CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Biogas is seen as one of the most important renewable energy resources that can

replace part of the fossil fuel-based energy used today, and it shows great potential

and many advantages, including both climate and economic benefits (Meyer-

Aurich et al., 2016). A biogas process can be implemented in small or large scale,

which is important when designing flexible and sustainable energy solutions in

both industrialised and developing countries (Holm-Nielsen et al., 2019). Materials

that can be used for biogas production include various types of waste products,

such as manure, straw, municipal wastewater, food waste etc., and dedicated

energy crops (Vasco-Correa et al., 2018). Among these substrates, lignocellulosic

materials, such as agricultural residues, are of great interest due to their high

abundance and potential for biogas production (Azman et al., 2015). By controlled

use of wastes in a biogas process rather than e.g. dumping household waste in

landfill or storing farm manure in open tanks, it is possible not only reduce the

number of waste deposits, but also to decrease emissions of carbon dioxide and

other greenhouse gases (Borjesson & Mattiasson, 2018). The biogas produced,

containing the energy carrier methane, can be used for production of heat,

electricity and vehicle fuel after upgrading (removal of carbon dioxide and trace
1
gases) (Holm-Nielsen et al., 2019). The residues left after biogas production are

rich in mineral nutrients and can be used as a fertiliser during crop production to

replace fossil energy-requiring mineral fertilisers, thus enabling recycling of

nutrients between urban and rural areas (Vasco-Correa et al., 2018).

Microorganisms are essential for degrading organic material to biogas, in a process

that involves various anaerobic digestion pathways and requires the combined

activity of several groups of microorganisms with differing metabolic capacities

(Angelidaki et al., 2011). To obtain a stable biogas process, all these conversion

steps and microorganisms must work in a synchronised manner (Vanwonterghem

et al., 2014). When plant-based materials (e.g. agricultural residues) are used for

biogas production, the first step of the microbiological process, hydrolysis,

becomes rate-limiting. It has been suggested that the crystalline structure of the

lignocelluloses obstructs degradation in the initial step, and thus the hydrolysis of

these insoluble compounds becomes slow (Mulat & Horn, 2018). Some of the

obstacles with degradation of these types of materials can be overcome by various

pre-treatment methods, making the material more accessible to microbial and

enzymatic attack (Martínez-Gutiérrez, 2018). An alternative strategy is to increase

the efficiency of the active microbial community. Numerous studies have been

devoted to examining anaerobic cellulose-degrading bacteria and their enzymatic

capabilities, in efforts to clarify the degradation mechanisms and identify ways to

2
enhance degradation rates. Most of these studies have been performed on samples

from gut and soil ecosystems (Tsavkelova & Netrusov, 2018) (Do et al., 2018),

while only a few have examined cellulose-degrading bacteria in biogas digesters

(Jia et al., 2018). Consequently, insufficient information is available on cellulose-

degrading communities in biogas processes and on possibilities to enhance the

degradation rate by ‘microbial steering’, i.e. by supporting the growth of highly

efficient cellulose-degrading bacteria or communities.

1.2 Statement of the Problem

Biogas production is influenced by a number of process parameters including

substrate retention time, total solids, and temperature for different digester designs

and management regimes (including batch, semi continuous and continuous

feeding of the organic matter) in the field, and industrial, household and

experimental or laboratory digesters or bioreactors. One of the most pressing

problems of our industrialized society is the uncertainty of maintaining an adequate

supply of energy. Fossil fuels continue to decline in availability, because it is not

renewable so the search for alternative sources of energy has to be intensified. One

alternative source of energy is the production of methane gas (Biogas) by the

anaerobic digestion of various types of biomass.

3
1.3 Objective of the Study

The main aim of this study is to investigate the effect of anaobiidea gut microbes to

degrade lignocellulose in plant for biogas production using gastrointestinal of cow

i. To enhance the methane production from different lignocelluloses with the

help of different pretreatment methods

ii. To characterize the physical changes in the lignocelluloses after

pretreatment, using substrate characteristic analysis.

iii. To study the long-term co-digestion effects of pretreated and untreated

lignocelluloses together with the organic fraction of gastrointestinal of cow.

iv. To evaluate the economic feasibility of a full-scale pretreatment and

anaerobic digestion plant using lignocelluloses as a substrate.

1.4 Significance of Study

The dependence on fossil fuels as primary energy source has led to global climate

change, environmental degradation, and human health problems. Moreover, the

recent rise in oil and natural gas prices may drive the current economy towards

alternative energy sources such as biogas. Security of energy supply especially

sustainable energy and reduction of CO2 emission are priorities and biogas fuel

helps to reduce greenhouse gas emissions. Domestic biogas installations can

reduce greenhouse gas (GHG) emissions in three ways: by changing the manure

management modality; by substituting fossil fuels and non-renewable biomass for

4
cooking (and to a smaller extent for lighting) with biogas, and; by substituting

chemical fertilizer with bio slurry. Utilizing biogas as an alternative to fossil based

fuels reduces the net amount of carbon dioxide emitted to the atmosphere. Biogas

is regarded as carbon neutral fuel because CO2 released is utilized by plants for

photosynthesis which creates organic matter from it. The use of renewable energy

sources can contribute to solve the present and future energy problems. Among the

