Researcho microoranisms in wet anaerobic fermentation pilot to treat domestic waste masters thesis major sciences and management of the environment

SCIENCES AND MANAGEMENT OF THE ENVIRONMENT BETWEEN INDUSTRIAL UNIVERSITY OF HO CHI MINH CITY AND LIÈGE UNIVERSITY NGUYEN DUY RESEARCH MICROORGANISMS IN WET ANAEROBIC FERMENTATION PIL

SCIENCES AND MANAGEMENT OF THE ENVIRONMENT BETWEEN INDUSTRIAL UNIVERSITY OF HO CHI MINH CITY AND LIÈGE

UNIVERSITY

NGUYEN DUY

RESEARCH MICROORGANISMS

IN WET ANAEROBIC FERMENTATION PILOT

TO TREAT DOMESTIC WASTE

Major: EXECUTIVE MASTER IN SCIENCES AND MANAGEMENT OF THE ENVIRONMENT

MASTER’S THESIS

The project was completed at The Industrial University of Ho Chi Minh City

(Write full name and signature)

COMMITTEE CHAIR DEAN OF INSTITUTE OF ENVIRONMENTAL

SCIENCE, ENGINEERING AND MANAGEMENT

ACKNOWLEDGMENT

First of all, I would like to thank Dr Trinh Ngoc Nam and Prof Eppe, who were give me many useful instructions, suggestions and advices to complete this thesis And they were also give me many documentation related to my research, which are really exciting and provide me many knowledge in my thesis

Then, I really appreciate the support of Prof Le Hung Anh and Mr Thang (a manager of Nam Binh Duong Domestic Waste Treatment Plant), who gave me many advices and helped me contact with Nam Binh Duong Domestic Waste Treatment Plant to installed the wet anaerobic fermentation pilot and implemented the research

Third, I would like to thank Industrial University of Ho Chi Minh and University of Liege gave me opportunity to complete this thesis and the master course

Finally, sincerely thank my team members (Mr Rodolfo, Mr Trung, Mr Danh, Ms Van, Ms Chau), who were cooperate with me to had this successful research

Author

Nguyen Duy

ABSTRACT

Domestic waste is a current worrying situation in Vietnam Together with the economic development, population growth and the waste of resources in the living habits of people, the quantity of waste is increasing, composition increasingly complex and potentially more risk noxious to the environment and human health

In fact, most domestic waste only land filled in the normal landfill with many disadvantages (waste the land, the stench affect to residential areas, could become the source of disease) The consequences have serious impacts to the environment and does not save this renewable materials

One of the methods to recover energy and reuse organic waste with highly effective and applied in many developed countries is wet anaerobic fermentation system

Wet anaerobic fermentation system has the opportunity to be an integral part of the solution to two of the most pressing environmental concerns of urban centers: waste management and renewable energy Through wet anaerobic fermentation system, organics are decomposed by specialized bacteria in an oxygen-depleted environment to produce biogas and a stable solid The biogas, which consists of up to 65% methane, can

be combusted in a cogeneration unit and produce green energy

The most important factor in wet anaerobic fermentation system is anaerobic microorganisms, so this thesis will focus on:

- Isolated microorganisms in anaerobic sludge after fermentation domestic waste

- Identify some species of anaerobic microorganisms that predominate during fermentation

