Prasun Kumar

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.

Evolution of Resistance to Quorum-Sensing Inhibitors

The major cause of mortality and morbidity in human beings is bacterial infection. Bacteria have developed resistance to most of the antibiotics primarily due to large-scale and “indiscriminate” usage. The need is to develop novel mechanisms to treat bacterial infections. The expression of pathogenicity during bacterial infections is mediated by a cell density-dependent phenomenon known as quorum sensing (QS). A wide array of QS systems (QSS) is operative in expressing the virulent behavior of bacterial pathogens. Each QSS may be mediated largely by a few major signals along with others produced in minuscule quantities. Efforts to target signal molecules and their receptors have proved effective in alleviating the virulent behavior of such pathogenic bacteria. These QS inhibitors (QSIs) have been reported to be effective in influencing the pathogenicity without affecting bacterial growth. However, evidence is accumulating that bacteria may develop resistance to QSIs. The big question is whether QSIs will meet the same fate as antibiotics.

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.

Challenges and Opportunities for Customizing Polyhydroxyalkanoates.

Abstract Polyhydroxyalkanoates (PHAs) as an alternative to synthetic plastics have been gaining increasing attention. Being natural in their origin, PHAs are completely biodegradable and eco-friendly. However, consistent efforts to exploit this biopolymer over the last few decades have not been able to pull PHAs out of their nascent stage, inspite of being the favorite of the commercial world. The major limitations are: (1) the high production cost, which is due to the high cost of the feed and (2) poor thermal and mechanical properties of polyhydroxybutyrate (PHB), the most commonly produced PHAs. PHAs have the physicochemical properties which are quite comparable to petroleum based plastics, but PHB being homopolymers are quite brittle, less elastic and have thermal properties which are not suitable for processing them into sturdy products. These properties, including melting point (Tm), glass transition temperature (Tg), elastic modulus, tensile strength, elongation etc. can be improved by varying the monomeric composition and molecular weight. These enhanced characteristics can be achieved by modifications in the types of substrates, feeding strategies, culture conditions and/or genetic manipulations.

Genome Wide Analysis for Searching Novel Markers to Rapidly Identify Clostridium Strains

Abstract Microbial classification is based largely on the 16S rRNA (rrs) gene sequence, which is conserved throughout the prokaryotic domain. The Ribosomal Database Project (RDP) has become a reference point for almost all practical purposes. The use of this gene is limited by the fact that it can be used to identify only to the extent to what has been known and is available in the RDP. In order to identify an organism whose rrs is not present in the RDP database, we need to generate novel markers to place the unknown on the evolutionary map. Here, sequenced genomes of 27 Clostridium strains belonging to 9 species have been used to identify two sets of genes: (1) common to most of the species, and (2) unique to a species. Combinations of genes (recN, dnaJ, secA, mutS, and/or grpE) and their unique restriction endonuclease digestion (AluI, BfaI and/or Tru9I) patterns have been established to rapidly identify Clostridium species. This strategy for identifying novel markers can be extended to all other organisms and diagnostic applications.

Biofouling Control by Quorum Quenching

Bacteria above a threshold cell density regulate the expression of a specific group of their genes in response to small signal molecules called autoinducers. This twitter communication phenomenon is called quorum sensing (QS). Among the various genetic expressions mediated by QS , biofilm formation and expression of virulence factors are the most prominent. These phenotypes are a major cause of concern for health departments and a wide range of industries dealing especially with drinking water, waste water reclamation, and desalination. This phenomenon leads to heavy economic losses. Efforts to disrupt biofilms have met with little success. In fact, bacteria within the biofilms are 1000 Open image in new window times more resistant to antibiotics than their planktonic counterparts. Biosensors or reporter strains have been developed to screen QS inhibitors. A potential approach to inhibit the process of biofouling is to interfere with bacterial QS signals. QS inhibiting enzymes and molecules have been isolated from a wide range of organisms such as bacteria, fungi, algae, and marine organisms. Efforts to disrupt biofilm formation have been carried out through designing antifouling agents that primarily reduce surface adhesion of the organisms. These include fouling release coatings such as nanocomposites. In contrast to removing biofilms, there are certain areas where it is desirable to regenerate biofilm for a fresh round of biotransformation.

Potential Applications of Quorum Sensing Inhibitors in Diverse Fields

All organisms are susceptible to attack by other organisms, and it may even spell death for the recipient. However, each organism is also bestowed with an inherent ability to protect itself by developing self-defence mechanisms. Human beings have a strong immune system but are quite susceptible to infections by fungi, bacteria and viruses. Microbial infections have been a constant worry for health departments (Kalia 2013, 2014). The economy of a nation is dramatically affected by the health of its residents. The discovery of antibiotics was a great boon to mankind. However, microbes have been developing resistance against antibiotics. So much so that during the last seven to eight decades, there has been a need to find new antibiotics. Now the scenario is quite depressing as almost all antibiotics are proving ineffective. The evolution of multidrug resistance among pathogenic microbes has taken a new dimension (Davies and Davies 2010). Bacteria expressing their virulent behaviour through the phenomenon of quorum sensing (QS) develop a biofilm. Bacteria inside the biofilm are up to 1000 times more resistant to antibiotics compared to their planktonic counterparts (Kalia et al. 2014a, b). Research efforts during the last four decades have brought hope by providing alternatives and supplements to antibiotics. Quorum sensing inhibitors (QSIs) are seen as novel drugs especially against infectious bacteria. Although, the search for QSIs was intended for helping human beings to fight against diseases, the applications can be extended to other fields as well: agriculture, aquaculture, water treatment, fisheries, etc. A few examples of applications of QSIs have been described below (Table 1).

Searching Biomarkers in the Sequenced Genomes of Staphylococcus for their Rapid Identification

Abstract Bacterial identification using rrs (16S rRNA) gene is widely reported. Bacteria possessing multiple copies of rrs lead to overestimation of its diversity. Staphylococcus genomes carries 5–6 copies of rrs showing high similarity in their nucleotide sequences, which lead to ambiguous results. The genomes of 31 strains of Staphylococcus representing 7 species were searched for the presence of common genes. In silico digestion of 34 common genes using 10 restriction endonucleases (REs) lead to select gene-RE combinations, which could be used as biomarkers. RE digestion of recA allowed unambiguous identification of 13 genomes representing all the 7 species. In addition, a few more genes (argH, argR, cysS, gyrB, purH, and pyrE) and RE combinations permitted further identification of 12 strains. By employing additional RE and genes unique to a particular strain, it was possible to identify the rest 6 Staphylococcus aureus strains. This approach has the potential to be utilized for rapid detection of Staphylococcus strains.

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).