the CSTR. Results and Discussion The assay was applied to test the treatability of aerated and non-aerated industrial stream samples neutralized using sodium hydroxide. In oculum for the batch test was obtained from CSTR4 effluent. Measurements were taken afte r 4, 8, and 24 hours. The analyses from batch test done non ion-exchanged industrial sample are shown in Figures 4-1 through 4-6. Aerated industrial sample y = -8.7571E-04x + 2.0296E+00 R2 = 9.4477E-01 0 0.5 1 1.5 2 2.5 05001000150020002500 SnoAbsorbance Figure 4-1. Absorbance vs. nitrate concentration, aerated sample non aerated industrial sample y = -2.0796E-03x + 4.0101E+00 R2 = 7.8896E-01 0 0.2 0.4 0.6 0.8 1 150015501600165017001750180018501900 Snoabsorbance Figure 4-2. Absorbance vs. nitrate concentration, non-aerated sample

PAGE 57

57 synthetic nitrate solution y = -9.1872E-04x + 2.2502E+00 R2 = 9.9483E-01 0 0.5 1 1.5 2 2.5 0 500 1000 1500 2000 2500 SnoAbsorbance Figure 4-3. Absorbance vs. nitrate concentration, synthetic nitrate solution Aerated industrial sample y = 2.0728x 0.8511 R2 = 0.8101 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0 0.5 Yno*tln(beta-YnoSno(t)) Figure 4-4. ln( -YNOSno(o)) vs. YNO*time, aerated sample y = 5.6921E-01x 1.0997E+00 R2 = 6.9921E-01 -1.3 -1.2 -1.1 -1 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0 0.2 0.4 0.6 0.8 11 2 Figure 4-5. ln( -YNOSno(o)) vs. YNO*time, non-aerated sample

PAGE 58

58 Synthetic nitrate solutiony = 3.0266x 1.1197 R2 = 0.961 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 00.10.20.30.40.5 Yno*tln(beta-YnoSno(t)) Figure 4-6. ln( -YNOSno(o)) vs. YNO*time, synthetic nitrate solution Figures 4-1, 4-2, and 4-3 show the plots of absorbance vs. nitrate concentration for aerated high nitrate i ndustrial stream, non-aerated high nitr ate industrial stream, and synthetic nitrate solution respectively. From the plots, the YNO was obtained to be 8.76E-4 for aerated stream, 2.08E-3 for non-aerated stre am, and 9.19E-4 for synthetic nitrate solution. The plots in Figures 4-4, 4-5, and 4-6 were used to obtain maxNO values for aerated sample, non-aerated sample, and synthetic nitrate solution respectively. maxNO values were obtai ned to be 2.073 for aerated sample, 0.569 for non aerated sample, and 3.027 for synthetic nitrate. From maxNO values, the biotreatability i ndex, BI was calculated. BI for aerated sample was 0.635 and for non-aerated sample was 0.188. The assay showed that the aerated industrial stream can be treated biologically. The results were verified by using aerated industrial stream as the feed for a suspended growth reactor. High denitrification rates and absorban ce values were observed. The assay was done on other industrial samples as we ll. The results obtained from the industrial samples are given in Figures 4-7 through 4-12. The BI values are summarized in Table 4-1.

PAGE 59

59 Sample A YNO y = -1.1341E-03x + 1.7336E+00 R2 = 9.5532E-01 0 0.5 1 1.5 2 0200400600800100012001400 Snoabsorbance Figure 4-7. Absorbance vs. SNO for sample A Sample A y = 0.868x 0.7094 R2 = 0.9042 -1.5 -1 -0.5 0 0.5 1 0 12 Yno*tln(beta-Yno*Sno(t)) Figure 4-8. ln( -YNOSNO(0)) vs. YNO t for sample A Aerated Sample C YNO y = -1.4015E-05x + 5.3903E-01 R2 = 1.8517E-03 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 10001100120013001400150016001700180019002000 SnoAbsorbance Figure 4-9. Absorbance vs. SNO for aerated sample C

PAGE 60

60 aerated Sample C y = 1.1444x 0.5501 R2 = 0.3737 -0.8 -0.6 -0.4 -0.2 0 0.2 0 0.5 Yno*tln(beta-YnoSno(t)) Figure 4-10. ln( -YNOSNO(0)) vs. YNO t for aerated sample C aerated sample D, YNO y = -1.4607E-03x + 6.7163E-01 R2 = 8.7487E-01 0 0.1 0.2 0.3 0.4 0.5 0.6 0 50 100 150 200 SnoAbsorbance Figure 4-11. Absorbance vs. SNO for aerated sample D Sample D, aerated y = 5.0297E-01x 1.0017E+00 R2 = 8.9919E-01 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.00E+00 4.00E-01 8.00E-01 Figure 4-12. ln( -YNOSNO(0)) vs. YNO t for aerated sample D

