ABSTRACT
The aim of this study is to ascertain the biocide effect and microbial population in formulated fluid. A total of four (4) biocides namely: sodium hexametaphosphate, hydrogen peroxide, ciprofloxacin and fluconazole were treated on microorganisms isolated from the formulated fluid. A total of three (3) bacteria spp and two (2) fungal specie were isolated belonging to the genera of Escherichia coli, Salmonella specie, Staphylococcus aureus, Aspergillus flavus and Aspergillus niger respectively. From this study, the biocidal effect of sodium hexametaphosphate on Escherichia coli ranged from 0.2x 105 cfu/g to 0.47x105cfu/g followed by salmonella sp which ranged from 0.19x 105 cfu/g to 0.46x105cfu/g. The biocidal effect of Hydrogen peroxide on Escherichia coli ranged from 0.32x105cfu/g to 0.37x105cfu/g followed by Salmonella sp which ranged from 0.22x105cfu/g to 0.35x105cfu/g. The biocidal effect of sodium hexametaphosphate on A. niger ranged from 0.32x105cfu/g to 0.33x105cfu/g and A. flavus ranged from 0.25x105cfu/g to 1.07x105cfu/g’ While the biocidal effect of hydrogen peroxide on A. niger ranged from 0.04x105cfu/g to 0.08x105cfu/g and A. flavus ranged from 0.01x105cfu/g to 0.12x105cfu/g.
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgments iv
Table of Contents v
List of Tables vi
List of Figures vii
Abstract viii
1.0 CHAPTER
ONE
1.1 INTRODUCTION 1
1.1.1 Biocide 1
1.2 Types of Biocides 3
1.3 Uses of Biocides 5
1.4 Sources of Biocides 7
1.5 Application of Biocides 7
1.6 Household Products 8
1.6.1 Biocides as Disinfectants 9
1.6.2 Biocides as Food
Preservatives 9
1.6.3 Biocides in Animal
Husbandry 10
1.6.4 Biocides as Teat Dips 10
1.6.5 Biocide Use in Fish Farming 10
1.7 Factors Affecting Microbial Population of
Change of Fat to Liquid 10
1.7.1 pH and Acidity 10
1.7.2 Nutrient Content 12
1.7.3 Moisture 13
1.7.4 Temperature 14
1.7.5 Elements
Present 14
1.7.6 Fats 14
1.8 Aim and Objectives 16
1.8.1 Objectives 16
2.0 CHAPTER
TWO
2.1 LITERATURE
REVIEW 17
2.1.1 Effect of Biocides on Microorganisms 17
2.1.1.1 Chlorine Compounds 17
2.1.2 Alcohols 17
2.1.3 Cationic Biocides 19
2.1.4 Phenols 20
2.1.5 Triclosan as a Biocide 21
2.2 Microorganisms and Biocide 22
2.1.1 Intrinsic Resistance to Biocides 22
2.1.2 Mycobacteria 24
2.1.3 Other Gram-Positive Bacteria 24
2.1.4 Bacterial Endospores 25
2.1.5 Physiological (phenotypic) Adaptation 25
2.3 Microorganisms Associated with Fats 26
2.4 Microorganisms Isolated From Oils from Fat 27
2.4.1 Yeast 27
2.4.2 Microalgae 27
2.4.3 Bacteria 28
2.5 Microorganisms Associated with Fluids from
Fats 29
3.0 CHAPTER
THREE
3.1 Materials and Methods 33
3.1.1 The
Study Area 33
3.2 Sample
Collection 33
3.3 Media
Used 33
3.4 Sterilization 34
3.5 Microbiological
Analysis 34
3.6
Identification and Characterization of Isolates 34
3.7 Gram
Staining 34
3.8 Biochemical
Cultural Characteristics 35
3.8.1 Catalase Test 35
3.8.2 Coagulase
Test 35
3.8.3 Motility
Test 35
3.8.4 Indole
Test 35
3.8.5 Oxidase
Test 36
4.0 CHAPTER FOUR
4.1 RESULTS 37
5.0 CHAPTER FIVE
5.1 DISCUSSION, CONCLUSION AND RECOMMENDATION
5.1.1 Discussion 48
5.2 Conclusion 50
5.3 Recommendation 50
Reference 51
Appendix 54
LIST OF
TABLES
1
Biocidal Effects on the Isolated
Bacteria Population at
18, 24 and 48 hours
Incubations. 38
2
Biocidal Effect on the Isolated Fungi
Population at Days of Incubations 40
3
Identification and Characterization of Isolates 41
4
Identification of the Fungi Isolated 42
LIST
OF FIGURES
Page No. Figure Title
1.
