Sanjay K.S. Patel

Extending the limits of Bacillus for novel biotechnological applications

Bacillus, generally regarded as safe, has emerged as a robust organism that can withstand adverse environmental conditions and grows easily to very high densities. Bacillus has been recognized for its biotechnological applications on an industrial scale. Recent efforts have shown the potential of Bacillus to generate biofuels (hydrogen), biopolymers (polyhydroxyalkanoates), and bioactive molecules (acyl-homoserine lactonases). Bacillus can be considered the dark horse in the race to generate sustainable energy, ecofriendly non-fossil fuel-based polymers, and bioactive molecules for use as therapeutics.

Integrative approach to produce hydrogen and polyhydroxybutyrate from biowaste using defined bacterial cultures

Biological production of hydrogen (H2) and polyhydroxybutyrate (PHB) from pea-shell slurry (PSS) was investigated using defined mixed culture (MMC4, composed of Enterobacter, Proteus, Bacillus spp.). Under batch culture, 19.0LH2/kg of PSS (total solid, TS, 2%w/v) was evolved. Using effluent from the H2 producing stage, Bacillus cereus EGU43 could produce 12.4% (w/w) PHB. Dilutions of PSS hydrolysate containing glucose (0.5%, w/v) resulted in 45-75LH2/kg TS fed and 19.1% (w/w) of PHB content. Under continuous culture, MMC4 immobilized on coconut coir (CC) lead to an H2 yield of 54L/kg TS fed and a PHB content of 64.7% (w/w). An improvement of 2- and 3.7-fold in H2 and PHB yields were achieved in comparison to control. This integrative approach using defined set of bacterial strains can prove effective in producing biomolecules from biowastes.

Bioconversion of crude glycerol to polyhydroxyalkanoate by Bacillus thuringiensis under non-limiting nitrogen conditions

Glycerol has emerged as a cheap waste material due to blooming biodiesel manufacturing units worldwide. The need is to exploit the crude glycerol (CG) to produce useful products such as polyhydroxyalkanoate (PHA). Bacillus thuringiensis EGU45 was found to produce 1.5-3.5 gP HA L(-1) from feed containing 1-10% CG (vv(-1)) and nutrient broth (NB, 125 mL) without any acclimatization. B. thuringiensis EGU45 could produce PHA at the rate of 1.54-1.83 g L(-1), from 1% CG (vv(-1)) on media having high nitrogen contents: (i) NB, (ii) NB+0.5% NH4Cl (wv(-1)), and (iii) peptone+yeast extract+0.5% NH4Cl (wv(-1)). B. thuringiensis EGU45 was able to produce co-polymer of P(3HB-co-3HV) with 13.4% 3HV content on high N containing feed supplemented with propionic acid. This is the first report demonstrating the abilities of B. thuringiensis to convert CG into PHA co-polymer under non-limiting N conditions.

Dark fermentative bioconversion of glycerol to hydrogen by Bacillus thuringiensis

Biodiesel manufacturing units discharge effluents rich in glycerol. The need is to convert crude glycerol (CG) into useful products such as hydrogen (H2). Under batch culture, Bacillusthuringiensis EGU45 adapted on pure glycerol (PG, 2% v/v) resulted in an H2 yield of 0.646 mol/mol glycerol consumed on minimal media (250 mL) supplemented with 1% ammonium nitrate at 37°C over 4 days. Here, H2 constituted 67% of the total biogas. Under continuous culture, at 2 days of hydraulic retention time, B. thuringiensis immobilized on ligno-cellulosic materials (banana leaves - BL, 10% v/v) resulted in a H2 yield of 0.386 mol/mol PG consumed. On CG, the maximal H2 yield of 0.393 mol/mol feed consumed was recorded. In brief, B. thuringiensis could transform CG, on limited resources - minimal medium with sodium nitrate, by immobilizing them on cheap and easily available biowaste, which makes it a suitable candidate for H2 production on a large scale.

Improvement in methanol production by regulating the composition of synthetic gas mixture and raw biogas

Raw biogas can be an alternative feedstock to pure methane (CH4) for methanol production. In this investigation, we evaluated the methanol production potential of Methylosinus sporium from raw biogas originated from an anaerobic digester. Furthermore, the roles of different gases in methanol production were investigated using synthetic gas mixtures of CH4, carbon dioxide (CO2), and hydrogen (H2). Maximum methanol production was 5.13, 4.35, 6.28, 7.16, 0.38, and 0.36mM from raw biogas, CH4:CO2, CH4:H2, CH4:CO2:H2, CO2, and CO2:H2, respectively. Supplementation of H2 into raw biogas increased methanol production up to 3.5-fold. Additionally, covalent immobilization of M. sporium on chitosan resulted in higher methanol production from raw biogas. This study provides a suitable approach to improve methanol production using low cost raw biogas as a feed containing high concentrations of H2S (0.13%). To our knowledge, this is the first report on methanol production from raw biogas, using immobilized cells of methanotrophs.

Protein–inorganic hybrid system for efficient his-tagged enzymes immobilization and its application in L-xylulose production

The facile synthesis of protein–inorganic hybrid nanoflowers was evaluated for the efficient immobilization of recombinant his-tagged enzymes, which have a broad range of potential applications. In this study, we report the preparation of a metal–protein hybrid nanoflower system for efficient immobilization of the recombinant enzymes L-arabinitol 4-dehydrogenase from Hypocrea jecorina (HjLAD) and NADH oxidase from Streptococcus pyogenes (SpNox). Compared with free enzymes, synthesized hybrid nanoflowers exhibited enhanced enzymatic activities of 246 and 144% for HjLAD and SpNox, respectively. We have demonstrated that immobilized enzymes retained high catalytic activity and improved the tolerance towards pH and temperature changes. Synthesized nanoflowers also retained high storage stability and reusability. In addition, the immobilized enzymes exhibited significantly enhanced L-xylulose production under co-factor regeneration conditions than the free enzyme combination. These results demonstrate that variations in the concentration of metals and synthesis conditions of nanoflowers can be extended to efficiently immobilize recombinant his-tagged enzymes.

