Preface Microorganisms are interesting live forms not only for morphological types but also for its unique physiological traits and biotechnological applications. In comparison to aerobes; anaerobic Bacteria, Fungi and Archaea are understudied. Earlier studies on anaerobic bacteria were mostly confined to anaerobic pathogen like Clostridium. Development of improved handling protocols and specialized techniques expanded the study of anaerobes like fastidious anaerobic bacteria, methanogenic archaea, anaerobic fungi and human gut- resident. Parallel to these developments, we have realized that anaerobic microorganisms including bacteria fungi and archaea are the key component of clean-energy, global climate change, waste to energy generation, solid waste management, waste water treatment, bio-toilets, Human-gut-microbiome, anaerobic probiotics, fecal-microbiota-transplantation (FMT), fecal microbiome banking and emerging threat of anaerobic infection. Therefore; cultivation, bio-banking and In-depth-understanding of functionality and physiology of anaerobes are imperative to exploit them for biotechnological processes. Due to fastidious nature and need of special setup for cultivation, purification, characterization, handling and bio-banking in comparison to aerobic microorganisms very less work has been done on anaerobic bacteria, archaea and fungi. In addition, despite ecologically, industrially and clinically important group very little description is available on anaerobes and anaerobic process in most of the textbooks of microbiology. If we go through with most of the good textbooks available in microbiology we find that only 2-4 pages are dedicated to this important group of microbes. Most of the textbooks of microbiology only provide partial information about anaerobes and anaerobic process and these hidden heroes of the nature. We realized that a textbook in simple languages with ability to provide extensive knowledge about cultivation, purification, handling and bio-banking of different classes of anaerobes including the obligate, facultative, aero-tolerant and micro-aerophillic is very much essential. The aim of preparation of current text book is to provide the state-of-the-art knowledge about anaerobes and anaerobic process to undergraduate, graduate and PhD students. During the preparation of book we tried to incorporate contents from very basic like cultivation, handling, preservation and various important process related to anaerobes like biomethanation, anaerobic methanotrophy, solid waste management, anaerobic biohydrogen and biobutanol production, anaerobic nitrogen treatment, anaerobic solid waste management. In addition considering the importance of anaerobes in human health and disease we tried to dedicate a complete section like anaerobic probiotic, anaerobes and human gut microbiology and anaerobes and human infection. Finally we tried to incorporate a chapter on role of anaerobes in animal rumen and food digestion. We believe that current book will serve its aim to satisfy the hunger of knowledge about anaerobes and anaerobic process for the UG, PG and PhD students of Microbiology, Biotechnology, Environmental Science and Biochemistry background.

Abstract Based on oxygen requirement, microorganisms have been classified into different groups, like facultative anaerobes, micro-aerophilic, aerotolerant and strict/obligate anaerobes. Obligate or strict anaerobes are the key components of anoxic environments and provide valuable ecosystem services like methanogenesis, sulphate reduction, metal reduction, degradation of pollutants etc. Apart from being valuable for the ecosystem, they are causative agents of human infections. They are the key players of human-gut microbiology, and can be used in bio-toilets, solid waste management, anaerobic digestion of waste, wastewater treatment processes and generation of waste to energy etc. Only few anaerobic microorganisms are pathogenic in nature. A mechanistic understanding about physiology and metabolism of anaerobic microorganisms is imperative for their industrial applications. Cultivation of these organisms is the first and very important step for the basic and applied study of anaerobes. Unlike cultivation of aerobic microorganisms anaerobic cultivation needs special care and facilities for successful handling of anaerobes. In the current chapter, We tried to incorporate the components and technicalities of anaerobic pre-reduced media preparation, cultivation and successful isolation of strict anaerobes. In addition, we also tried to discuss the different tools used for cultivation of anaerobes.

Abstract Microbes are key ingredients of various ecosystems and backbone of modern biotechnology and industry. In addition to their cultivation, purification and characterization, preservation of microorganisms is equally important. Different preservation methods and protocols have been developed for long term preservation of microbes. Microbial Resource Centers (MRCs) are providing active services in the field of life sciences. National Centre for Microbial Resource (NCMR) at National center for Cell Science (NCCS), Pune is one of the MRCs. Unlike aerobes little work has been done in the area of cultivation and preservation of anaerobic microbes. In the previous chapter we have discussed the technicalities and problems of anaerobic cultivation. The aim of current chapter is to provide the recent overview of preservation of anaerobic microorganisms like methanogens, sulfate and metal reducers and the obligate anaerobes of clinical and industrial importance with fermentative mode of life style.

