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Effectiveness of High Salinity Anerobic Digestion of Marine Microalgae

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Title:
Effectiveness of High Salinity Anerobic Digestion of Marine Microalgae
Creator:
Maharaj, Kavir
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[Gainesville, Fla.]
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University of Florida
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English

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Subjects / Keywords:
Algae ( jstor )
Anaerobic bacteria ( jstor )
Anaerobic digestion ( jstor )
Bioreactors ( jstor )
Fresh water ( jstor )
Methane ( jstor )
Methane production ( jstor )
Rubber ( jstor )
Salinity ( jstor )
Syringes ( jstor )
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Undergraduate Honors Thesis

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Abstract:
Most of the research conducted in anaerobic digestion of microalgae to produce methane gas has done so using digesters that are very low in salinity level. It has been researched that high salinity levels are responsible for sever inhibition of anaerobic bacteria in these digesters. With freshwater a considered nonrenewable recourse due to it high demand, it would be irresponsible if freshwater is diverted to perform anaerobic digestion. In this experiment anaerobic bacteria that have been adapted to salinity levels similar to that measured from the ocean were tested to determine how effective they were at digesting a cyanobacterium called Synechococcus sp. Synechococcus sp is a microalgae that can grow rapidly in high saline environments, require very little nitrogen nutrient, and is known to produce extracellular biomaterials that are easily metabolized by anaerobic bacteria. The experiments performed by other researches required that they wash the harvested algae substrate before pouring into the anaerobic digesters in order to remove the sodium ion content. In this experiment the micro algae Synechococcus sp is harvested and thermochemical pretreated and placed into digesters with no change in salinity, specifically 37g/l. The bioreactors ran at this same salinity level for 20 days and produced a methane yield of 107 ml/g VS. Based on other experiment that ran their anaerobic digesters in low saline environments, our results show similar performances which demonstrates that micro algae grown in the ocean and harvested could directly be digested, without the need of washing them, using anaerobic bacteria that have been adapted to metabolize in high saline environments. ( en )

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UNIVERSITY OF FLORID A Effectiveness of High Salinity Anaerobic Digestion of M arine Microalgae . Honors Thesis Kavir Maharaj 12/5/2015

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Page number 1 Contents List of Figures ................................ ................................ ................................ ................................ 2 Abstract ................................ ................................ ................................ ................................ ........... 3 Introduction ................................ ................................ ................................ ................................ ..... 3 Objective ................................ ................................ ................................ ................................ ......... 4 Preparation of the Algae ................................ ................................ ................................ ................. 4 Physical Apparatus Setup ................................ ................................ ................................ ........... 4 Inoculation of Algae ................................ ................................ ................................ .................... 5 Preparation of the Anaerobic Bacterial Communities ................................ ................................ .... 5 Experimental Procedure ................................ ................................ ................................ .................. 5 Equipment Used ................................ ................................ ................................ .......................... 5 Experimental Preparation of Algae ................................ ................................ ............................ 6 Experimental Preparation of Anaerobic Bacteria ................................ ................................ ...... 6 Experimental Setup of Batch Bioreactors ................................ ................................ ................... 6 Data Collection ................................ ................................ ................................ ........................... 7 Results and Discussion ................................ ................................ ................................ ................... 8 Conclusion ................................ ................................ ................................ ................................ .... 11 Acknowledgement ................................ ................................ ................................ ........................ 11 References ................................ ................................ ................................ ................................ ..... 12

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Page number 2 List of Figures Figure 1 Accumulated Methane Gas Production Over 20 Days ................................ ..................... 8 Figure 2 Real Methane production over 20 Days vs Theroretical Amount Possible ...................... 9 Figure 3 Percent Yield of Methane Gas from each Bioreactor ................................ ..................... 10

