DIGESTION OF FATS, OIL AND GREASE (FOG) FOR BIOGAS PRODUCTION

ABSTRACT

A study on the anaerobic digestion of Fats, oil and grease (FOG) that are being
indiscriminately dumped in drainage channels in Uyo metropolis was undertaken. Fresh cow
dung was introduced into a vessel and incubated at 45±2°C in a water-bath until no biogas
was detected. The FOG sample was mixed in a ratio of 50:10 for the control experiment and
25:10:25 for the treatment which was spiked with Costus afer extract/slurry to serve as a
natural buffer and anaerobically digested in two separate 100ml amber serum bottles
(Gerresheimer 61020G, USA) with a 20mm alumminium cap with central moulded septum
combination seal (FB 67567, Fisher Scientific, UK) and tightened with a crimper (JG
Finneran 9300-20, USA) to ensure a complete anaerobic environment. The bottles were
incubated at a temperature of 35±0.2°C for 28 days. Results obtained showed that the
treatment had a cumulative biogas production of 59.49cm³, two times greater than the
control with a cumulative biogas production of 29.25cm³ at initial total and volatile solids
concentrations of 8.755mgL⁻¹ and 1.33mgL⁻¹ respectively. However, final analysis showed
that the TS and VS reduced tremendously at a difference of 4.4mgL ⁻¹ and 0.5mgL⁻¹
respectively, explaining the drop in biogas production towards the end of the digestion
process. Also, microbiological analysis of the sample aerobically showed a microbial count
of 6.85(Log₁₀CFU/ml) and 6.75(Log₁₀CFU/ml) for the treatment and control experiments
whereas the anaerobic culturing had a microbial count of 6.8(Log₁₀CFU/ml) and
6.85(Log₁₀CFU/ml) for both set-up. From the results obtained, Costus afer can be said to
have enhanced the biogas produced from fats, oil and grease and this can further be
improved through the process of co-digestion with other substrates.
 CHAPTER ONE

Introduction

Fats, oil, and grease (FOG) refers to any material either liquid or solid composed primarily of

fats, oils and grease from animal or vegetable sources. They are produced by Food Service

Establishments (FSE) like restaurants, motels, cafeterias in schools, hospitals, retirement

centres, prisons, and military bases (Iman et al., 2014). Large amounts of oil and grease in the

wastewater system cause problems in collection pipes. Grease is a major contributor to sewer

system overflows (SSOs). Fats, oil and grease decreases pipe capacity and, therefore, requires

that piping systems be cleaned more often and/ or some piping to be replaced sooner that

otherwise expected (USEPA, 2013). Grease leads to stoppages and backups into residential

and commercial properties. Oil and grease also hamper effective treatment at the wastewater

treatment plant. Grease in a warm liquid may not appear harmful, but as the liquid cools, the

grease or fat congeals on the surface of settling tanks, digesters, and the interior of pipes and

other surfaces which may cause a short down of wastewater treatment units. FOG also build

up in a city’s wastewater collection system that could lead to clogging or blocking of sewer

lines, causing backup and flooding of streets, residences, and commercial buildings, resulting

in potential liability to the city (Iman et al., 2014). Fats, oils and grease (FOG) can cause

debilitation of any hydrolytic system through coagulation within internal piping and overtime

cause manhole overflows and sewage spills into homes and business premises (Alade et al.,

2011). As a result, sewer back-ups and overflows occur creating health hazards and harmful

environmental impacts, sometimes even entering storm-water drains flowing into streams or

oceans. These contaminants can cause depletion of oxygen within natural waterways causing

harm to the aquatic life. Stemming from the highlighted impacts of FOG build-up in the

environment, it has become pertinent to research into ways its negative impact on the
environment can be effectively mitigated through the collection of fat, oil and grease from the

food service industry, treatment and use in renewable energy production through the process

of anaerobic digestion. FOG has been reported to increase biogas production by 30% or more

when added to the anaerobic digester and may allow wastewater treatment plants to meet

50% of their electricity demand through on-site generation (Long et al., 2012)

Anaerobic digestion is a biological process that utilizes microorganisms to breakdown

organic matter in the absence of oxygen (Divya et al., 2015). Through this process, methane

and carbon dioxide, collectively known as biogas, are produced from organic matter fed to

the digester. Anaerobic processes occur both naturally and in controlled environments, such

as waste management facilities (Long et al., 2012). There are four stages to anaerobic

digestion, which are carried out by different sets of microorganisms. During the first stage

which is hydrolysis, bacteria breakdown organic matter into simple sugars and fatty acids.

The second stage involves acidogenic bacteria that consume the products of the first stage to

produce short chain volatile acids (propionic and butyric acid) among other products such as

alcohols, hydrogen and carbon dioxide. In the third stage, acetogenic bacteria transform the

products from both the first and second stages into acetic acid and hydrogen. The fourth stage

(methanogenesis), methanogenic bacteria convert hydrogen and acetic acid into methane and

carbon dioxide which is known as biogas (Weiland, 2010).

The biogas produced is a by-product of anaerobic decomposition of organic matter

and is a renewable energy source which can be used for cooking, heating, lighting, and in

larger institutions for power generation (Angelidaki and Ellegaard, 2003). Despite the fear of

depletion of fossil fuels and their attendant ecological effects, the high cost of non-renewable

energy technology in Nigeria has triggered a need to develop alternative sources of energy,

among which is biogas production (Sambo et al., 2015). Biogas is a mixture of gases mainly

methane and carbon dioxide used in domestic cooking. The use of biofuel technology for the
production of biogas is a veritable means of curbing the environmental concerns associated

with the use of fossil fuels for electricity generation (Divya et al., 2015).

1.1 Aim of the study

This project aim to use fats, oil and grease (FOG) also known as restaurant waste, to

produce biogas through the process of anaerobic digestion.

1.2 Objectives of the study

 To isolate, characterize and identify microorganisms associated with the

anaerobic digestion of FOGs

 Evaluate the anaerobic digester performance in relation to biogas production

 Assess the effect of a natural organic buffer on the process performance

 CHAPTER TWO

Literature review

2.1 Definition of FOG

Fats, oil and grease (FOG) are the by-products of cooking. Typically, FOG includes

matter such as food scraps, meat fats, lards, tallow, cooking oil, butter, margarine, sauces

gravy, deep-fried food, baked goods, and cheeses. FOG can be solid or a viscous liquid

depending on the saturation of the carbon chain. Oils and fats are a subsection of lipids that

are composed of fatty acids, triacylglycerols, and lipid-soluble hydrocarbons that are minor

but important components of FOG (Iman et al., 2014).

2.2 Chemical composition of FOG

2.2.1 Free Fatty Acids (FFAs)

Free fatty acids are simply carboxylic acids with long-chain hydrocarbon side chains

and are usually found in the esterified form as major components of lipids. There are over

1000 identified natural fatty acids; however, only 20 of these are common in food sources.

