Citation
Use of Banana Peduncle as Feedstock for Ethanol and Biogas Production

Material Information

Title:
Use of Banana Peduncle as Feedstock for Ethanol and Biogas Production
Creator:
Pazmino, Marco Antonio
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (68 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Agricultural and Biological Engineering
Committee Chair:
PULLAMMANAPPALLIL,P C
Committee Co-Chair:
PORTER,WENDELL A
Committee Members:
KOOPMAN,BEN L
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Anaerobic digestion ( jstor )
Bananas ( jstor )
Biogas ( jstor )
Ethanol ( jstor )
Fermentation ( jstor )
Juices ( jstor )
Methane ( jstor )
Peduncle ( jstor )
Raw materials ( jstor )
Sugars ( jstor )
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
banana -- bioethanol -- biofuel -- biogas -- fermentation
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Agricultural and Biological Engineering thesis, M.S.

Notes

Abstract:
Tropical and sub-tropical countries in Latin America, Caribbean and Asia grow bananas in large quantities. Ecuador is the world's largest exporter of bananas, producing 7.4 million tonnes annually. More than 12% by weight of the harvested banana cluster is banana peduncle, which is discarded. The idea of this project is to evaluate the feasibility of using the banana peduncle as a feedstock to produce ethanol by fermentation and/or biogas by anaerobic digestion. The analyses considered will characterize the composition, structure, chemical properties, fermentability and anaerobic digestibility of banana peduncle fiber, concentrated juice and stillage (after distillation). Results show that the extract from banana peduncle contains high concentrations of glucose (7 g l^(-1)) , sucrose (3 g l^(-1)) and fructose (8 g l^(-1)). Fermentation of concentrated peduncle extract (5x) resulted in an ethanol yield of 41% g ethanol/ g sugars. These results are promising indications on the usability of peduncle for fermentation. In addition, all parts of the peduncle including the remaining fiber after juice extraction, banana peduncle juice and the stillage (after distillation), were successful used as feedstock for anaerobic digestion. Herein, the methane yield were 0.263, 0.043, 0.034L CH4 at STP g VS-1, respectively, showing that especially the peduncle fiber could serve as a rich feedstock for biogasification. For large scale ethanol production from peduncle juice and biogas synthesis from peduncle fiber, helping to meet the growing demand of biofuels and energy consumption in countries where banana is a principal crop. ( en )
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, 2014.
Local:
Adviser: PULLAMMANAPPALLIL,P C.
Local:
Co-adviser: PORTER,WENDELL A.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-05-31
Statement of Responsibility:
by Marco Antonio Pazmino.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
5/31/2016
Resource Identifier:
908645682 ( OCLC )
Classification:
LD1780 2014 ( lcc )

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USE O F B ANANA PEDUNCLE AS FEEDSTOCK FOR ETHANOL AND BIOGAS P RODUCTION By MARCO A. PAZMINO HERNANDEZ 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 2014

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2014 Marco A. Pazmino Hernandez

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To my parents, Ma rco Pazmino and Maria Hernandez, to my sisters, Fernanda Sonia, Diana Pazmino and to my brother Andres Pazmino Without your support I would not have been able to complete this dream.

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4 ACKNOWLEDGMENTS First, I would like to thank God for challenging me and for every lesson that I learned. Through him, I grew to a mature person Thanks to Dr. Pratap P ullammanappallil, my committee chair, who gave me the opportunity to do my master under his supervision. Thanks for invaluable advice and suggestions throughout my research work at University of Florida. Also, I would like to express my sincere gratitude to Dr. Wendell Porter and Dr. Ben Koopman for their inspiring lectures which helped me to be come a better student. I will always be grateful for everything that they have done for me. I give special thanks to Gloria and Dave Chyn oweth They were like a second family since day one on this master journey dream. Special thanks to Paul Lane who gave me the opp ortunity to work and meet this big family of the Agricultural and Biological Engineering Department. Cesar Moreira and Jaime Chavez who were like brothers and supported me on all my personal and professional issues. I also give thanks to Inga Wessels. She has been part of my life since 2012 and has been ve ry important during these past t wo years. I do not know if without her, I would have been able to complete especially Juan Martin Tanca Special thanks go o ut to all department staff Agricultural and Biological Engineering Department whom were my friends, support ed and help ed me many times during my master s tudies Last but not least, I would like to thank my whole family and friends in Ecuador for being ther e to support me anytime.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 A BSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 1.1 Biofuels ................................ ................................ ................................ ............. 13 1.2 Banana Production in the World ................................ ................................ ....... 16 1.3 Banana Production in Ecuador ................................ ................................ ......... 18 1.4 Morphology of the Banana Plant ................................ ................................ ....... 20 1.5 Research Objectives ................................ ................................ ......................... 23 2 BANANA PEDUNCLE EXTRACT FOR ETHANOL PRODUCTION ....................... 25 2.1 Introductory Remarks ................................ ................................ ........................ 25 2.2 Experimental Material and Methods ................................ ................................ .. 29 2.2.1 Feedstock ................................ ................................ ................................ 29 2.2.2 Extraction of Banana Peduncle Juice by Grinding and Milling ................. 29 2.2.3 Uniformity Test and Extract Generation ................................ ................... 30 2.2.4 Fermentation Process ................................ ................................ ............. 31 2.2.4.1 The microorganism ................................ ................................ ........ 31 2.2.4.2 The fermenter ................................ ................................ ................ 32 2.2.4.3 Inhibition analysis ................................ ................................ ........... 32 2.2.5 Chemical Analyses ................................ ................................ .................. 32 2.2. 5.1 Gas c hromatography ................................ ................................ ...... 33 2.2.5.2 High p erformance l iquid c hromatography ................................ ...... 33 2.2.5.3 Statistical analyses ................................ ................................ ......... 34 2.3 Results ................................ ................................ ................................ .............. 34 2.3.1 Characteri zation of the Banana Peduncle ................................ ............... 34 2.3.2 Comparison of Different Extraction Techniques: Crushing and Grinding ................................ ................................ ................................ ......... 35 2.3.3 Uniformity Test ................................ ................................ ........................ 36 2.3.4 Characterization of the Banana Peduncle Extract ................................ ... 39 2.3.5 Fermentation of Banana Peduncle Extract ................................ .............. 40 2.4 Discussion ................................ ................................ ................................ ........ 41

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6 3 BANANA PEDUNCLE FIBER AND EXTRACT FOR BIOGAS PRODUCTION ....... 45 3.1 Introductory Remarks ................................ ................................ ........................ 45 3.1.1 Anaerobic Digestion ................................ ................................ ................ 45 3.1.2 Banana Peduncle as Anaerobic Digestion Feedstock ............................. 47 3.2 Experimental Material and Methods ................................ ................................ .. 48 3.2.1 Feedstock ................................ ................................ ................................ 48 3.2.1.1 Banana p eduncle f iber ................................ ................................ ... 48 3.2.1.2 Banana p eduncle c oncentrated e xtract ................................ .......... 49 3.2 .1.3 Banana p eduncle s tillage ................................ ............................... 49 3.2.2 Anaerobic Digestion ................................ ................................ ................ 49 3.2.3 Anaerobic Digestion Protocol ................................ ................................ .. 51 3.2.4 Chemical Analyses ................................ ................................ .................. 51 3.2.5 Statistical Analyses ................................ ................................ .................. 52 3.3 Results ................................ ................................ ................................ .............. 52 3.3.1 Characteristics of Banana Peduncle Fiber and Digested Residue .......... 52 3.3.2 Biogasification of Banana Peduncle Fiber ................................ ............... 53 3.3.3 Characteristics of Banana Peduncle Juice Concentrate (5x) ................... 56 3.3.4 Biogasification of Banana Peduncle Juice Concentrate (5x) ................... 56 3.3.5 Characteristics of Banana Peduncle Stillage ................................ ........... 58 3.3.6 Biogasification of Banana Peduncle Stillage ................................ ........... 58 3.4 Discussion ................................ ................................ ................................ ........ 60 3.4.1 Biogasification Efficiency ................................ ................................ ......... 60 3.4.2 Inhibitory Factors ................................ ................................ ..................... 60 4 CONCLUSIONS ................................ ................................ ................................ ..... 62 APPENDIX A DISPLACEMENT GAS METER ................................ ................................ .............. 64 LIST OF REFERENCES ................................ ................................ ............................... 65 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 68

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7 LIST OF TABLES Table page 1 1 Automobile fleet in Ecuador. (modified from AIHE annual report, 2011) ................. 15 1 2 Fuel consumption in Ecuador (modified from AIHE annual report, 2011) ............... 16 2 1 Peduncle properties. ................................ ................................ ............................... 34 2 2 Banana peduncle properties. ................................ ................................ .................. 40 2 3 Banana peduncle extract (5x) minerals. ................................ ................................ .. 40 2 4 Characteristics between feedstock ................................ ................................ .......... 42 2 5 Comparison of fermentation substrates ................................ ................................ .. 43 3 1 Loading and unloading parameters. ................................ ................................ ........ 53