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Page number 3 Abstract Most of the research conducted in anaerobic digestion of microalgae to produce methane gas has done so using digesters that are very low in salinity level. It has been researched that high salinity levels are responsible for sever inhibition of anaerobic bacteria in these dige sters. With freshwater a considered nonrenewable recourse due to it high demand, it would be irresponsible if freshwater is diverted to perform anaerobic digestion. In this experiment anaerobic bacteria that have been adapted to salinity levels similar to that measured from the ocean were tested to determine how effective they were at diges ting a cyanobacterium called Synechococcus sp . Synechococcus sp is a microalga e that can grow rapidly in high saline environments, require very little nitrogen nutrient , and is known to produce extracellular biomaterials that are easily metabolized by anaerobic bacteria. The experiments performed by other researches required that they wash the harvested algae substrate before pouring into the anaerobic digesters in order t o remove the sodium ion content. In this experiment the micro algae Synechococcus sp is harvested and thermochemical pretreated and placed into digesters with no change in salinity, specifically 3 7 g/l . The bio reactor s ran at this same salinity level for 20 days and produced a methane yield of 107 ml/g VS. Based on other experiment that ran their anaerobic digesters in low saline environments , our results show similar performances which demonstrates that micro algae grown in the ocean and harvested could dir ectly be digested , without the need of washing them , using anaerobic bacteria that have been adapted to metabolize in high saline environments. Introduction Producing biofuels from first generation crops, such as sugar cane and corn , is an established in dustrial process that is economically feasible and is produced in large quantities in countries like Brazil, where it is competes with petroleum gasoline at a competitive price. To provide land for sugar cane cultivat ion many acres of forest in the Amazon are cleared, which destroys the natural habitats of many species of plants and animals. Growing crops require s fertilizers such as nitrogen, potassium, and phosphorus. The mining off theses nutrients causes sever land degradation and contamination. Large q uantities of fresh water have to be used to sustain these crops. In effect biofuels from land cultivated crops put an enormous strain on the environment. To reduce the environmental impact that cultivation of first generation feedstock causes, research in a new feedstock, particular microalgae has gained prominence. Microalgae have very low lignin and hemicellulose content and are primarily composed of cellulose and polysaccharides. Some species are native to the ocean and thus can be grown using ocean wat er. Lastly m icroalgae are k nown to have high growth rates, which is beneficial because first generation biofuels crops have long harvesting cycles as oppose to microalgae which has a harvesting cycle of 1 10 days.

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Page number 4 Of interest to researchers is the anaer obic digestion of saltwater algae feedstock to produce methane gas . The reason for this is because algae grown using ocean water will reduce freshwater use and also reduce the strain on arable land for food production. The challenge presented is that the a naerobic bacteria used to metabolize algae biomass to produce methane gas live in very low saline conditions. Naturally algae grown using saltwater would need to be washed and neutralized of its salt content before being digested in anaerobic fermenters. R inzema [2 ] documented that sodium concentration of 5, 10, and 14 g/l causes 10, 50, and 100% inhibition of methanogenic bacteria receptively . In this experiment anaerobic bacteria that were acclimatized to a salinity of 37 g/l where used to digest an alga e feedstock. The micro algae used in this experiment is named Synechococcus sp and is a cyanobacterium that grows in a salinity of 37 g/l , has a high grow rate, and fixes its own nitrogen. The experiment aims to validate the methane yield performance of the anaerobic diges tion of algae feedstock in a saline environment of 37 g/l . Objective To measure the methane yield from high salinity anaerobic digestion of wet algae grow n in a saline environment of 37 g/l . Co mpare this methane yield with other yields in literature to gauge performance. Preparation of the Algae The proposed experiment required that Algae be incubated and cultivated in enough quantities as to be used as a substrate in eight different anaerobic b atch reactors. Physical Apparatus Setup An improvised refrigerator was used to contain the lights and algae growth vessel . The vessel used to grow the algae was made out of glass with a diameter of 18.5 cm and a height of 36 cm . At the top of the vessel i s a constricted opening with diameter 5 cm . Flanked on three sides of the growth chamber where florescent lights, with two on either sides of the vessel and one located directly behind it provide a light flux of 25.58 . The growth chamber was placed onto a laboratory hot plate that provided rotational convection within the growth chamber using a magnetic stirrer with a length of 3cm. A t the top of the algae growth chamber are two glass tubes attached to a rubber cap. Th e first tube is used to inject air into the growth chamber. The air used was atmospheric air that was pumped into the vessel with a gas flow rate of 1 l/min . The pump used in this setup was a peristaltic pump. The inlet air tubed terminated near the bottom of the growth vessel using a porous material that served the purpose of reducing the diameters of the gas bubbles formed. The second tube on the rubber cap served as a small vent to allow excess gas that was pumped into the growth vessel to escape.

