Rakesh Bajpai

Microbial lipids from renewable resources: production and characterization.

A number of microorganisms belonging to the genera of algae, yeast, bacteria, and fungi have ability to accumulate neutral lipids under specific cultivation conditions. The microbial lipids contain high fractions of polyunsaturated fatty acids and have the potential to serve as a source of significant quantities of transportation fuels. This paper reviews the current state of the art of this field. It summarizes the various microorganism used, feed stocks available, environmental factors that influence growth of cells and accumulation of lipids, major fatty acid composition of lipids, and the technology.

Relevance of microbial coculture fermentations in biotechnology

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro‐organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.

Aerobic biodegradation of 2,4-DNT and 2,6-DNT: performance characteristics and biofilm composition changes in continuous packed-bed bioreactors.

This manuscript deals with continuous experiments for biodegradation of individual dinitrotoluenes by a defined mixed culture in packed-bed reactors (PBRs) containing either poraver or fire-clay as packing material. Removal efficiencies and volumetric biodegradation rates were measured as a function of the loading rate of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) under steady-state conditions. The poraver reactor showed higher removal efficiencies for both the DNTs. The removal efficiency for 2,4-DNT remained greater than 90% in the poraver reactor whereas it dropped steadily from 85 to 65% in the fire-clay reactor as the organic loading rates were increased from 19 to 60 mg L(-1)day(-1). Similar trends were seen for the volumetric degradation rate as well. In both the reactors, 2,4-DNT degraded more effectively than 2,6-DNT. The microbial consortium was characterized both in the inoculum as well as in the operating PBR. Cell numbers per gram dry packing material were similar in the two reactors. However, there was a distinct difference in the nature of microorganisms that were found in the two packings. The fire-clay contained a larger number of cells that were not primary degraders of DNTs.

Dual feeding strategy for the production of α-amylase by Bacillus caldolyticus using complex media

In this study, the objective was to investigate an exponential feeding strategy for fed-batch production of thermostable alpha-amylase (E.C. 3.2.1.1.) from the Bacillus caldolyticus (DSM405). The parameters for establishing compositions of feed media and feeding rate were obtained by statistical analysis of batch and continuous shake flask experiments. These parameters were casitone to starch ratio of 2.67g(casitone)g(starch)(-1), maintenance coefficient 0.174g(casitone)g(DW)(-1)h(-1), cell yield 0.62g(DW)g(casitone)(-1) and mu(opt)=0.2h(-1). The exponentially fed fermentation resulted in yield of 120Uml(-1) alpha-amylase that was thermostable up to 105 degrees C. Results of the exponentially fed fermentation have been discussed in the light of a feed-back controlled fed-batch fermentation reported earlier by the authors. A comparison of the temperature and pH effects on amylase produced by B. caldolyticus and on several other commercially available amylases has also been presented.

Biodegradation of a mixture of mononitrophenols in a packed-bed aerobic reactor

Aerobic biodegradation of individual mononitrophenols (4-, 3- and 2-NPs) and their mixture in simulated wastewater was investigated in a packed-bed bench scale bioreactor continuously operated in a flow mode, with a mixed microbial culture adsorbed on expanded slate. Under a low, suboptimal hydraulic retention time (HRT) of 30 min the reactor removed more than 3 g.L−1.day−1 of the NP mixture while maintaining a > 85–90% removal efficiency (RE). Under higher HRT values, starting at 45 min, more than 2 g.L−1.day−1 of the NP mixture were removed with an RE > 98%. Significant substrate interactions were observed; the addition of other NPs caused the saturation of 2-NP catabolic capacity whereas the addition of 2-NP caused the de-saturation of the 4- and 3-NP catabolic capacity. 3- and 4-NPs appeared to be removed independently, i.e., by different enzyme systems. After ten months of operation, the biofilm composition was significantly altered to become predominantly bacterial. Only one originally inoculated strain remained indicating microbial contamination followed by a genetic material exchange.

