REVIEW OF RELATED LITERATURE

2.1 INTRODUCTION

Our focus in this chapter is to critically examine relevant literature that would assist in explaining the research problem and furthermore recognize the efforts of scholars who had previously contributed immensely to similar research. The chapter intends to deepen the understanding of the study and close the perceived gaps.

2.2 CONCEPTUAL FRAMEWORK

Concept of Environment

Environments are components of ecosystems. An ecosystem is a community of micro-organisms and their physical and chemical environment that functions as an ecological unit5. The ecosphere or biosphere, organise the entirety of living organisms on Earth and the abiotic surroundings they occupy. It can be divided into atmosphere, hydrosphere and litho-ecosphere to define the shares of the global expense inhabited by living things in air, water and soil environments respectively. Micro-organisms lives within the habitats of the ecospheres. The habitat is one component of a comprehensive concept of the ecological niche, which includes not only where an organism lives but also the functions it performs. The niche is the functional role of an organism within an ecosystem. Micro-organisms may be autochthonous or indigenous or allochthonous or foreign6.

Environment is defined as the circumstances or conditions that surround an organism or group of organisms. Environment is the complex of social or cultural conditions that affects an individual or community. Since humans inhabit the natural world as well as the built or technological, social, and cultural world, all constitute an important part of our environment. Environmental studies need to understand the life processes at the microscopic level and ecologist levels from species to ecosystem7. Species refer to organisms of the same kind that are genetically similar enough to breed in nature and produce live, fertile offspring. Population consists of all members of the same species living in a given area in the same time. All the populations of organism living and interacting in a particular area make up a biological community. An ecological system or ecosystem is composed of a biological community and its physical environment. The environment includes abiotic factor such as climate, water, minerals, and sunlight as well as biotic factors such as organisms, their products, and effect in a given area8.

Types of environment and their communities

The groups of similar species create population which results in a community. Microbial community means all microbial populations in a habitat. The activities of complex communities of microbes affect biogeochemical transformations in natural, managed, and engineered ecosystem. Microbial communities are very important for the rigorous progress in the field of agriculture which increases the rate of crop production. Microbial community may be terrestrial or aquatic9.

Terrestrial Communities

A community of microbes and their environment that occurs on the landmasses of continents and islands form a terrestrial microenvironment. Terrestrial microenvironment is distinguished from the aquatic microenvironment ecosystems by the lower availability of water and the consequent importance of water as a limiting factor. Rain forests are the most diverse and productive terrestrial microenvironment, but their soil is nutrient deficient due to extensive leaching by rainwater.

Soil

Soil formation is a slow process that involves physicochemical weathering and biological processes over millions of years. Microbes play an important role in soil aggregate formation and soil stability that confer fertility and productivity to soil10. The soil microbes participate in these processes through many ways, e.g., filamentous microbes assemble clay particles using extensive network of hyphae resulting into soil aggregates. Additionally, some microbes secrete exopolysaccharides or cause compaction of clay particles that promote soil aggregation. The surface soil is always rich in indigenous population of bacteria (including actinomycetes), fungi, algae, and protozoans. Additionally, human and animal activities also introduce specific microbes in the soil by several ways. Human activity directly adds bacteria as biodegradative agents or applying sewage sludge to agricultural fields. Animals introduce microbes through bird dropping or excretion.

Air

The atmosphere is an inhospitable climate for microbes because of stress due to dehydration. This results in a limited time frame for microbes to be active; however, some microbes get resistance to these stresses through specific mechanisms promoting loss of their biological activity. Spore-forming bacteria, molds, fungi, and cyst-forming protozoans all have specific mechanisms through which they are protected from these harsh gaseous environments11. Therefore, viability is highly dependent on the environment, time they spend in the environment, and type of microbes. However, many other factors also influence the viability of microbes such as humidity, temperature, oxygen content, specific ions, UV radiation, various pollutants, and other air-associated factors (AOFs).

