Review of Literature
Materials and Methods
I express my gratitude towards many people who saw me through this book; to all those who provided support, talked things over and assisted in the editing, proofreading and design.
Above all, I thank the Management of Charotar University of Science and Technology for continuous encouragement. In addition, I thank the Principal and Dean, PD Patel Institute of Applied Sciences for the support. I also thank Department of Biological Sciences for the critical comments to improve.
My special gratitude to Prof. S.P. Singh, Professor (Microbiology) and Head, Saurashtra University, Rajkot and Dr. Rajesh K. Sani, Associate Professor, South Dakota Schools of Mines and Technology, USA for continuous support, guidance and timely support.
At this moment, I seek blessings of my parents and in-laws. At the same time, I thank my family members, my wife and my little son who sacrificed more than me to see me achieving the goals.
I also wish to thank GRI PUBLICATIONS for the opportunity.
Last and not least: I beg forgiveness of all those who have been with me and whose names I have failed to mention."
Date: February 22, 2018 Bhavtosh A. Kikani, PhD
Life thrives well in range of adverse environmental conditions, for example extremes of salinity, acidity, alkalinity, temperature or pressure. It always fascinates the scientific fraternity to explore the microbial diversity and phylogeny under these inhospitable habitats. Their possible adaptive measures may provide clues to various evolutionary pathways (Austin, 1988; Herbert and Sharp, 1992; Singh, 2006). Among them, the thermophilic bacteria are common in soil and volcanic habitats with limited species composition. Yet, they possess all the major nutritional categories similar to their mesophilic counterparts. Among, the genus Bacillus and related genera are widely distributed in the nature, including thermophilic, psychrophilic, acidophilic, alkaliphilic and halophilic bacteria, which can be able to utilize a wide range of carbon sources for the heterotrophic or autotrophic growth (Claus and Berkeley, 1986; Nazina et al., 2001).
On the other hand, cellulose is the most abundant biomass on Earth, being the primary product of photosynthesis in the terrestrial environment and the most plentiful renewable bioresource. Cellulases, a group of enzymes commonly breaks cellulose, are produced by several microorganisms, mainly by bacteria and fungi. They are inducible enzymes which are synthesized by microorganisms during their growth on cellulosic materials. The complete enzymatic hydrolysis of cellulosic materials needs different types of cellulases: namely endoglucanase (1,4-β-d-glucan-4glucanohydrolase), exocellobiohydrolase (1,4-β-d-Glucan glucohydrolase) and β-glucosidase (β-d-glucoside glucohydrolase). The endoglucanase randomly hydrolyzes β-1,4 bonds in the cellulose molecule, whereas the exocellobiohydrolases in most cases release a cellobiose unit, showing a recurrent reaction from chain extremity. Lastly, the cellobiose is converted to glucose by β-glucosidase.
Bacteria, having quite higher growth rates as compared to fungi contribute significantly in cellulase production. Cellulase yields rely on various factors, mainly inoculums size, pH, temperature, presence of inducers, medium additives, aeration, growth time, and so on. Enormous cellulosic wastes are getting accumulated day by day. Therefore, it is of considerable economic interest to develop the processes for effective treatment and utilization of cellulosic wastes as inexpensive carbon sources.
The cellulose-degrading enzymes can be used, for example, in the formation of washing powders, extraction of fruit and vegetable juices, and starch processing. Cellulases are used in the textile industry for cotton softening and denim finishing; in laundry detergents for colour care, cleaning; in the food industry for mashing; in the pulp and paper industries for drainage improvement and fibre modification, and they are even used for pharmaceutical applications. In nutshell, the cellulose enzymes is commonly used in many industrial applications, whereas the demand for more stable, highly active and specific enzymes will also grow rapidly in near future. Therefore, continuous research for advances in speckled aspects for cellulose production (such as cost, substrate specificity, and specific activity) is desired to achieve improved techno-economic feasibility (Sethi et al., 2013).
In the present study, we intended to isolate potent cellulase producing thermophilic bacteria, followed by analysis of its growth and morphological properties. We optimized its production followed by partially purification of the cellulase by ammonium sulphate fractionation. The study focused on some interesting aspects related to production of the cellulases which would be helpful for its commercialization.