A
graph of TVC and Concentration for A.
flavus against Time 43
2. A
graph of TVC and Concentration for A.
niger against Time 44
3. A
graph of TVC and Concentration for E.
coli against Time 45
4. A
graph of TVC and Concentration for Salmonella
sp. against Time 46
5. A
graph of TVC and Concentration for S.
aureus against Time 47
1.0 CHAPTER
ONE
1.1 INTRODUCTION
1.1.1 Biocide
A biocide is defined in the European
legislation as a chemical substance or microorganism intended to destroy,
deter, render harmless, or exert a controlling effect on any harmful organism.
The US Environmental Protection Agency (EPA) (2011) uses a slightly different
definition for biocides as "a diverse group of poisonous substances
including preservatives, insecticides, disinfectants, and pesticides used for
the control of organisms that are harmful to human or animal health or that
cause damage to natural or manufactured products". When compared, the two
definitions roughly imply the same, although the US EPA definition includes
plant protection products and some veterinary medicines (Lester, 2013).
The terms "biocides" and
"pesticides" are regularly interchanged, and often confused with
"plant protection products". To clarify this, pesticides include both
biocides and plant protection products, where the former refers to substances
for non-fat and feed purposes and the latter refers to substances for fat and
feed purposes. The biocidal active substances are mostly chemical compounds,
but can also be microorganisms (e.g. bacteria). Biocidal products contain one
or more biocidal active substances and may contain other non-active
co-formulants that ensure the effectiveness as well as the desired pH,
viscosity, colour, odour, etc. of the final product. Biocidal products are available
on the market for use by professional and/or non-professional consumers.
Although most of the biocidal active substances have a relative high toxicity,
there are also examples of active substances with low toxicity, such as carbon
iv oxide (CO2), which exhibit their biocidal activity only under
certain specific conditions such as in closed systems. In such cases, the
biocidal product is the combination of the active substance and the device that
ensures the intended biocidal activity, i.e. suffocation of rodents by Carbon
iv oxide (CO2) in a closed system trap. Another example of biocidal
products available to consumers are products impregnated with biocides (also
called treated articles), such as clothes and wristbands impregnated with
insecticides, socks impregnated with antibacterial substances etc. Biocides are
commonly used in medicine, agriculture, forestry, and industry. Biocidal
substances and products are also employed as anti-fouling agents or
disinfectants under other circumstances: chlorine, for example, is used as a
short-life biocide in industrial water treatment but as a disinfectant in
swimming pools. Many biocides are synthetic, but there are naturally occurring
biocides classified as natural biocides, derived from, e.g., bacteria and
plants (Zhou, 2010).
In Europe the biocidal products are divided
into different product types (Product Type), based on their intended use. These
product types, 22 in total under the BPR, are grouped into four main groups,
namely disinfectants, preservatives, pest control, and other biocidal products.
For example, the main group "disinfectants" contains products to be
used for human hygiene (Product Type 1) and veterinary hygiene (Product Type
3), main group "preservatives" contains wood preservatives (Product
Type 8), the main group "for pest control" contains rodenticides (Product
Type 14) and repellents and attractants (Product Type 19), while the main group
"other biocidal products" contains antifouling products (Product Type
21). It should noted that one active substance can be used in several product
types, such as for example sulfuryl fluoride, which is approved for use as a
wood preservative (Product Type 8) as well as an insecticide (Product Type 18).
Biocides can be added to other materials (typically
liquids) to protect them against biological infestation and growth. For
example, certain types of quaternary ammonium compounds (quats) are added to
pool water or industrial water systems to act as an algicide, protecting the
water from infestation and growth of algae. It is often impractical to store
and use poisonous chlorine gas for water treatment, so alternative methods of
adding chlorine are used. These include hypochlorite solutions, which gradually
release chlorine into the water, and compounds like sodium
dichloro-s-triazinetrione (dihydrate or anhydrous), sometimes referred to as
"dichlor", and trichloro-s-triazinetrione, sometimes referred to as
"trichlor". These compounds are stable while solids and may be used
in powdered, granular, or tablet form. When added in small amounts to pool
water or industrial water systems, the chlorine atoms hydrolyze from the rest
of the molecule forming hypochlorous acid (HOCl) which acts as a general
biocide killing germs, micro-organisms, algae, and so on. Halogenated hydantoin
compounds are also used as biocides.
