Rajesh K. Sani

Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains.

The composition of thermophilic (60 degrees C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of sequences related to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera Geobacillus, Thermobacillus, Cohnella, and Thermus. A compost isolate WSUCF1 (Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75 degrees C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had K(m) values of 3.11 and 1.08mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 degrees C for 1day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels.

Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes.

Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ≤ 50 °C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed.

Copper-Induced Inhibition of Growth of Desulfovibrio desulfuricans G20: Assessment of Its Toxicity and Correlation with Those of Zinc and Lead

ABSTRACT The toxicity of copper [Cu(II)] to sulfate-reducing bacteria (SRB) was studied by using Desulfovibrio desulfuricansG20 in a medium (MTM) developed specifically to test metal toxicity to SRB (R. K. Sani, G. Geesey, and B. M. Peyton, Adv. Environ. Res. 5:269–276, 2001). The effects of Cu(II) toxicity were observed in terms of inhibition in total cell protein, longer lag times, lower specific growth rates, and in some cases no measurable growth. At only 6 μM, Cu(II) reduced the maximum specific growth rate by 25% and the final cell protein concentration by 18% compared to the copper-free control. Inhibition by Cu(II) of cell yield and maximum specific growth rate increased with increasing concentrations. The Cu(II) concentration causing 50% inhibition in final cell protein was evaluated to be 16 μM. A Cu(II) concentration of 13.3 μM showed 50% inhibition in maximum specific growth rate. These results clearly show significant Cu(II) toxicity to SRB at concentrations that are 100 times lower than previously reported. No measurable growth was observed at 30 μM Cu(II) even after a prolonged incubation of 384 h. In contrast, Zn(II) and Pb(II), at 16 and 5 μM, increased lag times by 48 and 72 h, respectively, but yielded final cell protein concentrations equivalent to those of the zinc- and lead-free controls. Live/dead staining, based on membrane integrity, indicated that while Cu(II), Zn(II), and Pb(II) inhibited growth, these metals did not cause a loss of D. desulfuricans membrane integrity. The results show that D. desulfuricans in the presence of Cu(II) follows a growth pattern clearly different from the pattern followed in the presence of Zn(II) or Pb(II). It is therefore likely that Cu(II) toxicity proceeds by a mechanism different from that of Zn(II) or Pb(II) toxicity.

Molecular Techniques to Assess Microbial Community Structure, Function, and Dynamics in the Environment

Culture-based methods are important in investigating the microbial ecology of natural and anthropogenically impacted environments, but they are extremely biased in their evaluation of microbial genetic diversity by selecting a particular population of microorganisms. With recent advances in genomics and sequencing technologies, microbial community analyses using culture-independent molecular techniques have initiated a new era of microbial ecology. Molecular analyses of environmental communities have revealed that the cultivable fraction represents <1% of the total number of prokaryotic species present in any given sample. A variety of molecular methods based on direct isolation and analysis of nucleic acids, proteins, and lipids from environmental samples have been discovered and revealed structural and functional information about microbial communities. Molecular approaches such as genetic fingerprinting, metagenomics, metaproteomics, metatranscriptomics, and proteogenomics are vital for discovering and characterizing the vast microbial diversity and understanding their interactions with biotic and abiotic environmental factors. This chapter summarizes recent progress in the area of molecular microbial ecology with an emphasis on novel techniques and approaches that offer new insights into the phylogenetic and functional diversity of microbial assemblages. The advantages and pitfalls of commonly used molecular methods to investigate microbial communities are discussed. The potential applications of each molecular technique and how they can be combined for a greater comprehensive assessment of microbial diversity has been illustrated with example studies.

Microbial Diversity in Uranium Mining-Impacted Soils as Revealed by High-Density 16S Microarray and Clone Library

Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.

Toxic Effects of Chromium(VI) on Anaerobic and Aerobic Growth of Shewanella oneidensis MR‐1

Cr(VI) was added to early‐ and mid‐log‐phase Shewanella oneidensis ( S. oneidensis) MR‐1 cultures to study the physiological state‐dependent toxicity of Cr(VI). Cr(VI) reduction and culture growth were measured during and after Cr(VI) reduction. Inhibition of growth was observed when Cr(VI) was added to cultures of MR‐1 growing aerobically or anaerobically with fumarate as the terminal electron acceptor. Under anaerobic conditions, there was immediate cessation of growth upon addition of Cr(VI) in early‐ and mid‐log‐phase cultures. However, once Cr(VI) was reduced below detection limits (0.002 mM), the cultures resumed growth with normal cell yield values observed. In contrast to anaerobic MR‐1 cultures, addition of Cr(VI) to aerobically growing cultures resulted in a gradual decrease of the growth rate. In addition, under aerobic conditions, lower cell yields were also observed with Cr(VI)‐treated cultures when compared to cultures that were not exposed to Cr(VI). Differences in response to Cr(VI) between aerobically and anaerobically growing cultures indicate that Cr(VI) toxicity in MR‐1 is dependent on the physiological growth condition of the culture. Cr(VI) reduction has been previously studied in Shewanella spp., and it has been proposed that Shewanella spp.may be used in Cr(VI) bioremediation systems. Studies of Shewanella spp. provide valuable information on the microbial physiology of dissimilatory metal reducing bacteria; however, our study indicates that S. oneidensis MR‐1 is highly susceptible to growth inhibition by Cr(VI) toxicity, even at low concentrations [0.015 mM Cr(VI)].