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Page number 5 Inocu lation of Algae The algae growth vessel was filled to a height of 25 cm with a nutrient medium containing minerals necessary for the growth of the algae. The growth vessel was inoculated with algae, and for 127 days was grown , maintaining a pH of 7.5 8.0 and a salinity of 3.7 grams per 100 ml . Preparation of the A naerobic Bacterial Communities The initial anaerobic bacterial community was harvested from a dairy anaerobic treatment vessel. This bacterial community was used to inoculate 4 batch bioreactors that were 5 liters in volume. Each of the bioreactors was placed on laboratory hot plate to provi de rotational agitation. Two of the bioreactors were incubated in thermophilic conditions at a temperature of 55 deg rees Celsius. The other two bio reactors where incubated at mesophilic conditions at a temperature of 37 degrees Celsius. The c h amber used to provide and maintain these temperature conditions were improvised refrigerat ors. The anaerobic community harvested and subsequently used as an ino culant was adapted to low saline environments . Over an incrementally increased in small amounts with the objective off slowly acclimatizing the bacteria community to the new conditions. At the same time molasses or table sugar were regularly injected into the bioreactors for the bacteria to consume for maintenance and growth. The gas produced from each of these bior eactors where measured and their methane composition determined. The pH of each react or was maintained at levels between 6 and 7.5. The volatile fatty acid levels were measured regularly, and based on these readings the appropriate amount of sugar was injected into each bioreactor with the purpose of maintaining a low VFA concentration . Hi gh VFA levels were observed to inhibit methane production from anaerobic communities. After the time interval of 2 years the 4 bioreactors where acclimatized to a saline environment of 3.7 gram per 100 ml or more with each bioreactor competent in producin g methane gas using sugar as a substrate. Experimental Procedure Equipment U sed Measurements on the gas produced from the experiment were performed using a GOW MAC Series 580 Gas c hromatograph (GC). To measure the volume of gas produced from each batch digester a 50 ml syringe with a needle attached was used. A total of 8 batch digesters were used. Each bat ch reactor was a transparent g l ass bottle that could contain 250 ml of fluid. Each batch reactor used where operating in anaerobic conditions. A rubber cap was used to properly seal each reactor. The rubber seal also allowed the ability to sample the biogas produced without having to replace the seal each time.