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8 LIST OF FIGURES Fi gure page 1 1 Global fruit production ................................ ................................ ........................ 16 1 2 Schematic of banana plant. ................................ ................................ ................ 17 1 3 Banana exports per country. ................................ ................................ ............... 18 1 4 Condition of banana plantation in Ecuador. ................................ ........................ 20 1 5 Banana plant morphology ................................ ................................ ................... 21 1 6 C urrent utilization of peduncle ................................ ................................ ............ 22 1 7 Hypothesis of banana peduncle. ................................ ................................ ........ 23 1 8 Project overview. ................................ ................................ ................................ 24 2 1 Schematic representation of ethanol pathway ................................ .................... 25 2 2 Schematic presentation of the initial steps of the banana production process .. 27 2 3 Fermentation of banana peduncle extract overview ................................ ........... 28 2 4 Comparison of extraction techniques ................................ ................................ 30 2 5 Sample preparation for the uniformity test of the banana peduncle .................... 30 2 6 User manual for Saccharomyces cerevisiae ex bayanus ................................ .. 31 2 7 Airlock S Curve trap for fermentation purpose ................................ .................... 32 2 8 Comparison of different extraction te chniques ................................ ................... 35 2 9 Uniformity of different peduncle parts: ................................ ................................ 37 2 10 Uniformity test of sugars profile from different parts of banana peduncle extract. ................................ ................................ ................................ ................ 38 2 11 Analysis of sugar composition of raw banana peduncle extract. ........................ 39 2 12 Ethanol production from peduncle extract fermentation. ................................ .... 41 3 1 Banana as agricultural nonfood feedstock. ................................ ......................... 47 3 2 Biogasification of banana peduncle fiber ................................ ............................ 48

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9 3 3 Schematic of digesters 1 and 2 set up ................................ ............................... 5 0 3 4 Digester set up. Digester 1 with banana peduncle fiber and lava rocks after digestion. ................................ ................................ ................................ ............ 53 3 5 Methane Production and Cumulative Methane yield. ................................ ......... 54 3 6 Pro file of pH, and sCOD performance during banana peduncle digestion ......... 56 3 7 Methane Production and Cumulative Methane yield of bana na peduncle concentrated juice (5x) ................................ ................................ ....................... 57 3 8 Methane Production and Cumulative Methane yield of banana peduncle stillage ................................ ................................ ................................ ................ 59 3 9 Crucial points for improvement the efficiency in biogas production. ................... 61

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10 LIST OF ABBREVIATIONS AD Anaer obic Digestion AEBE Asociacion de bananeros del Ecuador APHA American Public Health Association AWWA American Water Works Association COD Chemical oxygen demand DSFF Down flow stationary fixed film FAOSTAT Food and Agriculture Organization of the United Nations GC Gas Chromatography GHG Greenhouse gas GLP L iquefied petroleum gas HPLC High performance liquid chromatography sCOD Soluble chemical oxygen demand TS Total solids VS Volatile solids

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11 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 USE OF BANANA PEDUNCLE AS FEEDSTOCK FOR ETHANOL AND BIOGAS P RODUCTION By Marco A. Pazmino Hernandez May 2014 Chair: Prata p Pulla mmanappallil Major: Agricultural and Biological Engineering Tropical and sub tropical countries in Latin A merica, Caribbean and Asia grow exporter of bananas, producing 7.4 million tonnes annually. More than 12% by weight of the harvested banana cluster is banana peduncle, which is discarded. The idea of this project is to evaluate the feasi bility of using the banana peduncle as a feedstock to produce ethanol by fermentation and/or biogas by anaerobic digestion. Th e analyses considered will characterize the composition, structure, chemical properties, fermentability and anaerobic digestibility of banana peduncle fiber, concentrated juice and stillage (after distillation) R esults show that the extract from banana pe duncle contains high concentrations of glucose (7 g ) sucrose (3 g ) and fructose (8 g ) Fermentation of concentrated peduncle extract (5x) resulted in an ethanol yield of 41% g ethanol/ g sugars. These results are promising indications on the usability of peduncle for fermentation. In addition, all parts of the peduncle including the remaining fiber after juice extraction, banana peduncle juice and the stillage (after distillation), were

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12 successful used as feedstock for anaerobic digestion Herein, the m ethane yield were 0.263, 0.043, 0.034L CH 4 at STP g VS 1 respectively, showing that especially the peduncle fiber could serve as a rich feedstock for biogasification. For large scale ethanol production from peduncle juice and biogas synthes is from peduncle fiber helping to meet the grow ing demand of biofuels and energy consumption in countries where banana is a principal crop

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13 CHAPTER 1 INTRODUCTION In recent years there has been significant effort to use agro industrial residues to develo p new bioprocesses, for energy generation, especially biofuels, as environmentally friendly alternatives as the increasing industrialization and motorization of the world has led to a steep rise for the demand of petroleum based fuels (Singh, et al., 2010) Today fossil fuels m ake up 80% of the primary energy consumed in the wor ld, of w hich 58% alone is consumed by the transport sector (Singh, et al., 2010). I t may be impossible to meet the energy requirement of the world by consuming fossil fuels to fulfill the energy demand without causing many negative effects including urban pollution, greenhouse gas emission (GHG), and loss of biodiversity because of oil reserves depletion. 1.1 Biofuels Energy alternatives such as biofuels; including bio gas, syngas (synthesis gas), hydrogen and ethanol from biorenewable resources have be en developed as suitable sustainable alternative fuel source s Several processes have been developed for effective fermentation of agricultural waste. These processes can be subdivided into first (1) and second (2) generation processes: For first generation processes, simple sugars extracted from agricultural waste are used for large scale yeast fermentation. Thereafter, ethanol is separated by disti llation During second g eneration processes, lignocellulose from fiber from agricultural biomass is used to produce ethanol by biochemical steps. T hose steps include pretreatment, enzymatic hydrolysis and fermentation. Research is underway to produce ethanol from cellulosic feed stock like agricultural and forestry residues, and energy crops like grass.

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14 Ethanol, in particular, has been considered as one of the most environmentally friendly, biodegradable and sulfur free energy sources; as long as i t comes from a vegetable origin or from another renewable feedstock ( Velasquez Arredondo, et al., 2009; Wang, et al., 1999). Ethanol constitutes an alternative fuel for spark ignition engines that are currently used. It will not contribute to an increase of carbon monoxide emissions, du ring the combustion process. Moreover, e thanol fuel provides many advantages such as reduction of fossil fuel consumption, and increase of energy security as well as social benefits derived from its production (Velasquez Arredondo et al. 2009) T he latter benefits include the creation of local jobs A liter of ethanol contains 66% of the energy provided by a liter of gasoline but has a higher octane level making it qualitative ly a better additive than regular additives; such as, t etraethyllead for gasoline (Singh, et al., 2010). Biogasification is a biochemical process that allows to transform organic matter into biogas (methane and carbon dioxide), by anaerobic digestion. Anaerobic digestion for biogas production has become a worldwide focus of research, because it produces energy that is renewable and environmentally friendly. Recent results are encouraging for the use of animal waste available to produce renewable energy and clean environment. For example Ounaar et al. obtai ned biogas production of 26.9 m 3 with an average methane content of 61 % during the anaerobic digestion of 440 kg of cow dung with an energy equivalent of 164.5 kWh (Ounaar et al., 2012). Special emphasis was initially focused on anaerobic digestion of municipal solid waste for bioener gy production about a decade ago. Anaerobic biological treatment can be an acceptable solution because it reduces and stabilizes solid wastes volume,

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15 produces biogas comprising mainly methane and carbon dioxide, and traces amount of other gases (Stroot et al., 2001). In addition to biogas, a nutrient rich digestate is also produced which provide either fertilizer or soil conditioner properties. Anaerobic biological treatment can be an acceptable solution because it reduces and stabilizes solids and also pro duces energy, serving two purposes at the same time (Nasir et al., 2012). A n example underlining the need for further research in the biofuel sector is given in table 1 1 which shows the fuel consumption of Ecuador ( Asociacion de la Industria Hidrocarburif era del Ecuador, AIHE et. al 2011 Table 1 1). T he national automotive fleet in 2011 consisted of established 1,830,717 million of cars. Thus using approximately 22,386 thousand barrels of gasoline is consumed by these vehicles per year (Table 1 2). Those numbers show clearly the high demand of gasoline. I n addition Ecuador started the distribution of bioethanol produced form sugar cane juice called in Guayaquil. It was planned that the bioethanol should replace 5 ate by 2020. To reach this goal, the government of Ecuador is encourag ing the production of bioethanol from sugar cane. Table 1 1. Automobile f leet in Ecuador (modified from AIHE annual report, 2011) The automotive fleet in Ecuador 2010 2011 Cars 727,481 790,077 Bus 25,704 27,455 Truck 170,319 181,093 Pick up 465,434 493,004 SUV 281,024 312,867 VAN 20,126 26,221 Total 1,690,088 1,830,717

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16 Table 1 2. Fuel c onsumption in Ecuador (modified from AIHE annual report, 2011) Fue l consumption in Ecuador ( thousands of Barrels) Gasoline Diesel GLP 2007 16,138 22,740 11,093 2008 17,549 23,409 11,389 2009 18,794 26,519 11,228 2010 20,418 29,911 11,370 2011 22,386 28,440 11,782 1.2 Banana Production in the World Bananas are one of the most widely grown tropical fruits, cultivated in over 130 countries, along the tropics and subtropics of Capricorn (Mohapatra et al ., 2010) making it an important cash crop grown on large plantations for export. Banana s are mainly cultivated for its seedle ss fruit, which can be eaten green as well as ripe. For instance, banana is the second most p roduced fruit after citrus fruits and comprises the Food and Agriculture Organization of the United Nations (FAOSTAT 2011, Figure 1 1). India is the largest producer, contributing 27% of the banana producti on Additionally, b anana s are grown and harvested all year round and are ready to be harvested 8 to 10 months after planting. Fig ure 1 1. Global fruit production ( modified from FAOSTAT, 2011)