1.2 Types of Biocides
There are many different types of biocides,
divided in 4 groups and 23 product-types:
MAIN GROUP 1: Disinfectants and general biocidal products
Product-type 1: Human hygiene biocidal
products
Product-type 2: Private area and public
health area disinfectants and other biocidal products
Product-type 3: Veterinary hygiene biocidal
products
Product-type 4: Fat and feed area
disinfectants
Product-type 5: Drinking water disinfectants
MAIN GROUP 2: Preservatives
Product-type 6: In-can preservatives
Product-type 7: Film preservatives
Product-type 8: Wood preservatives
Product-type 9: Fibre, leather, rubber and
polymerised materials preservatives
Product-type 10: Masonry preservatives
Product-type 11: Preservatives for
liquid-cooling and processing systems
Product-type 12: Slimicides
Product-type 13: Metalworking-fluid
preservatives
MAIN GROUP 3: Pest control
Product-type 14: Rodenticides
Product-type 15: Avicides
Product-type 16: Molluscicides
Product-type 17: Piscicides
Product-type 18: Insecticides, acaricides and
products to control other arthropods
Product-type 19: Repellents and attractants
MAIN GROUP 4: Other biocidal products
Product-type 20: Preservatives for fat or
feedstocks
Product-type 21: Antifouling products
Product-type 22: Embalming and taxidermist
fluids
Product-type 23: Control of other vertebrates
1.3 Uses of Biocides
Biocides are used extensively in healthcare
settings for different applications: the sterilization of medical devices; the
disinfection of surfaces and water; skin antisepsis; and the preservation of
various formulations. In addition, there are now numerous commercialized
products containing low concentrations of biocides, the use of which is
controversial. Some professionals believe that the indiscriminate usage of
biocides in the healthcare environment may not be justified and is detrimental
in the long term, for example, by promoting the emergence of bacterial
resistance to specific antimicrobials (Russell et al., 2013; Russell, 2012). The indiscriminate use of
disinfectants in the hospital environment is not a new problem as it was raised
in the 1960s, but it remains a current issue. There are diverging opinions
regarding the use of biocide formulations and products for noncritical surface disinfection.
While some view such use as unnecessary (Fraise 2016), others support such a
practice (Rutala and Weber 2016).
The use of biocidal products may be more appropriate
only in specific situations where the risk of spreading health-care associated
infections (HAIs) is high (Bloomfield et
al., 2014; Russell 2014). Biocides
are widely used in fat preservation, fluid treatment, healthcare sanitation,
textile, and other industries, during past decades, a wide variety of bioactive
organic chemicals have been developed for disinfection, sterilization, and
preservationpurposes, including quaternary ammonium compounds, alcoholic and
phenolic compounds, aldehydes, halogen-containing compounds, quinoline and
isoquinoline derivatives, heterocyclic compounds, and peroxygens (Windler,
2012).
Biocides have also been applied in Oil
reservoirs for many decades, particularly in fluid flooding operations during
secondary Oil recovery. Likewise, biocides are among the most common chemical
additives used to protect fluid from biological infestation and growth, a
process in which fluid is used to help induce cracks in Oil and/ or natural
gas-containing unconventional formations such as shale rock. At total
concentrations of up to >500 mg/L (McCurdy, 2011) and total fluid volumes
surpassing 10 million L per horizontal well, (Nicot, 2012) total amounts of
biocide(s) used per hydraulic fracturing event can exceed 1,000 gallons.
Bacterial control is necessary in hydraulic fracturing operations to prevent
excessive biofilm formation down hole that may lead to clogging, consequently
inhibiting gas extraction (Aminto, 2012).
Biocides inhibit growth of sulfate-reducing
bacteria (SRB), which anaerobically generate sulfide during the organisms’
respiration process. Sulfide species created in the subsurface may pose a risk
regarding occupational safety and health when the fluid returns along with
produced H2S gas. Furthermore, Sulphate reducing bacteria and
acid-producing bacteria (APB) may induce corrosion of the production
casing/tubing underground, potentially leading to casing failure and
environmental contamination by petroleum product (Fitcher, 2008; Gieg,
2011; Lester, 2013).