TABLE OF CONTENTS

LIST OF FIGURES 6

LIST OF TABLES 7

ABBREVIATIONS 8

INTRODUCTION 9

1 The reason for choosing the topic 9

2 Objectives of the study 10

3 Subjects and scope of the study 11

4 The methodology 11

CHAPTER 1 LITERATURE REVIEWS 12

1.1 Solid waste situation 12

1.1.1 In developed countries 12

1.1.2 In Vietnam 14

1.2 Anaerobic digestion process 15

1.2.1 Stages of anaerobic digestion 16

1.2.1.1 Hydrolysis 16

1.2.1.2 Acidogenesis 17

1.2.1.3 Acetogenesis 17

1.2.1.4 Methanogenesis 18

1.2.2 Microorganisms in anaerobic digestion 19

1.2.2.1 Hydrolytic bacteria 20

1.2.2.2 Acetogenic bacteria 23

1.2.2.3 Methanogenic microorganisms 24

1.2.3 Factors that affect the anaerobic digestion process 25

1.2.3.1 pH 25

1.2.3.2 Temperature 26

1.2.3.3 Carbon/Nitrogen ratio 26

1.2.3.4 Retention time 27

1.2.3.5 Loading rate 27

1.4 Conclusion 30

CHAPTER 2 MATERIAL AND METHODS 31

2.1 Materials, pilot modeling and equipment 31

2.1.1 Materials 31

2.1.2 Pilot modeling 31

2.1.3 Equipment used to analysis samples in laboratory 32

2.2 Methods 33

2.2.1 Coning and quartering method to calculate solid waste composition 33

2.2.2 COD determined 35

2.2.3 Experiments implementation 35

2.2.3.1 DNA extraction 35

2.2.3.2 Gel electrophoresis .37

2.2.3.3 Microorganism identify by phylogenetic tree 39

2.2.3.4 Experiments conducting 40

CHAPTER 3 RESULTS AND DISCUSSION 41

3.1 Solid waste composition 41

3.2 COD 41

3.2.1 COD in experiment 1 41

3.2.2 COD in experiment 2 42

3.3 Temperature 43

3.3.1 Temperature in experiment 1 43

3.3.2 Temperature in experiment 2 44

3.4 pH 44

3.4.1 pH in experiment 1 44

3.4.2 pH in experiment 2 45

3.5 Anaerobic microorganisms 46

3.5.1 Anaerobic bacteria density 46

3.5.2 Anaerobic yeast density 46

3.5.3 Anaerobic bacteria density and anaerobic yeast density conclusion 47

3.6 Anaerobic bacteria isolated 47

3.6.1 K1 bacteria strain 47

3.6.2 K2 bacteria strain 48

3.6.3 K3 bacteria strain 48

3.6.4 K4 bacteria strain 49

3.6.5 K5 bacteria strain 49

3.6.6 K6 bacteria strain 49

3.7 Microorganism identification 50

3.7.1 16S - rDNA characteristics of K1 bacteria strain 50

3.7.2 16S - rDNA characteristics of K3 bacteria strain 52

3.7.3 16S - rDNA characteristics of K4 bacteria strain 53

3.7.4 Microorganism identification result 55

CHAPTER 4 CONCLUSIONS AND RECOMMENDATIONS 56

4.1 Conclusions 56

4.2 Recommendations 56

REFERENCES 58

LIST OF FIGURES

Figure 1.1 Total AD installed capacity per country 13

Figure 1.2 Total AD installed capacity per million inhabitants 14

Figure 1.3 Composition of solid waste in Vietnam in 2008 and expected 14

Figure 1.4 Metabolic pathways and microbial groups involved in anaerobic digestion 16

Figure 1.5 Overall process of anaerobic decomposition 21

Figure 1.6 Effect of the loading rate above the sustainable 28

Figure 2.1 Large pilot model in Nam Binh Duong Waste Disposal Plant 31

Figure 2.2 Small pilot model in laboratory of Industrial University Of Ho Chi Minh City 32

Figure 2.3 Thermo meter 33

Figure 2.4 pH meter 33

Figure 2.5 Coning and quartering method 33

Figure 2.6 Municipal waste was divided into 4 part 34

Figure 2.7 Waste was classified 34

Figure 2.8 Gel electrophoresis 37

Figure 2.9 Restriction enzyme 38

Figure 2.10 Automation 39

Figure 2.11 Sequencing modify by FinchTV and SEAView software 39

Figure 2.12 Phylogenetic tree by MEGA software 40

Figure 3.1 Percentage of components in domestic waste 41

Figure 3.2 COD concentration in experiment 1 42

Figure 3.3 COD concentration in experiment 2 43

Figure 3.4 Temperature in experiment 1 43

Figure 3.5 Temperature in experiment 2 44

Figure 3.6 pH in experiment 1 45

Figure 3.7 pH in experiment 2 45

Figure 3.8 Anaerobic bacteria density 46

Figure 3.9 Anaerobic yeast density 46

Figure 3.10 Colonies (left) and Gram’s method (right) of K1 bacteria strain 47

Figure 3.11 Colonies (left) and Gram’s method (right) of K2 bacteria strain 48

Figure 3.12 Colonies (left) and Gram’s method (right) of K3 bacteria strain 48

Figure 3.13 Colonies (left) and Gram’s method (right) of K4 bacteria strain 49

Figure 3.14 Colonies (left) and Gram’s method (right) of K5 bacteria strain 49

Figure 3.15 Colonies (left) and Gram’s method (right) of K6 bacteria strain 50

Figure 3.16 Similarity ratio showed by color of K1 strain 51

Figure 3.17 Phylogenetic tree of K1 strain 51

Figure 3.18 Similarity ratio showed by color of K3 strain 52

Figure 3.19 Phylogenetic tree of K3 strain 53

Figure 3.20 Similarity ratio showed by color of K4 strain 54

Figure 3.21 Phylogenetic tree of K4 strain 54

LIST OF TABLES

Table 1.1 Acetogenic dehydrogenation reactions 19

Table 1.2 Methane producing reactions 19

Table 1.3 Groups of bacteria according to their response to free molecular oxygen 20