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Page number 6 Four of the batch reactors were expose d to thermophilic conditions. To accomplish this an incubator chamber at 55 degrees Celsius was used. The other four batch reactors were exposed to mesophilic conditions at a temperature of 37 degrees Celsius using a similar chamber. Experimental Preparat ion of Algae least 480 ml of it was extracted to be used for the experiment. The extract ed Algae were measured to have a volatile solid consistency of 3.7 g/l . The algae sample was placed into a 500 ml glass bottle for thermochemical pretreat ment . The algae sample was first reduced to a pH of 5 using hydrochloric acid. After the sample was placed into an autoclave which subjected it to temperatures of 121 degrees Celsius using saturated steam for 30 minutes . Experimental Preparation of Anaerobic Bacteria Sinc e the inception of the four bio Preparation of the Anaerobic Bacterial Communities s were collected from each bio reactor and stored. The objective was to collect at least 180 ml of the oldest bacterial sampled broth from each main bio reactor. The reason for this strategy was because the older sample broths would be depleted of sugar su bstrate to a larger extent compared to the more recently collected broth. This would ensure that the bacteria is dormant and producing no gas due to metabolism. Experimental Setup of Batch Bioreactors As m entioned previously, 180 ml of bacterial broth was collected from each of the four main bio reactors. The two bioreactors exposed to thermophili c conditions were named T1 and T2; the two mesophilic bio reactors were name d M1, and M2 . Eight glass bottles of vol ume 250 ml each were prepared by autoclave ste rilization at a temperature of 121 degrees Celsius for 1 hour. From the T1 sample , 90 ml out of the 180 was placed into one of the eight glass bottles and labeled T11. The remaining 90 ml was placed into an additional glass bottle and labeled T12. The same procedure was replicated for the sample broths of bioreactors, T2, M1, and M2 with the 6 remaining sterilized glass bottles. The remaining 6 glass bottles were named T21, T22, M11, M12, M21, and M22. Each of the eight experimental bioreactors prepared had 60 ml of pretreated algae fluid poured into them. 100 ml of deionized water (DI) was introduced into each experimental bioreactor as well. Summarizing every experimental bioreactor received 90 ml of a naerobic bacteria broth, 60 ml of pretreated algae, and 100 ml of DI water. The final salinity of each bioreactor was at 35 g/l at a pH of 7. This volume of substance culmina tes to a total fluid volume of 2 50 ml. The volume of the glass bottle used is 280 ml, which leaves a 30 ml head space in each experimental bioreactor at this point . All experimental bioreactors were agitated thoroughly to achieve a uniform suspensio n and solution. Bio reactors T12, T22, M12, and M22 had 50 ml of uniform fluid removed f rom each with the intention of testing the volatile fatty acid content of each of them . T his resulted in Bio

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Page number 7 reactors T12, T22, M12, and M22 having a larger head space of 80 ml rather than 30 ml originally. During this entire procedure of preparing the ex perimental bioreactors , sufficient quantities of dissolved oxygen (DO) had contaminated the fluid used to fill the bioreactors. To reduce DO concentrations pure nitrogen gas was sparged into the all of the experimental bioreactors. To accomplish this rubbe r caps were placed on top off all eight experimental bioreactors to achieve an airtight seal. Two clinical needles were injected into each of the rubber caps. One needle was injected deep enough into the rubber cap until the tip was submerged into the flui d inside the bioreactor , the other needle was injected to the height of the head space of the bioreactor . Pure nitrogen gas at a flow rate of 1 L/min was pumped through the submerged needle which caused the fluid inside the bioreactor to be agitated as a r esult of the nitrogen bubbles forming below the maintained a pressure of 1 atm in the head spaces of each bioreactor. After one minute of nitrogen flow the sparging was stopped and the needles removed. T11, T12, T21, and T22 were placed into the thermophilic incubation chamber . M11, M12, M22, and M22 were placed into th e mesophilic incubation chamber . Data Collection 50 ml syringe. The syringe would be injected into the rubber cap on top of each bioreactor into the head space. The gas produced during incubation would cause the pressure inside the bioreactor to increase; this would cause the piston inside the syringe to expand when injected into the bioreactor. When the internal pressure of the bioreactor equaled that of the outside environment, approximately 1atm, the piston wou ld stop expanding and the volume of gas produced would be read based on where the piston stopped on the syringe. The volume of the bioreactor would be recorded and the gas accumulated in the syringe would be reintroduced into the bioreactor , this would ins ure that enough gas would be present to perform a gas composition test using a gas chromatograph. A 1 ml sample of gas would be collected using a smaller syringe rated for 1 ml of fluid, and injected into the bio reactor to collect the sample. The sample o f gas collected by the 1 ml syringe was placed into the GC to measure its carbon dioxide and methane composition and was recorded. After this measurement was made the 50 ml syringe would be injected into the bioreactor and the piston allowed to equilibriat e with the atmosphere. The volume of gas accumulated into the syringe would be removed which would reset the internal pressure of the bioreactor to atmospheric pressure .