Abstract Anoxygenic phototrophic bacteria (APB) are having the genetic potential of converting radiant energy into chemical energy useful for growth and metabolic activities without evolving oxygen. They are physiologically, metabolically and phylogenetically versatile group of organism spread in five distinct phyla; Proteobacteria (purple bacteria), Chlorobi (green-sulfur bacteria), Chloroflexi (green non-sulfur bacteria), Fir micutes (heliobacteria) and Acidobacteria. Different group of APB harbors distinct photosynthetic pigments, carotenoids, and a photosynthetic reaction center. These group of bacteria succeeds in a natural environment with light availability and presence of anoxic conditions as the pigment, and the molecular oxygen inhibits bacteriochlorophyll synthesis whereas few APB can grow in hypooxic as well as in aerobic conditions. Calvin-Benson-Bassham (CBB) pathway, reverse tricarboxylic acid (rTCA) cycle, and hydroxypropionate cycle are reported pathways for carbon assimilation in APB to fix carbon dioxide. Photosynthesis occurs in either intracytoplasmic membrane (ICM) or membrane bound chlorosomes and in the cytoplasm for few APB. APB have a wide range of potential applications including removal of H2S from wastewater and gas streams, hydrogen production, degradation of xenobiotic, toxic metals and radioisotopes, work as host for the production of challenging membrane proteins, source of carotenoids and terpenoids, single cell protein, extensive applications in agriculture and production of bioactive compounds with antimicrobial, antioxidant and anticancer activity. The chapter is focused on the diversity of different types of APB, habitats, their growth requirements, photosynthetic machinery and biotechnological applications of APB.

Abstract The anaerobic fungi are one of the early organisms in evolution which are commonly found in the digestive tract of ruminating and non-ruminating animals, (including herbivores) deep sea and earth, dumping sites, etc. In animals, these are considered the primary colonizers of the plant biomass ingested by them. Still their taxonomy was unclear for a long period of time. However, with the help molecular techniques, they are now classified under a separate phylum Neocallimastigomycota. There are 38 species known so far belonging to 11 genera. These have been identified based on both morphological data (like thallus, rhizoids, flagella per zoospore, etc.) and phylogenetic analysis. There is a need to explore the undiscovered anaerobic fungi and look for their potential in industrial applications and solutions to the present environmental problems. It can be achieved by improving techniques of sampling, isolation, maintenance and long term preservation. Animals like herbivores do not have the ability to produce enzymes for degrading the plant biomass and anaerobic fungi help them by producing a range of extracellular enzymes. This ability of anaerobic fungi are of use to humans in bioenergy (biogas production) and biomass degradation (by producing cellulases). Recent advances in metagenomics coupled with new methods of cultivation are helping immensely to know the uncultivated anaerobic fungal populations from the animal gut. The present book chapter discusses the past and present of anaerobic fungi, lacunaes and need for more work on this group of fungi, It focuses on the progress in sampling, isolation and preservation techniques, simultaneously discussing the advances in the application these fungi in biotechnology.

Abstract Microorganisms that produce methane as their metabolic end product are known as methanogens. Methanogens are prokaryotic, unicellular, obligate anaerobes having fastidious growth requirements making them difficult to isolate and preserve. They catalyse the terminal step in anaerobic breakdown of organic substances. Even though methanogenic archaea or methanogens show very limited metabolic diversity, they are one of the most phylogenetically and ecologically diverse group of Archaea. Methanogenic community varies greatly with respect to temperature. Methanogens have been reported from permanently frozen habitats like permafrost to hyperthermophilic environments such as hydrothermal vents. However, the metabolic and phylogenetic diversity of methanogens in these habitats vary greatly. Methanogens are the biocatalysts, having the ability to provide a solution to help solve energy crisis by producing methane as storable energy carrier. Methane being a flammable gas, can act as a clean and renewable source of energy. The biogenic methane gas thus produced may serve as an efficient alternative for fossil fuels in the future.