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17 Approximately 106.54 million tonnes of banana fruit are produced every year to meet the world demand, (FAOSTAT, 2011 ) Unused byproducts of the banana ind ustry include the banana pseudo stem, leaf, peduncle and those bananas that do not meet the high quality standards for packaging the fruits. Recent l iterature shows that especially the banana fruit and peel contain high amounts of sugar ( Velasquez Arredondo, et al., 2010; Cordeiro, et al., 2003; Hammond, et al., 1996) In contrast to that, there is currently no information on the composition of the banana peduncle and its extract As the banana peduncle provides all the sugars for the banana fruit ; it can be assumed, t hat it also contains a lot of rest sugars, usable f or fermentation ( Fig ure 1 2 ) Therefore, the peduncle may provide a cheap and easily accessible waste feedstock for the production of biofuels, including bioethanol and biogas Fig ure 1 2. Schematic of banana plant (modified from El U niverso Ecuadoria n newspaper from 07/24/12 )

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18 1.3 Banana Production in Ecuador Ecuador has been endowed with great natural resources in areas such as forestry and agriculture. In the agriculture sector, Ecuador, the largest banana exporter in the world, produces around 7.5 million tonnes per year of which 5.2 million tonnes of banana s are exported (FAOSTAT, 2011 ; AEBE, 2011 Figure 1 3 ). Moreover, about 285 million boxes of banana are exported an nually, with a weight of over 18 kg per unit ( AEBE 2011 ). For Ecuador it is estimated that 10 12% of all economically active people obtain some benefit from banana production and 80% of the total export pr oduction comes from growers who cultivate areas smaller than 30 ha (Graefe et al. 2011). As a consequence, bananas are the sec ond most export product after crude oil in Ecuador Fig ure 1 3 Banana e xports per country ( modified from FAOSTAT, 2011 )

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19 Banana s are mostly grow n a long the coast of Ecuador. Specifically, two states (Los Rios and Guayas) are responsible for 67% of the banana production. These two states are neighbors, which makes logistics of transportation between the more than 5000 banana plantations easy. Most of the edible bananas are either Musa Cavendish or Musa Paradisiaca variety M. Cavendish, the pur e triploid acuminate (AAA) group known as dessert banana, is sweeter and less starchy than M. Paradisiaca (Mohopatra et al., 2010). Also M. Cavendish is the more exploited type equaling about 33.3% of worldwide production (Oliveira et al ., 2007). Besides all the positive effects that banana plantations have on Ecuador economy there are some areas that concern the population living near the banana plantations: water quality availability of drinking water and air pollution. Banana plant ations require a substantial amount of fresh wat er, fertilizer and pesticides. Like m ost agricultural crops bananas need a large input of nitrogen to maximize production. This leads to significant energy costs for synthesis, transport and application of fertilizer, as well as water pollution because of excess nitrogen run off and nitrification of water courses (Wheals et al., 1999). Moreover 48% 52% of the total nitrogen applied is not used by the banana plant but rather lost into the aquifer from a banana crop with sprinkler fertigation system ( Mu noz Carpena et al. 200 2 ) Another potential source of pollution is the usage of large amounts of fuel for power generation. Many plantations in Ecuador are located in insulated areas, lacking local e nergy generation and fresh water supplies. For example, 146 he ctares and plant densit y of 1,420 banana trees per

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20 hectare That causes a use of 20,000 gallons of diesel annually for irrigation and drainage Therefore, the farmers have to transport and store large amounts of fuel in big tanks. However, no security standar ds are followed in m ost cases, d ue to lack of material, mon ey and regulations This causes costly spills and leakage of fuel, and thereby pollution of the area ( Figure 1 4 (A)). Furthermore, massive diesel generators are used to power the irrigation system and drainage Lack of regulations and inspections is a potential risk for pollution, too ( Fig 1 4 ( A B)) A. B. Fig ure 1 4 Condition of banana plantation in Ecuador Example of condition of Irrigation pipes (A). A fuel tank and diesel engine (B) used in banana plantations in Ecuador ( July 10, 2013. Courtesy of Marco Pazmino ) 1.4 Morphology of the Banana Plant The banana plant is described as a monocotyledonous, her baceous, evergreen perennial. It is herbaceous because after fruit harvest the aerial parts die down to the ground and there are no woody components. It is perennial because new suckers grow up from the base of the mother plant to replace aerial parts whic h have died (Robinson et al. 1996; Ennos et al. 2000). A suitable banana climate is a mean temperature of

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21 80 o F (26.67 o C) and mean rainfall of 4 inches (10 cm) per month ( Tock et. A l ., 2009 ). Moreover, the bana na plant can be subdivided into the root syst em, pseudostem, sucker, leaf, cigar leaf, and inflorescence (Figure 1 5 ). The inflorescence is a complex structure that emerges upwards through the center of the pseudostem before bending down under the weight of the developing flowers that will develop into fruits T he inflorescence can be subdivided in: peduncle, bunch and rachis. This study will focus on the peduncle. Peduncle is the stalk that supports the inflorescence and attaches it to the rhizomes as well as the individual fruits (also called fingers) that are arranged in hands Fig ure 1 5 Banana plant morphology ( modified from Champion et al 1963 ) During the process of harvesting banana fruits large amounts of agricultural waste are produced. Around 20% 40% of the bananas that are produced do not meet export standards and quality demands of local markets, and are usually discarded in open air dumps. The banana peduncle arrives with the bunch of fruit at the end of the production line. Thereafter, the cutter (who is assigned to cut down the banana fruit)

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22 cut s the finger s and hands apart (denoted as banana fruit and group of fruit). Then, the banana peduncle is discarded. Most banana peduncle s are left o n the soil for degradation which improves the conditions for growing bananas or other types of crops by giving nutrients to the soil but also brings pollution from fertilizers to the soil (Fig ure 1 6(A)(B) ) A B Fig ure 1 6 Current utilization of pedun cle (A) Banana peduncle at the end of the production line. (B) Banana peduncle left in the field ( July 10, 2013. Courtesy of Marco Pazmino ) The processing of 1 box of banana fruit (18 kg) for exporting approximately produces 3.26 kg of banana peduncle residues ( AEBE, 2011 plantation annual report et al. 2012 ) In 201 1, Ecuador exported 285 million boxes This repre sented just 6% of the banana production worldwide (FAOSTAT, 2011). Just in Ecuador this banan a production volume would generate over one million ton nes of banana peduncle annually. This amount of organic matter can be potentially used as feedstock for ethanol production.

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23 This example shows the high significance of banana peduncle and the usage th at peduncle for bio ethanol and biogas pr oduction could have, if this byproduct turns out to be a viable feedstock for fermentation and biogasification 1.5 Research Objectives The b anana peduncle is an abundant agricultural feedstock around Asia, Africa, Central and South America is examined as a resource for biofuel production. The aim of this study is to investigate the feasibility of using banana peduncle extract for ethanol production by fermentation. As well as, for biogas production f rom t he banana fiber and stillage by anaerobic digestion The hypothesis of this study were: Because of its simi larity to sugarcane (Figure 1 7 ), the juice can be extracted from banana peduncle using crushing equipment for sugar industry The banana pedunc le extract juice should contain sugars which can be fermented to ethanol using commercially available yeast. Banana peduncle fiber remaining after juice extract should be a good feedstock for biogasification as it might contain residual sugars and the fib ers would be rich in carbohydrate The stillage remaining after fermentation of the extract could be used for biogas production by anaerobic digestion. Fig ure 1 7 Comparison of sugar cane and banana peduncle. ( A) Cross section of sugar cane stalk and (B) Cross section of banana peduncle. (modified: h ttp://www.njcharters.com/nj_charters_blog/uploaded_images/bigst ockphoto_Tropical_Raw_Sugar_Cane_1785700 720055.jpg) (July 10, 2013. Courtesy of Marco Pazmino)

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24 Th ose hypotheses result in the following r esearch objectives (Figure 1 8 ) : To characterize the quantity and composition of the extracted juice from the banana peduncle emphasizing on sugar content. Determine ethanol yie ld for fermentation of the extract from different concentration samples Biogas ification of the banana peduncle fiber left after banana peduncle was squeezed Biogasification of the stillage that is left after fermentation of the extract Figure 1 8. Project overview

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25 CHAPTER 2 BANANA PEDUNCLE EXTRACT FOR ETHANOL PRODUCTION 2.1 Introduct o ry Remarks The biofuel industry has greatly expanded over the past decade due to concern about increasing dependence on foreign oil and increasing levels of atmospheric carbon dioxide. Bioethanol is one of the most common biofuels due to its pro perties that make this biofuel really attractive to the market. This liquid fuel is usually produced out of organic based matter with high contents of sugars which can be fermented by yeast enzymes The yeast convert s six carbon sugars to ethanol and carb on dioxide (Nigam et al 2011 ; Figure 2 1 ). Figure 2 1. Schematic representation of ethanol pathway The challenge for large scale bioethanol production is to find a cheap, broadly available feedstock with a high sugar content and low amounts of inhibito rs. Th e latter includes aliphatic acid; for example acetic acid and byproducts of the sugar degradation such as phenols, furans and carboxylic acids. In addition, suboptimal sugar contents and compositions as well as high amounts of ethanol itself can inhi bit yeast activity. Moreover, th e fermentability of a feedstock depends on its pH value and the