alternative energy sources, biogas production from green energy crops and organic

wastes has world wide application as it yields a good quality fuel and fermented

slurry, which may be used as a manure or soil conditioner

5
 CHAPTER TWO

LITERATURE REVIEW

2.1 Biogas

Biogas is the name given to a biologically produced specific gas mixture mainly

composed of methane (52-85%), carbon dioxide (14-48%) and some small

quantities of nitrogen, oxygen, hydrogen, hydrogen sulphide, ammonia and

hydrocarbons (C2-C7) and some traces of organic compounds of sulphur, chlorine,

fluorine, silicon etc. (Zamorska-Wojdyła et al., 2022). Methane (CH4) is an

energy-rich and economically valuable energy resource. Methane can be produced

through anaerobic digestion, a complex microbiological process requiring the

combined activity of several groups of microorganisms with different metabolic

capacities (Schnürer, 2016). At least four different groups of microorganisms (i.e.

performing hydrolysis, acidogenesis, acetogenesis and methanogenesis) are

involved (Schnürer, 2016). The substrate fed to a biogas process, such as manure,

crop residues, food wastes or municipal sewage sludge, is mainly composed of

polysaccharides (such as starch, cellulose, hemicellulose, pectin etc.), proteins and

lipids. Most of these complex organic compounds are too large for a single

organism to bring into the cell for its metabolism. Thus, in the first degradation

step, the compounds are degraded (hydrolysed) to soluble sugars, peptides, amino

acids and fatty acids, by the action of extracellular enzymes produced by

6
microorganisms (Adekunle & Okolie, 2015). In the second step, the fermenting

bacteria use these monomers as carbon and energy sources in their metabolism

and, as a result, they produce alcohols, organic acids, carbon dioxide, hydrogen,

hydrogen sulphide and ammonia (sometimes called intermediate products). These

compounds can then be utilised by acetogens in the third step, producing mainly

acetic acid, hydrogen and carbon dioxide. In the last step, methanogens (archaea)

use mainly acetate, formate, methyl compounds, hydrogen and carbon dioxide as

carbon and energy sources, forming carbon dioxide and methane (biogas) as the

final products. According to the known methanogenic pathways, these

methanogens can be categorised as hydrogenotrophic methanogens, acetoclastic

methanogens and methylotrophic methanogens (Kleinsteuber, 2018). The

hydrogenotrophic methanogens perform a very important role, as they ‘pull’ many

of the preceding oxidation reactions, e.g. oxidation of acids. These oxidation

reactions are endergonic under standard conditions and can only proceed at a low

partial pressure of hydrogen, i.e. in the presence of hydrogenotrophic

methanogens. The hydrogen and carbon dioxide produced during the acidogenesis

and acetogenesis steps can be converted to acetate through homoacetogenesis,

which can also affect the partial pressure of hydrogen (Collet et al., 2020). The

conversion of acetate to methane can proceed through two different pathways,

7
depending on prevailing environmental conditions such as ammonia and volatile

fatty acid (VFA) level and temperature:

i. The acetoclastic pathway, which involves acetoclastic methanogens cleaving

acetate into methane and carbon dioxide.

ii. The syntrophic acetate oxidation (SAO) pathway, where acetate is first

metabolised into hydrogen and carbon dioxide by syntrophic acetate-

oxidising bacteria (SAOB) and is later used by hydrogenotrophic

methanogens for methane production (Zinder & Koch, 2017).

8
Fig2.1: Grass bedding mixed with cattle manure, an agricultural waste with

high potential for biogas production

2.2 Advantages of biogas

1. Biogas is Eco-Friendly

Biogas is a renewable, as well as a clean, source of energy. Gas generated through

biodigestion is Biogas is a renewable, as well as a clean, source of energy. Gas

generated through biodigestion is non-polluting; it actually reduces greenhouse

emissions (i.e. reduces the greenhouse effect). No combustion takes place in the

9
process, meaning there is zero emission of greenhouse gasses into the atmosphere;

therefore, using gas from waste as a form of energy is a great way to combat global

warming.

Unsurprisingly, concern for the environment is a major reason why the use of

biogas has become more widespread. Biogas plants significantly curb the

greenhouse effect: the plants lower methane emissions by capturing this harmful

gas and using it as fuel. Biogas generation helps cut reliance on the use of fossil

fuels, such as oil and coal.

Another biogas advantage is that unlike other types of renewable energies, the

process to create the gas is natural, not requiring energy for the generation process.

In addition, the raw materials used in the production of biogas are renewable, as

trees and crops will continue to grow. Manure, food scraps, and crop residue are

raw materials that will always be available, which makes it a highly sustainable

option.

2. Biogas Generation Reduces Soil and Water Pollution

Overflowing landfills don’t only spread foul smells- they also allow toxic liquids

to drain into underground water sources.

Subsequently, another advantage of biogas is that biogas generation may improve

water quality. Moreover, anaerobic digestion deactivates pathogens and parasites;

thus, it’s also quite effective in reducing the incidence of waterborne diseases.
10
Similarly, waste collection and management significantly improve in areas with

biogas plants. This in turn, leads to improvements in the environment, sanitation,

and hygiene.