CH···π Interactions Do Not Contribute to Hydrogen Transfer Catalysis by Glycerol Dehydratase

The role of the nonbonded CH···π interaction in the hydrogen abstraction from glycerol by the coenzyme B(12)-independent glycerol dehydratase (GDH) was examined using the QM/MM (ONIOM), MP2, and CCSD(T) methods. The studied CH···π interaction included the hydrogen atom of the -C(2)H(OH)- group of the glycerol substrate and the tyrosine-339 residue of the dehydratase. A contribution of this interaction to the stabilization of the transition state for the transfer of a hydrogen atom from the adjacent terminal C(1)H(2)(OH) group to cysteine 433 was determined by ab initio HF, MP2, and CCSD(T) calculations with the aug-cc-pvDZ basis set for the corresponding methane/benzene, methanol/phenol, and glycerol radical/phenol subsystems. The calculated CH···π distance, defined as the distance between the H atom and the center of the phenol ring, shortened from 2.62 to 2.52 Å upon going from the ground- to the transition-state of the GDH-catalyzed reaction. However, this shortening was not accompanied by the expected lowering of the CH···π interaction free energy. Instead, this interaction remained weak (about -1 kcal/mol) along the entire reaction coordinate. Additionally, the mutual orientation of the CH group and the phenol ring did not change significantly during the reaction. These results suggest that the phenol group of the tyrosine-339 does not contribute to lowering the activation barrier in the enzyme, but do not exclude the possibility that tyrosine 339 facilitates proper orientation of glycerol for the electrostatic catalysis, or inhibits side-reactions of the reactive glycerol radical intermediate.

The diversity and molecular modelling analysis of B 12-dependent and B 12-independent glycerol dehydratases

To broaden our knowledge on the diversity of glycerol dehydratases, comprehensive sequence and molecular modelling analyses of these enzymes were performed. Our sequence analysis showed that B 12-dependent and B 12-independent glycerol dehydratases are not related, suggesting that they evolved from different ancestors. Second, our study demonstrated that a gene fusion event occurred between α and β subunits of B 12-dependent glycerol dehydratases in several bacteria during enzyme evolution. In addition, our sequence and molecular modelling analyses revealed more B 12-independent glycerol dehydratases including hypothetical proteins. Furthermore, we found that some microorganisms contain both B 12-dependent and B 12-independent glycerol dehydratases in their genomes.

Glycerol Dehydratases: Biochemical Structures, Catalytic Mechanisms, and Industrial Applications in 1,3-Propanediol Production by Naturally Occurring and Genetically Engineered Bacterial Strains.

To date, two types of glycerol dehydratases have been reported: coenzyme B12-dependent and coenzyme B12-independent glycerol dehydratases. The three-dimensional structure of the former is a dimer of αβγ heterotrimer, while that of the latter is a homodimer. Their mechanisms of reaction are typically enzymatic radical catalysis. Functional radical in both the glycerol dehydratases is the adenosyl radical. However, the adenosyl radical in the former originates from coenzyme B12 by homolytic cleavage, and that in the latter from S-adenosyl-methionine. Until some years ago, Clostridium butyricum VPI 1718 was the only microorganism known to possess B12-independent glycerol dehydratase, but since then, several other bacteria with this unique capability have been identified. This article focuses on the glycerol dehydratases and on 1,3-propanediol production from glycerol by naturally occurring and genetically engineered bacterial strains containing glycerol dehydratase.

Positive effect of reduced aeration rate on secretion of alpha-amylase and neutral proteases during pressurised fermentation of thermophilic Bacillus caldolyticus

The thermophilic microorganism Bacillus caldolyticus was incubated in laboratory scale stirred bioreactors under pressurised conditions at different aeration rates. Increased amounts of CO2/bicarbonate were solubilised under the chosen conditions. A reduction in aeration rate from 1 vvm to 0.1 vvm resulted in accumulation of CO2 and bicarbonate up to 126 mg l(-1) and 733 mg l(-1), respectively and also increased secretion of α-amylase and neutral proteases (increases of 123% and 52%, respectively). In this paper, the effect of reduced aeration rate on CO2/bicarbonate concentration and enzyme activities is presented. The selected fermentation conditions are closely related to those prevalent in large scale bioreactors and may offer the possibility of achieving high enzyme yields at reduced aeration costs on an industrial scale.

Screening for ω−1-hydroxy fatty acid over-producing mutants for bioconversion of oleic acid by combining general mutagenesis and specific selection

Chemical mutation of a strain producing hydroxy-fatty acid from oleic acid (OA) using NTG (N-methyl-N’-nitro-N-nitrosoguanidine) resulted in a high percentage of improved mutants. A positive screening procedure yielded several high producers, specifically the strain Bacillus pumilus M-F641 (BP M-F641). This strain produced predominantly ω−1-hydroxy fatty acid and could utilize higher concentrations of OA than the parent strain. In shake flask culture, the best ω−1-hydroxy fatty acid concentration and yield (the ratio of ω−1-hydroxy fatty acid accumulation to OA consumption) reached 570 mg L−1 and 11.5%, respectively. Repeated tests showed that the mutant BP M-F641 was genetically stable.