(Iman et al., 2014) FFAs are very important constituents of FOG because of their chemical

reactivity. Moreover, the FFAs content of restaurant effluent can reach over 15%, which

reduces the pH and serves as indicator of the chemical activity of FOG (Iman et al., 2014)

2.2.2 Triacylglyycerols

Fatty acids occur mainly as glycerol esters known as triacylglycerol (TAGs) which

are nonpolar, water-insoluble substances, and have been known to be a class of abundant

lipids. Fats and oils are complex mixtures of triacylglycerols, whose fatty acid composition

varies on the basis of the organism of origin. Plant oils are generally richer in unsaturated
fatty acid residues than animal fats, as indicated by the lower melting points of the oils. Thus,

oils are typically liquids whereas fat is solid at room temperature (Gunstone, 2004).

2.2.3 Ester waxes

Wax comprises several types of medium and long chain compounds, including

hydrocarbons, alcohols, aldehydes, acids, and esters. Waxes are of both vegetable (e.g.

carnauba and jojoba) and animal origin (e.g. beewax and woolwax) (Madan, 2004).

2.2.4 Phospholipids

The amphiphilic nature of phospholipids impacts unique properties to them that make

them important in foods, cosmetics and pharmaceuticals. Although phospholipids are

removed during oil refining, they are present in small quantities in vegetable oils and thus

will appear in the FOG mixture (Solomons and Fryhle, 2013).

2.2.5 Sterols and sterol esters

Sterols (e.g. cholesterol) are not lipids; they occur in many oils and fats and exhibit

certain similar physical properties. Sterols can be esterified to long chain fatty acids through

oxidation reactions. Most crude oils contain 0.1-2.2% of phytosterol, partly as free sterols and

partly as esterified with FFAs. The ratio of esterified to free sterols varies where the free

sterols (40-80%) are generally predominant (Patrick, 2012)

2.3 Physical properties of FOG

FOG may occur as liquid or solid and is characterized by a greasy texture. In its pure

state, FOG is colourless, odourless and tasteless. Moreover, FOG is insoluble in water but

soluble in organic solvents such as hexane, ether, and chloroform (Sincero, 2003). FOG has a

density less than water (specific gravity < 1) and thus, it floats on the water surface.
However, FOG will form emulsions with aqueous media in the presence of soap or other

emulsifying agents. Generally, FOG has high viscosity that varies based on the fatty acid

composition and the presence of double bonds. The more double bonds present in the carbon

chain, the lower the viscosity of FOG (Firestone, 2006). The presence of a large amount of

FFAs in FOG, which are generally formed by the hydrolysis and oxidation reactions of oils

during deep frying of food, results in a characteristically low pH of FOG (Sharoba and

Ramadan, 2012).

2.4 Sources of fats oil and grease (FOG)

FOG is usually produced at food service establishments, residences, and

slaughterhouses. FOG components are introduced into the sewer system either by direct

dumping into the sewer or by escapes from grease traps (GTs) that are usually installed in

restaurants. The high concentration of FOG in wastewater can be attributed to fast food

restaurant effluent. The menu of these restaurants consists mainly of fried chicken, seafood,

and salad dressing that contain a large amount of FOG (Long et al., 2012).

2.5 Chemical and biological reactions related to FOG

FFAs are chemically active and readily under saponification in the presence of

sodium hydroxide and potassium hydroxide which act as strong agents for generating

metallic soap. To understand the mechanism by which FFAs form during frying, it is very

important to understand the role of sodium and potassium in the reactions. Sodium and

potassium are naturally present in raw food; during deep frying, some sodium ions may be

extracted by the FFA present in the frying oil to form sodium oleate. Sodium oleate decreases

the interfacial tension between the frying oil and the thin layer of water on the surface of the

fried food, causing migration of polar lipids from the frying oil to the fried food (Marchetti et

al., 2007)
2.6 Effects of FOG deposition

FOG tends to stick to the surface of drain and sewer pipes causing clogging that

restricts the flow of sewage and may lead to sanitary sewage overflows (SSOs). SSOs cause

unpleasant odours and insect and rat infestation, and the sewage may make its way into water

sources causing ground and surface water pollution (Suratman et al., 2011). They are very

unpleasant and require quick action from the municipalities to clear the deposition to allay

public concerns. Moreover, FOG deposition can cause corrosion of sewer pipes under

anaerobic conditions, thus reducing the lifetime of the pipe and demanding earlier repair and

replacement of the pipe.

On the other hand, the biological treatment of wastewater with a high concentration of

FOG suspended on the surface may be hindered by sticking of FOG to the pipes and clogging

of the strainer and filters, thus affecting the treatment unit operations. At the last stage of the

wastewater treatment process, FOG is deposited in the sludge making it viscous and waxy,

and thereby reducing the sludge dewatering efficiency (Alade et al., 2011).

2.7 Anaerobic Digestion Process

Anaerobic biodegradation of organic material proceeds in the absence of oxygen and

the presence of anaerobic microorganisms (Weiland, 2010). Anaerobic digestion is the

consequence of a series of metabolic interactions among various groups of various

microorganisms. It occurs in four stages: hydrolysis, acidogenesis, acetogenesis and

methanogenesis. In order for the microorganisms in anaerobic digesters to access the

chemical energy potential of the organic material, the organic matter macromolecular chains

must first be broken down into their smaller constituent parts. These constituent parts or

monomers such as sugars are readily available to microorganisms for further processing. The

process of breaking these chains and dissolving the smaller molecules into solution is called
hydrolysis (Benegas et al., 2007). Therefore hydrolysis of high molecular weight molecules

is the necessary first step in anaerobic digestion. It may be enhanced by mechanical, thermal

or chemical pretreatment of the waste. Hydrolysis step can be merely biological (using

hydrolytic microorganisms) or combined: bio-chemical (using extracellular enzymes),

chemical (using catalytic reactions) as well as physical (using thermal energy and pressure) in

nature. Acetates and hydrogen produced in the first stages can be used directly by

methanogens. Other molecules such as volatile fatty acids (VFA’s) with a chain length that is

greater than acetate must first be catabolised into compounds that can be directly utilised by

methanogens. The biological process of acidogenesis is where there is further breakdown of

the remaining components by acidogenic (fermentative) bacteria. Here VFA’s are generated

along with ammonia, carbon dioxide and hydrogen sulphide as well as other by-products. The

third stage anaerobic digestion is acetogenesis. Here simple molecules created through the

acidogenesis phase are further digested by acetogens to produce largely acetic acid (or its

salts) as well as carbon dioxide and hydrogen. The final stage of anaerobic digestion is the

biological process of methanogenesis. Here methanogenic archaea utilise the intermediate

products of the preceding stages and convert them into methane, carbon dioxide and water. It

is these components that makes up the majority of the biogas released from the system.

Methanogenesis is sensitive to both high and low pH values and performs well between pH

6.5 and pH 8 (Divya et al., 2015). The remaining, non-digestible organic and mineral

material, which the microbes cannot feed upon, along with any dead bacterial residues,

constitutes the solid digestate (Benegas et al., 2007).

2.8 Biological process and microorganisms associated with AD

Since the knowledge of the ecology and function of methanogenic consortia is a

prerequisite for control of AD processes, considerable effort has been made to understand the

microbiology of AD using both culture-dependent and culture-independent molecular

approaches. These analyses (e.g. targeting the 16S rRNA gene), a comprehensive view of the

composition of methanogenic consortia is emerging (McHugh et al., 2003). Recently, several

functionally important anaerobes that play key role in the AD process have been cultivated

(Ziemińsk and Frąc, 2012). Furthermore, the eco-physiology of yet to be cultured organisms

has begun to be elucidated (Collins et al., 2006; Narihiro and Sekiguchi, 2007).