3. Biogas Generation Produces Organic Fertilizer

The by-product of the biogas generation process is enriched organic digestate,

which is a perfect supplement to, or substitute for, chemical fertilizers. The

fertilizer discharge from the digester can accelerate plant growth and resilience to

diseases, whereas commercial fertilizers contain chemicals that have toxic effects

and can cause food poisoning, among other things.

4. it’s A Simple and Low-Cost Technology That Encourages A Circular

Economy

The technology used to produce biogas is quite cheap. It is easy to set up and needs

little investment when used on a small scale. Small biodigesters can be used right

at home, utilizing kitchen waste and animal manure. A household system pays for

itself after a while and the materials used for generation are absolutely free. The

gas produced can be used directly for cooking and generation of electricity. This is

what allows the cost of biogas production to be relatively low.

Farms can make use of biogas plants and waste products produced by their

livestock every day. The waste products of one cow can provide enough energy to

power a lightbulb for an entire day.

11
5. Healthy Cooking Alternative for Developing Areas

Biogas generators save women and children from the daunting task of firewood

collection. As a result, more time is left for cooking and cleaning. More

importantly, cooking on a gas stove, instead of over an open fire, prevents the

family from being exposed to smoke in the kitchen. This helps prevent deadly

respiratory diseases. Sadly, 4.3 million people a year die prematurely from

illnesses attributed to the household air pollution caused by the inefficient use of

solid fuels for cooking.

2.3 Disadvantages of biogas

1. Few Technological Advancements

An unfortunate disadvantage of biogas today is that the systems used in the

production of biogas are not efficient. There are no new technologies yet to

simplify the process and make it accessible and low cost. This means large scale

production to supply for a large population is still not possible. Although the

biogas plants operating today are able to meet some energy needs, many

governments are not willing to invest in the sector.

2. Contains Impurities

After refinement and compression, biogas still contains impurities. If the generated

bio-fuel was used to power automobiles it could corrode the metal parts of the
12
engine. This corrosion would lead to increased maintenance costs. The gaseous

mix is much more suitable for kitchen stoves, water boilers, and lamps.

3. Effect of Temperature on Biogas Production

Like other renewable energy sources (e.g. solar, wind), biogas generation is also

affected by the weather. The optimal temperature bacteria needed to digest waste is

around 37°C. In cold climates, digesters require heat energy to maintain a constant

biogas supply.

4. Less Suitable For Dense Metropolitan Areas

Another biogas disadvantage is that industrial biogas plants only make sense where

raw materials (food waste, manure) are in plentiful supply . For this reason, biogas

generation is much more suitable for rural and suburban areas.

2.4 Lignocellulosic materials as a substrate for biogas production

When lignocellulosic materials are used as a substrate for anaerobic digestion, the

first step, hydrolysis, usually becomes rate-limiting for the whole process, due to

the recalcitrant structure of the plant cell wall (Mulat & Horn, 2018; Lynd et al.,

2002a). Moreover, lignocellulosic materials are characterised by low nutrient

content, giving low methane yield compared with other substrates, such as food

and municipal wastes (Li et al., 2013; Chynoweth et al., 1993). To overcome this

disadvantage of using lignocellulosic materials, many approaches have been

suggested, involving both substrate optimisation (e.g. pre-treatment and co-

13
digestion) and optimisation of process configuration (e.g. improved process

design). These are discussed in more detail below.

2.5 Structure of lignocellulose

Lignocellulose is widely present in plants in the form of microfibrils in the cell

wall, which makes plants strong (Li et al., 2009). It is abundant in most kinds of

plants, comprising e.g. around 100% in cotton flower parts (Bayané & Guiot,

2010) and around 40-50% in different agriculture residues (e.g. rice straw, rice

husk, maize stalks etc.) (Gani & Naruse, 2007). In the linear structure of

microfibrils, acetal bonds provide a strong binding force between each cellulose

unit. Each linear cellulose strain interacts with the neighbouring strains forming a

sheet structure, which is similar to the β-sheet structure in the DNA molecule.

These cellulose strains are covered by hemicellulose, which has several branched

glucose structures that are further reinforced by the mesh of lignin (Figure 4).

Lignin is a complex aromatic structure that cannot be significantly degraded by

microorganisms in the anaerobic environment (Prochazka et al., 2012). The rigid

structure of the plant cell wall, lignocellulose, is almost unreachable by enzymes

produced by microorganisms (Akin, 1988), thus restricting the degradation

efficiency (Bayané & Guiot, 2010). Consequently, the hydrolysis rate is the main

limitation in biogas production using lignocellulosic materials (Mulat & Horn,

2018).
14
2.6 Methods for investigating the community structure of cellulose-degrading

bacteria

As mentioned in the previous section, lignocellulosic materials are available in

large amounts, but the intricate structure and imbalanced nutrient content limit the

efficiency of AD of these materials. Although different strategies such as pre-

treatment and co-digestion strategies have been investigated and applied, there is

no conclusive solution for improving the biodegradability at a reasonable cost.

Moreover, the microbiology of the AD process is still somewhat of a ‘black box’.

The microbial community, especially the cellulolytic community, needs further

investigation.