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Page number 8 Results and Discussion Figure 1 is a bar graph for the accumulated biogas production of bioreactors M11, M12, M21, M22, T11, T12, T21, and T22 over a 20 day interval at STP . Each bar re presents the total amount of methane gas produced on that day since the inception of the experiment . Figure 1 Accumulated Met h ane Gas Production Over 20 Days At the start of digestion for each reactor o nly small amounts of methane had accumulated , but after two day s methane production drastically increased as can be seen in M11, T21, M22, T12, and T22 in figure 1. Bioreactors M21 , T11, and M12 exhibited relatively small methane production over the 20 day interval shown in figure 1. Bioreactor T12 in figure 1 had produced the most methane gas over the 20 day interval. For the purpose of comparison the maximum theoretical amount of methane gas that can be produced was calculated for all eight experimental bioreactors. As discussed in the removed. This resulted in these bioreactors containin g only 48 ml of the original pretreated algae instead of 60 ml, therefore the maximum theoretical methane production of bioreactors T12, T22, M12, and M22 is going to be less compared to the other 4 experimental bioreactors. Before calculation of the maxi mum theoretical methane production, a few assumptions were made for sim plifying the calculations. It was assumed that the algae subst rate for anaerobic digestion possessed the Redfield elemental composition of , which is the average el emental composition in marine systems. It was assumed also that the growth and maintenance of the anaerobic bacterial community was directly coupled with methane production. Further simplifying the above assumption, growth and maintenance are neglected as well . With these assumptions incorporated the theoretical methane yield can be calculated using the following Buswell equation [3 ] . 0.0 5.0 10.0 15.0 20.0 M11 M21 T11 T21 M12 M22 T12 T22 Methan Gas STP ml Experimental Bioreactor Accumulated Methane Gas Production Over 20 Days Day 2 Day 8 Day 14 Day 20

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Page number 9 Equation 1 , , and are methane, carbon dioxide, water, and ammonia respectively. is the generic form of the biomass substrate where a , b , c , and d are the carbon, io. The maximum theoretical specific methane yield at STP can now be calculated by using equation 2 below. Equation 2 Since the concentration of VS is known for the algae substrate the maximum theoretical methane volume was calculate using equation 2 for bioreactors with 48 ml and 60 ml of pretreated algae using the ideal gas law under STP conditions. Results of these calculations are presented in figure 2 . Figure 2 Real Methane production over 20 Days vs Theroretical Amount Possible Figure 2 compares the real methane volume over 20 days for each bioreactor with the maximum theoretical methane production possible as predicted by equation 2 . As depicted in figure 2 only a small fraction of potential methane production is actually produced in this experiment , this is because the bioreactors were measured for only 20 days. If the bioreactors were given more time to ferment then more methane woul d have been measured . Notice that the maximum theoretical 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 M11 M21 T11 T21 M12 M22 T12 T22 Methane Gas STP ml Experimental Bioreactor Real Methane Production in 20 Days vs Theoretical Amount Possible Produced CH4 ml Theoretical CH4 ml

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Page number 10 methane produced is smaller for bioreactors M12, M22, T12, and T22, this is because these reactors contained less algae substrate. T he performance of each bioreactor was quantified by calculating the % yield, and the results graphed in figure 3. Figure 3 Percent Yield of Methane Gas from each Bioreactor Figure 3 illustrates that bioreactor T12 had the highest % yield . An observation noted is that bioreactors M22, T12, an d T22 exhibited superior performance over M21, T11, and, T21 as illustrated in figure 3. This was a surprising observation because bioreactors M22, T12, and T22 as discussed contained less algae substrate. One possible explanation for this superior perform ance by M22, T12, and T22 over M21, T11, and, T21 could be due to the effects of different headspace volumes . Bioreactors M22, T12, and T22 had larger head spaces compared to M21, T11, and, T21. The performance of these eight experimental bioreactors that were done under saline conditions needs to be compared to other experiments conducted using freshwater bioreactors for the purpose of validating the effectiveness of high saline anaerobic digestion over freshwater digestion. One experiment conducted under freshwater conditions used a n untreated cyanobacterium named Microcystis spp as the substrate for anaerobic digestion [4 ] . The anaerobic bacteria inoculum was harvested and filtered from actively digested dairy cattle manure slurry from an 800 size bio gas plant. Three experimental 250 ml bioreactors were used each with a different bacterium inoculum /substrate ratio ( ISR ) of 2, 1, and 0.5 calculated based on the VS ratio of the bacterium inoculum and the substrate. The three bioreactors were sealed with rubber caps and ran for 30 days. At the end of the experiment the cumulative methane yield at STP were 140.48, 132.44, and 94.42 for bioreactors with ISR s of 2.0, 1.0, and .5 respectively. B ioreactor T12 performed the best as shown in figures 1, 2, and 3 and 0 5 10 15 20 25 30 35 M11 M21 T11 T21 M12 M22 T12 T22 Percent Yield % Experimental Bioreactors Percent Yield of Methane Gas from each Experimental Bireactor % Yield