Microorganisms from two biological domains, the bacteria and the archaea, carryout

several interdependent, sequential, and complex biological reactions during the anaerobic

digestion process. The various biological conversion processes in AD must remain balanced

to avoid the accumulation of inhibitory intermediates such as volatile fatty acids. For

simplicity, four phases can be identified: hydrolysis, acidogenesis, acetogenesis,and

methanogenesis. Representatives of over 20 bacterial phyla have been detected in AD sludges

(Narihiro and Sekiguchi, 2007), including Proteobacteria, Chloroflexi, Firmicutes,

Spirochaetes and bacterioidetes. The classes Methanomicrobia, Methanobacteria, and

Thermoplasmata, forming the archaea, are also typical phylotypes (McHugh et al., 2003).

Complex Organic Matter

Carbohydrates, proteins,
fats
Hydrolysis

Soluble Organic Molecules

Sugars, amino acids, fatty
acids
Acidogenesis

Volatile Fatty
acids

Acetic acids H2 + CO2

Biogas Methanogenesis
CH4 + CO2

Figure 1: Schematic flow diagram of anaerobic digestion process (Divya et al., 2015)
2.8.1 Hydrolysis

Hydrolytic fermentative bacteria (including both facultative and obligatory

anaerobic species) facilitate the enzymatic hydrolysis of complex organics such as proteins

and polysaccharides (hydrolases e.g. cellulase, amylase, protease, and lipase). Hydrolases

may be secreted into the environment or be bound to the cell surface (Kaseng et al., 1992).

Polysaccharides are generally converted into simple monomeric or dimeric sugars. For

example, starch is degraded into glucose units by a number of enzymes. Hydrolysis of

cellulose by the cellulase enzyme complex yields glucose. Hemicellulose is biodegraded to a

variety of monosaccharides, including galactose, arabinose, xylose, mannose, and glucose

(Elefsiniotis and Oldham, 1994). The Firmicutes are the best characterized carbohydrate

degraders during anaerobic digestion (Sekiguchi and Kamagata, 2004). Organisms from

subphylum I of the Chloroflexi, which are major populations in AD sludges, were isolated,

and reports suggests that they may also play a key role in the primary degradation of

carbohydrates and cellular materials (such as amino acids) in methanogenic digestion

processes (Nahihiro and Sekiguchi, 2004).

Lipids are hydrolyzed into long and short chain fatty acids and glycerol

moieties by lipases and phoholipases. Lipases catalyze the stepwise hydrolysis of the fatty

acid-ester bonds in triglycerides to release corresponding fatty acids and eventually glycerol.

Clostridia and the Micrococci appear to be responsible for most extracellular lipase

production (Sekiguchi and Kamagata, 2004). Phospholipid metabolism by phospholipases

results in the production of fatty acids and a variety of other organic compounds, depending

on the substrate used. Proteins are broken down into amino acids, small peptides, ammonia,

and carbon dioxide (CO2) by proteases, mainly secreted by Bacteriodes, Butyrivibrio,

Clostridum, Fusobacterium, Selenomonas, and Streptococcus species (Sekiguchi and

Kamagata, 2004). Further fermentation of the sugars, long chain fatty acids, and amino acids
by the hydrolytic and other non-hydrolytic fermentative bacteria results in the generation of a

wide variety of fermentation end-products including acetic, propionic, butyric, and other

short-chain fatty acids, alcohols, ketones, aldehydes, hydrogen and carbon dioxide.

Organisms involved in this stage include: Clostridium thermocellum, Acetivibrio,

Cellulomonas, Bacillus, Bacteroides, Butyrivibrio, Ruminococcus and Eubacterium sp.

(Demirel and Yenigun, 2002).

2.8.2 Acidogenesis

The monomers formed in the hydrolytic phase are taken up by the acidogenic bacteria to be

further degraded into short chain organic acids, alcohols, hydrogen and carbon dioxide. At

this stage, fermentative bacteria process products of hydrolysis into acetate, carbon dioxide,

hydrogen and volatile fatty acids. The intermediate products formed by the process of

hydrolysis are further broken down during acidogenesis (the acidification phase) by

fermentative (acid-forming) bacteria to form lower fatty acids (acetic, propionic and butyric

acid) along with carbon dioxide and hydrogen. In addition, small quantities of lactic acid and

alcohols are also formed. The nature of the products formed at this stage is influenced by the

concentration of the intermediate hydrogen (Sekiguchi and Kamagata, 2004). Specific

microbial population involved here include: Butyrivibrio, Acetivibrio and Propionibacterium

(Ahring et al., 1992).

2.8.3 Acetogenesis

In this stage, acetogenic bacteria, also known as acid formers, convert the

products of the first phase to simple organic acids, carbon dioxide and hydrogen. The

principal acids produced are acetic acid, propionic acid, butyric acid, and ethanol. The

products formed during acetogenesis are due to a number of different microbes, e.g.

Syntrophobacter wolinii, a propionate decomposer and Sytrophomonas wolfei, a butyrate

decomposer. Other acid formers are Clostridium sp, Peptococcus, Anerobus, Lactobacillus,

and Actinomyces (Imachi et al., 2006). Example of microorganisms that are involved here

are: Clostridium and Acetivibrio; Syntrphobacter, Methanobacterium suboxydans (Aiyuk et

al., 2006).

2.8.4 Methanogenesis

Finally, in the last stage, methane is produce by bacteria called methane formers

(also known as methanogens) in two ways: either by means of cleavage of a acetic acid

molecules to generate carbon dioxide and methane, or by reduction of carbon dioxide with

hydrogen. Methane production is higher from reduction of carbon dioxide but limited

hydrogen concentration in digesters results in that the acetate reaction is the primary producer

of methane (Sekiguchi and Kamagata, 2004). The methanogenic bacteria include

Methanobacterium, Methanobacillus, Methanococcus and Methanosarcina. Methanogens

can also be divided in two groups: acetate and H 2/CO2 consumers. Methanosarcina sp and

Methanothrix spp are considered to be important in anaerobic digestion as both acetate and

H2/CO2 consumers (Ferry, 1999).

2.9 Conditions and variables influencing AD

The rate at which the microorganisms grow is of paramount importance in the

AD process. The operating parameters of the digester must be controlled so as to enhance the

microbial activity and thus increase the anaerobic degradation efficiency of the system. Some

of these parameters are discussed below:

2.9.1 Total solid (TS) content and organic loading rate (OLR)

The anaerobic digester design usually will dictate loading rates and contents,

but experience has shown that uniform loading, on a daily basis, of manure with 6 to 10
percent solids generally works best (Akwaka et al., 2014). The load’s retention time in the

digester will typically range from 15 to 30 days. The loaded manure needs to be mixed

regularly to prevent formation of scum or sediments and to maintain contact between the

bacteria and the manure. The agitation also helps to prevent temperature differentiation

between different portions of substrate in digester. Slow agitation over 2 to 3 hours a day is

considered adequate, as too frequent, long and intensive agitation is harmful to the digestion

process. Above all, appropriate mixing action facilitates release of the biogas (Baki et al.,

2004).