2.6.1 Culture-dependent methods

Enrichment, isolation and cultivation of microorganisms in pure culture is a

labour- and time-consuming task, but is also an essential step in studying the

morphology, physiology and genetics of specific microorganisms. Furthermore,

available pure culture makes the development of molecular tools feasible, based on

genomic information. In order to cultivate anaerobic microorganisms, special

equipment and techniques are required to provide an anaerobic environment, and

the agar shake or role tube method is typically used for isolation (Schnürer et al.,

1996; Hungate & Macy, 2013). In this thesis, in order to isolate cellulose-

degrading bacteria, cellulose or cellobiose was used as the sole carbon source
15
during the whole isolation procedure. The isolation started with enrichment of the

bacterial consortia in a reduced mineral medium (Schnürer et al., 1994), with the

purpose of enriching the bacteria able to metabolise cellulose/cellobiose. Serial

dilution of the enrichment cultures was then performed in the same mineral

medium (Schnürer et al., 1994). For the highest dilution at which growth (visual)

occurred, the agar shake method was applied for picking single colonies (Schnürer

et al., 1996) and cultivation in pure culture. The industrial biogas plants in Paper

IV were used as the inoculum source for isolation and from this, two cellulose-

degrading bacteria were isolated (unpublished data). The 16S rDNA sequence

revealed the two isolates to be closely related to Clostridium straminisolvens CSK1

(1368 bp, 98% identity) and Clostridium clariflavum DSM 19732 (1500 bp, 97%

identity) respectively (unpublished data). Other cellulose-degrading bacteria have

been isolated in a similar way, including Clostridium cellulolyticum ATCC 35319

(Petitdemange et al., 1984), Clostridium cellulovorans 743B (Sleat et al., 1984),

Clostridium papyrosolvens (Madden et al., 1982), Clostridium populeti (Sleat &

Mah, 1985) and Clostridium stercorarium (Madden, 1983)

2.6.2 Culture-independent methods

The majority of microorganisms in the AD process have not yet been cultivated,

and it is estimated that 5% or less of the microbial diversity in the biosphere is

cultivable using standard cultivation techniques (Curtis et al., 2002; Amann et al.,
16
1995). Consequently, understanding of the microbial ecology and physiology

associated with AD is most likely incomplete and biased. There are many factors

that co-exist in this complex environment and affect microbial activity which

cannot be studied when using culture-based methods. Moreover, functions related

to competition and interaction between microorganisms is difficult to determine

when using isolated microorganisms (Vanwonterghem et al., 2014). Based on

available genomic data, a variety of molecular methods have been invented and

developed for use in further investigating the microbial community structures

within AD processes. Clone library is a culture-independent method that enables

investigation of DNA extracted from an environmental sample by cloning and

subsequent sequencing (Chouari et al., 2005). In this thesis, clone libraries were

constructed using products generated after polymerase chain reaction (PCR)

amplification of genes encoding glycosidase hydrolase family 5 and 48 (II and IV).

This technique has also been used to target 16S rDNA of the microbial community

in various anaerobic digesters, such as digesters processing beet silage (Krakat et

al., 2011), crops and cow manure (Wang et al., 2009b), grass silage (Wang et al.,

2010a), pig manure (Liu et al., 2009) and organic solid waste (Sasaki et al., 2011).

Terminal restriction fragment length polymorphism (T-RFLP) is a method based

on PCR technology where a selected target gene is amplified with PCR using the

total DNA extracted from digester samples, However, different from clone library
17
method, the primer in the PCR reaction is labelled with a fluorescent dye such as

6-carboxyfluorescein (FAM). In a second step, the amplicon generated from the

PCR is digested with a selected restriction enzyme appropriate for the sequence of

interest. The digestion products, which are called fluorescently labelled terminal

restriction fragments (T-RFs) are separated and detected using capillary

electrophoresis. The T-RFLP profile is visualised as the relative abundance of each

T-RF at a specific length. As different sequences possibly have different restriction

sites, in the assay each T-RF could represent a unique sequence, but occasionally

the same T-RF can be represented by two different organisms (II and IV).

Differences in the TRFLP profile indicate the differences in structure between the

communities. This method is usually combined with a clone library. Once the

sequence for the environmental sample is available, the restriction site of each

sequence can be analysed in silico and thus the corresponding sequence for each T-

RF can be decided. This technique has been used in combination with clone

libraries in several previously mentioned studies (Wang et al., 2010b; Wang et al.,

2009b), as well as in this thesis work (II and IV).

2.7 Cellulose degradation in anaerobic environments

2.7.1 Anaerobic cellulose-degrading bacteria

Globally, around 5-10% of cellulose is degraded in anaerobic environments

(Leschine, 1995). Anaerobic cellulose degradation occurs in various environments,

18
such as soil, aquatic and animal gut environments (Morrison et al., 2009; Lynd et

al., 2002; Leschine, 1995). Cellulosic biomass degradation in anaerobic

environments can be performed by physiologically diverse taxa of microorganisms

(Lynd et al., 2002; Schwarz, 2001; Leschine, 1995). For example, cellulose-

degrading bacteria have been found within the genera Clostridium, Ruminococcus,

Caldicellulosiruptor, Acetivibrio, Butyrivibrio, Halocella, Fibrobacter, Bacteroides

and Spirochaeta (Azman et al., 2015; Tsavkelova & Netrusov, 2012). In the rumen,

Fibrobacter, Ruminococcus, Butyrivibrio, Prevotella and Eubacterium have been

identified as the dominant cellulolytic bacterial genera, with Clostridia a minor

player (Ransom-Jones et al., 2012; Koike & Kobayashi, 2009; Leschine, 1995). In

municipal waste landfill sites, Clostridium is the most commonly reported genus

(Burrell et al., 2004; Van Dyke & McCarthy, 2002). However, new evidence has

shown the importance of bacteria from the genera Fibrobacter for cellulose

degradation at landfill sites (McDonald et al., 2012). Within anaerobic digesters,

the class Clostridium, belonging to the phylum Firmicutes, is commonly found and

is suggested to be involved in the hydrolysis of cellulosic materials (Lebuhn et al.,

