Kavish Kumar Jain

Solid state bioconversion of wheat straw into digestible and nutritive ruminant feed by Ganoderma sp. rckk02

Solid state fermentation (SSF) of wheat straw with Ganoderma sp. rckk02 was carried out for 15 days for improving its digestibility and nutrients. Fungal growth caused a significant (P<0.05) decrease in acid detergent fiber (ADF), neutral detergent fiber (NDF), hemicellulose, lignin and cellulose content till 15th day. In vitro gas production (IVGP) test revealed that 10th day fermented feed possessed higher metabolizable energy (ME: 4.87 MJ/kg), in vitro organic matter digestibility (OMD: 334 g/kg) and short chain fatty acids (SCFAs: 1.82 mmol/g Dry Matter). The fermented feed was also evaluated in vivo in goats fed with either untreated wheat straw (T1) or fungal treated straw (T2). Dry matter intake (DMI), digestible crude protein (DCP), total digestible nutrients (TDN) and nitrogen (N) intake were found significantly (P<0.05) increased in T2 group. The study shows that fermentation of wheat straw with Ganoderma sp. rckk02 holds potential in improving its nutritive value.

Bioprocessing of wheat straw into nutritionally rich and digested cattle feed

Wheat straw was fermented by Crinipellis sp. RCK-1, a lignin degrading fungus, under solid state fermentation conditions. The fungus degraded 18.38% lignin at the expense of 10.37% cellulose within 9 days. However, when wheat straw fermented for different duration was evaluated in vitro, the 5 day fungal fermented wheat straw called here “Biotech Feed” was found to possess 36.74% organic matter digestibility (OMD) and 5.38 (MJ/Kg Dry matter) metabolizable energy (ME). The Biotech Feed was also observed to be significantly enriched with essential amino acids and fungal protein by fungal fermentation, eventually increasing its nutritional value. The Biotech Feed upon in vitro analysis showed potential to replace 50% grain from concentrate mixture. Further, the calves fed on Biotech Feed based diets exhibited significantly higher (p<0.05) dry matter intake (DMI: 3.74 Kg/d), dry matter digestibility (DMD: 57.82%), total digestible nutrients (TDN: 54.76%) and comparatively gained 50 g more daily body weight.

Cellulases and Their Biotechnological Applications

For a long-range solution to the global issues of energy, chemical and food, the most abundant, renewable and sustainable bioresource cellulose could be a feasible solution. The depolymerisation of cellulose by a group of enzyme cellulases could potentially lead to the development of various value-added products. Due to their immense potential, cellulases are involved in various industrial and biotechnological applications related to pulp and paper, textile, fuel and other organic chemical synthesis industries. However, to further economise the cellulase production, extensive research is being carried out using various approaches including genetic manipulation and process engineering. In this chapter, a brief overview of cellulases and their potential applications are being discussed.

Multiple Genes in a Single Host: Cost-Effective Production of Bacterial Laccase (cotA), Pectate Lyase (pel), and Endoxylanase (xyl) by Simultaneous Expression and Cloning in Single Vector in E. coli

This study attempted to reduce the enzyme production cost for exploiting lignocellulosic materials by expression of multiple genes in a single host. Genes for bacterial laccase (CotA), pectate lyase (Pel) and endoxylanase (Xyl), which hold significance in lignocellulose degradation, were cloned in pETDuet-1 vector containing two independent cloning sites (MCS). CotA and xyl genes were cloned in MCS1 and MCS 2, respectively. Pel gene was cloned by inserting complete cassette (T7 promoter, ribosome binding site, pel gene, His tag and complete gene ORF) preceded by cotA open reading frame in the MCS1. IPTG induction of CPXpDuet-1 construct in E. coli BL21(DE3) resulted in expression of all three heterologous proteins of ~65 kDa (CotA), ~45 kDa (Pel) and ~25 kDa (Xyl), confirmed by SDS-PAGE and western blotting. Significant portions of the enzymes were also found in culture supernatant (~16, ~720 and ~370 IU/ml activities of CotA, Pel and Xyl, respectively). Culture media optimization resulted in 2, 3 and 7 fold increased secretion of recombinant CotA, Pel and Xyl, respectively. Bioreactor level optimization of the recombinant cocktail expression resulted in production of 19 g/L dry cell biomass at OD600nm 74 from 1 L induced culture after 15 h of cultivation, from which 9, 627 and 1090 IU/ml secretory enzyme activities of CotA, Xyl and Pel were obtained, respectively. The cocktail was also found to increase the saccharification of orange peel in comparison to the xylanase alone. Thus, simultaneous expression as well as extra cellular secretion of these enzymes as cocktail can reduce the enzyme production cost which increases their applicability specially for exploiting lignocellulosic materials for their conversion to value added products like alcohol and animal feed.

Biofuels: The Environment-Friendly Energy Carriers

Escalating globalisation, high demand for energy, increasing greenhouse gas emissions and depleting fossil fuel reserves have necessitated the search for alternative and sustainable energy carriers such as biofuels. Worldwide, the laboratories are engaged in extensive research for the development of different biofuels such as bioethanol, biodiesel, biohydrogen, biogas and advanced bioalcohols. This chapter provides an overview of bioprocessing of various types of biofuels.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

Cellulases: Application in Wine and Brewery Industry

Abstract Cellulase is a complex of three enzymes that work synergistically to hydrolyze native cellulose, these are endoglucanases (EC 3.2.1.4), exoglucanases (cellobiohydrolases EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). A large number of microorganisms, such as bacteria, actinomycetes, and fungi, are known to degrade cellulose. Cellulolytic enzymes from soft rot and white rot fungi have been studied in model fungi such as Trichoderma viride and Phanerochaete chrysosporium, respectively. Cellulase has major application in the bioconversion of lignocellulosic materials. In the recent years apart from saccharification of lignocellulose, there has been an increasing interest in the production of beverages using cereal blends based upon a complex endogenous enzymology occurring during the malting of grain, mashing of grist, and fermentation, which can be connected way back historically. The present chapter discuss the use of this enzyme cellulase in brewing industry specially focussing on the methodologies and its role in beer and wine making.

‘Omics’ Approaches to Understand and Manipulate Rumen Microbial Function

Diverse populations of rumen microorganisms in gut contribute to develop ability of breaking down fibrous foods, which are mostly unusable by humans (Owens FN, Goetsch AL (1988) Ruminal fermentation. In: Church DC (ed) The ruminant animal, digestive physiology and nutrition. Prentice-Hall, Englewood Cliffs, p 160). Rumen is having a larger population of microorganisms, more than a trillion organisms and wide diversity (hundreds of species and thousands of subspecies), per ounce of rumen contents (Xu et al., J Anim Sci 85:1024–1029, 2007). There are various traditional approaches through which overall performance of the rumen has been attempted to improve, e.g. plant secondary metabolites, microbial feed additives, chemical feed additives, selective stimulation of beneficial rumen microbes and selective inhibition of harmful rumen microbes. In spite of these, nowadays various new approaches are being used to improve our understanding of the relationships among the various rumen microorganisms and towards how they interact with their hosts (Chaucheyras-Durand and Ossa, Prof Anim Sci 30:1–12, 2014). To better characterize species in the rumen, new advanced technological aids such as gene sequencing and study of gene (genomics), protein (proteomics) and metabolite (metabolomics) expression are being frequently used. This chapter will majorly emphasize on recent tools used in exploring the diversity of rumen.