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Page number 11 had cumulative methane yield of 107.05 ; however the ISR is not known for T12. Based on observation the inoculum used for T12 exhibited the same viscosity as pure water which w ould indicate that its VS concentration is relatively low compared to the pretreated algae which exhibited a higher viscosity compared to pure water. With this into consideration the ISR should be between .5 and 0. Our methane yield is greater than the met hane yield from the referenced experiment because at an ISR of .5 their methane yield was 94.42 ; however since cyanobacterium Microcystis spp was not pretreated before digestion, the reduced performance can be attributed to this. Another study experimented with a cyanobacterium called Arthrospira platensis and used it as a substrate for anaerobic digestion in a salinity level close to 0 g/l [1 ] . The experiment was conducted using 250 ml batch bioreactors at 38 º C for 32 days . Their methane yiel d was 440 which is much greater than our measured methane yield; however our methane yield was calculated for 20 days. Also to note for this referenced experiment the ISR was assumed to be much higher than our experiment, which would also expl ain their higher methane yields. Conclusion If th e can be concluded that anaerobic digestion of pretreated algae in saline conditions of 3 7 g/l has proven to be just as effective compared to anaerobic digestion in a digester with salinity levels close to 0 g/l . The implications of this experiment shows that algae cultivation using sea water and afterwards direct anaerobic digestion using bacteria adapted to high saline conditions is feasible. The benefits of this would be reduced strain on arable land and freshwater resources. Acknowledgement This experiment would not have been possible without the training and constant guidance of Nguyet Doan and Dr Pratap Pullammanappallil .

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Page number 12 References [1] J. H. Mussgnug, V. Klassen, A. Schlüter and O. Kruse. Microalgae as substrates for fermentative biogas production in a combined biorefinery concept. J. Biotechnol. 150(1), pp. 51 56. 2010. Available: http://www.sciencedirect.com/science/article/pii/S016816561000369X . DOI: http://dx.doi.org/10.1016/j.jbiotec.2010.07.030 . [2] A. Rinzema, J. van Lier and G. Lettinga. Sodium inhibition of acetoclastic methanogens in granular sludge from a UASB reactor. Enzyme Microb. Technol. 10(1), pp. 24 32. 1988. Available: http://www.sciencedirect.com/science/article/pii/0141022988900944 . DOI: http://dx.doi.org/10.1016/0141 0229(88)90094 4 . [3] B. Sialve, N. Bernet and O. Bernard. Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnol. Adv. 27(4), pp. 409 416. 2009. Available: http://www.sciencedirect.com/science/article/pii/S0734975009000457 . DOI: http://dx.doi.org/10.1016/j.biotechadv.2009.03.001 . [4] S. Zeng, X. Yuan, X. Shi and Y. Qiu. Effect of inoculum/substrate ratio on methane yield and orthophosphate release from anaerobic digestion of microcystis spp. J. Hazard. Mater. 178(1), pp. 89 93. 2010. Available: http://www.sciencedirect.com/science/article/pii/S0304389410000804 . DOI: http://dx.doi.org/10.1016/j.jhazmat.2010.01.047 .