Relative Humidity

The relative humidity or relative water content of the air is critical for survival of airborne microbes. Most of the gram-negative bacteria associated with aerosols are able to survive for longer period at low relative humidity, whereas in contrast gram-positive bacteria remain viable longer in association with high relative humidity. The ability of microbes to survive in aerosol is related to the organism’s surface biochemistry12. One possible explanation of this fact could be a structural change in lipid bilayers of the cell membrane in response to very low humidity. During loss of water, the cell membrane bilayer changes from the typical crystalline structure to a gel phase and affects the surface protein configuration resulting into inactivation of the cell. The viruses with enveloped nucleocapsids (e.g., influenza virus) have longer airborne survival in low relative humidity below 50 %, whereas viruses without nucleocapsids (e.g., enteric viruses) are able to survive in high relative humidity above 50 %13.

Temperature

Temperature is a critical factor influencing the activity of microbes. In general, high temperature leads to inactivation due to desiccation and protein denaturation, whereas lower temperature promotes longer survival rates. At very low temperature, some microbes lose viability because of ice crystal formation on their surface due to freezing14.

Radiation

Mostly radiation at low wavelengths, e.g., UV radiation and ionic radiation (X-rays), is harmful for microbes causing DNA damage. These radiations target DNA by producing single or double strand breaks and changing the structure of nucleic acid bases15. UV radiation causes damage by forming intra-strand thymidine dimers causing inhibition of biological activity such as replication of genome, transcription, and translation. Several mechanisms including association of microbes with large airborne particles, pigments or carotenoids, high relative humidity, cloud cover, etc. protect microbes from these harmful radiations. However, many microbes (e.g., Deinococcus radiodurans) have evolved mechanisms to repair DNA damage caused by UV radiation.

Oxygen, OAF, and Ions

Oxygen, open-air factors (OAF), and ions combine to inactivate many species of airborne microbes. Some reactive forms of oxygen including superoxide radicals, hydrogen peroxide, and hydroxide radicals are produced due to lighting, UV radiation, or pollution and cause DNA damage by producing mutations. Similarly, OAFs (mixture of ozone and hydrocarbons) also cause inactivation of microbes by damaging nucleic acids and enzymes. In addition to these factors, positive ions cause only physical decay, e.g., inactivation of cell surface proteins, whereas negative ions confer both physical and biological damages such as DNA damage12.

Aquatic Communities

Aquatic microenvironments occupy more than 70 % of the earth’s surface including mostly ocean but also others such as estuaries, harbors, river, lakes, wetlands, streams, springs, aquifers, etc4. The microbiota, living in aquatic environment, are the primary producers (responsible for approximately half of all primary production on earth) and primary consumers as well. A large variety of microbial communities live in aquatic environments such as the planktonic, sediment, microbial mat, biofilm communities, etc. Planktons refer to photoautotrophic microbial community including both eukaryotes (algae) and prokaryotes (cyanobacteria) and heterotrophic community including bacteria (bacterioplankton) and protozoans (zooplankton)8. Phytoplanktons are the primary producers in the food web using their ability to fix CO2 into organic matter through photosynthesis. Aquatic microenvironment is further classified into three microenvironments, occupied by microbes living in freshwater, brackish water, and marine water.

Freshwater

The study of freshwater microenvironment is known as micro-limnology. There are two types of freshwater environment: standing water or lentic habitats (e.g., lakes, ponds, bogs) and running water or lotic habitats including springs, rivers, and streams16. Lentic habitats are dominated by phytoplankton, forming distinct community gradients based upon the wavelength and the amount of light that penetrates to a depth, e.g., Chlorobium. Chlorobium can utilize longer wavelength than other phototrophs and survive with little or no oxygen by consuming H2S instead of H2O for photosynthesis17. In freshwater environment, two types of lakes are present: eutrophic and oligotrophic lakes. Oligotrophic lakes have higher rate (20–120 mg carbon/m3/day) than eutrophic lakes (1–30 mg carbon/m3/day) because eutrophic lakes have much higher levels of organic matter causing turbidity and interfering with light penetration. However, in terms of secondary productivity, eutrophic lakes have much higher rates (190–220 mg carbon/m3/day) as compared to oligotrophic lakes (1–80 mg carbon/m3/day)18.