2.9.2 Temperature

One of the most important factors in biogas production is maintaining optimal

temperature regime (Oyeleke et al., 2013). Biogas production is possible for temperatures

between 0oC and 95oC. However, with regards to gas fuel and biofertilizer production certain

differentiations in temperature have become necessary. There are two distinct temperature

ranges most suitable for biogas production, and different bacteria operate in each of these

ranges. Mesophilic bacteria optimally function in the 32oC to 43oC range (Atuanya and

Ajuzie, 2005). Thermophilic bacteria are most productive in the 49 o to 60oC range (Atuanya

and Ajuzie, 2005). The benefits of thermophilic range of digestion include higher speed of

substrate digestion and therefore higher biogas yield as well as practically total destruction of

pathogenic bacteria present in the substrate. On the contrary, thermophilic digestion has

higher costs due to maintaining higher temperatures, and thermophilic digesters may be less

stable. Mesophilic digestion range allows for higher amino acid content of fertilizer, but with

incomplete disinfection of the substrate (Ezekoye, 2013). Temperature within the digester is

critical, with maximum conversion occurring at approximately 35°C in conventional

mesophilic digesters. Bacterial digestion in covered lagoons at temperatures below 32oC is

called pschrophilic (meaning a preference for lower temperatures). However, digestion slows
down or stops completely below 15° or 21°C, so these digesters do not produce methane all

of the time (Ezekoye, 2013) .

2.9.3 Retention time

Retention time in the AD reactors, refers to the time that feedstock stays in the

digester. It is determined by the average time needed for decomposition of the organic

material, as measured by the chemical oxygen demand (COD) and the biological oxygen

demand (BOD) of the influent and the effluent material. The longer the substrate is kept

under proper reaction conditions, the more complete its degradation will be. However, the

rate of the reaction decreases with longer residence time, indicating that there is an optimal

retention time that will achieve the benefits of digestion in a cost effective way. The

appropriate time depends on the type of feedstock; environmental conditions and intended

use of the digested material (Oyeleke et al., 2013)

2.9.4 pH

Though all micro-organisms have their optimal pH, in anaerobic digestion the

methanogens are the most sensitive with a working range of 6.5-7.5 and optimal range of 7.0-

7.2 (Bitton, 1999). Usually anaerobic processes are thus operated in the optimal pH range for

methanogens. While 24 the formation of degradation intermediates (VFA) tends to lower the

process pH, ammonia (NH3), formed during degradation of proteins, may increase process pH

and affect the non-adapted micro-organisms. A balanced and adequate content of proteins

and organic acids in the raw materials enhances the ion content and buffering capacity of the

anaerobic process and thus increases its resistance toward organic overloads and enhances the

treatment equilibrium (Alvarez and Liden, 2008).

2.9.5 Carbon to Nitrogen ratio (C: N)

The relative proportions of carbohydrates, proteins and lipids affects the quality

and amount of degradation intermediates (i.e. VFA, LCFA, NH4+-N, NH3) during anaerobic

digestion. Ideal C: N ratio for the growth of micro-organisms is reported to be 25–30:1, but in

practice the C: N ratios are often considerably lower or higher than this (Kizilkaya and

Bayrakli, 2005). Optimal ratio of chemical oxygen demand (COD), nitrogen and phosphorus

for the anaerobic micro-organisms is reported to be 600:7:1 (Hobson and Wheatley 1993;

Mata-Alvarez, 2003).

2.9.6 Mixing

The purpose of mixing in a digester is to blend the fresh material with digestate

containing microbes. Furthermore, mixing prevents scum formation and avoids temperature

gradients within the digester. However excessive mixing can disrupt the microbes so slow

mixing is preferred. The kind of mixing equipment and amount of mixing varies with the type

of reactor and solids content in the digester (Parry et al., 2009).

2.9.7 Presence of toxic substances

Some of the toxic materials that might inhibit the normal growth of pathogens in the

digester include mineral ions, heavy metals and detergents. However, low concentrations of

the mineral ions, such as sodium, potassium, calcium, magnesium, ammonium and sulphur,

are needed for stimulation of bacterial growth. At the same time, if the concentration of these

ions were too high, it would lead to a toxic effect on the growth of methanogenic bacteria.

Addition of substances including soap, antibiotics, and organic solvents should be avoided,

since this would lead to inhibition of the activity of methane producing bacteria (Kossmann

et al., 2003).
2.9.8 Availability of nutrients

Apart from providing a source of carbon and energy through organic substances for the

bacteria to be able to grow, they require other mineral nutrients as well. Except from carbon,

oxygen and nitrogen for the production of biomass a sufficient amount of nitrogen, sulphur,

phosphorous, potassium, calcium, magnesium and a little amount of trace elements such as

manganese, molybdenum, cobalt, zinc, selenium and nickel etc. are also needed. Generally

agricultural residue and municipal sewage as substrate have enough amounts of these

elements. However each of these substances at high concentrations typically would have an

inhibitory effect. So it is important to investigate the nutrient characteristics in order to decide

the amount of the nutrients needed (Bjornsson and Mattiasson, 2007).

2.9.9 Presence of xenobiotic compounds

Inhibitory substances are often found to be the leading cause of anaerobic reactor

upset and failure since they are present in substantial concentration in wastewaters and

organic solid wastes. A wide variety of substances have been reported to be inhibitory to

methanogenic bacteria. Among these inhibitory compounds, organic compounds are

mentioned and more especially aromatic compounds (Kalombo et al., 2012). Aromatic

compounds are naturally present in the environment as degradation products of lignin,

tannins, phenolic amino acids, pigments and other aromatic compounds from plants. Human

activity also contributes to the presence of aromatic compounds in the environment: waste

incineration, petrochemical effluents, industries of paper manufacturing, pharmaceutical and

chemical industries, pesticides etc. are very important sources of aromatic pollution

(Hechtand and Greihl, 2009).

2.9.10 AD Configuration

Digesters can either be batch or continuous depending on the substrate being treated. Batch

systems are simple, cheaper and require less equipment. Continuous digestion allows for

constant gas production. A single (one step digestion) or multiple digesters may be used. For

one step digestion, all stages in the microbial breakdown process, i.e. hydrolysis,

fermentation, anaerobic oxidation and methane production take place simultaneously

particularly for completely mixed processes. It is mainly applicable for the treatment of

sludge, food waste and manure. In some cases, process liquid is returned to the system and

this increases retention time and allows more microbes to remain in the process (Nordberg et

al., 2007). In a two – stage digestion, the first step involves loading material into a digestion

tank where hydrolysis, acetogenesis and acidogenesis occur. It is then introduced into the

methanogenic reactor for methane production. The two-stage process results in fast and

efficient formation of biogas in the second stage with methane concentrations of up to 85%

(Verrier et al., 1987).