Abstract Methane is the second most important green-house gas and has a major role in global warming. Aerobic oxidation of methane is known for more than 100 years. Anaerobic methane oxidation (AOM) was recently discovered before ~25 years, first in marine sediments. The first discovered AOM was found to be mediated by anaerobic methanotrophic archaea (ANME) in association with sulphate reducing bacteria (SRB) and termed as sulphate dependent anaerobic methane oxidation (S-DAMO). Three main groups of ANME are present which perform methane oxidation by reverse methanogenesis pathway. Later on, methane oxidation by using nitrate or nitrite as the electron acceptor was discovered from a freshwater sediment. N-DAMO or nitrate/ nitrite dependent AMO can be carried out by an archaeal or a bacterial member, respectively. Members of Ca. Methylomirabilis oxyfera are bacteria, and use nitrite as the electron acceptor and methane monooxygenase to carry out methane oxidation. Whereas, members of Ca. Methanoperedens nitroreducens are archaea, and use reverse methanogenesis coupled to nitrate reduction. Metals like FeIII or Mn IV can also be used as electron acceptors for AOM, a process recently discovered (M-DAMO). In this case, one archaeal partner and one bacterial partner could be involved, although more research is going in this area. S-DAMO is the main methane oxidation process in the marine sediments and accounts for the major methane oxidation in the marine sediments. In fresh water sediments, aerobic methane oxidation and processes like N-DAMO seem to play an important role.

Abstract Anaerobic sulfur compound reduction processes make use of sulfur, thiosulfate, trithionate, tetrathionate, and sulfite during the reduction process. Complete reduction of these sulfur compounds leads to production of H2S. Isolation of sulfur compound reducers requires a suitable electron donor and one of the sulfur compound as a sole electron acceptor during the reduction process carried out in anaerobic conditions. Anaerobic sulfur, thiosulfate and tetrathionate reductases identified to date are molybdopterin binding oxidoreductases that extract out electron during the reduction process. The electron transport chain during the reduction process involves cytochromes and menaquinones. Dissimilatory sulfite reductases include conventional siroheme dissimilatory sulfite reductase and the nonconventional sulfite reductases including ASR sulfite reductase, F420 dependent sulfite reductase and the MccA sulfite reductase. Anaerobic sulfur compound reduction process are almost ubiquitous in nature and are important part of the biogeochemical cycle. They occurs in aquatic systems, are found in extreme environments such as hydrothermal vents, acid mine drainage sites and are also found in important human and animal pathogens.

Abstract Grazing animals have developed symbiotic relationship with anaerobic microorganisms, which survive in their rumen and help in the oxidation of complex molecules of feed containing lignocellulosic materials into volatile fatty acids. The consortium comprises anaerobic bacteria, fungi and protozoans. The rich microbial diversity of rumen remains constant after its establishment and does not get affected by intruding contaminants along with feed or water. The rumen contains a wide range of microbes such as Fibrobacter spp., Ruminococcus spp., Clostridium spp., Prevotella spp., Eubacterium spp., Methanobacterium spp., Treponema sp. Lachnospira sp., Bacterioides s pp., fungi: Anaeromyces s pp., Neocallimastix spp., and protozoa: Enoplastron sp., Eudiplodinium spp., Ostracodinium sp. They often have a variety of enzymes, glucanases, xylanase, glucuronidase, pectinase and amylase, etc. These enzymes inhabit the microbes and even break down complex molecules into simpler forms like; glucose and volatile fatty acids. The present chapter discusses on the role of microorganisms of rumen.

Abstract Hydrogen synthesis from bioprocesses a good approach for generation clean of fuel for world's development. Throughout the ancient and current periods, most of the developmental activities are completed by consumption of non-renewable fossil fuel energy that which has resulted in multiple environmental issues or challenges (Climate change and pollution by toxic gases or byproducts formation) at global level. Biohydrogen synthesis from anaerobic bacterial fermentation could be a good effort for sustainable energy production without any toxic byproduct formation. For production of hydrogen, lignocellulosic material can be utilized as cheap raw substrates. This would help the farmers in providing surplus benefits to farmer for their agriculture residues that normally burnt and cause the serious air pollution. Utilization of agro-waste residues can help in development of sustainable biofuels for world energy need. Burning of agrowaste residues by farmer's can be discouraged via utilization as raw carbon substrates thereby reducing air pollution.