Table 2.1 List of equipment used in laboratory 32

ABBREVIATIONS

INTRODUCTION

1 The reason for choosing the topic

Waste is a current worrying situation in Vietnam Together with the economic development, population growth and the waste of resources in the living habits of people, the quantity of waste is increasing, composition increasingly complex and potentially more risk noxious to the environment and human health

While many countries around the world such as Japan, America, Britain invested millions of dollars in waste recycling They were successful in both work: environmental protection and economic recovery through waste recycling For them, waste is a resource Meanwhile, Vietnam is among the top countries that are wasting this energy source

In fact, most domestic waste only land filled in the normal landfill with many disadvantages (waste the land, the stench affect to residential areas, could become the source of disease) The consequences have serious impacts on the environment and does not save this renewable materials

Composting is one of the effective method to treat domestic waste Currently, there are some domestic waste treatment plant apply composting method such as: Nam Binh Duong (Binh Duong Province), Cam Xuyen (Ha Tinh Province), Trang Cat (Hai Phong Province), Nam Thanh (Ninh Thuan Province), … But the technology is not effective because: the domestic waste treatment process is not suitable for Vietnam, the rate of solid waste is land filled or burned after treatment is very large from 35-80%, high operating and maintenance costs In addition, the composting fertilizer is difficult to consume, only suitable for some kind of tree

Some domestic waste treatment facilities use burning technology, but efficiency is not high, air emissions are not strictly controlled, release dioxin and furan leading to air pollution

One of the methods to recover energy and reuse organic waste with highly effective and applied in many developed countries is wet anaerobic fermentation (Anaerobic

renewable energy Through AD, organics are decomposed by specialized bacteria in an oxygen-depleted environment to produce biogas and a stable solid The biogas, which consists of up to 65% methane, can be combusted in a cogeneration unit and produce green energy The solid digestate can be used as an organic soil amendment As a waste management strategy employed in over 20 countries, AD has been successful in reducing the volume of waste going to landfill, decreasing emissions of greenhouse gases and creating organic fertilizer, all at a profit [11]

With a total of 244 plants and a capacity of almost 8 million ton of organics treatment capacity, anaerobic digestion is already taking care of about 25 % of the biological treatment in Europe In The Netherlands and Belgium, it is expected that 80 % of the composting plants will have anaerobic digestion as the primary treatment technology by the year 2015 Long term successful experience has made anaerobic digestion the preferred treatment technology for the municipal solid waste organics, making use of a variety of technological approaches and systems It can only be expected that anaerobic digestion will continue to increase on a steady basis, not only because of the production

of renewable energy but also because of the reduction in odor potential and surface area required [15]

With the large amounts of organic domestic waste generation and demand recover energy from waste, Vietnam completely could apply anaerobic wet fermentation model for waste management in the future [15]

2 Objectives of the study

Aims to apply the model of wet anaerobic fermentation to treat domestic waste in Vietnam, this study will be based on the actual pilot located in Nam Binh Duong Waste Treatment Plant (Binh Duong Province, Vietnam) to analysis sludge, water samples and components of microorganisms to achieve the desired results follow:

- By empirical, isolated microorganisms in anaerobic sludge after fermentation domestic waste

- Identify some species of anaerobic microorganisms that predominate during fermentation

3 Subjects and scope of the study

- Step 1: Use large pilot located in Nam Binh Duong waste disposal plant (Binh Duong province, Vietnam) to implement anaerobic fermentation domestic waste to produce anaerobic microorganisms In this period, we will monitor temperature, pH and COD

to control progress in the pilot

- Step 2: Use small pilot located in Industrial university of Ho Chi Minh city to culture the anaerobic microorganisms, which we collected from the large pilot at Nam Binh Duong The purpose of this step is create an completely anaerobic pilot to provide the best condition for anaerobic microorganisms Then we will collect biogas to analysis percentage of methane (CH4) and identify some kind of dominate anaerobic bacteria

