Butanol production from wheat straw hydrolysate using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation 48.9 g L⁻¹ glucose (initial sugar 62.0 g L⁻¹) was used to produce 20.1 g L⁻¹ ABE with a productivity and yield of 0.28 g L⁻¹ h⁻¹ and 0.41, respectively. In a similar experiment where WSH (60.2 g L⁻¹ total sugars obtained from hydrolysis of 86 g L⁻¹ wheat straw) was used, the culture produced 25.0 g L⁻¹ ABE with a productivity and yield of 0.60 g L⁻¹ h⁻¹ and 0.42, respectively. These results are superior to the control experiment and productivity was improved by 214%. When WSH was supplemented with 35 g L⁻¹ glucose, a reactor productivity was improved to 0.63 g L⁻¹ h⁻¹ with a yield of 0.42. In this case, ABE concentration in the broth was 28.2 g L⁻¹. When WSH was supplemented with 60 g L⁻¹ glucose, the resultant medium containing 128.3 g L⁻¹ sugars was successfully fermented (due to product removal) to produce 47.6 g L⁻¹ ABE, and the culture utilized all the sugars (glucose, xylose, arabinose, galactose, and mannose). These results demonstrate that C. beijerinckii P260 has excellent capacity to convert biomass derived sugars to solvents and can produce over 28 g L⁻¹ (in one case 41.7 g L⁻¹ from glucose) ABE from WSH. Medium containing 250 g L⁻¹ glucose resulted in no growth and no ABE production. Mixtures containing WSH + 140 g L⁻¹ glucose (total sugar approximately 200 g L⁻¹) showed poor growth and poor ABE production. Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Induction of carboxymethylcellulase inMacrophomina phaseolina

Macrophomina phaseolina, the well-known jute pathogenic fungus produces very low levels of both extra- and intracellular carboxymethylcellulase even in the absence of any cellulose as carbon source in the medium. However, the production of these enzymes is greatly induced by soluble carboxymethylcellulose. The carboxymethylcellulase inM.phaseolina is repressed by glucose.     

The Influence of Polyvinylpyrrolidone on Freezing of Bovine IVF Blastocysts Following Biopsy

A study was conducted to develop a better freezing protocol for in vitro developed biopsied bovine blastocysts. Biopsied blastocysts were exposed to 1.8 M ethylene glycol (EG) + 0.05 M trehalose (T) and different concentration (5, 10, and 20%) of polyvinylpyrrolidone (PVP). Exposure to the solutions alone did not affect their in vitro development (Experiment 1). Experiments 2, 3, and 4 tested the viability of biopsied blastocysts cryopreserved in 1.8 M EG + different concentrations of T (0, 0.05, 0.1, and 0.3 M), 1.8 M EG + different concentrations of PVP (0, 5, 10, and 20%), and 1.8 M EG + 0.05 M T + different concentrations of PVP (0, 5, 10, and 20%), respectively. The proportion of biopsied blastocysts that reexpanded following cryopreservation in 1.8 M EG + 0.05 M T (38.5%) and 1.8 M EG + 0.1 M T (36.1%) was significantly (P < 0.05) higher than the proportion that reexpanded in 1.8 M EG + 0.3 M T (13.9%) (Experiment 2). The viability and the percentage of embryos that developed to >250 μm in diameter in the 5, 10, and 20% PVP groups (77.8 and 50.0%, 78.1 and 43.8%, 76.9 and 65.4%, respectively) were significantly higher than those that developed cryopreserved without PVP (55.1 and 20.7%) (Experiment 3). Optimum development of in vitro culture of frozen-thawed biopsied blastocysts was obtained using 1.8 M EG + 0.05 M T and 20% PVP. Analysis of blastocysts >250/μm in diameter showed that the number of ICM cells of biopsied blastocysts cryopreserved in 1.8 M EG + 0.05 M T with or without PVP was not different from the number of unfrozen biopsied blastocysts. These results indicate that PVP has some beneficial effect on freezing of biopsied bovine blastocysts.     

Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating <Emphasis Type="Italic">Brevundimonas diminuta</Emphasis> in drinking water

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100–200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 × 10³ colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5′ 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (<4 h) enumeration of B. diminuta and may be viable alternatives to plating when validating drinking water filtration systems.     

Genetic and Genomic Resources of Small Millets

Small millets are very promising agricultural entity to ensure global food security. They gained remarkable importance in agriculture due to their resilience to climatic changes and increasing demand for nutritious food and feed. The genetic variability in the core and mini-core germplasm of small millets was characterized for nutritional composition and capacity to tolerate abiotic stresses that can be infused in breeding programs. Other than the foxtail millet, availability of genomic information in small millets is far below the mark for use in marker-assisted breeding and other genetic improvement programs. The genome sequence of foxtail millet has recently triggered a plethora of post-genomic analysis and envisaged foxtail millet as a model organism for the C₄ grasses and bioenergy research. Recent developments in the next-generation sequencing technologies enabled us, with the simultaneous discovery of high-throughput markers and multiplexed genotyping of germplasm, to speedup marker-assisted breeding. In this context, an in-depth analysis of the wealth of diverse germplasm resources and future perspectives of integrating genomics in genome-wide marker-trait association and breeding in small millets is worthy.     

Oxidation of D-glucose in the presence of 2,2'-bipyridine by CrVI in aqueous micellar media: a kinetic study

The kinetics of Cr(VI) oxidation of D-glucose to the corresponding lactone in the presence and absence of 2,2'-bipyridine (bipy) has been carried out under the conditions, [D-glucose](T) > [Cr(VI)](T) at different temperatures in aqueous micellar media. The monomeric Cr(VI) species has been found to be kinetically active in the absence of bipy whereas in the bipy-catalysed path, the Cr(VI)-bipy complex has been found to be the active oxidant. In the bipy-catalysed path, the Cr(VI)-bipy complex undergoes nucleophilic attack by the substrate to form a ternary complex. The ternary complex spontaneously experiences a redox decomposition (through two-electron transfer) in the rate-determining step leading to the product lactone and Cr(IV)-bipy complex. The Cr(IV)-bipy complex then takes part in faster steps in the further oxidation of D-glucose and is ultimately converted into a Cr(III)-bipy complex. In the uncatalysed path, the Cr(VI)-substrate ester experiences acid catalysed redox decomposition (two-electron transfer) in the rate-determining step. The uncatalysed path shows a second order dependence on [H(+)] and a first order dependence on each of the reactants [D-glucose](T) and [Cr(VI)](T). In contrast, the bipy-catalysed path shows a first order dependence on each of the reactants [H(+)], [D-glucose](T) and [Cr(VI)](T). The bipy-catalysed path is first order in [bipy](T). These observations remain unaltered in the presence of externally added surfactants. The effect of the cationic surfactant, N-cetylpyridinium chloride (CPC) and anionic surfactant, sodium dodecyl sulfate (SDS) on both the uncatalysed and bipy-catalysed path has been studied. CPC inhibits both the uncatalysed and bipy-catalysed path, while SDS catalyses these reactions. The observed micellar effects have been explained by considering hydrophobic and electrostatic interactions between the surfactants and reactants.     

Lactose-Enhanced Cellulase Production by Microbacterium sp. Isolated from Fecal Matter of Zebra (Equus zebra)

A cellulase-producing bacterial strain designated Z5 was isolated from the fecal matter of Zebra (Equus zebra). The strain was identified as Microbacterium sp. on the basis of 16S rDNA sequence analysis. The effect of substrates like CMC, avicel, starch, maltose, sucrose, glucose, fructose, galactose, and lactose on cellulase production was also determined. Lactose as the sole carbon source induced cellulase production in this bacterial strain and a positive synergistic effect of lactose and CMC was also observed with enhancement of 3-4 times in cellulase activity. The optimum cellulase production was recorded with 3% CMC and 1% lactose when added individually in the Omeliansky's medium. The optimum temperature and time for cellulase production by this bacterial strain was 37°C and 10 days, respectively. To our knowledge this is the first report on enhancement of cellulase production by lactose in the Microbacterium sp.