Likewise, bacteria can thrive in stored
produced formulated fluid that was recycled for use in future (Lysnes, 2013;
Shaffer, 2013). The increased temperatures in produced fluids are exposed to
different atmospheric condition which may also favor microbial growth and
therefore many bacterial species (including anaerobic species that are native
to shale formations) (Struchtemeyer, 2012; Strong, 2013) may proliferate
underground durin production of animal waste fluid. A diverse array of bacteria
including those within the taxa γ-proteobacteria, α-proteobacteria,δ-proteobacteria, Clostridia, Synergistetes,
Thermotogae, Spirochetes,
Bacteroidetes, and Archaea have all been found in untreated flow back fluid
samples (Murali Mohan, 2013).
1.4 Sources of Biocides
Because biocides are intended to kill living
organisms, many biocidal products pose significant risk to human health and
welfare. Great care is required when handling biocides and appropriate
protective clothing and equipment should be used. The use of biocides can also
have significant adverse effects on the natural environment. Anti-fouling
paints, especially those utilising organic tin compounds such as TBT, have been shown to have severe and
long-lasting impacts on marine eco-systems and such materials are now banned in
many countries for commercial and recreational vessels (though sometimes still
used for naval vessels).
1.
Pesticides: fungicides, herbicides, insecticides, algicides, molluscicides, miticides, rodenticides,
and slimicides
2.
Antimicrobial: germicides, antibiotics, antibacterials, antivirals, antifungals, antiprotozoals,
and antiparasites.
1.5 Application of Biocides
Biocides
used to control the growth of pathogenic microorganisms or to eliminate them from
inanimate objects, surfaces or intact skin, are classified on the basis of the
level of inactivation reached. Low-level disinfectants inactivate most
vegetative bacteria, some fungi and some viruses (enveloped viruses);
intermediate-level disinfectants inactivate vegetative bacteria, mycobacteria,
most viruses and most fungi, but do not necessarily kill bacterial spores;
high-level disinfectants inactivate all micro-organisms (vegetative bacteria,
mycobacteria, fungi, enveloped and non-enveloped viruses) except large numbers
of bacterial spores. High-level disinfectants can inactivate spores when
applied with prolonged exposure times and are called chemical sterilants.
1.6 Household products
Although biocidal products as defined by the Biocide Directive
98/8/EC are not commonly used in household products, the active ingredients of
the biocidal products in categories 1-9 of the Directive are widely used in
household products and other consumer products. Regular use of household
products such as laundry detergents, cleaning products, pet disinfectants and
general disinfectants are the major sources of exposure to biocides in home
settings. Biocides present in these products may be from different chemical
groups, but their mechanism of action may be similar. Biocides/antimicrobial
agents used as preservatives in household cleaning products and
laundry detergents may contain the same active ingredients as
cosmetic products. However, the use of biocides/antimicrobial agents in
household products is not regulated. Furthermore, certain biocides present as
preservatives in diverse household products may also be present in household
cleaning products, where they may serve as disinfectants.
Many of the ingredients used in detergent products, such as
cationic surfactants, quaternary ammonium compounds and fragrances, possess
antimicrobial properties. In a survey of industrial and institutional cleaning
products in Denmark, only a limited number of biocides, besides antimicrobial
surfactants and other ingredients, were found (Madsen et al. 2017). Cleaning
product formulations for private homes may be similar to those used in industry
and in public and private buildings. Disinfectants in consumer products are
used to control or to prevent growth of microorganisms. There is a great diversity
in use and application types for these products e.g. liquids, granulates,
powders, tablets, gasses etc.
1.6.1 Biocides as disinfectants
Disinfection is regarded as a crucial step in achieving a defined,
desired hygiene status in food production and processing areas, and in food
processing plants. A variety of biocides are commonly used for the disinfection
of equipment, containers, surfaces or pipework associated with the production,
transport and storage of food or drink (including drinking water).
Disinfectants intended for use in the food-processing industry are regulated
within the scope of Directive 98/8/EC on the placing of biocidal products on
the market. The use of disinfectant in water quality intended for human
consumption is regulated by the so-called Drinking Water Directive 98/83/EC18.