4 The methodology

This research will apply some methods as below:

- Theoretical research methods: research scientific articles, research projects and teaching materials related to treatment domestic waste, wet anaerobic fermentation, microorganisms in wet anaerobic fermentation,

- Empirical research method on pilot: use actual pilot to treat domestic waste by wet anaerobic fermentation method located in Nam Binh Duong waste disposal plant (Binh Duong province, Vietnam) and a smaller pilot located in Industrial university of Ho Chi Minh city

- Analysis method: from actual pilot and anaerobic microorganisms collected, we will culture, analyze and identify some dominate bacterias

- Analyzing data method

- Comparision method

- Synthesizing data method

CHAPTER 1 LITERATURE REVIEWS

1.1 Solid waste situation

The management of municipal solid waste (MSW) has been subject to major developments during the past 20 years At the end of the ‘80s, land filling and mass burn incineration were still the major methods by which MSW was disposed of Composting made up a small percentage of the disposal and was on the decline because of major quality challenges due to heavy metals and inert materials in the final end-product Recycling was limited to paper and glass and easily recoverable materials [15]

Major progress was made in all areas of waste management but the introduction of anaerobic digestion into the treatment of MSW is one of the most successful and innovative technology developments observed during the last two decades in the waste management field Anaerobic digestion has become fully accepted as a proven and an even preferred method for the intensive biodegradation phase of organic fractions derived from MSW [15]

Even though continued progress has been made with other alternative treatment technologies (gasification pyrolysis, plasma, biological drying, etc.), these technologies have by far not seen the same wide spread implementation that anaerobic digestion has been able to achieve In Europe alone, 244 installations dealing with the organic fraction

of MSW as a significant portion of the feedstock have been constructed or are permitted and contracted to be constructed The cumulative capacity of all of these anaerobic digestion plants amounts to 7,750,000 ton per year of organics going into the digestion phase If one assumes 300 kg of biodegradable waste generated per person and per year, this capacity represents about 5% of the biodegradable waste generated across Europe (excluding former USSR-states) by 550 million inhabitants In addition, this capacity represents 25% of all biological treatment, which is estimated at around 20% of all municipal solid waste disposal in Europe However, it should be noted that probably 10

to 15% of the plants are no longer in operation This could be partially compensated by the too low (inventoried) capacity to be constructed in 2014, as there are undoubtedly projects that are not included in the assessment yet [15]

Countries having the largest capacity installed are Germany with about 2 million tons of annual capacity, and Spain with 1.6 million tons However, if one adjusts for the number

of inhabitants, then countries like Netherlands and Switzerland become the highest in installed annual capacity of respectively 52,400 tons per million people and 49,000 tons per million people Netherlands have implemented a strategic initiative in order to promote anaerobic digestion of MSW-derived organics during the last three years The country has a very well developed infrastructure for natural gas but as the gas wells are running dry in the North Sea, the government is intent on producing a large amount of bio-methane which can be distributed across the country Netherlands have the ambition

to replace 15 to 20% of the natural gas by green gas by 2030 [15]

One big difference between the two countries is that the sizes of the plants are very different The average size of an anaerobic digester is 31,700 tons per year in Europe but there exists a big variation Netherlands has large plants (average capacity = 54,000 tons), while Switzerland installed many small plants (average capacity = 14,000 tons) This reflects the dense population in Netherlands and the drive to lower costs, while in Switzerland the split is due to geographical complications in transporting waste from one area to another [15]

Figure 1.1 Total AD installed capacity per country [15]

1.1.2 In Vietnam

In 2008, the volume of domestic solid waste produced in urban and rural areas through out Vietnam was 35,100ton/day and 24,900 ton/day respectively In most urban areas, domestic solid waste accounts for 60% - 70% of the total amount of municipal solid waste (even 90% in some areas) Domestic waste generation in urban areas increases by 10% to 16% annually on average [22]

The amount of domestic solid waste generated annually in major municipalities like Ho Chi Minh City has risen sharply but a slight increase is reported in smaller urban areas including Thai Nguyen Province, Thai Binh Province and Nam Dinh Province, where urbanization process remains slow.[22]

Figure 1.2 Total AD installed capacity per million inhabitants [15]

Figure 1.3 Composition of solid waste in Vietnam in 2008 and expected [22]