2014; Hanreich et al., 2013; Wang et al., 2009b; Krause et al., 2008; Klocke et al.,

2007). Bacteria belonging to this class were also detected in the laboratory-scale

digesters using mono-digestion of manure or co-digestion with wheat straw in

Paper II. In the same series of digesters, Bacteroidetes was identified as the
19
dominant bacterial phylum, with its members possibly engaged in cellulose

degradation, while the genus Ruminococcus was a minor group. However,

Ruminococcus has previously also been identified in some anaerobic digesters

operating with plant-based materials and/or manure (Wirth et al., 2012; Schlüter et

al., 2008). To date, only limited numbers of bacteria capable of degrading cellulose

have been isolated from anaerobic digestion processes and these mainly belong to

the genus Clostridium, but also include some members of the Bacteroides.

Clostridium aldrichii was first isolated from a wood-fermenting anaerobic digester

and is able to utilise cellulose, xylan and cellobiose at temperatures between 20

and 45 °C (optimum 35 °C) (Yang et al., 1990). Acetate, propionate, butyrate,

isobutyrate, isovalerate, lactate, succinate, hydrogen and carbon dioxide are

products of cellobiose fermentation.

2.7.2 Enzyme system of anaerobic cellulose-degrading bacteria

In nature, the enzymatic degradation of cellulose is generally a slow and

incomplete process. The degradation proceeds through the action of extracellular

enzymes and, due to the heterogeneity of native cellulose, multiple cellulolytic

enzymes are required in order to achieve efficient degradation (Bayer et al., 2004;

Lynd et al., 2002). Thus many anaerobic cellulose-degrading bacteria possess an

extracellular multi-enzyme complex, called cellulosome (Lynd et al., 2002;

Leschine, 1995). This distinguishes them from the aerobic cellulose-degrading

20
microorganism, which instead secretes numerous individual extra-cellular enzymes

(Schwarz, 2001). The cellulosome is a large extracellular enzyme complex

(including catalytic modules containing enzymes such as glycoside hydrolases). In

fact, it is probably the largest enzyme complex in nature, with a molecular weight

ranging from 650 000 Da to 2.5 MDa (Doi et al., 2003). It has been observed in

various anaerobic bacteria, such as Clostridium, Acetivibrio, Bacteroides and

Ruminococcus (Doi et al., 2003). The common structure of the cellulosome

consists of large non-catalytic scaffolding proteins (also called scaffoldins) and

numerous catalytic modules. The scaffolding proteins usually contain a

carbohydrate-binding module (CBM), surface layer homology (SLH) modules and

a number of cohesin domains (Bae et al., 2013). The catalytic module consists of

the catalytic domain, which exhibits hydrolytic activity, and the dockerin domain,

which interacts with the cohesin domain. This interaction plays an important role

in assembly of the cellulosome (Doi et al., 2003). The number of cohesins on the

scaffoldins is limited compared with the number of enzyme subunits present in the

cellulosome, which suggests a heterogeneous population of cellulosomes. This

heterogeneous population contributes to the efficient degradation of plant materials

in nature (Murashima et al., 2002; Pohlschröder et al., 1994).

2.8 Bacterial community within anaerobic digestion processes

21
Lignocellulosic biomass has a complex structure, which requires a range of

enzymes and microorganisms working in a synergistic way to achieve effective

degradation (Kostylev & Wilson, 2012). Various culture-independent studies have

been used to investigate the general bacterial community structures in biogas

digesters and have revealed dominance of the phyla Firmicutes and Bacteroidetes

(III) (Sundberg et al., 2013; Riviere et al., 2009). When lignocellulosic materials

are specifically included in the substrate, Clostridiales from the phylum Firmicutes

and Bacteroidales from the phylum Bacteroidetes are commonly found as two

dominant orders, e.g. in: A production-scale biogas plant fed maize, green rye and

chicken manure (Schlüter et al., 2008); batch fermentation of straw and hay

(Hanreich et al., 2013); batch fermentation of cellulose (Lu et al., 2013); a

hydrolysis/acidogenesis reactor of a two-stage AD process fed straw and hay

(Lebuhn et al., 2014); and laboratory-scale reactors degrading manure and straw

(II, III). In addition to identification of Clostridiales and Bacteroidetes in biogas

processes operating with lignocellulose material, one or both of these taxa have

also been shown specifically to be responsible for cellulose degradation in different

AD processes: (1) Batch fermentation of 13C-labelled cellulose and glucose, where

members of Clostridiales and Bacteroidetes were dominant in the heavy fraction

resulting from 13C-labelled cellulose and glucose, respectively (Li et al., 2009);

(2) batch cultivation processing straw and hay, in which the relative abundance of
22
Clostridiales was higher on day 5 than day 30, when the lignocellulosic material

was depleted, while the Bacteroidales presented in the opposite way (Hanreich et

al., 2013); (3) this thesis, where a higher level of Bacteroidales was recorded in a

digester fed wheat straw and cattle manure than in a digester mono-digesting cattle

manure (III); (4) studies using culture-dependent techniques revealing that bacteria

isolated from different AD processes and belonging to Clostridiales and

Bacteroidales, are capable of degrading crystal cellulose (Koeck et al., 2014; Lynd

et al., 2002).