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26 temperature, both of which should remain constant. Currently bioethanol is produced commercially from sugarcane juice, sugar beets and corn starch. In that cont ext some countries are using different types of crops to make biofuel, such as, sugar cane (Brazil), sugar beet (Canada) m aize (Canada and United states) (Kim et al 2011 ) For example Brazil which is the biggest producer of sugarcane, is able to produce bioethanol from sugarcane fermentation in large scale On the contrary, there is the constantly debate on whether a food supply should be part of energy production, implying the need for alternative feedstock After apple, the banana fruit is the ne xt most abundant fruit produced worldwide. The massive demand makes banana production a favorable trade causing some tropical and sub tropical countries, like Ecuador, to grow the banana as primary export products. In Ecuador, the production of banana, Mus a Cavendish type, has a major economic importance. Indirectly, the production of banana generates inversion of $4,500 million dollars between cultivation, infrastructure, packing, and more (AEBE, 201 1) The large production of banana generates a large amo unt of residues such as, pseudo stem leafs, and peduncles This organic waste has between 90% 57 % of dry matter depending of the organic matter. Besides e ach banana plant prod uce s only one bunch of bananas. The banana bunch arrives at the production facility including fruit and peduncle. The fruit are processed for export, while the peduncle is discarded, generating a valuable immense amount of feedstock for fermentation at no cost. (Fig ure 2 2 ). After it is harvesting most of the organic waste (leaf, ps e udo stem and peduncle ) is left o n the soil of the plantation to be used as fertilizer for other crops that are planted on that farm

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27 F ig ure 2 2. Schematic presentation of the initial steps of the banana production process (modified from : Wheals et al ., 1999) (July 10, 2013. Courtesy of Marco Pazmino) The amount of banana agricultural waste in Ecuador has been increased over the past decade. This rise requires a good agricultural waste management. As discarded peduncle causes pollution, indicated earlier. Alternatively, the peduncle could be used as a feedstock for ethanol fermentation at no cost. These agricultural residues are so far not used for energy production, but a good managing of the banan a agricultural waste could be suitable to support the energy supply for Ecuador Several compounds can be found in the juice from the banana peduncle such as carboh ydrates, protein, and sugars ( Mohpatra et al., 2010 ; Oliveira, et al., 2006 ) By u sing a com mercial sugar cane cru sher the juice of the banana peduncle (denoted as extract further in the text) may be extracted readily, followed by its fermentation to

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28 produce ethanol T he fiber remaining after extraction may be suitable for biogasification proces s ing (Clarke et al. 2007 ; Chapter 3 ) As the biofuel industry faces many challenges, there is a nee d for more research and development on renewable energies I t is necessary to develop an economically viable, and sustainable process that fits easy into the current infrastructure In this context, we have recently started a research program aiming to understand the chemical and structural constitution of banana peduncle and testing the usability of its extract in ethanol production. This type of feedstock has the advantages that raw materials can be produced eve ry 8 to 10 months in large quantities and all year around The objective of this study was to produce ethanol from agricultural waste such as banana peduncle extract which ar e abundant and do not in terfere with food security using a commercial Saccharomyces cerevisiae ex bayanus yeast for fermentation (Figure 2 3 ) Fig ure 2 3 Fermentation of banana peduncle extract overview

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29 2.2 Experimental Material and M ethods 2.2.1 Feedstock The banana peduncle s of M. cavendishi were harvested from the banana in Vinces ( Los Rios, Ecuador ) Fresh peduncle s were randomly selected and hand il y s eparated from the bunch B anana peduncle s were obtained from the plantation Thereafter, th ree banana peduncle s were selected for uniformity test The remaining banana peduncle s where moved to the sugar cane press for the milling process. 2.2.2 Extraction of Banana Peduncle Juice by Grinding and M illing Fresh banana peduncles were treated using a Henglian YZ 28 sugarcane presser equipment As well as, corn grinder was used for analyzing extraction yield production (Figure 2 4 (A )( B)) (A) Samples were milled at 25rpm through a mesh in to a 1000mL pot containe r E ach peduncle (cut into less than 40 mm of thickness) was milled one at the time. (B) Samples were crushed manually into a 500mL pot container. Each peduncle (cut into less than 40 mm x 20mm of thickness) was milled one at the time. The resulting peduncle e xtract was collected after passage through each press and sampled for quantity of liquid (mL). The pH of the extracts was measured for each banana peduncle using pH meter (Oakton pH meter, model ECOtestr pH.2). In addition, the sugar composition of the raw extract was determin e d us ing a refract o meter for brix concentration After extraction, the banana extract was concentrated at 85C to 1.25x, 2.5x, 5x, [%] and pH were an alyzed for each concentration.

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30 Figure 2 4 .Comparison of extraction techniques : (A) Henglian YZ 28 sugarcane presser equipment ; (B) Corn grinder http://www.china foodmachine.com/products/YZ 28A 28B.htm#.Uvg_i_ldV E ; http://nbsunshineproduct.com/Showpro.aspx?P_id=768&P_Tid=49 2.2.3 Uniformity T est and Extract G eneration Banana peduncles of three different plants were subdivided into three parts (upper, medium and lower part). Each piece had a length of 5 cm (Figure 2 5 ) The weight and diameter of each piece was determined for later ca lculations. All pieces were hand chopped and squeezed individually using a standard corn mill grinder as described ( 2.2.2 ) The resulting extract was collected and stored at rther analyses. The sample characteristics of the banana peduncl e were calculated as a weighted, pH, brix concentration with the devices mentioned before Figure 2 5 Sample preparation for the u niformity test of the banana peduncle (July 20, 2013. Co urtesy of Marco Pazmino)

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31 2.2. 4 Fermentation P rocess 2.2.4 .1 The m icroorganism Fermentation was carried out using a commercial Saccharomyces cerevisiae ex bayanus (LAL VIN EC 1118 wine yeast), according to the manufacturers protocol (Figure 2 6 ) The banana peduncle extract was pre heated to 33 o C and agitated with a speed of 1 20 rpm using Queue Orbital shaker 4730 Subsequently, the yeast ( S cerevisiae ex bayanus ) was added. After 20 min the temperature in the Queue Orbital shaker 4730 was adjusted to 30 o C and the agitation speed was set to 140 rpm. These set tings were kept for the next 5 days of fermentation. No more nutrients or medium were added to the culture. Under anaerobic conditions, yeast can be used i n the fermentation process. Y east can c onvert six cabon sugars into ethanol according to Equation 2 1 (2 1) Figure 2 6 User manual for Saccharomyces cerevisiae ex bayanus (LALVIN EC 1118 wine yeast)

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32 2.2.4 .2 The f ermenter Batch fermentation experiments were carried out in Erlenmeyer flask s each 125mL, previously sterilized. The working vol ume of each fermenter was 50mL. In addition, five concentration of each banana peduncle extract were used ( 1.25x, 2.5x, 5x, 10x, and 15x ) and run in triplicate. Moreover, a fermentation airlock S Curve trap wit h a rubber stopper (Figure 2 7 ) was used to measure CO 2 production as a marker of occurring fermentation Figure 2 7 Airlock S C urve trap for fermentation purpose (modified from http://alaskawine.blogspot.com/2011/07/basic home wine making step one.html ) 2.2.4 3 Inhibition a nalysis Because no data w as available from t he literature, the peduncle extract was tested for possible inhibitors. T hree concentration s were analyzed 1.25x, 2.5x, and 5x using HPLC for possible acids contained in the extracts. 2.2.5 Chemical A nalyse s Total solids (TS) of raw banana peduncle and concentrated extract (5x) were determined gravimetrically after drying overnight at 105C. Volatile solids (VS) content was determined by ashing a dried sample at 550C for 2h and measuring the ash free

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33 dry weight Those two experim ents were performed using American Water Works Methods. The analysis of mineral s par t and nitrate demand (sCOD) w as analyzed using a Hach kit (Loveland, Colorado). 2.2.5 .1 Gas C hromatography The e thanol concentration was measured by Gas chrom atography (GC), using a n Agilent Technologies 7890A chromatograph equipped with a polystyrene divinylbenzene (DVB) capillary column J&W HP PLOT /Q (15 m x 0.53 5 mm, 40.00 m film thickness). Before the samples were injection ed calibration curves for methan e, ethylene, acetylene, ethane, propylene, propane, and propyne were obtained using high purity commercial standards. 7890A Infinity Series HPLC systems with OpenLab CDS ChemStation software. The analysis of ethanol production w as carried out at the 2.2.5 .2 High P erformance Liquid C hromatography Sugar composition was d etermined by High performance liquid chromatography ( HPLC ) analysis, using Agilent technologies 1260 Infinity chro matograph equipped with a polystyrene divinylbenzene (DVB) sufonic acid resin capillary column Aminex HPX 87P. The column was eluted with 1 NaOH and Milli Q water 20C during analysis. S amples were diluted 1:1, 1:5, and 1:10 in n anowater. Daily reference curves were obtained for glucose, fructose and sucrose by injecting calibration standards with concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03215 and 0.015625 [M] of each sugar was used for data generation 1260 Infinity Series H PLC systems with OpenLab CDS ChemStation software. All analyses and fractionation

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34 experiments were carried out, in duplicate. The analysis of sugar composition w as 2.2.5.3 Statistica l a nalyses The statistical evaluation of experiment a test using Graph Pad Prism software version 5 (Gra phPad software, La Jolla, CA, USA). Experimental data are expressed as means + standard error (SEM). Statis tical significances are referred to as *p < 0.05, **p < 0.01, and ***p < 0.001. 2 .3 Result s 2 3 .1 Characterization of the Banana P eduncle As a first step general dimensions of the banana peduncle were assessed. Table 2 1 shows that the average peduncle h ad a length of 136 cm and a weight of 3.05 kg, which is 13 % of the weight of one harvested banana bunch. Assuming that 5.2 millions of tonnes of banana bunches are harvested for export per year in Ecuador, a total amount of 1 millions of tonnes would be av ailable for biofuel 2012 ) approximately The t otal solids from the banana peduncle were 6 .23% and 77.16% volatile solids, a llowing a first speculation about high sugar contents of the peduncle. Table 2 1. Peduncle p roperties. Peduncle Properties Wet weight (kg) 3.05 Dry matter (kg) 0.19 Volatile matter (kg) 2.35 Average length (m) 1.36 Peduncle weight per 13 Total bunch of banana harvesting (%)

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35 2. 3 .2 Comparison of Different E xtraction Techniques: Crushing and G rinding Two different methods were compared to extract the juice out of the banana peduncle. The firs t approach was using a commercial sugar cane crusher, resulting in a yield of 0.358 w/w (Figure 2 8A ), which was only about 50% of the expected yield. The crusher is built for sugar cane extraction which is much harder than banana peduncle. This lead to slipping of the peduncle through the rolls of the crusher, explaining the lower yield during the process As a second approach a corn grinder was tested Figure 2 8A shows that the yield for the grinder was significantly higher reaching almost 0.6 w/w. T he numbers obtained for pH and b rix [%] of the extracts were also significantly different betwee n the two approaches (Figure 2 8 (B )(C)). F igure 2 8. Comparison of different extraction techniques: crushing and grinding. (A) Difference between from crusher and grinder yield. (B) Difference on pH between from crusher and grinder. (C) Difference on brix [%] between crusher and grinder.