2.9.11 Potential substrates for AD

Potential substrates include food waste, manure, crop residue, slaughterhouse waste as well

as stillage and other sulphur containing material. Stillage waste is rich in protein and can

possibly lead to ammonia inhibition. Thus stillage should be co-digested with more

carbohydrate rich material. Food waste is a good feed source for biogas production as it

contains proteins, fats, carbohydrates and various trace elements, this promote a balanced

process (Gunaseelan, 1997). Food waste must not contain a lot of proteins as this will lead to

ammonia inhibition (Fricke et al., 2007). Pigs and chicken manure contain more protein

compared to cattle manure. This is because most of the organic material in the feed has

already been converted into methane in the stomach of ruminants. Various crops and plant
materials such as corn, grain, sugar beets, potatoes, fruit, grass maybe used for biogas

production (Lehtomaki et al., 2007). Many bioenergy crops have a high C: N ratio and

mixing with more nitrogen-rich material can achieve optimum process conditions. Co-

digestion of energy crops with manure can increase methane recovery by 16- 65%

(Lehtomaki et al., 2007). Slaughterhouse waste has high protein and fats contents, thus very

energy rich hence high biogas production potential. Stable process operation can be achieved

with co-digestion (Cuetos et al., 2008).

2.10 Methanogens as key organisms involved in the anaerobic digestion process

Methanogens are ancient organisms that are key players in the carbon cycle accounting for

about one billion tons of biological methane produced annually (Issazadeh et al., 2013).

Methanogens are strict anaerobes which share a complex biochemistry for methane synthesis

as part of their energy metabolism. A number of studies have provided evidence that they are

of economic value. The successive petroleum crisis since 1973 has led to great interest in

alternative forms of energy, including recovery of methane via anaerobic digestion of

wastes .Improvements in the design of digestors have been made possible by advances in

understanding the ecology and physiology of methanogens.

2.10.1 Morphology

Methanogens exhibit a wide variety of shapes and sizes, including rods, regular and irregular

cocci, long-chained rods, spirilla, sarcina and irregular unusual flattened plates. Motility is

sometimes present. Some species can aggregate in clusters. Several species of

Methanosarcina and Methanosaeta contain gas vacuoles. Gram reaction may be positive or

negative even within members of the same genus (Deppendmeier et al., 1998).
2.10.2 Types of methanogens

83 species of methanogens have been described so far (including six synonymous) and are

separated into three main nutritional categories: (a) 61 species (including five synonymous)

of hydrogenotrophs which oxidizes H2 and reduce CO2 to form methane and among them 38

species (including three synonymous) of formatotrophs which oxidize formate to form

methane. (b) Twenty species (including one synonymous) of methylotrophs uses methyl

compounds as methanol, methylamines, or dimethylsulphide and of which 13 species are

obligate methylotrophs (Oremland et al., 1989).

2.10.3 Physiology of methanogens

The metabolic characteristic that unites the rather diverse species of methanogenic

bacteria is the capacity to couple hydrogen oxidation with the concomitant reduction of

carbon dioxide. Furthermore, the ability of many species to grow autotrophically indicates the

enormous biosynthetic capabilities of these microbes. Methanogens differ from other

autotrophs (organisms that proliferate with CO2 as the sole carbon source) in that their CO 2

metabolism involves both fixation to cell carbon and reduction to methane.

2.10.4 Nutritional characteristics of methanogens

The growth of virtually all methanogens is stimulated by acetate, and for some species, by

certain amino acids. Many methanogens require complex additions in the culture medium,

such as yeast extract or casein digests, and some rumen methanogens require a mixture of

branched chain fatty acids. Vitamins such as riboflavin, pantothenic acid, thiamin, biotin and

p-aminobenzoate are also required by some methanogens. All use NH 4+ as a nitrogen source

and a few species are known to fix molecular nitrogen. Trace metals, such as nickels, a
compound of certain enzymes and coenzymes, iron and cobalt are required by all

methanogens (Stetter and Gaag, 1983).

2.10.5 Ecology of methanogens

Methanogens are widely distributed in nature, but confined to strictly anaerobic

environments. In addition to aquatic sediments (ponds, marshes, swamps, rice soils, lakes,

and oceans), other methanogenic habitats include the intestinal tracts of man and animals

(especially the rumen of herbivores), sewage digesters, landfills, heart wood of living trees,

hot springs, decomposing algal mats, oil wells, and midocean ridges. In these habitats, the

methanogens occupy the terminal niche in the transfer of electrons generated by the

anaerobic degradation of organic matter (Issazadeh et al., 2013).

2.11 By-products of anaerobic digestion

Anaerobic digestion is a cost-effective way to manage biodegradable waste

because it produces biogas and digestate. A wide range of application is common for biogas,

which can be used like any other fuel gas for household energy and industrial use. Some

common applications include gas cookers, refrigerators, engines, incubators, radiant heaters

and biogas lamps (Bjornsson et al., 2000). Biogas covers a variety of markets, including

electricity, heat and transportation fuels. Whereas using the gas for direct combustion in

household stoves and gas lamps is common in some countries, producing electricity from

biogas is still relatively rare in most developing countries.

2.11.1 Biogas

Biogas produced during anaerobic digestion is primarily composed of methane and

carbon dioxide, with small amounts of hydrogen sulphide and ammonia. Trace amounts of

hydrogen, nitrogen, carbon monoxide, saturated or halogenated carbohydrates and oxygen are
occasionally present in the biogas. Usually the mixed gas is saturated with water vapour and

may contain dust particles and siloxanes. There are several different options for converting

biogas to energy. Numerous factors such as project goals, local energy policies, infrastructure

availability, and markets for renewable energy products will dictate what end use best fits the

project (Christensen, 1995). Unlike intermittent renewable energy alternatives such as wind

and solar power, biogas delivers a continuous source of energy with a very high capacity

factor. The flexibility and reliability of biogas systems are very important assets. Currently 37

states recognize biogas in their state renewable energy goals, and the U.S. government has set

a target for 20 percent of the electricity consumed by Federal agencies to be from renewable

energy by 2020 (Weiland, 2003). Biogas can assist in achieving these goals and provide

many energy benefits. Specific commercially proven energy uses for biogas include:

1. Thermal applications:

Biogas is used directly on-site to heat digesters and buildings/maintenance shops, to fuel

boilers or kilns, and to generate heat or steam. Electricity is produced through an internal

combustion engine, gas turbine, or microturbine technologies for on-site use or sale to the

electric grid. Combined heat and power (CHP) systems increase overall energy efficiency of

electricity systems by producing heat and electricity at the same time, which can be used for

heating, cooling, dehumidification or other process applications. Unlike intermittent

renewable energy sources, biogas systems are providing continuous dispatchable electricity

onto the grid (Weiland, 2003).

2. Industrial application:

Biogas can be used in industrial applications to offset use of natural gas, propane, fuel oil, or

other fossil fuels. Many industries such as sugar refineries, distilleries, dairies, and paper

mills generate processing and waste water that can be digested directly on site. The resulting
biogas can then be used for fuel in equipment such as boilers, kilns (e.g., cement, pottery, and

brick), sludge dryers, infrared heaters, paint shop oven burners, tunnel furnaces, process

heaters, and blacksmithing forges or for other direct thermal applications.

3. Application as vehicle fuel

Upgraded biogas can be converted to various vehicle fuels including compressed natural gas,

liquefied natural gas, hydrogen, and liquid transportation fuels.