23
Fig 2.2: Microstructure of a typical plant cell wall, indicating the relationship
between cellulose, hemicellulose and lignin
2.9 Lignocellulosic substrate optimisation

The biodegradability of lignocellulosic materials can be increased by a pre-

treatment with the purpose of removing lignin, hydrolysing hemicellulose,

decreasing cellulose crystallinity, increasing the porosity of materials and making

the material more accessible to microbial and enzymatic attack (Monlau et al.,
24
2013). Different pre-treatment methods for lignocellulosic materials have been

explored, for example mechanical, thermal, chemical and biological methods

(Monlau et al., 2013). However, most pretreatment methods require expensive

specialist equipment with substantial energy requirements. In addition, toxic

products such as furfurals, 5- hydroxymethylfurfural (HMF), organic acids and

phenols may be formed and cause inhibition of the microbial process

(Sawatdeenarunat et al., 2015). Lignocellulose-rich materials typically also have a

high carbon to nitrogen (C/N) ratio, low levels of micronutrients and, often, a low

energy content (Li et al., 2013). However, through co-digestion, the substrate

mixture can be designed to optimise the composition of nutrients, balance the C/N

ratio etc. and achieve higher methane yields (Ebner et al., 2016; Macias-Corral et

al., 2008; Lehtomäki et al., 2007; Sosnowski et al., 2003). Many substrates have

been tested for co-digestion in biogas production from lignocellulose-rich material.

For example, lignocellulose-rich cattle manure has been evaluated in co-digestion

with food waste (Awasthi et al., 2018; Ebner et al., 2016) and stillage (Westerholm

et al., 2012) and co-digestion has been shown to give enhanced methane yield

compared with mono-digestion of the manure. Process stability and volumetric

biogas yield from lignocellulose-rich materials with a low C/N ratio, such as corn

stovers (Li et al., 2014), switchgrass (Zheng et al., 2015) and other agricultural

residues, have been shown to improve when these materials are co-digested with
25
nitrogen-rich animal manure (Neshat et al., 2017; Zhang et al., 2013). When

diluted agricultural residues (such as liquid manure) are used, codigestion with

lignocellulosic materials can also be applied to achieve a higher organic loading

rate (OLR), with only minor effects on hydraulic retention time (HRT). This is

particularly important, as relatively long hydraulic retention time is typically

needed for degrading lignocellulose-rich materials (Neshat et al., 2017; Mata-

Alvarez et al., 2014). Positive effects, such as increased methane yield, of

combining lignocellulose-rich agricultural substrates with various high-energy co-

substrates, including protein- and sugarrich materials, have been demonstrated in

several different studies (AhlbergEliasson et al., 2017; Neshat et al., 2017; Mata-

Alvarez et al., 2014).

An issue to consider when selecting a co-substrate for lignocellulosic materials is

that some co-materials can result in decreased degradation efficiency. For example,

a negative effect of proteins on anaerobic digestion of carbohydrate-rich materials

has been observed and has been attributed to high ammonia levels (Breure et al.,

1986). Similar results are presented in this thesis, with negative effects, specifically

on cellulose degradation, observed following high levels of ammonia release

during degradation of proteins. A decrease in the degree of degradation efficiency

and specific methane production was also observed in this thesis work when

digesting lignocellulose-rich material with milled feed wheat, resulting in elevated

26
ammonia levels (III). The low degree of degradation efficiency is unfavourable, as

it represents a loss of energy and could potentially lead to higher methane

emissions during digestate storage (Liebetrau et al., 2013); (III).

2.10 Biogas Production Technology

Agricultural biogas plants typically consist of a number of low digesters built

either from concrete or metal. They are often topped by a twin-skinned gas storage

bag, giving them a characteristic appearance. Majority of the biogas is produced

from the first digestion tank with a lower gas yield being attained in the secondary

digestate storage tank. The common technology for biogas production is the

digestion of feedstock in specially designed digesters. These digesters must be

strong enough to withstand the buildup of pressure and must provide anaerobic

conditions for the bacteria needed in the digestion process. Today, there are many

different technologies and digester types available. Generally, the size of biogas

plants can vary from a small household system to large commercial plants of

several thousand cubic meters. Digester size also influences logistics and vice

versa. Therefore, they are often built near the source of the feedstock. The water

content of substrate influences the design and type of digester. One of the most

common classifications regarding the water content of the substrate is wet

digestion (which is fed with dry mass contents lower than 15% and into dry

digestion which is fed with dry mass content between 20 and 40%). Wet digestion
27
is usually applied to manure and sewage sludge whereas dry digestion is often

applied to the fermentation of energy crops. The removal of H2S from the biogas is