Biological methanol production by immobilized Methylocella tundrae using simulated biohythane as a feed

Biohythane may be used as an alternative feed for methanol production instead of costly pure methane. In this study, methanol production potential of Methylocella tundrae immobilized through covalent immobilization, adsorption, and encapsulation was evaluated. Cells covalently immobilized on groundnut shells and chitosan showed a relative methanol production potential of 83.9 and 91.6%, respectively, compared to that of free cells. The maximum methanol production by free cells and cells covalently immobilized on groundnut shells and chitosan was 6.73, 6.20, and 7.23mM, respectively, using simulated biohythane as a feed. Under repeated batch conditions of eight cycles, cells covalently immobilized on chitosan and groundnut shells, and cells encapsulated in sodium-alginate resulted in significantly higher cumulative methanol production of 37.76, 31.80, and 25.58mM, respectively, than free cells (18.57mM). This is the first report on immobilization of methanotrophs on groundnut shells and its application in methanol production using biohythane as a feed.

Potential of Immobilized Whole-Cell Methylocella tundrae as a Biocatalyst for Methanol Production from Methane.

Methanol is a versatile compound that can be biologically synthesized from methane (CH4) by methanotrophs using a low energy-consuming and environment-friendly process. Methylocella tundrae is a type II methanotroph that can utilize CH4 as a carbon and energy source. Methanol is produced in the first step of the metabolic pathway of methanotrophs and is further oxidized into formaldehyde. Several parameters must be optimized to achieve high methanol production. In this study, we optimized the production conditions and process parameters for methanol production. The optimum incubation time, substrate, pH, agitation rate, temperature, phosphate buffer and sodium formate concentration, and cell concentration were determined to be 24 h, 50% CH4, pH 7, 150 rpm, 30°C, 100 mM and 50 mM, and 18 mg/ml, respectively. The optimization of these parameters significantly improved methanol production from 0.66 to 5.18 mM. The use of alginate-encapsulated cells resulted in enhanced methanol production stability and reusability of cells after five cycles of reuse under batch culture conditions.

Heterologous Expression of Quorum Sensing Inhibitory Genes in Diverse Organisms

The discovery of antibiotics was a wonderful solution to provide relief to human beings from infectious diseases. However, indiscriminate usage of antibiotics turned out to be counterproductive. It was observed that patients were not getting cured in spite of the systematic use of antibiotics. In fact, microbes had developed resistance to antibiotics. This perturbation has been in operation even with antibiotics subsequently developed during the next 6–7 decades (D’Costa et al. 2006). Pharmaceutical companies are no longer interested in investing money into this business (Spellberg et al. 2004; Courvalin 2008). It obliged scientists to look for alternative drugs and new drug targets. It was realised that more than 80 % of the infectious diseases are caused by microbial pathogens, through specialised structures – biofilms. It enables bacteria to survive the lethal effect of drugs, as they “become” up to 1,000 times more resistant to antibiotics (Kalia 2013; Gui et al. 2014; Kalia et al. 2014a, b). These biofilms are developed by bacteria in a population density-dependent process called quorum sensing (QS) (Dong and Zhang 2005). Most Gram-negative bacteria operate through a QS system termed as LuxR/I-type, where acylated homoserine lactones (AHLs) acts as signals. QS signals consist of the lactone ring with varying acyl chains (Yang et al. 2012; Shang et al. 2014). QS regulates the expression of virulence factors, antibiotic production, nitrogen fixation, sporulation, conjugation, swarming, etc. (Borlee et al. 2008; Kalia and Purohit 2011; Kalia 2013; Wang et al. 2013; Zhang et al. 2013; Kalia et al. 2014a, b). These properties allow such bacteria to dominate the community structure. It is thus no surprise that the competing organisms have also developed mechanisms to interfere with the QSS and degrade these signals – a phenomenon termed as quorum quenching (QQ) (Kalia and Purohit 2011; Annapoorani et al. 2012; Bakkiyaraj et al. 2013; Kalia 2013; Agarwala et al. 2014).

A green chemistry approach for synthesizing thermostable antimicrobial peptide-coated gold nanoparticles immobilized in an alginate biohydrogel

Developing rapid, green, and cost effective approaches for synthesizing metal nanoparticles with biochemical and biomedical applications is currently a top priority. Here, we report a rapid synthesis of gold nanoparticles (AuNPs) in an alginate polymer using thermostable antimicrobial peptides. At a high temperature, these antimicrobial peptides acted as reducing agents and converted Au3+ to Au0 in 15 min while retaining their antimicrobial activity. The alginate polymer acted as a stabilizing agent for this reaction. The resulting Au–peptide–alginate biohydrogel was characterized by UV-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). TEM analysis showed AuNPs of less than 25 nm in diameter, with peptides present on their surfaces. The Au–peptide–alginate biohydrogel showed effective catalytic activity in reducing 4-nitrophenol and hexacyanoferrate(III) in the presence of sodium borohydride with durable reusability. The Au–peptide–alginate biohydrogel was also demonstrated to have antimicrobial activity against pathogenic bacteria. The method presented here for the synthesis of a metal–polymer conjugate is eco-friendly and robust. Large-scale production of the Au–alginate polymer would be possible in a very short time using this method, which makes it potentially useful for biomedical and industrial applications.