4. Biogas drives economic growth

Biogas systems offer a wide range of potential revenue streams, growing jobs and boosting

economic development in the community. These systems can also improve rural

infrastructure for waste management and distributed energy delivery improving community

health, resiliency, and viability. Biogas systems can produce high-quality, concentrated liquid

organic fertilizer for improved land management and increased crop yield, building and

maintaining healthy and productive soils needed for sustainable food production. Along with

generating revenues from the sale of renewable energy products, outputs from biogas systems

can offer avoided costs of on-site electricity, heat, and transportation fuel. Renewable

electricity can be sold into the power grid, and is often the primary driver for many biogas

project investments. However, energy off take contracts are often insufficient to fully finance

a biogas system, and to be feasible many projects must realize the broader value of co-

products, such as separated nutrients, marketable fertilizers and soil amendments. Separated

fibers from the effluent stream can also reduce operational expenses or increase revenue

through the production and sale of animal bedding (Bjornsson and Mattiasson, 2007).
2.11.2 Digestate

Digestate liquids and solids (what remains after digestion) can produce additional economic

benefits. The digestate has soil enhancement qualities and can be applied to growing crops,

making it a marketable and valuable soil amendment. Reducing the need for synthetic

fertilizers, the digestate delivers nutrients in a form that is more consistent, more readily

absorbed, and more concentrated than raw manure. The use of digestate could provide a cost-

savings to the farmer when compared to the purchase of synthetic fertilizers. Storage, mixing,

pumping, and spreading digestate are easier than handling undigested organic materials,

which can reduce energy demand and handling costs (Bove and Lunghi, 2006). Biogas

production facilities designed to process landfill gas or source-separated organics (SSOs)

provide economic benefits to the municipalities or waste management companies that own

these facilities, as well as the broader community. Direct revenue sources include commercial

tipping fees for SSOs. An emerging benefit associated with biogas systems that use anaerobic

digesters is the extraction of valuable nutrients, which supports environmentally and

economically sound waste management. A number of systems, technologies, and procedures

are available for nutrient recovery. The degree to which nutrients are removed depends on the

value of the recovered nutrients, the need to produce clean water, and the economics of the

technology used. Recovered nutrients offer an opportunity to create a value-added product

that can be sold off-site as an organic amendment or as an organic fertilizer (Bove and

Lunghi, 2006).

2.12 Advantages of anaerobic digestion

Anaerobic digestion contributes to reducing the greenhouse gases. A well-

managed AD system maximizes methane production without the release of any gas to the

atmosphere, thereby reducing overall emissions. AD provides a source of energy with no net
increase in atmospheric carbon which contributes to climate change (Angelidaki and Sanders,

2004).

The feedstock for AD is a renewable source, and therefore is sustainable.

Energy generated through this process can help reduce the demand for fossil fuels. The use of

the digestate also participates in its reduction by decreasing synthetic fuels use in fertilizer

manufacturing, which is an energy intensive process. AD creates an integrated management

system which reduces the likelihood of soil and water pollution to happen, compared to

disposal of untreated animal manure/slurries. In terms of generating income, the advantage of

AD is to convert residues into marketable products: biogas, soil conditioner, liquid fertilizer.

It can also contribute to the economic viability of farms by keeping costs and benefits within

the farm if the products are used on-site (Hill and Bolte, 2000)

2.13 Costus afer as a natural buffer for anaerobic digestion

Costus afer which belongs to the family Zingiberaceae is a monocot and a

relatively tall, herbaceous, unbranched tropical plant with creeping rhizome. It is commonly

found in moist or shady forest of West and Tropical Africa (Iwu, 2009). Diogenin is a very

important raw material found in C. afer used as a precursor in the synthesis of a number of

steroid drugs including corticusteroids, sex hormones, oral contraceptive and anabolic agents

(Aweke, 2007). In this study, Costus afer extract and slurry were used in the anaerobic

digester to maintain a stable pH and ensure optimum conditions for methanogenic bacteria to

produce biogas.
 CHAPTER THREE

Materials and Methods

3.1 Collection of sample

Fats, oil and grease (FOG) sample was collected from the drains of some

restaurants located along Ikot Ekpene Road, in Uyo, Akwa Ibom State and transported to

microbiological laboratory for analysis.

3.2 Equipment and Supplies

The following equipment and supplies were used in the study: 100ml amber serum bottles

(Gerresheimer 61020G, USA), Crimper (JG Finneran 9300-20, USA), 20mm aluminium cap

with central moulded septum combination seal (FB 67567, Fisher Scientific, UK), an active

seed inoculum (cow dung), needle/syringe, crucibles and gas pak.

3.3 Experimental set-up

Fresh cow dung in reaction vessel

incubated at 45 ± 2°C until no biogas was detected

To fume hood

Blender
Waste + inoculum (cow dung) + water

t°

Substrate + inoculum equilibrated at 37 ± 2°C

Batch reactor in thermostat Graduated reverse cylinder device
and spiked with Costus afer extract/slurry
regulated waterbath

Figure: Experimental Set-up for the anaerobic digestion of Fats, oil and grease (FOG)
The biomethane potential assay (BMP) is used as an indication of the anaerobic

biodegradation ability because it shows the experimental value of the maximum quantity of

methane produced per gram of volatile solid. The BMP is measured with BMP test, which

consists of measuring the biomethane or biogas produced by a known quantity of waste in

batch and anaerobic condition (Esposito et al., 2012). To measure the sample digestion and

biogas production, a modified method of Owen et al., (1993) and Angelidaki et al., 2003 in

which FOG pretreated with Costus afer was used as substrate. The experiment was set up in a

batch mode and involved the use of 100ml amber serum bottles, (Gerresheimer 61020G,

USA) and 20mm aluminium cap with central moulded septum combination seal (FB 67567,

Fisher Scientific, UK) as reactor. 60ml of blended substrate (FOG) inoculated with 5mg cow

dung as seed inoculum will be allowed to equilibrate for one hour at 350C, introduced into the

reactors and sealed using standard hand operated crimper, 20mm cap size (JG Finneran 9300-

20, USA). The experiment was carried out in duplicate reactors incubated at 35±0.2 0C with

continuous agitation at 120rpm for 28 days. Gas volume was determined by volumetric

method (Vakke and Verstraete, 1983) by connecting the reactor to a graduated reverse

cylinder device containing water as a barrier solution and the liquid displacement measured

and converted to biogas volume using the formular:

Biogas (mL) = π r 2 h × K + H−h

K
Where:

r = internal radius of the column (cm);

h = production of biogas as water level decreased in the column (cm);

H = working length of gas collection column (cm);

k = standard atmospheric pressure (1033cm water gauge).

The total solids (TS) and Volatile Solids (VS), and pH were determined before and after the

incubation period using standard methods (APHA, 2005). Also, culture-dependent

microbiological analysis of the fresh sample and digestate was carried out using appropriate

methods.