Brackish Water

Brackish water environment is more saline than freshwater but less saline than marine water environment. An estuary, a part of river that meets with sea, is the best example of brackish water environment. Estuaries are highly variable environments because salinity changes drastically over a relatively short distance. Despite this, estuaries are highly productive environments, e.g., mangrove swamps in the Everglades of Florida, USA. Estuaries are generally turbid due to the large amount of organic matter brought by rivers and the mixing action of tides; therefore, light penetration is poor19. Primary producers vary from 100 to 107organisms/ml and in relation to depth and proximity to littoral zones. Despite the low primary productivity, substrate availability is not limited, and heterotrophic activity is high ranging from 150 to 230 mg carbon/m3/day.

Marine Water

Marine water environments are highly diverse and contain 33–37 % salinity. The ocean is divided into two zones on the basis of light availability: photic zone, where light can penetrate, and aphotic zone with lower light. Marine microenvironment is further divided into four habitats: neuston, pelagic, epibiotic, and endobiotic. Habitat at the surface of sea (air-water interface) is termed as neuston20. On the basis of the precise depth, pelagic habitat is subdivided into epipelagic and benthopelagic zones. Epipelagic zone is found in upper 100 m of the water column, and a large proportion of organisms living in it are photosynthetic, whereas benthopelagic zone is sea-sediment interface. The third major habitat is the epibiotic habitats referring to surfaces on which attachment of communities occur, while the fourth is the endobiotic habitat with organisms (e.g., Epulopiscium) found within the tissues of other larger organisms such as fish.

Extremophilic Communities

The organisms living in physically or geochemically extreme conditions that are detrimental to most life on earth are termed as extremophiles13. Most of the extremophiles are microbes and belong to the domain Archaea. Here are some extreme environmental conditions where extremophiles survive.

High Temperature

Environments with high temperature (>70 °C) including terrestrial and submarine springs with a temperature of 100 °C, hydrothermal vents with a temperature more than 300 °C are inhospitable for most forms of life except some bacteria and archaebacteria, e.g., Thermus, Methanobacterium, Sulfolobus, Pyrodictium, and Pyrococcus. Pyrodictium and Pyrococcus are capable of surviving at temperature >100 °C. Another example of such renowned extremophiles is Thermus aquaticus having thermotolerant DNA polymerase, which is widely used in the polymerase chain reaction (PCR). These thermophiles have developed such characteristic mechanisms facilitating proteins in folded state even at high temperatures due to increased salt bridges (cations that bridge charges between amino acid residues)21.

High Solute

Some organisms require salt concentrations substantially higher than that found in seawater for their growth, and they are known as halotolerant. Halobacterium and Halanaerobium are two examples of halotolerant bacteria; however, some algae and fungi also exhibit halotolerance feature. The main mechanism of salt tolerance operates by internal sequestration of high balancing solute (K+ in bacteria and glycerol in halotolerant eukaryotes) equal to external salt concentration22. A second mechanism of salt tolerance involves proteins with acidic and low proportion of nonpolar amino acids. These proteins require high salt concentrations to balance their charge for their optimal activity. Therefore, some obligate halophiles are unable to survive in the environment lacking high salt concentration due to these macromolecular modifications.