Biocides are used at the waterworks to maintain the microbiological quality of
the water before and during its distribution, by sustaining the total counts of
micro-organisms at an acceptable level and eliminating pathogenic
micro-organisms.
For drinking water treatment, chlorine has been used worldwide for
the past century for pre-chlorination at the point of entrance of raw water,
disinfection and post-disinfection in the water distribution system. However,
because of the formation of halogenated byproducts, pre-chlorination is no
longer recommended and other oxidising agents such as ozone or chlorine-dioxide
are more commonly used for disinfection. In some countries, post-disinfection
is always performed with chlorine or chloramine.
1.6.2 Biocides
as food preservatives
Preservatives are substances which prolong the shelf-life of
foodstuffs by protecting them against deterioration caused by micro-organisms.
These compounds are considered food additives and are regulated by the Food
Additives Directive 89/107/EEC19. Their use in food must be explicitly
authorised at European level and they must undergo a safety evaluation before
authorisation for using the preservative as intended.
1.6.3 Biocides
in animal husbandry
Proper cleaning and disinfection play a vital role in protecting
food animals from endemic and zoonotic diseases, and thus indirectly protecting
human health. It is impossible to give detailed accounts of all applications,
but uses can essentially be divided into four broad categories:
• Cleaning and disinfection of farm buildings, particularly
between batches of animals.
• Creating of barriers, such as in the use of foot dips outside
animal houses and disinfecting vehicles and materials during outbreaks of
infectious diseases.
1.6.4
Biocides as teat dips
The
udders of animals used for milk production may be contaminated with faecal and
other materials. Therefore, prior to milking, udders are cleaned with water
that may contain biocides, although this is less common. More frequently, after
the milking process, so-called teat dips are applied to protect the milk duct
and the entire udder from invading pathogens. Various chemicals are used for this
purpose including chloroisocyanurates, which are organic chloramines, bronopol,
quaternary ammonium compounds and iodine-based compounds
1.6.5
Biocide use in fish farming
Under
the prerequisites of Directive 98/8/EC a range of disinfectants are permitted
for decontamination in fish farming, for example for fish eggs, ponds and
equipment. These include iodophores, metallic salts, haloorganic compounds, aldehydes,
hydrogen peroxide, quaternary ammonium compounds and antimicrobial dyes.
1.7 Factors Affecting Microbial Population of Change of Fat to Liquid
1.7.1 pH and acidity
Increasing
the acidity of fat, either through fermentation or the addition of weak acids,
has been used as a preservation method since ancient times. In their natural
state, most fats such as meat, fish, and vegetables are slightly acidic while
most fruits are moderately acidic. A few fats such as egg white are alkaline. The
pH is a function of the hydrogen ion concentration in the fat: Another useful
term relevant to the pH of fluids is the pKa. The term pKa describes the state
of dissociation of an acid. At equilibrium, pKa is the pH at which the
concentrations of dissociated and undissociated acid are equal. Strong acids
have a very low pKa, meaning that they are almost entirely dissociated in
solution (ICMSF, 2012). For example, the
pH (at 25 °C [77 °F]) of a 0.1 M solution of HCl is 1.08 compared to the pH of
0.1 M solution of acetic acid, which is 2.6. This characteristic is extremely
important when using acidity as a preservation method for fats. Organic acids
are more effective as preservatives in the undissociated state. Lowering the pH
of a fat increases the effectiveness of an organic acid as a preservative.
It is well known that groups of
microorganisms have pH optimum, minimum, and maximum for growth in fats. As
with other factors, pH usually interacts with other parameters in the fat to
inhibit growth. The pH can interact with factors such as aw, salt, temperature,
redox potential, and preservatives to inhibit growth of pathogens and other
organisms. The pH of the fat also significantly impacts the lethality of heat
treatment of the fat. Less heat is needed to inactivate microbes as the pH is
reduced (Mossel et al., 2015).
Another important characteristic of a fat to
consider when using acidity as a control mechanism is its buffering capacity.