3.4 Inoculation and Incubation of the digesters

50ml of the FOG sample was mixed with 10mg cow dung for the control

experiment while 25ml of FOG was blended with 10mg cow dung and 25g of Costus afer

slurry for the treatment and were introduced into the 100ml amber serum bottles

(Gerresheimer 61020G, USA). They digesters were then sealed using standard hand operated

crimper, 20mm cap size (JG Finneran 9300-20, USA). The inoculated digesters were then

incubated in a water bath at a temperature of 35±0.20C for 28 days.

3.5 Biogas Measurement

The volume of gas produced was measured by volumetric method which involved

connecting the reactor to a graduated reverse cylinder device containing water as a barrier

solution and the liquid displacement measured and converted to biogas volume on a 3 day

interval for 28 days.

3.6 Measurement of total solids (TS), volatile solids (VS)

3.6.1 Determination of Total solids: Determination of total solid is an effective way of

finding out the amount of nutrient that will be available for bacterial action during digestion.

It is made up of digestible and non-digestible materials. Meynell (1982) method was used. 5g

of the raw waste were dried in an oven at 180 0C for 5hrs. The dried sample was cooled in a

desiccator and then weighed. The weight obtained after all moisture loss is the total solid.
3.6.2 Determination of Volatile solids: Volatile solid is the true organic matter

available for bacterial action during digestion. The analysis was carried out using Meynell

(1982) method. The sample from the total solid determination was heated in a muffle furnace

at 6000C for 30minutes. After this, the heated residue was cooled in a desiccator and

weighed.

3.7 Microbiological analysis

Microbial analysis involves the isolation, identification and number of micro-organism

present in the set up. This is the Miles and Misra (1935) method.

3.7.1 Media and reagents

The media used for this research was nutrient agar and reinforced clostridia agar. The

medium was prepared according to the manufacturer’s instructions and sterilized in an

autoclave at 121°C for 15 min.

3.7.2 Isolation and characterization of microorganisms

The pour plate method was adopted where 1ml of the mixture inoculated into the digesters

(FOG, cow dung and Costus afer slurry) for experiment and (FOG, cow dung) for control

was added to 9ml of sterile water in a test-tube. 1ml from this was then transferred to another

test-tube (10-2) and properly mixed. The transfer continued until the seventh (10 -7) test-tube.

After the serial dilution, 1ml was aseptically transferred from 10-5 and 10-6 tubes into sterile

petri dishes in duplicate. Molten nutrient agar and reinforced clostridia agar (for anaerobes)

that has cooled to about 450C was then poured aseptically into the petri dishes. These were

gently swirled for proper mixing, covered and allowed to set after which the plates were

incubated at 370C for 24-28hrs for aerobes and the anaerobic jar with gas pak was used to

incubate the anaerobic microorganisms for 24-28hrs (Esposito et al., 2012).

3.7.3 Purification and maintenance of microbial isolates

Developed single, discrete colonies representing different organisms were respectively sub-

cultured into sterile nutrient agar slant and preserved as stock cultures for identification

purpose.

3.8 Identification tests for bacterial isolates

Following tests was carried out for the identification of the isolates:

3.8.1 Gram staining

This test was performed for the identification of bacterial isolates as either

Gram positive or Gram negative based on stain retention capabilities. A heat-fixed smear of

the bacterial culture was prepared on a sterile glass slide by emulsifying a loopful of the test

organism with distilled water using a sterile wireloop. The smear was flooded with crystal

violet for one minute. It was rinsed with slow running water and flooded with Lugol’s iodine

solution for one minute. The iodine solution was drained off and the slide was gently rinsed

with water. The smear was decolourized with 75% alcohol and rinsed with distilled water

after 30 seconds. The smear was counterstained with safranin for one minute and rinsed with

water. The slides were allowed to drain, blotted dry and observed under oil immersion

objective of the microscope. The bacterial cell which retained the crystal violet appeared

purple (Gram positive) while the one which retained the safranin counter stain appeared red

(Gram negative).

3.8.2 Catalase test

A drop of hydrogen peroxide (3%) was made on a clean grease free slide. Using a sterile

inoculating loop, a 24hrs colony of each of the test organisms was picked and smeared on the
hydrogen peroxide. Bubbles of gas indicated positive result whereas no bubble shows

negative test result (Etok et al., 2004).

3.8.3 Motility test

This test demonstrates the ability of an organism to move due to the presence of flagella. This

test is carried out in a semi-solid agar using a sterile inoculating wire to pick the test

organism and stabbed through the center of the semi-solid nutrient agar. This was incubated

for 24hrs at 370C and examined for motility indicated by a diffuse, hazy growth that spread

throughout the medium (Etok et al., 2004).

3.8.4 Indole test

Indole test is performed to determine the ability of the organism to split tryptophan molecule

into Indole. Indole is one of the metabolic degradation product of the amino acid tryptophan

Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating

tryptophan with the production of Indole, Pyruvic acid and ammonia . A loopful of the test

organisms were cultured in peptone water (5ml) for 24-48hrs. After incubation, 0.5ml of

Kovac’s reagent was added to the culture broth and shaken gently. This was examined for a

red colour ring on the surface layer within 10minutes, which is indicative of a positive result.

3.8.5 Citrate test

This is based on the ability of the organism to utilize citrate as its sole source of carbon. In

this test, Simmon’s citrate agar was prepared and sterilized according to manufacturer’s

instruction, poured into sterile petri dishes and allowed to solidify before inoculation using

streak plate method. The plates were incubated at 370C for 24-48hrs. After incubation, a

bright blue colour indicates a positive test result and no change in colour of medium indicates

a negative test result.

3.8.6 Oxidase test

This test is used to identify organisms which produce the enzyme, oxidase. A piece of filter

paper impregnated with oxidase reagent was placed in a clean petri dish and a colony of test

organism was then smeared on it and observed for the development of a blue-purple colour

within few seconds (Etok et al., 2004).

3.8.7 Sugar fermentation test

This test demonstrates the ability of some organisms to ferment certain sugars (glucose,

sucrose, lactose and mannitol) with the production of acid and gas or acid only or no

fermentation at all. The various sugar media containing peptone water base, phenol red

indicator and Durham tubes (inverted) prepared and sterilized in the autoclave. After cooling,

the test organisms were inoculated at 37 0C for 24-48hrs. The tubes were observed for

production of acid (indicated by a change in colour of the medium to yellow) and gas (as

collected in the inserted Durham tubes).

3.8.8 Spore staining

Bacteria in genera such as Bacillus and Clostridium produce quite a resistant structure

capable of surviving for long periods in an unfavourable environment and then giving rise to

a new bacterial cell. This structure is called endospore since it develops within the bacterial

cell. The isolate was aseptically transferred to a sterile grease free slide, allowed to air dry

and then heat fixed. The slide was then placed on a boiling water bath equipped with a

staining rack and covered with a paper toweling that has been cut the same size as the

microscope slide. The paper was soaked with malachite green solution and heated for 5-6min

until the malachite green began to steam. The paper was removed using forceps, allowed to

cool and rinsed with water for 30secs. It was then counterstained with safranin for 60secs
after which the slide was rinsed with water for 30secs. The slide was allowed to air dry and

then examined under oil immersion. The spores, both endospores and free spores, stain green;

whereas vegetative cells stain red (Etok et al., 2004).