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36 2. 3 .3 Uniformity T est During processing it was noticed that the peduncle does not have an equal diameter. However, the aspect of the banana peduncle is conical shape with a narrow bottom and a wide top. Therefore a uniformity test was performed to see if there are major differences in yield and other characteristics of the banana peduncle extract from three different parts of the peduncle: upper, middle, lower. The middle and upper parts were significantly wider that the lower parts. No difference was detected between middle and u pper parts diameter (Figure 2 9 A). The weight of the upper part was significantly higher than for the lower part, while the weights of upper and middle or lower and middle parts onl y slightly differed (Figure 2 9A ). No differen ces wh ere observed in the yields and b rix [%] fro m upper, middle, and lower on banana peduncle parts (Figure 2 9 (B)(C)). The comparison of the pH values of the three section revealed a slightly but significantly higher pH of the upper comp ared the middle p art (Figure 2 9D ). Result from HPLC analyses of the extract from upper, middle and lower parts showed the same pattern for all sample (Figure 2 10). This suggests a similar sugar composition throughout the peduncle.

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37 F ig ure 2 9 Uniformity of different peduncle parts : A ) Uniform ity test diameter vs. weight, B) Yield of banana peduncle extract depending on the section C ) Brix comparison of banana peduncle extract between sections, D) pH comparison of banana peduncle e xtract between sections

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38 Figure. 2 10 Uniformity test of sugars profile from different parts of banana peduncle extract. Plain banana peduncle extract were examined for uniformity to see if whole peduncle structure contains the same amounts of sugars or changes for the (Upper), (Middle), (Lower)

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39 2. 3. 4 Characterization of the Banana Peduncle E xtract Banana peduncles were chosen and cut into three 5 cm long pieces. The juice of each piece was extracted using a conventional grinder One aliquot was analyzed via HPLC Also, de nsity of the extract was calculated using weight of the extract. Total solids (TS) were determined gravimetrically after drying overnight at 105 o C. Volatile solids (VS) content was determined by ashing a dried sample at 550 o C for 2h and determining the ash free dry weight. Results are shown as mean + SEM for n = 12 (A) and n = 3 (B) independent samples. To a nalyze the amount and variety of sugars, available for fermentation, banana peduncle extracts were analyzed by HPLC in m ore detail. The amounts of Galactose, Arabinose and Xylose were negligible small (data not shown). In contrast, high concentrations of Fructose (7.12 g/l), Gl ucose (5.86 g/l), and Sucrose (2.18 g/l) were detected (Figure 2 11 A). The amount of sucrose, a di saccharide consisting of one glucose and one fructose molecule, was low (0.85 g/l), implying that it was degraded i nto its sugar monomers (Figure 2 11 A). The sugar concentrations of the three pe duncles, chosen for extraction and charac terizati on differed slightly between 17.3 g/l 16.18 g/l and 18.56 g/l, respectively (Fi gure 2 11 B). Figure 2 11 Analysis of sugar composition of raw banana peduncle extract.

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40 The peduncle extract (5x) had a density of 1.02 and the amounts of dry matter and volatile matter were 14.61 % and 57.12 % respectively shown ( Table 2 2 ). Table 2 2 Banana peduncle properties. Peduncle Properties Dry matter (%) 14.6 Volatile matter (%) 57.1 Density of juice (kg/L) 1. 02 pH of juice 5.4 5.6 The high amount of volatile matter explains the large amount of mineral s that banana peduncle extract (5x) contains (Table 2 3). Specially, potassium and magnesium with 34 g/L and 0.5 g/L, respectively. Table 2 3 Banana peduncle extract (5x) m inerals Banana Peduncle extract concentrated (5x) Minerals P 481.6 mg/L K 34 445.5 mg/L Ca 1.9 mg/L Mg 502.3 mg/L Si 97.3 mg/L Na 15.5 mg/L Nox N 462.6 mg/L S 477.5 mg/L In addition, a nalyses of volatile fatty acids were conducting in (1.25x, 2.5x, and 5x) by HPLC revealed that the extract does not contain any acids that could inhibit fermentation by yeast (data not shown) 2. 3. 5 Fermentation of Banana Peduncle E xtract As fermentation of peduncle extract was a completely new approach, no literature was available on the optimal condit ions and microorganisms for this purpose. Therefore, five different concentrates of the extract were tested (Figure 2 12 ).

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41 C ommercially available Saccharomyces cerevisiae ex bayanus (LALVIN EC 1118 wine yeast) was chosen as yeast as it is a well established microorganism for fermentation and known to ferment glucose and fructose (Berthels et al., 2004). Figure 2 12 shows a direct correlation of extract concentration and ethanol production for concentrates 1.25x to 5x from 30% to 41% g ethano l / g sugars Figure 2 12 Ethanol production from peduncle extract fermentation Maximal e thano l production was achieved for 5 x concentrated extract with (41% ethanol) However, it can be assumed that the optimal extract concentration for fermentation via Saccharomyces cerevisiae is 5x Concentrations higher than 10x of the original extract yielded minimum ethanol concentrations of all concentrations tested. 2.4 Discussion Analyses of banana peduncle showed that almost 734.4 tonnes of pedun cle would be available just from significant difference betwe en plantations within Ecuador; t he banana production in Ecuador is around 7.4 million tonnes per year that means around 1.34 million tonnes of banana peduncle would be available per year for extraction. In India, which is

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42 worldwide leading in banana production, 5.1 million tonnes of banana peduncle would be available. Table 2 4 Characteristics between feedstock Typical features of feedstock Properties Banana peduncle Sugar cane Sweet Sorghum World Production (million tonnes) 106 1 819 58 Crop cycle (months) 8 10 10 12 3.5 Number of cycle/year One One Two Brix (% juice) 2.5 3 13 15 11 13 pH 5.4 5.6 5.1 S ugar cane has a production every year of 1,819 million of tonnes per year worldwide (table 2 4) Worldwide amounts of sugar cane and peduncle should be compared. For example, Brazil main sources of fermentation ethanol are sugarcane juice, molasses, and to certain extent, starches derived from tuberous crops such as cassava (Jones et al. 1994 ) The yield, pH value and Brix of banana peduncle extract depends strongly o n the technique used for extraction. It turned out that the grinder is much better suited to the peduncle as a commercial crusher. This should be taken into account for large scale fermentation approaches using banana peduncle as feedstock Uniformity test c learly shows that there is a difference in extract properties, depending on the part of the peduncle that it originates from. Possible explanations for this include: The yield depends on the Surface/diameter/volume ratio? Alternatively, the yield of the ou ter parts, especially of the thinner bottom part could be lower due to faster air drying of those parts after cutting. However, the whole peduncle should be used for extraction because even in the lower parts the sugar concentration is still high and there

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43 is no difference in the sugar variety of the different parts. Additionally, no extract should be wasted/thrown away as it can easily be concentrated. Peduncle has mostly monosaccharides whereas sugar cane has mostly disaccharides. Compare the amounts and maybe also varieties of overall sugars in peduncle juice to the numbers that can be found in the literature for the sugarcane and sorghum (table 2 5) Table 2 5 Comparison of fermentation substrates Comparison of fermentation substrates Substrate constituent Banana peduncle juice cane juice* molasses* Fermentable sugars 1.91 22.4 57 glucose 0.95 0.20 10 fructose 0.88 0.29 13 sucrose 0.8 21.9 33.4 pH 5.4 5.1 Minerals P (mg/L) 96.3 72 600 K (mg/L) 6 889.1 647 27 200 Ca (mg/L) 0.4 200 10 600 Mg (mg/L) 100.5 120 4 200 Si (mg/L) 19.5 Na (mg/L) 3.1 < 1 1600 Nox N (mg/L) 92.5 S (mg/L) 95.5 123 3800 Jones et al., 1994 Concentrations higher than 10x of the original extract concentration result in minimal fermentation efficiency. (a) Ethanol is a major end product of glycolysis in S.cerevisiae and, therefore, the observed inhibition of growth and fermentation by ethanol m ay reflect its effect on enzymes involved in the glycolytic pathway ( Llorente and Sols et al. 1969) Yeast growth is also inhibited when the alcohol concentration of the fermenting must reach about 4% ( Handbook of fungal biotechnology et al. 200 4 )

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44 On th e other hand, inhibition by ethanol can be excluded, as the amount of ethanol after 5 days was lower in the highly concentrated fermentation bottle then in all others. (b) Glucose itself is inhibitory for yeast growth because of high osmotic pressure (Wang e t al., 2013). (c) Inhibitors that are available in all concentrations of the extract, but cross a threshold in extracts that have been more than 10x concentrated. (d). Because of initially higher fermentation rates due to higher available sugar levels, t oxic or inhibitory byproducts increase faster than in the lower concentrated extracts. Those might directly inhibit the yeast activity or change the density/pH which then inhibits yeast activity. (e) Considering, t he high amount of sugar induces osmotic pr essure can placed on the outside of the yeast cell wall. In this stage, cell tend to produce more glycerol inside of the cell, as well as acetic acid to try to decrease the viscos ity of the fluid.