Solid waste treatment in Vietnam still faces numerous challenges including in appropriate or substandard technologies and lack of scientific and practical background when selecting the site for landfills and transfer stations Therefore, it has not received the people’s support Solid waste treatment facilities remain scattered, making management difficult and resulting in a waste of land Landfilling is the main form of

landfills/treatment areas each in Hanoi and Ho Chi Minh City) Up to 85% of municipalities (from the township level up) apply unsanitary landfilling techniques There are 98 centralized landfills in operation in Vietnam but only 16 are sanitary landfills (mostly in major cities) In the remaining landfills, most of the solid waste is buried without care [22]

1.2 Anaerobic digestion process

The process of anaerobic digestion employs specialized bacteria to break down organic waste, convert it into a stable solid and biogas, a mixture of carbon dioxide and methane.[10]

In general terms, under completely anaerobic conditions, organic compounds are converted according to the overall reaction:

For example, carbohydrates (n = 1, a = 2, b = 1) give a 50 – 50 mixture of carbon dioxide and methane From this simplified reaction (which ignores important details such as microbial mass generation, as they will only slightly influence the stoichiometry), it becomes apparent that the constitution of the generated biogas will depend on the redox state of the organic carbon Therefore, hydrocarbons generate equal amounts of methane and carbon dioxide, methanol and lipids generate biogas rich in methane, oxalic acid will produce biogas low in methane and urea will produce no methane [13]

1.2.1 Stages of anaerobic digestion

Figure 1.4 Metabolic pathways and microbial groups involved in

anaerobic digestion [3]

1.2.1.2 Acidogenesis

During acidogenesis, soluble organic compounds, which have been generated through the action of hydrolytic enzymes originating from the same or other species of the microbial consortium are biodegraded, generating a mixture of volatile fatty acids (VFAs) (mainly acetate, propionate, butyrate, and isobutyrate) in relative amounts that depend on

(a) the organic composition,

(b) the available species, and

(c) the operational parameters of the reactor

The dominant microbes for this stage are bacteria, although small populations of protozoa, fungi, and yeasts have also been reported to carry out acidogenesis Until 1965, only very few bacterial species had been isolated from anaerobic digesters and it was thought that facultative microbes were larger in numbers than obligate anaerobes The acidogenic population accounts for approximately 90% of the total microbial population

in a digester [13]

Acetic acid is an important intermediate in the anaerobic metabolism of organic substrates, since it is further utilized for the generation of methane We generally distinguish between two different mechanisms for acetic acid production: acetogenic hydrogenations and acetogenic dehydrogenations Acetogenic hydrogenations take place during the growth of two microorganism groups: (a) obligate proton-reducing or obligate hydrogen-producing species and (b) those that can utilize various electron acceptors during the degradation of organic substrates The latter, depending on the prevailing hydrogen concentration, regulate their metabolism, producing more or less reduced products (facultative proton-reducing) Acetogenic dehydrogenations for the production

of acetate include reactions of carbon dioxide with hydrogen, of carbon monoxide and water, and of methanol and carbon dioxide Usually, these species have the ability to grow on other organic substrates such as sugars, lactic acid, etc also [13]

two orders of magnitude in comparison with the methanogens present in the same samples [13]

Methanogs are obligate anaerobic microorganisms that may be found in natural environments such as the rumen, the interior part of the stem of certain trees and in freshwater sediments Methane has also been found to be released from high-salt

isolated from thermal springs has an optimal growth temperature of 83◦C) [13]

Methanogens may use a relatively small number of organic compounds as an energy source, including carbon dioxide, fomic acid, acetic acid, methanol, methylamines, and dimethyl sulfide Some methanogens may also use carbon monoxide Table 1.1 presents the methane producing reactions on the basis of the use of these compounds and the corresponding free energy change [13]

Until recently, it was believed that all methanogens can generate methane from hydrogen and carbon dioxide However, it was shown that although most methanogenic species have this ability, there are some that use acetic acid as a substrate and thus they have been divided into two groups: (a) acetotrophs such as Methanothrix soehngenii,

which metabolize only methanol, methylamines, and dimethyl sulfide It has been shown that both acetotrophs and methylotrophs produce methane directly out of methyl groups

few species that metabolize formic acid (e.g., Methanococcus thermolithotrophicus,

every substrate among those referred to in the above discussion Methanosarcina barkeri

Bacteria may be divided further into three groups according to their response to free molecular oxygen (Table 1.3) These groups are 1) strict aerobes, 2) facultative anaerobes, and 3) anaerobes, including the methane-forming bacteria [18]