 CHAPTER FOUR

4.0 Results/Discussion

Total Solid Volatile Solids

10

9
8
7
Concentration (mg L⁻¹)

6
5
4

3
2
1

0
Initial final
Figure 3: Initial and Final Total and Volatile Solids

Control Treatment

70
Cummulative biogas (cm³)

60
50
40
30
20
10
0
0 4 6 9 12 15 18 21 26 28
Time (d)
Figure 4: Cumulative Biogas Production for against time (d)
 Treatment Control
6.86

6.84
Microbial counts (Log₁₀ CFU mL⁻¹)

6.82

6.8

6.78

6.76

6.74

6.72

6.7
Aerobic Anaerobic
Incubation
Figure 5: Microbial Count (Log₁₀CFU mL⁻¹)

Table 1: Morphological and Biochemical Characterization of Isolates

Cell shape

Oxidase

Glucose

Sucrose

Spore
Indole

Lactose

Mannitol
Catalase

Citrate

Motility
Isolates Probable Organism
ReactionGram

A Rod + + ⁻ + ⁻ A ⁻ A ⁻ + + Bacillus

B Rod + ⁻ ⁻ ⁻ ⁻ AG AG AG AG + + Clostridium

C Rod + + ⁻ + ⁻ AG A ⁻ ⁻ ⁻ ⁻ Propionibacterium

1 Cocci + ⁻ ⁻ + ⁻ AG AG A AG ⁻ ⁻ Streptococcus

2 Rod + ⁻ ⁻ ⁻ ⁻ AG AG A AG ⁻ ⁻ Micrococcus

3 Rod + + + ⁻ ⁻ AG A AG A ⁻ ⁻ Corynebacterium

4 Rod + ⁻ + + ⁻ A A AG AG ⁻ ⁻ Peptostreptococcus
Key: A = Acid, G = Gas, ⁻ = Negative, + = Positive
4.1 Discussion

Anaerobic digestion involves a sequence of complex biochemical process, in

which organic compounds are mineralized to biogas through a series of reactions mediated by

several groups of microorganisms. The amount of gas produced varies with biochemical

characteristics of organic wastes, consortia of microorganisms, pH, and temperature

(Gopinath et al., 2014). This study sought to determine the biogas potential of Fats, oil and

grease (FOG) as well as the effect of Costus afer slurry/extract on the process.

Microbiological analysis of the sample aerobically showed a microbial count of

6.85(Log₁₀CFU/ml) and 6.75(Log₁₀CFU/ml) for the treatment and control experiments

whereas the anaerobic culturing had a microbial count of 6.8(Log₁₀CFU/ml) and

6.85(Log₁₀CFU/ml) for both set-up as show in figure 5. It was observed that the digester with

the Costus afer slurry/extract had a cumulative biogas production of 59.49cm³ whereas the

control experiment had a cumulative biogas production of 29.25cm³, implying that the

treatment produced two times better than the control with a difference of 2.033cm³ as shown

in figure 4. This clearly shows that the Costus afer slurry/extract enhanced the biogas

production capacity of the substrate (FOG). Results of the total and volatile solids

determination showed a reduction in the value of total solids from 8.755mgL⁻¹ (initial value)

to 4.3mgL⁻¹ (final value) as shown in figure 3. Also, the volatile solids reduced to 0.85mgL⁻¹

at the end of the digestion from its initial value of 1.35mgL⁻¹, explaining the drop in the

volume of gas produced towards the concluding part of the experiment. This is due to the

depletion of organic matter available for bacterial action during the digestion (Bagi et al.,

2007). However, Long et al., (2012) reported that co-digestion of FOG with municipal

biosolids caused a 30-80% increase in biogas production. Also, Gujer and Zehnder, 1983,

showed that co-digestion of FOG and sewage sludge produced more methane than digesters

with sludge alone due in part to the lower (negative mean oxidation state) of carbon in fats as
compared to carbohydrates and proteins. From the above facts, it can be suggested that co-

digestion of FOG with other organic wastes can greatly improve the amount of biogas

produced as against digesting FOG alone as it is seen in this present study.

4.2 Biochemical Characterization of Bacterial Isolates

Biochemical characteristics of microorganisms were studied to identify the genus of

unknown bacteria. Microorganisms are extremely versatile and their range of metabolic

capacities is very large (Norrell and Messley, 2003). These characteristics can be used to

demonstrate the exceptional diversity of organisms. A biochemical characteristic of bacterial

isolates is shown in Table 1. Indole is a nitrogen metabolism test. Positive to indole test

indicates that bacteria can act upon amino acids and undergo deamination and hydrolysis to

form pyruvic acid and ammonia which leads to the production of methane and CO 2. The

negative results showed that isolates were unable to produce indole as a result of typtophan

breakdown due to lack of typtophanase in the cell (Hwang et al., 2001). Positive to citrate is

used to determine the capacity of microorganisms that can utilize citrate as a sole carbon

source, breaking down to oxaloacetate and acetate and further into pyruvate and carbon

dioxide and also produces ammonia with sodium citrate resulting in alkaline condition.

Alkalinity is an important parameter in anaerobic digestion because it provides enough

buffering capacity to neutralize any possible volatile fatty acids accumulation in the reactor

and to maintain pH around 6.7 to 7.4 for stable operation (Callander and Barford, 1983).

Carbohydrate fermentation test was used to determine the ability of the isolates to utilize four

sugars: glucose, lactose, sucrose and mannitol to produce either acid or gas, which is the main

function of anaerobic bacteria in biogas production. Catalase is an enzyme produced by many

organisms which converts hydrogen peroxidases into water and oxygen and cause foaming

due to release of oxygen. The bacteria that possess this enzyme are usually aerobic or

facultative anaerobes. Oxidase is an enzyme involved in oxidation reaction of

microorganisms due to the presence of cytochrome oxidase system in aerobic and facultative

anaerobic bacteria (Norrell and Messley, 2003). Based on the results of the biochemical tests

conducted the following bacterial were isolated from the substrate: Bacillus, Clostridium,

Proprionibacterium, Streptococcus, Micrococcus, Corynebacterium and Peptostreptococcus

as shown in table 1.
 CHAPTER FIVE

5.0 Conclusion

Anaerobic digestion has been, and continues to be, one of the most widely used process for

the stabilization of biosolid waste, such as those from agro and municipal wastes to industrial

waste. The widespread use of this technology stems from its potential advantages including

the production of energy (methane) reduction of waste volume requiring ultimate disposal

and ensuring a cleaner and greener environments. This study has shown that FOG wastes can

actually be converted to something useful for the benefit of humanity. Also, the process of

anaerobic digestion of FOG can be enhanced by the use of Costus afer as a natural buffer to

ensure the pH of the system is optimal for the methanogenic microorganisms involved in

biogas production.
5.1 Recommendation

Anaerobic digestion is a proven technology for processing source-separated organic wastes

and has experienced significant growth, hence the need for the government of Nigeria to look

towards this direction as a deliberate plan towards enhancing economic and health of its

citizens by establishing waste treatment plants across various parts of the country to utilize

the huge wastes generated on a daily basis into useful product like biogas which can serve as

an alternative source of electricity and also a means of reducing the teaming number of

unemployed graduates across the country.

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