The buffering capacity of a fat is its ability to resist changes in pH. Fats
with a low buffering capacity will change pH quickly in response to acidic or
alkaline compounds produced by microorganisms as they grow. Meats, in general,
are more buffered than vegetables by virtue of their various proteins. Titratable acidity (TA) is a better indicator
of the microbiological stability of certain fats, such as salad dressings, than
is pH. Titratable acidity is a measure
of the quantity of standard alkali (usually 0.1 M NaOH) required to neutralize
an acid solution (ICMSF, 2012). It measures the amount of hydrogen ions
released from undissociated acid during titration. Titratable acidity is a
particularly useful measure for highly buffered or highly acidic fats. Weak
acids (such as organic acids) are usually undissociated and, therefore, do not
directly contribute to pH. Titratable acidity yields a measure of the total
acid concentration, while pH does not, for these types of fats.
1.7.2 Nutrient content
Microorganisms require certain basic
nutrients for growth and maintenance of metabolic functions. The amount and
type of nutrients required range widely depending on the microorganism. These
nutrients include water, a source of energy, nitrogen, vitamins, and minerals
(Mossel et al., 2013). Varying amounts of these nutrients are present
in fats. Meats have abundant protein, Lipids, minerals, and vitamins. Most
muscle fats have low levels of carbohydrates. Animal fats have high
concentrations of different types of carbohydrates and varying levels of
proteins, minerals, and vitamins. Fats such as milk and milk products and eggs
are rich in nutrient. Microorganisms
found in fat can derive energy from carbohydrates, alcohols, and amino acids.
Most microorganisms will metabolize simple sugars such as glucose. Others can
metabolize more complex carbohydrates, such as starch or cellulose found in
plant fats, or glycogen found in muscle fats. Some microorganisms can use fats
as an energy source.
Amino acids serve as a source of nitrogen and
energy and are utilized by most microorganisms. Some microorganisms are able to
metabolize peptides and more complex proteins. Other sources of nitrogen
include, for example, urea, ammonia, creatinine, and methylamines. Examples of minerals required for microbial
growth include phosphorus, iron, magnesium, sulfur, manganese, calcium, and
potassium. In general, small amounts of these minerals are required; thus a
wide range of fats can serve as good sources of minerals (Struchtemayer, 2012).
In general, the Gram positive bacteria are
more fastidious in their nutritional requirements and thus are not able to
synthesize certain nutrients required for growth (Jay 2010). For example, the Gram
positive organisms, S. aureus requires amino acids, thiamine, and
nicotinic acid for growth (Jay 2010). Fruits and vegetables that are deficient
in B vitamins do not effectively support the growth of these microorganisms.
The Gram negative bacteria are generally able to derive their basic nutritional
requirements from the existing carbohydrates, proteins, Lipid s, minerals, and
vitamins that are found in a wide range of fat (Jay 2010). An example of a pathogen with specific
nutrient requirements is Salmonella enteritidis. Growth of Salmonella
enteritidis may be limited by the availability of iron. For example, the
albumen portion of the egg, as opposed to the yolk, includes antimicrobial
agents and limited free iron that prevent the growth of Salmonella Enteritidis
to high levels. Clay and Board (2011) demonstrated that the addition of iron to
an inoculum of Salmonella enteritidis in egg albumen resulted in growth
of the pathogen to higher levels compared to levels reached when a control
inoculum (without iron) was used (Struchtemayer, 2012).
The
microorganisms that usually predominate in fats are those that can most easily
utilize the nutrients present. Generally, the simple carbohydrates and amino
acids are utilized first, followed by the more complex forms of these
nutrients. The complexity of fats in general is such that several
microorganisms can be growing in a fat at the same time. The rate of growth is
limited by the availability of essential nutrients. The abundance of nutrients
in most fats is sufficient to support the growth of a wide range of
microorganisms found in fat. Thus, it is very difficult and impractical to
predict the pathogen growth or toxin production based on the nutrient
composition of the fat.
1.7.3 Moisture
The
free flow of water is vital to microorganisms for their cells to exchange
materials and for their metabolic processes. All microorganisms require some
level of water, but a few can survive in low-moisture conditions by conserving
all the water they find and by staying in a moisture-rich environment. As a
general rule, though, the more moisture, the more microorganisms there will be
found.
1.7.4 Temperature
In
general, the higher the temperature, the more easily microorganisms can grow up
to a certain point. Very high and very low temperatures both obstruct the
enzyme processes microorganisms depend on to survive, but individual species of
microorganisms have grown to prefer different levels of temperature. Scientists
usually divide them into three different groups: psychrophiles, mesophiles and
thermophiles. Psychrophiles prefer temperatures from 0 to 5 degrees Celsius;
mesophiles like it in the middle, 20-45 degrees Celsius; and thermophiles like
it hot, thriving in temperatures around or above 55 degrees..