Strict aerobes are active and degrade substrate only in the presence of free molecular oxygen These organisms are present in relatively large numbers in aerobic fixed-film processes, for example, trickling filters, and aerobic suspended growth processes, for example, activated sludge In the presence of free molecular oxygen they perform significant roles in the degradation of wastes However, strict aerobes die in an anaerobic digester in which free molecular oxygen is absent Facultative anaerobes are active in the presence or absence of free molecular oxygen If present, free molecular oxygen is used for enzymatic activity and the degradation of wastes If free molecular oxygen is absent, another molecule, for example, nitrate ion (NO3–), is used to degrade wastes such as methanol (CHOH) When nitrate ions are used, denitrification occurs and dinitrogen gas (N2) is produced [18]

Table 1.1 Acetogenic dehydrogenation reactions [13]

Table 1.2 Methane producing reactions [13]

1.2.2.1 Hydrolytic bacteria

Biodegradable polymers found in the MSW include lignocelluloses, proteins, lipids and starch Specialized microbial population of hydrolytic bacteria is responsible for depolymerization of these organic polymers towards their building compounds, monomers Usually this is found to be the slowest and the rate limiting step in the overall anaerobic digestion process Furthermore, the ultimate methane yield is directly depend

on the efficiency of this reaction [14]

Extracellular microbial enzymes catalyzing this reaction are known as hydrolyses or lyses Depending on the type of the reaction they catalyze, these hydrolyses can be esterase (enzymes that hydrolyze ester bonds), glycosidase (enzymes that hydrolyze glycosides bonds), or peptidase (enzymes that hydrolyze peptide bonds) For example, lipases hydrolyze the ester bonds of lipids to produce fatty acids and glycerol Lyses, on the other side, catalyze the non-hydrolytic removal of groups from substrates [14]

Table 1.3 Groups of bacteria according to their response to free molecular

oxygen [18]

Lignocellulose refers to the three major components of the plant tissue: cellulose, hemicelluloses and lignin The cellulose and hemicelluloses are biodegradable and make

up over 90% of the biochemical methane potential of the MSW, while the phenolic groups in lignin are even inhibitory to the enzymes Cellulose is degraded by hydrolyses

to yield a soluble disaccharide, cellobiose, which on further hydrolysis results in glucose The cellulolytic enzyme system is composed of endoglucanases, exoglucanases,

D-Figure 1.5 Overall process of anaerobic decomposition [14]

flavefaciens, Ruminococcus albus, Butyrivibrio fibrosolvens, Clostridium thermocellum,

Hemicelluloses are simpler structured and more readily degradable then cellulose by anaerobic microbes Despite that, its depolymerization requires complex enzyme system due to the various monomers comprising it The predominant bacteria found to degrade the hemicelluloses in the rumen are Bacterioides ruminicola B fibrisolvens, R

young plant tissues, berries and fruit Several Clostridium species have been identified as pectinolytic as well as rumen bacteria like Bacteroides rumenicola, and Streptococcus

monomers that can be used by the anaerobic bacteria in several anaerobic yielding processes such as anoxygenic photosynthesis, denitrification, sulfate reduction, fermentation and methanogenesis However, it is still doubtful whether lignin can be depolymerized to its monomers under AD conditions In anaerobic digesters, proteins serve as a source of carbon and energy for bacteria growth and a source of nitrogen Proteins are hydrolyzed by proteolytic enzymes to peptides, amino acids, ammonia, and carbon dioxide It has been shown that a specialized groups of anaerobic bacteria such as the proteolytic clostridia (e.g Clostridium perfringens, C bifermentans, C histolyticum,

these organisms, numerous other species of anaerobic bacteria such as Bacterioides,

further to simple fatty acids such as acetic, propionic and butyric acid as referenced in Archives of environmental protection [14]

Hydrolysis of the lipids is catalyzed by enzymes called esterase and leads to saturated and unsaturated long chain fatty acids and glycerol Glycerol is easily assimilated and metabolized by the bacteria while the long chain fatty acids undergo an intracellular betaoxydation mediated by a variety of enzymes, resulting in short chain fatty acids (e.g acetic and propionic acid) and hydrogen Anaerobic microorganisms capable to decompose lipids usually found in MSW anaerobic digesters are Anaerovibrio lipotyca

able to degrade lipids to Acetyl-coA as referenced in Archives of environmental protection [14]