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45 CHAPTER 3 BANANA PEDUNCLE FIBER AND EXTRACT FOR BIOGAS PRO DUCTION 3.1 Introduc tory Remarks In addition to bioethanol, biogas synthesized via anaerobic digestion can be used to replace fossil fuels such as natural gas, and l iquefied petroleum gas (GLP) This chapter will focus on the use of banana peduncle fiber, concentrated extracted juice and stillage for biogas production as an alternative t o bioethanol from fermentation. 3.1.1 Anaerobic Digestion Anaerobic digestion is a well known biochemical process, where organic matter (biodegradable material) is converted into biogas (a gas mixture of methane and carbon dioxide) in the absence of oxygen and by the action of microbial popula tion, according to equation 3 1. (3 1) The biomass can be unwanted waste such as slurry or left over food, also nonfood feedstock including forestry and agricultural residues. The output from anaerobic digestion is a mixture of 60% methane (CH4), and 40% carbon dioxide (CO2) and traces of other contamination gases. The le ftovers after biogasification are still rich in nutrients and minerals, applicable as fertilizers or for secondary processing. During has pollution potential, because of its high chemical oxygen demand (COD) ( Wilkie et al. 2000 ). The COD is the standard method for indirect measurement of the amount of pollution. It is based on the chemical decomposition of organic and inorganic contaminants, dissolved or suspended water (Pasztor et al. 2009 ). The higher the

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46 C OD, the higher the amount of pollution in the sample. Anaerobic digestion is a commonly used approach to decrease the COD in waste from; for example, fermentation which can exceed a COD of 100g/L. Therefore, biogasifica tion offers an environmentally friend ly waste disposal producing bioenergy and feedstock for further applications Y ard wastes, biosolids and veget ables municipal waste, sugar beet residues and fruit waste are examples for feedstock that have already successfully been used for anaerobic dige stion (Chynoweth et al. 1992 ; K oppar and Pullammanappallil et al. 20 1 3 ). Advantage of those feedstocks is that they do not require sh r edding, mixing agitation or hi gh pres s ure vessel s as they can be operated at low (ambient) pressure Also, they can be operated stably at both mesophilic (28 40 C) and thermophilic (50 60 C) temperatures. Although anaerobic digestion is a natural occur ring process, using it industrially for biogas production in large scale, has some challenges: Not all forestry or agr icultural residues are usable as feedstock, as the y often contain inhibitors of methanogenic bacteria or their carbohydrate content is not sufficient for anaerobic digestion. Another challenge is to find the optima l microorganism for a specific feedstock. Availabl e are for example mesophilic or thermophilic microorganism as indicated earlier Also a wet system (5 15% dry matter) or dry system ( > 15% dry matter) can be used. A big problem of anaerobic digestion is the wide variety of inhibitory substance present in substantial concentration in waste. Those include ammonia, sulfide, light metals, heavy metals and organics as reviewed in detail by Chen et al. (Chen et al. 2009).

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47 3. 1. 2 Banana Peduncle as Anaerobic D igestion F eedstock Banana peduncle as feedstock of anaerobic digestion offers several advantages: During the process of extraction of juice from banana peduncle large amount s of fiber remain The production of 1 tonnes of banana peduncle leaves about 300 kg of pressed banana peduncle fib er, with water content of 65% 70 %. This banana peduncle fiber c ould be a source of biomass for energy generation either in the form of thermal energy or biogas (Figure 3 1) An alternative option is to produce methane via anaerobic di gestion a s is currently done us in g nonfood biomass resources like forestry and agricultural residues. The amount of available peduncle is substantial, due to large pr oduction quantities every 8 10 months. The fiber which is left after extraction of the juice is o ne possible feedstock. On the other hand, a fter fermentation of the peduncle extract, the liq u or created is distilled, result ing in high amounts of stillage. Up to 20L of stillage may be generated for each liter of ethanol produced (Chen et al. 2013 Wilki e et al. 2000 ). Stillage waste has hi gh organic content, making it an amenable and attractive feedstock for anaerobic digestion. Figure 3 1 Banana as agricultural nonfood feedstock (July 20, 2013. Courtesy of Marco Pazmino)

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48 The effectiveness of employing a single stage batch process to biogasify banana peduncle fiber, concentrated banana juice and stillage after distillati on was investigated. T he performance of the process in terms of methane yield and extent of degradation w ere evaluate d and compared between the three feedstock (Figure 3 2) Figure 3 2 Biogasification of banana peduncle fiber, concentrated juice and stillage overview 3.2 Experimental Material and Methods 3.2.1 Feedstock 3.2.1.1 Banana P eduncle Fiber After ext raction of the juice for fermentation the banana peduncle s were air dried for 4 days. Subsequently, a liquots of 0.40kg (dry weight) of banana peduncle fiber were taken in Ziploc airtight plastic bags and stored in glass vacuum desiccators. Content of on e bag (i.e., 0.40kg) was loaded into a digester for each experimental run. To prevent

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49 compaction of the solids, 2 kg of bulking material layers (lava rocks fro m landscaping supplier, 0.025 m (in average size) w ere also mixed with the banana peduncle fiber an d the inoculum The inoculum used was collected from a laboratory scale anaerobic digester that had been digesting algae for a year. The liquid volume held in the digester 1 was 2 L (0.002 m 3 ) after the addition of bulking material. 3.2.1.2 Banana Peduncle Concentrated E xtrac t Banana peduncle extract was concentrated at 85C to 5x c oncentration of the original extract. The liquid volume held in the digester 2 was 4 L (0.004 m 3 ) after the addition the banana extract concentrate. 3.2.1.3 Banana Peduncle S till age The stillage was collected from a previous fermentation process of the banana peduncle extract A fter the distillation process ethanol was separated from banana peduncle fermentation liquor at 60C T he liquid volume held in the digester 2 was 4 L (0.004 m 3 ) after the addition the banana stillage. 3.2.2 Anaerobic D igestion Two digester s (Digester 1 and 2) each 5 L (0. 005 m 3 ), with a working volume of 2 L, (0.002 m 3 ) and 4 L (0.004 m 3 ) respectively, were constructed by modifying Pyrex glass jars. The height and inner diameter of the digesters were 0.406m (16in.) and 0.0610m (2.4in.), respectively. The digesters were sealed with a top lid, outer diameter of 0.0965m (3.8 in.) using an O ring fitted for gas and liquid tightness and clamped with a stainless steel clamp. T wo ports were provided at the top of the lid, one for gas outlet, and others for sample withdrawal. At the bottom of the digester s was an outlet port for draining. No additional external/internal mixing device was employed The reactor was then sealed and pressure tested to ensure air tightness. The digester set up is show in

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50 (Fig ure 3 3 ) Gas production from the digesters was measured using a positive displacement gas meter. The s ystem was calibrated per count was considered as that amount of gas read on syringe (in milliliters) for which the gas meter completes one whole number count (e.g. one count = 0.029 L then two counts = 0.058 L and continued on ) (Koppar et al. 2007) The pH was m easured every three day at the sampling outlet port of the digesters. A 10 ml sample was taking for COD and pH. The pH was measured on each digester using pH meter ( Accumet pH meter, model 805MP ). In addition, a CO 2 trap of soda lime w as placed int o a canister as a base for methane line Soda lime is a mixture of chemicals used to remove carbon dioxide breathing gases f r om anaerobic digestion Equation 3 2 shows t he overall reaction CO 2 + Ca(OH) 2 3 + H 2 O + heat (in the presence of water) (3 2 ) Fig ure 3 3 Schematic of d igester s 1 and 2 set up

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51 3.2.3 Anaerobic D igestion Protocol The Ziploc bags of air dried banana peduncle fiber were removed from the glass vacuum desiccators and loaded into t he digester. The experiments were carried out in duplicate runs on Digester 1, and 2 All digestion experiments were performed at mesophilic temperature s by placing the digesters in an incubation chamber set at 38 o C. The first run in each digester was inoculated with 2 L (0.002 m 3 ) and 4 L (0.004 m 3 ) of inoculum taken form mesophilic digester that had been digesting algae for over a year respectively Moreover, once the gas producti on from the first experimental run slowed down, the digester 1 was opened and the next (0.40kg) banana peduncle fiber charged from the top In addition, the digester 2 was fed from the feeding outlet port at the top lid. The duplicate run s in each digester 1 and 2 were initiated by digester liquor re m ain ing from the first run N o further inoculum was added. Afterwards, t he digesters were pla ced in the 38 o C incubator chamber and digestion was allowed to p roceed. 3.2.4 Chemical Analys e s Total solids (TS) were determined gravimetrically after drying of the sample overnight at 105 o C Volatile solids (VS) content was determined by ashing a dried sample at 550 o C for 2 h and determining the ash free dry weight. Volatile Fatty acids were measured by a gas chromatograph er using an Shimadzu GC Model GC 2014 equipped with Shimadzu SHRXI 5MS capillary column ( Dim: 15m ID:0.25mm DE:0.25 ) with a AOC 20s autosampler. The instrument was set to an injection volume of 1 L and an inlet split ratio of 100:1 The temperature program used for al l the analyses was as follows : 80 o C, 1 min; 20 o C /min to 220 o C, 4min. Quantification was assessed externally using calibration curves of peak area vs concentration covering the relevant concentration regime. Methane production was determined along the outline gas line