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REVIEW OF LITERATURE
Life at the extreme conditions
The majority of microbial populations, inhabiting in the extreme environments belong to the domains, archaea and bacteria. These habitats are uniquely noticeable due to existence of varieties of extreme conditions, such as salinity, temperature, pH and oxygen concentrations (Stetter, 1998). The microorganisms, optimally flourishing in these unusual environmental conditions, are broadly grouped under the term ‘ Extremophiles’. Besides, this microbes are classified based on the nature of the extremity in which they flourish. For instance, psychrophiles grow well in extremely cold environments. Similarly, the microbes which are inherently adapted to very high temperatures are referred to as thermophiles. Further to add, the microorganisms associated to the acidic environments are called acidophiles, whereas those found in the highly alkaline conditions are classified as alkaliphiles. In continuation, many of them uniquely flourish well in presence of two or more type of extreme conditions are known as polyextremophiles. Take an example of haloalkaliphiles, which can grow in the presence of high salt and alkaline conditions. Similar explanation is possible for the thermoalkalitolerant microorganisms. Fascinatingly, the adaptation to thrive in these conditions draws the attention, which may explain their evolution as well (Wiegel and Kevbrin, 2004).
The thermal environments
The most common thermal biotopes are the volcanically and geothermally heated hydrothermal systems, such as the solfataric fields, the natural hot springs and the submarine saline hot vents. The hot solfataric fields consist of one upper layer, containing significant amounts of oxygen, which reflects an ochre color owing to the presence of the ferric ions; while a layer below shows a blackish-blue color, due to the presence of ferrous ions. On the same note, the submarine thermal systems consist of the hot fumaroles, springs, sediments and hot vents with very high temperatures (Stetter, 1998). In addition, the other submarine hydrothermal systems usually contain high concentrations of NaCl, exhibiting a slightly acidic to alkaline pH range between 5 and 8 (Horikoshi, 1998). Interestingly, the thermal environments of the natural hot springs differ very widely with respect to the temperature, flow rate and chemistry of the water (Brock, 1994). The Yellowstone National Park (Wyoming, USA) covers maximum numbers of the hot springs in the World. In addition, the hot springs can also be found at Norris and Mud Vulcano in Italy, Kamchatka in Russia (Wiegel, 1990), along the western coast of India (Saha, 1993), Sao Michel in Azores, submarine hot springs in Iceland and Mount Grillo at Baia Naples in Italy (Romano et al., 2004). In addition to the above mentioned, other examples of thermal environments include hydrothermal vents at the Guaymas Basin and east Pacific Rise in Mexico. The hydrothermal vents are located in shallow and abyssal depths (Stetter, 1998). These environments possess a unique chemical composition, such as high content of sulfur and hydrogen sulphide, favouring growth of chemolithotrophs.
The biotic communities in the thermal environments
Taxonomically and ecologically, varieties of microorganisms can thrive in the thermal environments. Evidently, microbial life in these habitats represents all the three domains at the high temperatures. Reportedly, taxonomic studies on characterization of members belonging to the bacteria, archaea and eukarya had been conducted in the Yellowstone National Park (Ward, 1998). Specifically, these studies unfolded bacterial diversity, mainly cyanobacteria, anoxygenic phototrophs, aerobic and anaerobic chemoorganotrophs (Hugenholtz et al., 1998; Ward, 1998). In addition, several other unusual thermoacidophiles, such as Sulfolobus acidocaldarius were also reported from the Yellowstone National Park (Ward, 1998). Several taxonomic studies were also carried out and reported on the other diverse hot springs. For example, the Mono Lake in California – an alkaline, hypersaline and closed basin was explored by Gorlenko et al. (2004). A novel species, Anaerobranca californiensis was isolated from the sediment. Another hot spring that has been studied extensively is the Kamchatka at Russia, where Thermoproteus uzoniensis, an extremely thermophilic bacterium was isolated (Bonch-Osmolovskaya et al., 1990). Apart from that, numerous thermophilic microorganisms have also been isolated from the chimneys, sediments and the ambient water of the hydrothermal vent fields (Reysenbach et al., 2000).