Microbes in the Environment

Microbes are omnipresent in the biosphere, and their presence invariably affects the environment in which they grow. There is a wide range of microbes present in our biosphere depending on their physical and other characteristics8. Microbes fall into two groups, prokaryotes and eukaryotes, depending upon whether they have nucleus or not. Prokaryotes lack this membrane around their genetic material, and this group includes viruses, bacteria, and related archaea. The other category of microbes includes algae, fungi, protists, and other microscopic animals, having cell nucleus. For the scope of this study Bacteria will singly considered.

Concept of Bacteria

` Bacteria are also instrumental for understanding fundamental life processes that are required by all organisms, including central metabolism, replication, transcription, translation, protein targeting, assembly and structure of macromolecular complexes, protein folding, stress responses, error correction mechanisms, signal transduction, and developmental programs.Bacteria and archaea are the smallest free-living, unicellular organisms present on the earth23. Their cell sizes typically range from 0.5 to 1.0 μm in diameter. Both exist in various cell shapes, e.g., cocci, rods, or spirals, and some soil bacteria form branching filaments, e.g., actinomycetes24. Their DNA is found free in the cell cytoplasm and lack a true nuclear envelope, and the genome is mainly composed of single double-stranded DNA molecule with smaller DNA elements known as plasmids. The size of bacterial genome typically ranges from four to six million nucleotides in length and enable to code 3,000–4,000 genes. A bacterial cell envelope is composed of two layers, the inner layer is cell membrane made of phospholipids and the outer layer is cell wall made of proteins, carbohydrates, and lipids, but its composition varies based on the type of organism. Most of the microbes move through flagella (whiplike extensions from the cell) and file filaments, e.g., pili. The pili enable them to attach with each other or to soil particles. Additionally these pili are also involved in transfer of genetic material between bacterial cells, known as conjugation. These microbes usually reproduce asexually, e.g., binary fission, resulting in the formation of two genetically identical bacterial cells25. On the basis of gram staining, bacteria are of two types: gram positive and gram negative; both vary in cell structure and physiology (Fig. 3.3). Bacteria and archaea both require carbon as building blocks of their cellular materials and energy to drive the reactions involved in cell biosynthesis and metabolism. Most of the bacteria utilize oxygen, whereas some bacteria and archaea grow anaerobically by using alternative electron acceptors, e.g., nitrate and sulfate.s. These processes are more easily characterized in model bacteria and their phages than in other organisms because microbes provide such tractable experimental systems26. The large repertoire of genetic and biochemical tools and data that have been acquired from basic research on bacteria is crucial for dissecting the complex metabolic and regulatory networks that control these processes. This provides a launching point for understanding the enormous diversity in the bacterial world and facilitates the understanding of these processes in eukaryotes.

Classification of Bacteria

Basically microbes are classified into autotrophs and heterotrophs. Autotrophs utilize sunlight or inorganic compounds such as Fe2+, nitrate, or nitrite as energy source to fix atmospheric carbon dioxide to produce carbohydrates, fats, and proteins. However, heterotrophic bacteria use organic compounds as a source of carbon and energy. Archaea were originally known to be found in extreme environments and termed as “extremophiles,” but now they are widely distributed and are found in many environments including soil28.

It is hard to distinguish both archaea and bacteria on the basis of their morphology. But most recently their classification using molecular phylogenetic tools based on a comparison of 16S ribosomal rRNA sequences has revealed three separate domains of life: eukaryotes, bacteria, and archaea. Archaea are closely related to eukaryotes (all multicellular organisms) than the bacteria29.

The classification of bacteria serves a variety of different functions. Because of this variety, bacteria may be grouped using many different typing schemes. The critical feature for all these classification systems is an organism identified by one individual (scientist, clinician, epidemiologist), is recognized as the same organism by another individual. At present the typing schemes used by clinicians and clinical microbiologists rely on phenotypic typing schemes. These schemes utilize the bacterial morphology and staining properties of the organism, as well as O2 growth requirements of the species combined with a variety of biochemical tests. For clinicians, the environmental reservoir of the organism, the vectors and means of transmission of the pathogen are also of great importance30.