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52 M ethane was carried through a sealed reactor wi th the CO 2 trap soda lime base for removing CO 2 and moist ure Also, m ethane volume was reported at standar d temperature and pressure (STP ) conditions. For soluble COD (sCOD) analysis the leachate sample s withdrawn from the digesters were centrifuged (Fishe r Marathon micro H centrifuge), pipetted (2ml) in COD vials (range: 2 150 0 ppm, HACH) and placed in COD reactor (HACH) for 2h. The COD of the leachate was measured by using colorimeter (HACH DR/890 colorimeter). 3.2.5 Statistical A nalyses The statistical e valuation of experiment a test using Graph Pad Prism software version 5 (Gra phPad software, La Jolla, CA, USA). Experimental data are expressed as means + standard error (SEM). Statistical significances are referred to as *p < 0.05, **p < 0.01, and ***p < 0.001. 3.3 Result s 3 3.1 Ch aracteristics of Banana Peduncle Fiber and Digested R esidue The dry matter content of the banana peduncle fiber was 89.3% of which 76.4% was volatile matter For run 1, 0.040 kg (dry weight) containing 0.036 kg dry matter was loaded into the digester 1 For r un 2, 0.040 kg dry weight of banana peduncle was aga in added without removing solid residue from the previou s run. At the end the pH average of the system was 7.8 T a ble 3 1 shows the loa ding and unloading data for banana peduncle fiber. At the end of Run 2 the dry matter remaining in the digester was measured to be 21 g (0.021 kg) and 68% of dry matter was volatile. The dry matter and volatile solids reduction achieve biogasification was 91% and 95%, respectively (measured in the Bioprocess Engineering Research Laboratory, Agricultural and Biological Engineering Department, University of Florida).

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53 Table 3 1 L oading and unloading parameters Loading and unloading data for banana peduncle f iber Loading Dry weight Aliquots (kg) 0.040 Dry matter (kg) 0.036 Volatile matter (kg) 0.027 Inoculum added (L) 2.0 38 Unloading Dry weight (kg) 0.021 Volatile matter (kg) 0.00 21 Dry matter reduction (%) 91 Volatile matter (%) 95 3 3.2 Biogasification of Banana Peduncle F iber Profiles of cumulative methane yield s and methane prod uction rates from batch digester runs are shown in Figu re 3 4 for Digester 1 from both runs. Run 1 and 2 were slightly different in production. Run 1 was initiated by inoculum taken from a mesophilic digester that had been digesting algae for over a year. The first three days after start ing the digestions, the methane production rate peaked at 1.39 m 3 m 3 d 1 This indicates a heal thy onset of meth anogenesis in digester 1. After 9 days the methane production rate dropped to 0.9 m 3 m 3 d 1 Run 1 reached compl etion after 18 days, depicted in Figure 3 5, when the daily methane pro duction rate dropped to 0.0290 m 3 m 3 d 1 .The cumulativ e methane yield on run 1 was 0.2 9 0 L CH 4 at STP g VS 1 Figure 3 4 Digester set up. Digester 1 with banana peduncle fiber and lava rocks after digestion ,(January 15, 2014. Courtesy of Marco Pazmino)

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54 Figure 3 5 Methane Production and Cumulative Methane yield

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55 The next digestion run (Run 2) was initiate by adding a second batch of banana peduncle fiber (0.04 kg) into the digester after end of Run 1. Mixed liquor from Run 1 served as the inoculum. No alkalinity was added. Three days after starting run 2 the meth ane production rate was 1.27 m 3 m 3 d 1 After 9 days the methane production rate dropped to 0.7 m 3 m 3 d 1 The cumulative methane yield of run 2 was 0.235 L CH 4 at STP g VS 1 .The peak of methane gas productivity was 1.856 m 3 m 3 d 1 on j ust the first day after starting r un 2 in the digester 1 (Figure 3 4). In Run 1, the pH increased gradually from 7.7 to 8.4 until the end of the run In Run 2, the pH dropped from 8.4 to 7.96. The pH was not corrected but allowed to change (Figure 3 6). During the digestion of banana peduncle fiber, samples were withdrawn every three days ( 0, 2, 5, 8, 11, 14, and 17 ) for analysis of sCOD. Figure 3 6 shows the profile of sCOD change in digester 1. The sCOD at the end of Run 2 was <9.2 g L 1 In addition, measurement of volatile fatty acids (VFAs) was planned th roughout run 1. The first sample was taken before starting the run and showed a high level of acetic and propionic acid 63.5 mM and 5.6 mM respectively Samples were taken every three days. At day 9 the level of a cetic and propionic acid dropped to 1.2 mM and 0.5 mM (Data not shown).

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56 Figure 3 6 Profile of pH, and sCOD performance during banana peduncle digestion 3 3.3 Characteristics of Banana P eduncle Juice C oncentrate (5x) First, the feedstock was measured f or sCOD and pH values. The characteristics of the banana penducle extract (5x) as follows: pH 5.4 5.6, and sCOD 110.5 g/L. For run 1, 0.040 kg (concentrated juice) containing 0.006 kg dry matter was loaded into the digester 2. For run 2, 0.040 kg is in pro cess (measured in the Bioprocess Engineering Research Laboratory, Agricultural and Biological Engineering Department, University of Florida). 3 3. 4 Biogasification of Banana Peduncle Juice C oncentrate ( 5x ) Profiles of cumulative methane yields and methane production rates from batch digester runs of banana peduncle juice concentrated (5x) are shown in Figure 3 7, for Digester 1 from both runs. Run 1 was initiated by inoculum taken from a mesophilic digester that had been digesting algae for over a year. The first three days after starting the digestions, the methane production rate peaked at 0.34 m 3 m 3 d 1 This indicates a healthy onset of methanogenesis in digester 1 After 5 days the methane production ra te dropped to

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57 0.27 m 3 m 3 d 1 Run 1 reached compl etion after 6 days, depicted in Figure 3 7 when the daily methane pro duction rate dropped to 0.0290 m 3 m 3 d 1 .The cumulative methane yield on run 1 was 0. 043 L CH 4 at STP g VS 1 Figure 3 7 Methane Production and Cumulative Methane yield of banana peduncle concentrated juice (5x)

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58 3 3.5 Char acteristics of Banana Peduncle S tillage Banana peduncle stillage was obtained by evaporating the fermented concent rated extract (5X) liquor for 5 hrs at 60 o C. T he feedstock was measured for sCOD and pH values pH 6, and sCOD 35.7 g/L 8.1% and 31% is the Total and Volatile solids of the banana peduncle stillage, respectively. For digester 2 with a working volume of 4 L (0.004 m 3 ). R un 1 started with 0.040 k g ( banana peduncle stillage) containing 0.003 kg dry matter was loaded into the digester 2. For run 2, again 0.040 kg banana peduncle stillage was again added without removing solid residue from the previous run. At the end, the pH average of the system wa s 7.56 (measured in the Bioprocess Engineering Research Laboratory, Agricultural and Biological Engineering Department, University of Florida). 3 3.6 Biogasification of Banana P eduncle S tillage Profiles of cumulative methane yield s and methane prod uction rates from batch digester runs are shown in Figure 3 8 for Digester 2 from both runs. Run 1 and 2 were different in production. Run 1 was initiated by inoculum taken from a mesophilic digester that had been digesting algae for over a year. The first three days after starting the digestions, the methane production rate peaked at 0.125 m 3 m 3 d 1 After three days the methane production rate dropped to the minimum production 0.034 m 3 m 3 d 1 Run 1 reached compl etion after 3 days, depicted in Figure 3 8 .The cumulative methane yield on run 1 was 0.026 L CH 4 at STP g VS 1 The next digestion run (Run 2) was initiate by adding a second batch of banana peduncle stillage (0.04 kg) into the digester after end of Run 1. Mixed liquor from Run 1 served as the inoculum. No alkalinity was added. Three days after starting run 2, the methane production rate dropped to the minimum production 0.034 m 3 m 3 d 1 as well

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59 as Run 1. The average methane rate for the Run 2 was 0.159 m 3 m 3 d 1 The cumulative methane yield of run 2 was 0. 034 L CH 4 at STP g VS 1 .The peak of methane gas productivity was 0.272 m 3 m 3 d 1 on just the first day after starting r un 2 in the digester 2 In Run 1, the pH did not change during the process 7 6 until the end of the run. In Run 2, the pH dropped from 7.6 to 7. 4 The pH was not corrected but allowed to change Figure 3 8 Methane Production and Cumulative Methane yield of banana peduncle stillage

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60 3.4 Discussion 3.4 .1 Biogasification E fficiency Th e banana peduncle fiber turned out to be the best feedstock with average of 0.263L CH 4 at STP g VS 1 the stillage was the lowest with 0.034L CH 4 at STP g VS 1 However, probably the fermentation concentration of stillage was very low. Moreover, sCOD was 3500 mg L 1 that makes a suitable effluent for anaerobic digestion. Finally, banana peduncle juice on run 1 0.04L CH 4 at STP g VS 1 In all the reactors, the pH was stable and any VFAs build up. Compare the yield of Banana peduncle fiber to the yield of di gestion of waste Banana (Clarke et al., 2007) and the yields for manure fiber (Biswas et al., 2012) The extract before fermentation resulted in an intermediate biogas yield; compare it to other liquid biogas feedstock ; it will probably be lower which suggests that it should better be used for fermentation to bioethanol than for anaerobic digestion to produce biogas 3.4.2 Inhibitory F actors The concentration of Volatile Fatty acids (VFAs) was analyzed in the banana peduncle fiber (Run 1). It shows that acetic and propionic acid were high after start up the digestion. They dropped drastically into levels that I cannot inhibit the process. However, in butyric and acetic acid where the predominate products from the fermentation of glucose between pH 5 and & while acetic and propionic acids were favored at neutral to high pH conditions (Clarke et al., 2008). Propionate also decreased with the onset of metha nogenesis, it remained abo ve 1300mg L 1 throughout the digestion (Pullammanappallil et al., 2001) demons trated anaerobic digestion of glucose was unimpeded with propionate concentration up to 2750mg L 1 (Clarke et al., 2008).