Thermotoga maritime was isolated from the marine thermal vents at Vulcano (Huber et al., 1986). A similar organism, Thermotoga neapolitana was isolated from a submarine thermal vent at Lucrino, Italy (Belkin et al., 1986; Jannasch et al., 1988). Another specific group of thermophiles, including some bacilli, have also been reported from the natural and the artificial high temperature biotopes (McMullan et al., 2004). According to them, the thermophilic bacilli, belonging to the Bacillus Genetic Group-5, had been reclassified as the members of the recently named genus Geobacillus. Mostly, Geobacillus species are widely distributed in the continents, where geothermal areas occur. The geobacilli are also isolated from the shallow marine hot springs and from the deep-sea hydrothermal vents as well. Maugeri et al. (2002) previously described the isolation of three novel halotolerant and thermophilic Geobacillus strains from three separate shallow marine vents of the Eolian Islands, Italy. Besides, high temperature oil fields also yielded strains of Geobacillus, where two novel species, namely G. subterraneus and G. uzenensis were isolated from the Uzen oil-field in Kazakhstan (Nazina et al., 2001; Nazina et al., 2004). In addition, Geobacillus species were isolated from the temperate soils (McMullan et al., 2004) and other artificial hot environments, such as hot water pipelines, heat exchangers, waste treatment plants, burning coal refuse piles and bioremediation biopiles (Maugeri et al., 2002; Obojska et al., 2002). On the other hand, archaea is the least understood in terms of the diversity, physiology and ecological panorama of the three primary phylogenetic domains. Although many species of Crenarchaeota (Woese et al., 1990) have been isolated, they constitute a relatively tight-knit cluster of lineages in the phylogenetic analyses of the nucleic acid sequences. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture the organisms and not their absence. It obviates need for cultivation and identification of the organisms. This may have a definite impact on the concepts of the phylogenetic organization of archaea. Furthermore, use of molecular phylogenetic approaches in the microbial ecology has revolutionized the view of the microbial diversity and has led to the proposal of a new kingdom within the Archaea, namely the Korarchaeota (Reysenbach et al., 2000). The report consisted on the occurrence of another member of the archaeal group and a deeply rooted bacterial sequence from a thermal spring in the Yellowstone National Park. The phylotype is a lineage within the Aquificales. The In-situ hybridization with bacteria-specific and Aquificales- specific fluorescent oligonucleotide probes indicated that the bacterial populations dominated the community and contributed significantly to the biogeochemical cycling within the community (Reysenbach et al., 2000).
2.2.2 The adaptations against the elevated temperatures
Modifications to the protein structure for survival at the higher temperature have been extensively reviewed (Fields, 2001), with most research into the field of thermophilic enzymes (Kikani et al., 2010, Singh et al., 2013). As thermophilic microorganisms cannot shield their internal environment from the external temperature, the cellular components have get adapted at the elevated temperatures. The studies of the extremophilic proteins have revealed no structural motifs, covalent modifications or additional amino acids that would explain the ability of the proteins to function in the extreme conditions. The analysis of the structural data had shown that a redistribution of the some of the forces ensures the stability in the mesophilic environments and the changes in the protein-solvent interaction are sufficient to maintain the structural integrity at the high temperatures as well. In turn, the approach allowed the rapid modification in the enzyme stability against the environmental changes, by simply modifying the concentration of solutes, which help in the adaptation to a new thermal niche (Fields, 2001).
Besides, high temperatures increase the fluidity of the cellular membrane, which thereby lead to ‘leaky’ membranes and therefore consequently loss of the functioning of the membrane proteins. However, in the thermophilic bacteria, the fatty acids are more saturated and longer than the fatty acids in the mesophiles. The polar liquids are also enriched in the carbohydrates, containing a greater proportion of the methyl branched fatty acid chains (Mermelstein and Zeikus, 1998). The archaeal cytoplasmic membrane contains unique ether-linked lipids, which are very resistant to the high temperatures and do not degrade (De Rosa et al., 1994). They are also resistant to the mechanical degradation and very high salt concentrations, making these lipids more competent for the cellular membranes of the thermophiles than the eubacterial estertype lipids (Vossenberg et al., 1998). Evidently, the tetraether membrane lipids were reported in a thermoacidophilic euryarchaeota, Candidatus ‘Aciduliprofundum boonei’ (Schouten et al., 2008).
In addition, the cell wall of the extremely thermophilic archaea is reported to be composed of an ‘S-layer’ structure – a simple, regular two-dimensional lattice of the glycoproteins that covers the cell surface and leaves no periplasmic space. The glycoprotein layers are highly resistant to the mechanical and chemical degradation, while they spontaneously re-assemble due to very strong subunit interactions (Herbert and Sharp, 1993). The cell wall of an alkaliphilic Bacillus contained an acidic teichurono peptide polymer, which served as a barrier to ionic flux and played a role in the pH homeostasis (Kitada et al., 2000). Besides, cell wall of several alkaliphilic microbes also contain a large amount of the acidic amino acids. The acidic charges may act as charged membranes, reducing the pH on the cell surface between 8 and 9, allowing the cell to maintain a neutral internal pH (Horikoshi, 1998). Also, all hyperthermophiles contain the enzyme, reverse gyrases to induce the positive super-coiling of DNA that enhances its thermal stability (Forterre, 1996). In addition, the thermophilic microorganisms are reported to have proteins which are thermostable and resist denaturation and proteolysis (Kumar and Nussinov, 2001). The proteins and enzymes of the thermophilic microorganisms can also adapt to high temperatures by increased electrostatic, disulphide and hydrophobic interactions (Ladenstein and Ren, 2006; Pebone et al., 2008). Additionally, certain specialized proteins, known as ‘chaperons’, are produced by these organisms, which help to refold the proteins to their native form and restore their functions (Laksanalamai and Robb, 2001; Singh et al., 2010). Besides, certain thermophilic enzymes are stabilized by various conformational changes (Fitter, 2003). However, presence of certain metals, inorganic salts and substrate molecules are also reported to impart the thermostability (Vieille and Zeikus, 2001).