Starch from the food waste is readily biodegradable but requires multiple enzymes to complete its hydrolysis Three main types of enzymes that act synergistically are: alphaamylases, beta-amylases, gluco-amylases Some of the microbes found in anaerobic digesters capable of degrading starch are Streptococcus bovis, Bacteriodes amylophilus,

1.2.2.2 Acetogenic bacteria

Acetogenesis is the stage when the products of the hydrolysis are processed to hydrogen, carbon dioxide, formate and acetate This pathway occurs naturally in well balanced methanogenic systems However, in practice, there are cases of electron or hydrogen accumulation (e.g when methanogenesis is inhibited) when numerous other fermentation products may be formed (e.g propionate, butyrate, lactate, succinate, and alcohols) as a mechanism to remove the excess electrons or hydrogen Organisms that convert these fermentation products to acetate, generally exhibitobligate proton-reducing metabolism and are obligatory dependent on the hydrogen removal as referenced in Archives of environmental protection Because of this the acetogenic bacteria are also called obligatory hydrogenproducing acetogens (OHPAs) [14]

Despite the significant importance of synthrophs, the knowledge of their taxonomic position, diversity and physiology is insufficient, mainly because of the difficulties in isolating them Several important proton-reducing syntrophic bacteria such as butyrateoxidizers, propionate-oxidizers and even acetate-oxidizers have been successfully isolated and cultured from methanogenic communities in recent years as referenced in Archives of environmental protection Thermophilic acetate-oxydizin syntroph, Thermacetogenium phaetum, was isolated and characterized by Hattori et al

strains, and besides acetate it produces small amount of butyrate Thermophilic propionate-oxidizing bacteria have also been described, and two of these have been obtained in pure culture so far: Pelotomaculum thermopropionicum strain SI, and

al (2000) isolated a thermophilic butyrate-oxydizer capable of oxidizing saturated fatty acids with four to ten carbon atoms [14]

It has been documented that the bacterial species active in the polymer hydrolysis phase are also active during the acidogenic phase Hence, the hydrolytic and acidogenic bacteria are sometimes referred to as fermentative bacteria They can be either facultative anaerobic bacteria (i.e., can survive under both aerobic and anaerobic conditions) or strict anaerobes The family Enterobacteriaceae or enteric bacteria (a group of bacteria that inhabit the intestine of humans and other animals) are active fermenters and are among the organisms responsible for the first step in the bioconversion of carbohydrates to CH4 [4]

In addition, relevance to biomass deconstruction are the following microorganisms:

are found in the following genera; Aminobacterium, Psychrobacter, Anaerococcus,

The main route of methane production is through a syntrophic relationship between acetate-oxidizing bacteria and hydrogen-utilizing methanogenic Archea The acetoclastic and hydrogenotrophic methanogens contribute 70% and 30%, respectively, to the methane production in industrial wastewater treatment Numerous methanogens have been isolated and described so far, but the studies, mainly based on 16S rDNA cloning analyses, suggest that the most commonly found methanogens genera, in the biogas reactors, are Methanobacterium, Methanothermobacter (formerly Methanobacterium),

referenced in Archives of environmental protection Among the acetoclastic

methanogenic organisms, Methanosarcina and Methanosaeta species has been reported

to be dominated in large-scale mesophilic and thermophilic digesters treating wastewater and sewage sludge Its dominance comes mainly due to its wide tolerance for environmental factors such as nutrients and temperature [14]

A primary gauge of digester health is the pH level, which changes in response to biological conversions during the different processes of AD A stable pH indicates system equilibrium and digester stability A falling pH can point toward acid accumulation and digester instability Gas production is the only parameter that shows digester instability faster than pH The range of acceptable pH for the bacteria participating in digestion is from 5.5 to 8.5, though the closer to neutral, the greater the chance that the methanogenic bacteria will function Most methanogens function in a pH range between 6.7 and 7.4, and optimally between 7.0 and 7.2 [11]

The greatest potential for digester failure is a result of acid accumulation This would occur if the amount of volatile solids loaded into the digester as fresh waste increased sharply The acidogenic bacteria would then thrive, producing high volumes of organic acids and lowering the pH to below 5.0, a level lethal to methanogens This creates a positive feedback loop as a declining methanogen population will in turn lead to further acid accumulation as the methogens are responsible for consuming acids An acidic pH indicates that this process has already begun, and immediate action is required, such as

by recycling more water On the other hand, prolific methanogenesis may result in a higher concentration of ammonia, increasing the pH above 8.0, where it will impede acidogenesis This can be opposedby adding a greater amount of fresh feedstock, which will spur acidogenesis and acid formation [11]