Abstract Rise in global energy demand has led to an increase in consumption of fossil fuels. In recent years, there has been an increasing awareness about the consequences of pollution and global warming caused by burning of fossil fuels. This has led to an increased interest in the search for sustainable energy. Transportation sector is a major consumer of world’s energy resources. n-Butanol is one of the most promising alternative bio-fuels which has various advantages over other biofuels. The microbial n-butanol is produced by anaerobic conversion of the substrate to n-butyric acid and subsequently utilized to form n-butanol. The present chapter explores the scope of bacterial butanol production as need of the hour and its use as an alternative biofuel.

Abstract Solid Waste Management (SWM) has become a significant global concern, specifically for developing countries like India because of increasing population, rapid urbanization and high rate of municipal solid waste generation. Bio-composting and anaerobic digestion are the methods majorly used for solid waste management. Present chapter comprises detailed information on methods, microbiology involved and pros and cons of these methods to provide the detail information of solid waste management processes to the readers. Variable nature of substrates are available for bio-composting process. It includes sludge from effluent treatment plant (ETP), sewage treatment plant (STP) and bio-toilets, municipal solid waste, food waste and garden waste etc. Nutritional content, carbon /nitrogen (C/N) ratio of the substrate, available space and cost of the process are the main factors that affect selection method for waste management. Anaerobic digestion is the preferred method of solid waste management in comparison to other processes of composting due to methane generation as biogas and bioconversion of organic compounds to manure.

Abstract Anaerobes are a diverse groups of microorganisms which do not require oxygen or energy for respiration/metabolism and are one of the important components of nitrogen cycle. They are capable of using various other organic and even inorganic materials as electron acceptors during respiration. Anaerobic based wastewater treatment is a biological approach where microorganisms degrade organic contaminants (i.e. rich in carbon and nitrogen) in the absence of oxygen. In this chapter, the role of anaerobes in wastewater treatment has been discussed as it offers an alternative, cheap and eco friendly treatment technology among different biological treatment methods. Anaerobic biological treatment of wastewater is well developed, understood, and frequently used in anaerobic digesters to treat complex organic carbon and nitrogen compounds such as primary and secondary wastewater sludge. The anaerobic sludge contains diverse groups of anaerobic microorganisms that work together in association via a multistage processe consisting of the following biochemical reactions like hydrolysis, acidification, acetogenesis, and methanogenesis thereby converting the organic matter to biogas. The composition of biogas includes 70% methane (CH4) and 30% carbon dioxide (CO2) in addition to traces of other gases (e.g., H2 and H2S). The methane produced as a by-product can be used as an energy source. The potentiality of anaerobes in anaerobic based bioreactors for its maximum efficiency can be implemented in a variety of ways. The chapter discusses about the working principle, performance, treatment potential, and cost implication in the application of anaerobic treatment technology. The main bioprocess parameters such as pH, temperature, mixing, organic loading rate (OLR), and hydraulic retention time (HRT) have also been discussed in brief. Anaerobic treatment offers several benefits over aerobic treatment, including lower energy requirements, less chemicals, low capital, operation, and maintenance costs, and less sludge production. Hence, it saves money and the environment and overall simplifies the whole process.

Abstract The normal human microbial flora comprises of both aerobes and anaerobes at various sites. Anaerobes are recognised as pathogens causing simple abscesses to life threatening human infections like gas gangrene. They are also incriminated in a large number of mixed bacterial infections and their diagnosis and specific therapy is important for better patient outcomes. The emergence of Clostridioides difficile as an important pathogen causing antibiotic associated diarrhoeas and its emerging resistance to antimicrobial agents has brought anaerobic microbiology into the forefront. Unfortunately, anaerobic clinical microbiology is difficult to practice with long turnaround time, difficult to grow pathogens, new genera and species being discovered based on 16S rRNA sequencing methods and increasing incidence of antimicrobial resistance being reported in anaerobes. This chapter delves into the important aspects required to improve utilization of anaerobic microbiology services and the variety of human infections we encounter in clinical practice due to anaerobic bacteria.