Multiple Mechanisms of Uranium Immobilization by Cellulomonas sp. strain ES6

Removal of hexavalent uranium (U(VI)) from aqueous solution was studied using a Gram‐positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non‐growth conditions in bicarbonate and PIPES buffers. Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate‐based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X‐ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non‐uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)‐phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U‐free controls suggesting simultaneous precipitation of U and PO  43− . In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High‐resolution transmission electron microscopy (HR‐TEM) and energy dispersive X‐ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone‐2,6‐disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non‐uraninite U(IV) phase. Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6. Biotechnol. Bioeng. 2011;108: 264–276.

Assessment of lead toxicity to Desulfovibrio desulfuricans G20: influence of components of lactate C medium

Abstract The bioavailability and toxicity of lead (Pb) to Desulfovibrio desulfuricans G20 is greatly influenced by aqueous phase chemical composition. Apparent Pb toxicity is reduced by precipitation and complexation with chemicals found in standard growth media for sulfate-reducing bacteria (SRB). To determine the influence of medium composition on observed Pb toxicity, a new medium was developed to more accurately assess the toxicity of Pb to Desulfovibrio desulfuricans. The new medium, metal toxicity medium (MTM), eliminates abiotic Pb precipitation and minimizes formation of Pb complexes in solution. Significant growth of Desulfovibrio desulfuricans was observed on MTM in the absence of Pb, while no measurable growth was observed at 3 mg/l Pb as PbCl 2 . For comparison, in Lactate C medium ( Burlage et al., 1998 ) abiotic Pb precipitation was apparent, and the specific growth rate at 100 mg/l Pb was only reduced by 8.1% compared to the Pb-free control. Toxicity was measured in terms of longer lag times and slower growth rates (including no growth) as compared to Pb-free controls. This report describes the effects of specific medium components on Pb toxicity to Desulfovibrio desulfuricans and provides a better baseline for comparison of natural and industrial waters for observing heavy metal toxicity on SRB.

Novel thermostable endo-xylanase cloned and expressed from bacterium Geobacillus sp. WSUCF1.

A gene encoding a GH10 endo-xylanase from Geobacillus sp. WSUCF1 was cloned and expressed in Escherichia coli. Recombinant endo-xylanase (37kDa) exhibited high specific activity of 461.0U/mg of protein. Endo-xylanase was optimally active on birchwood xylan at 70°C and pH 6.5. The endo-xylanase was found to be highly thermostable at 50 and 60°C, retaining 82% and 50% of its original activity, respectively, after 60h. High xylan conversions (92%) were obtained with oat-spelt xylan hydrolysis. Higher glucan and xylan conversions were obtained on AFEX-treated corn stover with an enzyme cocktail containing WSUCF1 endo-xylanase (71% and 47%) as compared to enzyme cocktail containing commercial fungal endo-xylanase (64% and 41%). High specific activity, active at high pHs, wide substrate specificity, and higher hydrolytic activity on recalcitrant lignocellulose, make this endo-xylanase a suitable candidate for biofuel and bioprocess industries.

Toxic effects of uranium on Desulfovibrio desulfuricans G20

The toxic effects of U(VI) were studied using Desulfovibrio desulfuricans G20 in a medium containing bicarbonate or 1,4-piperazinediethane sulfonic acid disodium salt monohydrate (PIPES) buffer (each at 30 mM and pH 7). Uranium(VI) toxicity was dependent on the medium buffer and was observed in terms of longer lag times and, in some cases, no measurable growth. The minimum inhibiting concentration was 140 microM U(VI) in PIPES-buffered medium. This is 36-fold lower than that reported previously for D. desulfuricans. For all cases in which D. desulfuricans G20 grew in the presence of U(VI), the final cell protein yield was equivalent to that of the U(VI)-free control. In 24 h, D. desulfuricans G20 (total cell protein, 40 mg/L) removed 50 FiM U(VI) from solution in PIPES buffer, as compared to 96 microM U(VI) in bicarbonate buffer under anaerobic, nongrowth conditions. Even though the solubility of U(VI) was significantly lower in PIPES buffer than in bicarbonate buffer, U(VI) was much more toxic in PIPES buffer than in bicarbonate buffer. Analysis of thin sections of D. desulfuricans G20 treated with 90 microM U(VI) in medium containing PIPES buffer revealed that only a very small fraction of cells had reduced U precipitates in the periplasmic spaces. In the presence of bicarbonate buffer, however, reduced U was observed not only in the periplasm but also in the cytoplasm. Selected-area electron diffraction patterns and crystallographic analysis of transmission-electron microscopic lattice fringe images confirmed the structure of precipitated U in the cell periplasm and cytoplasm as being that of uraninite. These results suggest that U(VI) toxicity and the detoxification mechanisms of D. desulfuricans G20 depend greatly on the chemical forms of U(VI) that are present.