The Economic Importance of Bacteria in the Environment

The phrase "Economic Importance" is a double edged sword, it has it's advantages and disadvantages at the various area of applications. Putting into consideration the diverse areas of which includes bacteria display their economic importance which includes pharmaceutical, clinical, agriculture, food processing and manufacturing.etc.There are many kinds of bacteria without which we could not live. They are absolutely essential to the presence of life on earth. They make possible the continued existence of green plants and therefore of animals because the plants are the only source of food for animals.

Role in Agriculture

Scavenging Role: Saprophytic bacteria obtain food from organic remains such as animal excreta, fallen leaves, meat etc. They decompose these substances by action of digestive enzymes aerobically or anaerobically (known as fermentation). Thus they help in sanitation of nature, so also known as scavengers15. E.g. Pseudomonas

Nitrification:Rhizobium bacteria, living in root nodules of leguminous plant symbiotically, helps in fixing atmospheric nitrogen. Similarly, Nitrosomanas and Nitrococcus convert ammonium salt to nitrites. Nitrites are further changed to nitrates by Nitrobacter and Nitrocystis. It enables plants to uptake nitrogen.

Production of Organic Manure: As stated above, saprophytic bacteria help in breaking of complex organic substance to simpler forms. Thus, in this process, they help to convert farm refuse, dung and other wastes to manure.

Preparation of Ensilage:Ensilage is preserved cattle fodder prepared by packing fresh chopped fodder sprinkled with molasses. Fermentation activity of bacteria produces lactic acid that acts as preservative in ensilage16.

Production of fuel: Bacteria, while converting animal dung and other organic wastes to manure, help in production of fuel that is a must in gobar gas plant.

Disposal of sewage: Bacteria help in disposal of sewage by decomposing it and thus, help in environmental sanitation.

Role in Manufacturing/Food Industry

In the home and in industry, microbes are used in the production of fermented foods. Yeasts are used in the manufacture of beer and wine and for the leavening of breads, while lactic acid bacteria are used to make yogurt, cheese, sour cream, buttermilk and other fermented milk products. Vinegars are produced by bacterial acetic acid fermentation. Other fermented foods include soy sauce, sauerkraut, dill pickles, olives, salami, cocoa and black teas. Yeast are also involved in fermentations to convert corn and other vegetable carbohydrates into ethanol to make beer, wine or gasohol, but bacteria are the agents of most other food fermentations31.

Dairy Industry: Bacteria such as Streptococcus lactis convert milk sugar lactose into lactic acid that coagulates casein (milk protein). Then, milk is converted into curd, yoghurt, cheese etc needed for the industry.

Production of Organic Compounds: Fermentation (breakdown of carbohydrate in absence of oxygen) action of various bacteria produces organic compounds like lactic acid (by Lactobacillus), acetic acid (by Acetobacter aceti), acetone (by Clostridium acetabutylicum) etc32.

Fibre Retting: The action of some bacteria like Clostridium,

Pseudomonas etc. help in fibre retting i.e. separation of stem and leaf fibre of plants from other softer tissue.

Curing: The leaves of tea and tobacco, beans of coffee and coca are cured off their bitterness with the help of action of certain bacteria such as Bacillus megatherium.

Role in Medical, Pharmaceutical and Biotechnological Applications

In human and veterinary medicine, for the treatment and prevention of infectious diseases, microbes are a source of antibiotics and vaccines33.

Production of Antibiotics: Antibiotics are substances produced by microorganisms that kill or inhibit other microbes which are used in the treatment of infectious disease. Antibiotics are produced in nature by molds such as Penicillium and bacteria such as Streptomyces and Bacillus. Number of anti bacterial and anti fungal antibiotics such as Hamycin, Polymyxin, Trichomycin etc are obtained from mycelia bacteria (like Streptomyces). Similarly, Bacillus is used for production of antibiotics such as Bacitracin, Gramicidin etc.34

Production of Vitamins: Different kinds of vitamins are produced from bacteria like Riboflavin from Clostridium butylicum, Vitamin B12 from Bacillus megatherium and Vitamin K and B-complex from Escherichia coli35.