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61 In this case, all the concentration in this research were low in organic matter. Consequently, any reactor experience any type of inhi bition. Lots of options to improve biogasification of fiber can be found in the literature. Options will be discussed concerning application for peduncle fiber (Biswas et al., 2012; Frauenhofer 2011, Asam, 2011) (Figure 3 9) Figure 3 9 Crucial points for improvement the efficiency in biogas production

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62 CHAPTER 4 CONCLUSIONS The results of t he current study imply that the extract derived from banana peduncle can be f ermented to produce bioethanol using the commercial yeast Saccharomyces cerevisiae The amount and variety of sugars in the extract are sufficient for this purpose, while the occurrence of inhibitors seems to be low. However, the extract should not be concentrated more than 5 x. L arge amounts of peduncle are ava ilable at no cost as it is a waste produced of the banana production. Low transportation costs make s the banana peduncle juice a cheap alternative to e.g. Sugar Cane, Energy Cane or Sweet Sorghum The fiber, remaining after extraction can be used for anae robic digestion resulting in biogas (Methane and CO 2 ) synthesis. Finally, the stillage derived as a leftover after fermentation of the banana peduncle extract is an appropriated feedstock for anaerobic digestion.The use of banana peduncle juice for biogas and bioethanol production serves several purposes and solves various problems at the same time including: Increasing the amount of biofuels available in banana producing countries, which are at the same time often underdeveloped with high need of in countr y produced, cheap renewable energy sources Creation of jobs as a benefit for the economy A huge advantage of peduncle based bi ofuel synthesis over sugar cane dependent processes, is the non seasonal growth of banana plants. This guarantees continues produc tion flow in contrast to the usage of sugarcane as an energy crop,

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63 which pauses when the plant is out of season. Costs for ending and restarting processes can be saved. Employments are easier.

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64 APPENDIX DISPLACEMENT GAS METER Gas production from the diges ters was measured using a positive displacement gas meter. The device consisted of a clear PVC U tube filled with anti freeze solution solid state time delay (Dayton OFF Delay Model 6X153E), a float switch (Grainger), a counter (Redington Inc.) and a solen oid valve (Fabco Air). The U tube gas meter was calibrated in line to determine volume of biogas per count. A count was considered as that amount of gas read on syringe (in milliliters) for which the gas meter completes one whole number count (e.g. one cou nt = 0.029 L, then two counts = 0.058 L and continued on) (Koppar et al. 2007)

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6 5 LIST OF REFERENCES 1. Asam, Z. Z. Z., T. G. Poulsen, A. S. Nizami, R. Rafique, G. Kiely, and J. D. Murphy. 2011. How can we improve biomethane production per unit of feedstock in biogas plants? Applied Energy 88: 2013 2018. 2. Berthels, N. J., R. R. Cordero Otero, F. F. Bauer, J. M. Thevelein, and I. S. Pretorius. 2004. Discrepancy in glucose and fructose utilisation during fermentation by Sacc haromyces cerevisiae wine yeast strains. FEMS Yeast Res. 4: 683 689. 3. Bisson, L. F., and D. G. Fraenkel. 1983. Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A 80: 1730 1734. 4. Champion, J. 1963. Le Bananier. In Maisonneuve et Larose eds. Maisonneuve et Larose, Paris, France. 263. 5. Chen, Y., J. J. Cheng, and K. S. Creamer. 2008. Inhibition of anaerobic digestion process: a review. Bioresour. Technol. 99: 4044 4064. 6. Clarke, W. P., P Radnidge, T. E. Lai, P. D. Jensen, and M. T. Hardin. 2008. Digestion of waste bananas to generate energy in Australia. Waste Manag. 28: 527 533. 7. Cordeiro, N., M. N. Belgacem, I. C. Torres, and J. C. V. P. Moura. 2004. Chemical composition and pulping of banana pseudo stems. Industrial Crops and Products 19: 147 154. 8. Ennos, A. R., F. A. U. Spatz HC, and T. Speck. The functional morphology of the petioles of the banana, Musa textilis. 9. Geissen, V., F. Q. Ramos, J. B. B. de, G. Diaz Gonzalez, R. B ello Mendoza, E. Huerta Lwanga, and L. E. Ruiz Suarez. 2010. Soil and water pollution in a banana production region in tropical Mexico. Bull. Environ. Contam Toxicol. 85: 407 413. 10. Golub, K. W., S. R. Golub, D. M. Meysing, and M. T. Holtzapple. 2012. P ropagated fixed bed mixed acid fermentation: effect of volatile solid loading rate and agitation at near neutral pH. Bioresour. Technol. 124: 146 156. 11. Graefe, S., D. Dufour, J. A. s. Giraldo Jimnez, L. A. Muoz, P. Mora, H. Sols, H. n. Garcs, a nd A. Gonz¡lez. 2011. Energy and carbon footprints of ethanol production using banana and cooking banana discard: A case study from Costa Rica and Ecuador. Biomass and Bioenergy 35: 2640 2649. 12. Hammond, J. B., R. Egg, D. Diggins, and C. G. Coble. 1996 Alcohol from bananas. Bioresource Technology 56: 125 130.

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66 13. Kim, M., and D. F. Day. 2011. Composition of sugar cane, energy cane, and sweet sorghum suitable for ethanol production at Louisiana sugar mills. J. Ind. Microbiol. Biotechnol. 38: 803 807. 14. Llorente, P., and A. Sols. 1969. Ethanol inactivation of glycolytic enzymes in yeasts with different alcohol resistances. Spain. 123. 15. Mohapatra, D., S. Mishra, and N. Sutar. 2010. Banana and its by product utilisation. Journal of Scientific and I ndustrial Research 69: 323 329. 16. Munoz Carpena, R., A. Ritter, A. R. Socorro, and N. Perez. 2002. Nitrogen evolution and fate in a Canary Islands (Spain) sprinkler fertigated banana plot. Agricultural Water Management 52: 93 117. 17. Nigam, P. S., and A. Singh. 2011. Production of liquid biofuels from renewable resources. Progress in Energy and Combustion Science 37: 52 68. 18. Oliveira, L., N. Cordeiro, D. V. Evtuguin, I. C. Torres, and A. J. D. Silvestre. 2007. Chemical composition of different morp hological parts from 'Dwarf Cavendish' banana plant and their potential as a non wood renewable source of natural products. Industrial Crops and Products 26: 163 172. 19. Omar, R., R. Harun, T. Mohd Ghazi, W. Wan Azlina, A. Idris, and R. Yunus. 2008. Anae robic treatment of cattle manure for biogas production. Philadelphia, USA. 1 10. 20. Pasztor, I., P. Thury, and J. Pulai. 2009. Chemical oxygen demand fractions of municipal wastewater for modeling of wastewater treatment. Int. J. Environ. Sci. Technol. 6 : 51 56. 21. Shakeri, Y. S., A. Lindmark, U. Skyllberg, A. Danielsson, and B. H. Svensson. 2014. Importance of reduced sulfur for the equilibrium chemistry and kinetics of Fe(II), Co(II) and Ni(II) supplemented to semi continuous stirred tank biogas react ors fed with stillage. J. Hazard. Mater. 22. Tock, J. Y., C. L. Lai, K. T. Lee, K. T. Tan, and S. Bhatia. 2010. Banana biomass as potential renewable energy resource: A Malaysian case study. Renewable and Sustainable Energy Reviews 14: 798 805. 23. Velas quez Arredondo, H. I., A. A. Ruiz Colorado, and S. De Oliveira junior. 2010. Ethanol production process from banana fruit and its lignocellulosic residues: Energy analysis. Energy 35: 3081 3087. 24. Wang, L., X. Q. Zhao, C. Xue, and F. W. Bai. 2013. Impac t of osmotic stress and ethanol inhibition in yeast cells on process oscillation associated with continuous very high gravity ethanol fermentation. Biotechnol. Biofuels. 6: 133.

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67 25. Wheals, A. E., L. C. Basso, D. M. Alves, and H. V. Amorim. 1999. Fuel eth anol after 25 years. Trends Biotechnol. 17: 482 487. 26. Wilkie, A. C., K. J. Riedesel, and J. M. Owens. 2000. Stillage characterization and anaerobic treatment of ethanol stillage from conventional and cellulosic feedstocks. Biom ass and Bioenergy 19: 63 102. 27. Asociacion Ecuatoriana de Bananeros del Ecuador ( AEBE 2011) Annual report La Industria Bananera Ecuatoriana 28. Asociacion de la Industria Hidrocarburifera del Ecuador ( AIHE 2011) Annual report 29. El Universo 07/24/ 20 12 Ataque de la sigato ka preocupa a bananeros 30. International Plant Genetic Resources Institute ( IPGRI, 2010 ) Descriptors for Banana. 31. Food and Agriculture Organization of United Nations (FAOSTA 2011 ) Annual report 32. Food and Agriculture Organization of United Nation s (FAOSTA 2012 ) Annual report

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68 BIOGRAPHICAL SKETCH Marco A. Pazmino Hernandez was born in Guayaquil, Ecuador. In 2009, he received his B achelor of Economics in Business Administration at ESPOL University in Guayaquil, Ecuador. Thereafter he went to Unive rsity of Florida where he studied English. In the following year, he start ed his M aster of Science degree program at Agricultural and Biological Engineer ing D epartment, focusing on biofuels. In spring 2014, he completed his Master of Science degree at UF and plans to continue on in the doctorate program