achieved by air dosage into the digester gas phase. Water vapour is removed by

means of a water tap. Furthermore, digesters can be classified by the number of

process steps. Single-stage and two-stage digesters are the most common

technologies today. Single-stage digesters are characterized by no special

separation of different process steps (hydrolysis, acidification, methanisation). All

process steps are conducted in one single digester. Digesters can also be classified

according to the filling procedure and filling interval. Co-digestion refers to the

simultaneous anaerobic digestion of multiple organic wastes in one digester. Co-

digestion is used to increase methane production from low-yielding or difficult- to-

digest feed stocks. The co-digestion of manure, residuals of food processing and

energy crops in continuous stirred reactor systems is an effective and reliable way

for the production of biogas. The presence of too much NH3 or H2S can inhibit the

production of methane. Wastes from fruits, vegetables, and grains contain large

amounts of primary metabolites of lipids, proteins, and carbohydrates and a broad

range of high-value bioactive compounds of phenolics, terpenoids, and alkaloids.

Extraction techniques have been widely investigated to obtain valuable natural

compounds from plant-based processing wastes. Wet plant-based food processing

wastes, which are rich in sugars, vitamins, and minerals, have been used to produce
28
fuel alcohols, organic acids, biodegradable polymers, enzymes, antioxidants, and

vitamins in biological processes. Both high and low levels of nitrogen can inhibit

the biogas process. The substrate composition not only has an influence on the

process performance but also affect the composition of the produced biogas. For

instance, Table 8 depicts the various moisture contents of the feed stocks used in

the biogas production process that can guarantee success in biogas production

2.11 Bioreactor

A bioreactor refers to any manufactured device or system that supports a

biologically active environment. In one case, a bioreactor is a vessel in which a

chemical process is carried out which involves organisms or biochemically active

substances derived from such organisms (Eibl et.al, 2008). This process can either

be aerobic or anaerobic. These bioreactors are commonly cylindrical, ranging in

size from litres to cubic metres, and are often made of stainless steel. It may also

refer to a device or system designed to grow cells or tissues in the context of cell

culture. These devices are being developed for use in tissue engineering or

biochemical/bioprocess engineering.

29
Fig 2.3: Bioreactor (Eibl et.al, 2008)

Organisms or biochemically active substances growing in bioreactors may be

submerged in liquid medium or may be anchored to the surface of a solid medium.

Submerged cultures may be suspended or immobilized. Suspension bioreactors

may support a wider variety of organisms, since special attachment surfaces are not

needed, and can operate at a much larger scale than immobilized cultures.

However, in a continuously operated process the organisms will be removed from

the reactor with the effluent. Immobilization is a general term describing a wide

variety of methods for cell or particle attachment or entrapment. It can be applied

to basically all types of biocatalysis including enzymes, cellular organelles, animal

30
and plant cells and organs. Immobilization is useful for continuously operated

processes, since the organisms will not be removed with the reactor effluent, but is

limited in scale because the microbes are only present on the surfaces of the vessel.

2.11.1 Bioreactor Design and Operations

A good bioreactor design should address improved productivity, validation of

desired parameters towards obtaining consistent and higher quality products in a

cost effective manner. The design and mode of operation of a bioreactor depends

on the production of organism, optimum conditions required for desired product

formation, product value and its scale of production. The effective bioreactor is to

control uence the biological reaction and must prevent foreignand positively in

contamination. The capital investment and operating cost are also important factors

to be considered in bioreactor design. During the fermentation, monoseptic

conditions, optimal mixing with low, uniform shear rates should be maintained

throughout the process. A culture can be aerated by one, or a combination, of the

following methods: surface aeration, direct sparging, indirect and/or membrane

aeration, medium perfusion, increasing the partial pressure of oxygen and

increasing the atmospheric pressure (Eibl et.al, 2008). Adequate mass transfer

(oxygen), heat transfer, clearly de condition and appropriate feeding of substrate

avoiding under or overdosing would need to be maintained in a bioreactor. Proper

supply client substrate, salts for nutrition, vitaminsof suspension of solids, suf etc.
31
should be ensured with water availability and oxygen (for aerobic processes). Gas

evolution product and by-product removal need to be taken care of. The attributes

of a bioreactor should comply with design requirements such as sterilization,

simple construction and measuring, exibility inprocess control devices, regulating

techniques, scale-up, operations, compatibility with upstream and downstream

processes, antifoaming measures etc. are essential factors (Sharma, 2012) The

basic features of a bioreactor include headspace volume, agitator system, oxygen

delivery system, foam control, temperature & pH control system, sampling ports,

cleaning and sterilization system and lines for charging & emptying the reactor

(Alaghlavi, 2013).

32
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Materials Used

i. Fresh gut content of slaughtered cow (feedstock)

ii. Inoculum or Pretreatment or amendments (is powderpost beetle etc) 20mls

iii. 0.2g concentration

iv. Water

3.2 Equipment Used

i. Hand gloves / Nose Mask

ii. Weighing balance

iii. Masking tape

iv. Gas Collector (tubes)

v. Foil

vi. Adhesive (Bands)

vii. Digester

viii. Spatula

ix. 3liter Bucket

x. A Preparation of Inoculum (Strictly for Cockroach, powderpost beetle,

Poultry).