Maintaining pH is especially delicate in the start-up because fresh waste must undergo acid forming stages before any methane forming can begin, which will lower the pH To raise the pH during the early stages, operators must add a buffer to the system, such as

1.2.3.2 Temperature

Operating temperature is the most important factor determining the performances of the

AD reactors because it is an essential condition for the survival and optimum thriving of the microbial consortia Despite the fact that they can survive a wide range of temperatures, bacteria have two optimum ranges of the temperature, defined as mesophilic and thermophilic temperature optimum Mesophilic digesters have an operating temperature in the range of 25-40 °C and thermophilic digesters have operating temperature in the range of 50-65oC [11]

According to the reported experimental results as well as the operating performances of commercial scale AD plants, mesophilic and thermophilic reactors have different advantages and disadvantages [14]

Thermophilic digesters allow higher loading rate and yield higher methane production, substrate degradation and pathogen destruction Also, the higher temperature shortens the required retention time because it speeds up the reactions of degradation of the organic material However, the thermophilic anaerobic bacteria are very sensitive to toxins and small environmental changes Furthermore, these bacteria need more time (over a month) to develop redox population These systems are harder to maintain and are less attractive for commercial application because they require additional energy input for self-heating

Mesophilic AD reactors operate with robust microbial consortia that tolerate greater changes in the environment and are more stable and easier to maintain Another advantage is that usually these systems do not need any additional energy input for heating the system On the other hand, the disadvantages of the mesophilic AD systems are longer retention time and lower biogas production However, due to the fact that they are easier to operate and maintain, as well as the lower investment cost, they are more attractive for commercial scale plants [14]

The Carbon/Nitrogen Ratio is a measure of the relative amounts of organic carbon and nitrogen present in the feedstock This ratio may either be monitored explicitly or managers may simply keep track of the types of waste entering the facility, knowing the relative make-up of each For example, proteins such as meats are high in nitrogen while

paper products contribute relatively more carbon If a feedstock is high in carbon, manure can also be added to increase nitrogen As with composting, the optimum C/N ratio is between 20-30, with most sources citing 25 as the ideal level A low C/N ratio, or too much nitrogen, can cause ammonia to accumulate which would lead to pH values above 8.5 Additionally, the quality of the compost is lessened with high ammonia production A high C/N ratio will lead to a rapid consumption of nitrogen by the methanogenic bacteria and lower gas production rates [11]

Furthermore retention time in the AD system depends on process temperature and total solid content Mesophilic digesters have longer retention time (10-40 days) then thermophilic digesters Also the high solid content systems (“dry” processes) have longer retention time then low solid content systems (“wet” processes) Commonly used method for shortening the residence time in AD reactors is mixing the digester Usually

it is done by recirculation of the produced biogas back in the reactor [11]

Overloading of the system can results in low biogas yield This happens due to accumulation of inhibiting substances such as fatty acids in the digester slurry [14]

The events that would occur in the case of overloading the system are shown in the Figure 1.6 It would cause proliferation of the acidogenic bacteria further decreasing the

pH in the system and disturbing the population of the methanogenic bacteria Also there

is a definite relationship between the biogas yield and the loading rate This is the concept that we used in the design of the experimental part of this study The loading rate was at the point in favor of the acidogenesis avoiding the methane production and maximizing the VFA production in it [14]

Loading rate can be calculated using the following equation: [14]

1.3 Methods to identify microorganisms in Anaerobic Digestion

Traditional methods of investigating the microorganisms’ quantity and identification are based on microorganisms’ morphology and phenotypic features The studies of methanogenic microorganisms carried out by Grothenhius were based on microscopic techniques and consisted in identification of acetotrophic methanogenes based on their morphology Hydrogenotrophic methanogenes were demonstrated by autofluorescence,

at the wavelength of 420 nm

The culture of methanogenes is difficult because of a low rate of the growth of those microorganisms, specific nutritional requirements and restrictive environmental conditions Phylogenetic analysis allows the identification of microorganisms according

to molecular techniques This diagnostic method eliminates the need for the culture of these microorganisms This means that sequences of nucleic acids may be isolated from

Figure 1.6 Effect of the loading rate above the sustainable [14]