The enzymes of the thermophilic microorganisms can be used as models to understand the basis of thermostability. In addition, studies of the thermophilic organisms and their proteins has provided important insight into the mechanisms of the protein folding. Therefore, understanding how thermophilic proteins have evolved to be stable can yield information about the functional modulation of the folding landscapes. It has been shown, in studies comparing homologous proteins from the thermophilic and mesophilic organisms, those proteins from the thermophilic organisms have a lower change in the heat capacity upon unfolding. It is thought that this is due to residual structure in the unfolded state of the protein from the thermophilic organism.
2.3 The Biocatalytic potentials of thermophilic bacteria
As a consequence of the growth at high temperatures and unique macromolecular properties, the thermophilic bacteria also possess high metabolic rates, physically and chemically stable enzymes and lower growth but higher end product yields. Moreover, the reactions at very higher temperatures have benefits of the decreased viscosity and the enhanced diffusion coefficient of substrates, favoring equilibrium displacement in the endothermic reactions (Kumar and Swati, 2001). Thus, many possibilities for industrial processes have emerged with the thermostable enzymes (Haki and Rashit, 2003).
Biotechnology of cellulases and hemicellulases began in early 1980s, first in animal feed followed by food applications (Chesson, 1987). Subsequently, these enzymes were used in the textile, laundry as well as in the pulp and paper industries (Wong and Saddler, 1992). Currently, several commercial enzyme producers are marketing tailor-made enzyme preparations suitable for biotechnology (Bhat and Bhat, 1997).
MATERIAL AND METHODS
The present section focuses on the screening of these bacteria, with respect to their capabilities to produce various extracellular enzymes. Besides, various physicochemical environmental conditions were also optimized for the production of cellulases. At the end, cellulose was partially purified and characterized accordingly.
The Research site, Tulasi Shyam – A natural thermal habitat
The research site, Tulasi Shyam is a set of the natural hot spring reservoirs, located in the middle of the Gir Forest in the Gujarat State, India (Latitude: 21.051; Longitude: 71.025). Noticeably, the proposed site also has a religious significance. Various soil samples and water samples were collected from the site.
Characterization of the samples
Various physicochemical properties of the soil and water samples were measured. The parameters for the water sample included colour, temperature and pH values. The parameters for the soil samples included colour, pH, soil structure, soil texture and soil consistence (Marx et al., 1999; McSweeney and Grunwald, 1999).
Isolation of the thermophilic bacteria
The samples were serially diluted in the sterile distilled water. From the diluted samples, 5% (v/v) was inoculated into the modified thermophilic medium containing 0.7% (w/v) peptone, 0.5% (w/v) , malt extract and 0.5% (w/v) NaCl, along with 1% (w/v) CMC powder at different pH from 6-8. The flasks were incubated at 50°C for 24–48h. Thereafter, 0.1ml of the enriched culture was inoculated on the modified thermophilic agar plates with 5% (w/v) agar containing the same ingredients as mentioned above at pH 6-8. The inoculated plates were incubated at the temperatures between 37 and 70°C for 24–48h. The colonies were subsequently streaked repeatedly till pure colonies were obtained.
Screening for the various extracellular enzymes
In order to reveal the biocatalytic potentials, the bacterial isolates were individually spotted on different screening medium plates.
Preparation of activated culture
The thermophilic bacterial cultures were inoculated in the modified medium, containing 0.5% (w/v) peptone, 0.5% (w/v) yeast extract, 0.3% (w/v) malt extract, 0.2% (w/v) (NH4)2SO4, 0.5% (w/v) NaCl, 0.07% K2HPO4, 0.03% KH2PO4, 0.05% MgSO4.7H2O and 0.01% CaCl2. The cultures were incubated at their optimum growth conditions for 16-18h. The young actively growing culture, also commonly known as the activated culture was used further.