Abstract Gastrointestinal tract (GIT) harbors mixed microbial community, these are highly diversified, as revealed by molecular and phylogenetic studies. It is also estimated that microbiota of the GIT is composed of over 35,000 species and play a crucial role in human health and diseases. Thus are now of great interest to both the industrial and scientific communities. The microbes exhibit health benefits are known as probiotics. Anaerobic bacteria dominate in the GIT environment and genera like Bifidobacterium, Clostridium, are strict anaerobes while Lactobacillus, Lactococcus, is facultative anaerobes implicated in the probiotic effects. For developing anaerobic bacterial based bio-products, it is necessary to cultivate large-scale biomass production that could be done using a modern manufacturing practice. Information on detailed understanding of the perfect growth medium and other bioprocessing parameters for the growth and viability is must. Also high cell viability is a prerequisite during downstream processing and storage of probiotic formulation. In this work we review the latest information about the history, mechanism of action, benefits of probiotic, selection criteria, sources of anaerobic probiotic bacteria, industrial cultivation medium along with biomass production, downstream processing.

Abstract Hundreds of bacterial taxa makes up human gut microbiota and it is comprised primarily of anaerobes belonging to four main phyla which are Firmicutes, Bacteriodetes, Actinobacteria, and Proteobacteria. Anaerobes colonizes the human body, including oral cavity, small intestine, vagina, skin but the majority of them are housed in the colon. The composition and distribution anaerobes varies along the length of gastrointestinal tract because of variation in oxygen gradient, anatomy of intestinal tract and other host physiological factors. Human gets colonized by anaerobes during the birth and these initial colonizers have impact on infant’s health status. The colonization and development of the gut microbiome is responsive to variety of factors including the mode of delivery, feeding methods, environment etc. The human gut microbiota plays an important role in healthy status of host. It plays an important role in nutrient metabolism and absorption, synthesis of vitamins and production of short-chain fatty acids (SCFAs). In turn these metabolites have positive effect on host immunity and overall host fitness. Studying, the anaerobes using culturing method remains critical in microbiology. However, culturomics approach, combined with sequencing technology, provides new insights in studying anaerobes of human gut microbiota. In this chapter we have discussed that, how human gut is an ideal habitat for the colonization and growth of anaerobes and their impact on host health. We also tried to shed light on latest technical advancement in sequencing culturing technology and their application in studying gut anaerobes.

Abstract Anaerobic microorganisms occur in oxygen-free environments as they possess biomolecules and metabolic processes that have adapted to sustain them in the stressed environments. These biomolecules and metabolic processes are harnessed and employed in various biotechnological applications. Anaerobes are abundant in various ecological niches such as freshwater or marine sediments, subsurface aquifers, deep thermal vents etc. where, in the absence or limitation of oxygen, they utilise reduced ions such as nitrate, sulphate, ferric, carbonate and certain organic compounds like degrading organic matter, fatty acids or alcohols as electron acceptors during respiration/fermentation to produce more or fewer reduced products such as, alcohols, ammonia, organic acids, hydrogen and carbon dioxide as the oxidised product. Diversity of substrates that can be fermented and the wide range of products that can be formed make anaerobic processes useful in biotechnological applications. Their substrate versatility and flexibility, for example, they can utilize simple sugars as well as many of them can utilise complex renewable biomass make their biotechnological applications sustainable and environment friendly. Anaerobes have been employed in food, chemical and material industries for the production of food items, preservative, additives, chemical intermediates, solvents, catalysts and polymers. They find widespread usage in energy sector for generation of various kinds of biofuels that can mitigate our reliance on non-renewable sources for fuel production. An emerging aspect of anaerobic biotechnology is waste valorization that involves treatment of any kind of waste (industrial, municipal, agricultural, pharmaceutical), where organic fraction of the waste is converted into valuable products. Anaerobes play key role in maintaining the global cycles of carbon, nitrogen, and sulfur, and also facilitate bioremediation by breakdown of persistent compounds. Lately, role of anaerobes in medicine and health care sector is widely explored as they are found to produce several bacteriocins, antibiotics, immunomodulators, vitamins and anti-tumor compounds. Some of the anaerobes have also been employed as probiotics. In the present chapter, we discussed these and many more biotechnological applications of anaerobes in white, green, red and blue biotechnology.