33
3.3 Microbial biodegradation potential of insect gut microbes to degrade

lignocellulose in plants

3.3.1 Aim

To ascertain the ability of Insect gut microbes to degrade lignocellulose in plants.

3.3.2 Materials

Syringe, Hand gloves, distilled| deionized water, 95% alcohol, sample B

(ponderpost Beetles), bunsen burner, Needles, trypti case medium or soy bean

casein digest medium, 9 ml of peptone water, wire loop, forceps, Analytical

balance, cotton wool, masking tape, test tube, collection bottle, Incubator, micro

pipette

3.3.3 Procedure

i. The Sample (sample B- powderpost Beetles) was collected in a container.

ii. sample B (powderpost Beetle) wasput in in a Petri-dish and surface sterilized

using 95% alcohol for 30 seconds and washed off using distilled| deionized

water.

iii. The sample(Sample B-powderpost Beetles) was transferred into different

petri- dishes and dissected using sterilizedForceps

iv. The dissected Sample(B- powderpost Beetle) was then transferred to

different petri-dishes and weighed using an analytical balance.

34
 v. Sample B (powderpost beetles)weighed 0.2g.

vi. The dissected Sample B- powderpost Beetles) was then transferred to

different collection bottles containing distilled deionized water, and crushed

to pieces using a sterile wile loop, then swirled to get a homogenous solution

vii. The solution (from the crushed sampleB- powderpost Beetles) were

collected into different test tube using new syringe and needle, and labelled

in test tubesie test tube 2 contains broth culture of Powderpost Beetle).

viii. I Prepared a 1-10 dilution factor (1ml of our sample and 9ml of sterile

peptone water) to get a I in 10 dilution using the micro pipette for each tube.

ix. I took0.05ml of I in 10 dilution of each, broth culture (Sample B powderpost

Beetle) and transferred to a sterile trypti case in medium (9.95 mls)

x. After this, I incubated at prevailing room temperature.

3.3.4 Observation

I observed the effectiveness of insect gut microbes in breaking down

lignocellulose, a major component of plant biomass which could provide insights

into potential applications for biofuel production, waste management, and

sustainable agriculture practices.

3.4 Mode of preparation of peptone water

i. It is used for the cultivation of non-fastidious Organisms

ii. Add 15 gram powder to distilled deionized water

35
iii. Bring volume to 1m and mix thoroughly.

iv. Gently heat and bring to boiling

v. Autoclave at 15Ps (pressure per Square) at 121•c for 15mins

3.5 Mode of preparation of trypti case medium or soy bean casein digest

Medium

i. Anutrious general purpose medium used for wild variety of bacteria and

fungi

ii. Add 30 gram powder to distilled| deionized water

iii. Bring volume to 1m and mix thoroughly

iv. Gently heat and bring to boiling

v. Auto clave at 15ps, (Pressure per square) at 121°c for15 mins

3.6 A Sample collection

The fresh gut content or gastrointestinal content of slaughtered cow was collected

from a slaughter close to 34 artillery brigade, Obinze in Imo state. The Inoculum

which is powder post beetle was obtained from a beetle infested wooden bird cage

at Eziobodo, Imo state.

3.7 Proximate analysis of the substrate

Physiochemical parameter was determined in order to check the suitability of the

cow gastrointestinalor gut content. such parameters include; ph, ammonium,

available phosphorous, available potassium, lipids, crude protein, total

36
carbohydrate, ash content and moisture content were determined by the method

described by A0AC (2000)

3.8 Fabrication of the digester

The digester was constructed by method described by Ngumah et al (2013). Hole

of l/4mm diameter was made on the carer of each of the 25 litres capacity container

using the hot rod, and a hose of 1/4mm diameter and 50-60cm length was fitted

into the hole and sealed perfectly using epoxy glue to prevent gas leakage, the

other edge was then connected to a tube having an internal diameter of 29.0 cm

and external diameter of 51.0 cm with circumference as 27.0cm. The tube retains

the gas produced while degradation of substrate occurs.

3.9 Experimental Procedure

Identical biogas digesters were used for the study, and they served as the digester

unit, each digester unit was provided with an outlet port for gas flowing and to a

gas collecting system which is a tube. the bio digesters were setup as described by

Ngumah et al 2013, The substrate (gastrointestinal content of cow) in each of the

digester were labelled A and B (digester A as control and digester B containing

amendments (which is powderpost bettle). Both digester A and digester B contains

equal amounts of substrate which was measured using a 3 litres bucket. Four

buckets of the substrate is added into each digester using the 3 litres bucket, which

each of the substrate per bucket weighed 4.0gand in total 16.0g per digester. The
37
both digester labelled A and B contain equal amount of substrate which weighed

16.0 g. Using the three (3) litre bucket, four (4) bucket of water is then introduced

into each of the digesters labelled A and B to obtain slurry. The

inoculum( powderpost beetle) is then introduced into only digester B, leaving

digester A as control Using a spatula, both digester A and B were mixed

thoroughly and separately to become homogeneous. The digester labelled A and B

were then sealed with foil and held firm to the digester using a rubber band. The

sealed digester labelled A and B are then covered using its caps( covers). The tap

head are then opened for gas flow into the tube as degradation takes place.

Fig 3.1:

38