PAGE 61

61 Table 4-1. Biotreatab ility assay results Sample A maxNO = 0.868 Sample C aerated BI = 0.49 Sample C non-aerated Invalid BI value; bacteria did not grow Sample D aerated BI = 0.43 Sample D non-aerated Invalid BI value; bacteria did not grow As shown in Table 4-1, aerated sample C and sample D are treata ble biologically. The non-aerated sample C and non-aera ted sample D had negative YNO values, and thus gave invalid BI values. The absorbance for those samples d ecreased with nitrate consumption during batch test. Conclusions The objective of the assay is to quantitati vely analyze biological treatability of high nitrate solutions. The biotreatab ility index, BI is used to determine if the sample can be denitrified successfully. BI is obtained from the ratio, ( maxNO) test sample)/((maxNO) synthetic sample with same NO3-N concentration). YNO values can be obtained from the plots of absorbance vs. SNO observed during the batch tests, and maxNO is obtained from the plots of ln( YNOSNO(0)) vs. YNO*t. If the value of the ratio is closer to 1, the sample is treatable. A value much lower than 1 shows that high denitrification will be very difficult to achieve for that sample.

PAGE 62

62 CHAPTER 5 CONCLUSIONS AND FUTURE WORK The objective of the project wa s to establish high denitrificat ion rate for an industrial high nitrate stream using biological tr eatment. At the end of this study, it was concluded that the industrial stream was ind eed treatable. However, volatile contam inants and metals ions present in the stream must be removed prior to biological treatment. Volatile contaminants may be removed through aeration, and metal ions can be removed through an ion exchange process. During the treatment, micronutrients should be added to the reactor directly rather than in the feed, since precipitation can occur in the feed. It was also established that neutralization with sodium did not affect denitrifi cation rates, since the industrial stream sample neutralized with sodium hydroxide and the stream sample neutrali zed with potassium hydroxide had same results. CSTR with sodium hydroxide neutralized synt hetic nitrate feed was able to reach a denitrification rate of 4 mg NO3-N/L/min with a flow rate of 11.5 mL/min and an overall denitrification of 97%. This denitrification rate was twice the target denitrification rate. CSTR with the feed of sodium hydroxide neutralized industrial stream was able to reach the target denitrification rate of 2 mg NO3-N/L/min with a flow rate of 5.5 mL/min and an overall denitrification of 98%. The denitrification was performed usi ng potassium acetate as the exogenous carbon source. Thus, effects of carbon limitation on den itrification were not analyzed. For the next step, an industrial carbon sour ce may be used for the process. Studies using attached growth bioreactors can also be conducted with micronutrients added directly to the reactor instead of the feed. Attached growth bioreactor s can be more feasible since sl udge concentration in the reactor effluent will be very low.

PAGE 63

63 LIST OF REFERENCES Barber W P. and Stuckey D. C. (2000) N itrogen removal in a modified anaerobic baffled reactor (ABR): 2, Denitrification. Water Research 34, 2413-2422. Gasith A. J., Jop K. M., Dickson, K. L., Parker ton, T. F., and Kaczmarek, S.A. (1988). Protocol for the identification of toxic fractions in industrial wastewater effluents. Aquatic Toxicology and Hazard Assessment : 10th Volume, ASTM STP 971. Adams, W.J., Chapman, G.A., and Landis, W.G. Editors., American Society for Te sting and Materials, Ph iladelphia. pp. 204-215. Kanow, P.E., 1993. Denitrification Methods United States Patent 5211847. http://www.freepatentsonline.com/5211847.html March 25, 2008. Lee S., Maken S., Jang J., Park K., and Park J. (2006). Developm ent of physicochemical nitrogen removal process for high st rength industrial wastewater. Water Research 40 975-980 Leslie Grady C.P., Daigger, G.T., and Lim, H.C. 1999. Biological Wastewater Treatment 2nd ed. CRC Press, Boca Raton, USA. Tchobanoglous, G., Burton F.L., and Stensel, D.H. 2003. Wastewater Engineering: Treatment and Reuse McGraw-Hill, NY, USA. US EPA., 2005. Section 62-302.530, Criteria for Surface Water Quality Classifications (Florida) http://www.epa.gov/waterscience/stand ards/wqslibrary/fl/fl_4_62-302t.pdf March 30, 2008 Vishniac S., and Santer M. (1957). The Thiobacilli. Bacter iol Rev 21, 195-213 Wasser I.M., deVries S., Moenne-Loccoz P., Schroder, I., and Kavlin K. D. (2002). Nitric oxide in biological denitrification: Fe/Cu metall oenzyme and metal complex NOx redox chemistry. Chemical Reviews (Review) 102, 1201-1234

PAGE 64

64 BIOGRAPHICAL SKETCH Sherin Peter was born on February 23, 1984, in Muvattupuzha, India. S he received her Bachelor of Science degree in chem ical engineering from University of South Florida, Tampa, in 2005. She obtained her MS degree in chemical e ngineering from Univers ity of Florida in 2008.