1.7.5 Elements Present
In
addition to water, microorganisms usually require the presence of certain
elements in the air--gases that they absorb to produce needed nutrients.
Nitrogen is one necessary element, as is oxygen. There are many microorganisms
that require an oxygen-rich environment to survive, but others actually
flourish in low-oxygen surroundings. Between these two extremes is a wide
variety that may prefer more or less oxygen and that will be able to flourish
equally no matter how much oxygen is present.
1.7.6 Fats
Fats
are one of the three main macronutrients,
along with carbohydrates
and proteins
(Struchtemayer, 2012). Fat
molecules consist of primarily carbon and hydrogen atoms and are therefore hydrophobic
and are soluble in organic solvents and insoluble in water. Examples include cholesterol, phospholipids,
and triglycerides. The terms Lipid, Oil,
and fat are often confused. Lipid is the general term, though a Lipid
is not necessarily a triglyceride. Oil normally refers to a Lipid with short or unsaturated fatty
acid chains that is liquid at room temperature,
while fat (in the strict sense)
specifically refers to Lipid s that are solids at room temperature. However, fat (in the broad sense) may be used
in food science as a synonym for Lipid. Fat is an important foodstuff for many
forms of life, and fats serve both structural and metabolic functions. They are
a necessary part of the diet of
most heterotrophs
(including humans) and are the most energy dense, thus the most efficient form
of energy storage (Struchtemayer,
2012). Some fatty acids that are set free by the
digestion of fats are called essential because they cannot be synthesized in
the body from simpler constituents. There are two essential
fatty acids (EFAs) in human nutrition:
alpha-linolenic
acid (an omega-3
fatty acid) and linoleic
acid (an omega-6
fatty acid) (Struchtemayer, 2012). Other
Lipid s needed by the body can be synthesized from these and other fats. Fats
and other Lipid s are broken down in the body by enzymes called lipases
produced in the pancreas. Fats and Oils are categorized according to the
number and bonding of the carbon atoms in the aliphatic chain. Fats that are saturated
fats have no double bonds between the carbons in
the chain. Unsaturated fats
have one or more double bonded carbons in the chain. The nomenclature is based
on the non-acid (non-carbonyl) end of the chain. This end is called the omega
end or the n-end. Thus alpha-linolenic
acid is called an omega-3
fatty acid because the 3rd carbon
from that end is the first double bonded carbon in the chain counting from that
end. Some Oils and fats have multiple double bonds and are therefore called polyunsaturated
fats. Unsaturated fats can be further divided
into cis fats,
which are the most common in nature, and trans fats,
which are rare in nature. Unsaturated fats can be altered by reaction with
hydrogen effected by a catalyst. This action, called hydrogenation,
tends to break all the double bonds and makes a fully saturated fat. To make
vegetable shortening, then, liquid cis-unsaturated fats such as
vegetable Oils are hydrogenated to produce saturated fats, which have more
desirable physical properties e.g., they melt at a desirable temperature
(30–40 °C), and store well, whereas polyunsaturated Oils go rancid when
they react with oxygen in the air. However, trans fats
are generated during hydrogenation as contaminants created by an unwanted side
reaction on the catalyst during partial hydrogenation. Saturated fats can stack
themselves in a closely packed arrangement, so they can solidify easily and are
typically solid at room temperature. For example, animal fats tallow
and lard
are high in saturated fatty acid content and are solids. Olive and linseed Oils
on the other hand are unsaturated and liquid. Fats serve both as energy sources
for the body, and as stores for energy in excess of what the body needs
immediately. Each gram of fat when burned or metabolized releases about 9 food calories
(37 kJ =
8.8 kcal). Fats
are broken down in the healthy body to release their constituents, glycerol
and fatty acids.
Glycerol itself can be converted to glucose by
the liver and so become a source of energy.
1.8 AIM AND OBJECTIVES
The
aim of this study is to assess the biocide effect and microbial population in
animal waste fluid.
1.8.1 Objectives
1.
To isolate and identify microorganisms associated with animal waste fluid.
2.
To determine the effect of biocide on the microbial population of animal waste
fluid.
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