Production of Vaccines: Vaccines are substances derived from microorganisms used to immunize against disease. The microbes that are the cause of infectious disease are usually the ultimate source of vaccines36. Thus, a version of the diphtheria toxin (called toxoid) is used to immunize against diphtheria, and parts of Bordetella pertussis cells are used to vaccinate against pertussis (whooping cough). The use of vaccines such as smallpox, polio, diphtheria, tetanus and whooping cough has led to virtual elimination of these diseases in regions of the world where the vaccines have been deployed. Various antibiotics used in veterinary medicine37.

Biotechnology - Microbiology and the study of bacteria makes an important contribution to biotechnology, an area of science that applies microbial genetics to biological processes for the production of useful substances. Microorganisms play a central role in recombinant DNA technology and genetic engineering. Important tools of biotechnology are microbial cells, microbial genes and microbial enzymes38.

The genetic information for many biological products and biological processes can be introduced into microbes in order to genetically engineer them to produce a substance or conduct a process39. The genes can come from any biological source: human, animal, plant or microbial. This opens the possibility for microbial production of foods, fuels, enzymes, hormones, diagnostic agents, medicines, antibiotics, vaccines, antibodies, natural insecticides and fertilizers, and all sorts of substances useful in our civilization and society. Also, the microbial genes that encode for these substances, most of which are unknown, are a tremendous resource of information for application in medicine, pharmacy, agriculture, food science and biotechnology40.

2.2 THEORITICAL FRAMEWORK

A theoretical framework includes concepts and, accompanied by their definitions and reference to pertinent scholarly literature, existing theories used for a particular study. This demonstrates an understanding of theories and concepts that are relevant to the topic of a research paper and that relate to the broader areas of knowledge being considered (Labaree, 2009). The framework theories supporting the study is community ecology theory.

Theory of Environmental Microbiology

The widely used technique for the study of bacteria is the growth of a microbe of interest in a liquid nutrient medium, followed by dilution and plating on a solid agar medium. In this case, the theory is that one colony that arises from one organism. Each colony is then referred to as a Colony Forming Unit (CFU). In addition to providing an estimation of bacterial numbers, this procedure allows the opportunity to obtain pure culture isolates. Often times, researchers will measure the turbidity of the liquid culture at different time intervals, using a spectrophotometer. The comparison of turbidity with plating results allows for a rapid estimation of bacteria numbers in future studies (Pepper, & Gerba, 2004).

The techniques are used in all aspects of microbiology, including clinical and environmental microbiology. The growth of a bacterial isolate will be followed as a function of time to demonstrate the various stages of growth that occur in liquid culture. Spontaneously, one can recognize that bacterial growth through cell division in liquid media will continue to occur until nutrients would get limited or microbial waste products gather and constrain growth.

Community Ecology Theory

Community ecology theory can be used to understand biological invasions by applying recent niche concepts to alien species and the communities that they invade. These ideas lead to the concept of ‘niche opportunity’, which defines conditions that promote invasions in terms of resources, natural enemies, the physical environment, interactions between these factors, and the manner in which they vary in time and space. Niche opportunities vary naturally between communities but might be greatly increased by disruption of communities, especially if the original community members are less well adapted to the new conditions. Recent niche theory clarifies the prediction that low niche opportunities (invasion resistance) result from high species diversity. Conflicting empirical patterns of invasion resistance are potentially explained by covarying external factors. These various ideas derived from community ecology provide a predictive framework for invasion ecology. Community ecology is the study and theory of how populations of organisms including humans interact with each other and react to their non-living surroundings. As a subset of the general study of ecology, this field of specialization explores the organization and functioning of biological communities.