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Nitroalkanes are important toxic pollutants for which there is no effective removal method at present. Although genetic engineering bacteria have been developed as a promising bioremediation strategy for years, their actual performance is far lower than expected. In this study, important factors affecting the application of engineered Geobacillus for nitroalkanes degradation were comprehensively optimized. The deep-reconstructed engineered strains significantly raised the expression and activity level of catalytic enzymes, but failed to fully enhance the degradation efficiency. However, further debugging of a variety of key parameters effectively improved the performance of the engineering strains. The increased cell membrane permeability, trace supplementation of vital nutritional factors, synergy of multifunctional enzyme engineered bacteria, switch of oxygen-supply mode, and moderate initial biomass all effectively boosted the degradation efficiency. Finally, a low-cost and highly effective bioreactor test for high-concentration nitroalkanes degradation proved the multi-parameter optimization mode helps to maximize the performance of genetically engineered bacteria.

Ecological and efficient alternatives to industrial processes have sparked interest for using microorganisms and enzymes as biocatalysts. One of the difficulties is finding candidates capable of resisting the harsh conditions in which industrial processes usually take place. Extremophiles, microorganisms naturally found in "extreme" ecological niches, produce robust enzymes for bioprocesses and product development. Thermophiles like Geobacillus, Alyciclobacillus, Anoxybacillus, Pyrococcus and Thermoccocus are some of the extremophiles containing enzymes showing special promise for biocatalysis. Glutamate dehydrogenase used in food processes, laccases and xylanases in pulp and paper processes, nitrilases and transaminases for pharmaceutical drug synthesis and lipases present in detergents, are examples of the increasing use of enzymes for biocatalytic synthesis from thermophilic microorganisms. Some of these enzymes from thermophiles have been expressed as recombinant enzymes and are already in the market. Here we will review recent discoveries of thermophilic enzymes and their current and potential applications in industry.

The aim of this study was to investigate the effect of atmospheric pressure cold plasma on the microbial substrate utilization and biomass yield in a thermophilic strain. Geobacillus sp. strain WSUCF1, a thermophile capable of producing cellulolytic enzymes with higher activity was used for this investigation. Treatment with cold plasma for 4 min increased the rates of glucose utilization by 74% and biomass yield by 60% when compared with the control. WSUCF1 treated with plasma also displayed enhanced biofilm formation. This study for the first time, reports the use of cold plasma for enhancing the substrate utilization and biofilm formation in a thermophile.

In this study, a thermophilic facultative anaerobic strain Geobacillus thermoglucosidasius W-2 was found to degrade nitroalkane under both aerobic and anaerobic conditions. Bioinformatical analysis revealed three putative nitroalkane-oxidizing enzymes (Gt-NOEs) genes from the W-2 genome. The three identified proteins Gt2929, Gt1378, and Gt1208 displayed optimal activities at high temperatures (70, 70, and 80 °C, respectively). Among these, Gt2929 exhibited excellent degradation capability, pH stability, and metal ion tolerance for nitronates under aerobic condition. Interestingly, under anaerobic condition, only Gt1378 still maintained high activity for 2-nitropropane and nitroethane, indicating that the W-2 strain utilized various pathways to degrade nitronates under aerobic and anaerobic conditions, respectively. Taken together, the first revelation of thermophilic nitroalkane-degrading mechanism under both aerobic and anaerobic conditions provides guidance and platform for biotechnological and industrial applications.

The aim of the present work was to use a thermophilic consortium for H2 production using lignocellulosic biomass in a single pot. The thermophilic consortium, growing at 60 °C utilized both glucose and xylose, making it an ideal source of microbes capable of utilizing and fermenting both hexose and pentose sugars. The optimization of pH, temperature, and substrate concentration increased the H2 production from 1.07 mmol H2/g of prairie cordgrass (PCG) to 2.2 mmol H2/g PCG by using the thermophilic consortium. A sequential cultivation of a thermostable lignocellulolytic enzyme producing strain Geobacillus sp. strain WSUCF1 (aerobic) with the thermophilic consortium (anaerobic) further increased H2 production with PCG 3-fold (3.74 mmol H2/g PCG). A single pot sequential culturing of aerobic and anaerobic microbes can be sustainable and advantageous for industrial scale production of biofuels.

We constructed a novel thermophilic microbial consortium, TADC7, with stable and efficient aflatoxin B1 (AFB1) degradation activity. The microbial consortium degraded more than 95% of the toxin within 72h when cultured with AFB1, and the optimum temperature was 55-60°C. TADC7 tolerated high doses of AFB1, with no inhibitory effects up to 5000μgL-1 AFB1; moreover, the degradation kinetics fit well with the Monod model. The proteins or enzymes in the TADC7 cell-free supernatant played a major role in AFB1 degradation. AFB1 degradation by the cell-free supernatant was stable up to 90°C, with an optimal pH of 8-10. We performed 16S rRNA sequencing to determine TADC7 community structure dynamics; the results indicated that Geobacillus and Tepidimicrobium played major roles in AFB1 degradation.

Investigations on microbial electrocatalysis as a strategy for enhancing the rates of substrate utilization leading to enhanced yield of biomass and enhanced biofilm formation are reported. A thermophilic Geobacillus sp. strain WSUCF1 (60 °C), a potential lignocellulose degrading microorganism was used as the electrocatalyst. Glucose, cellulose, and corn stover were used as the feedstocks. The results of this investigation showed that applying the oxidation potential of −0.383 mV (vs PRE) increased the glucose utilization and COD removal by 25.5% and 29.7% respectively. The bioelectrocatalysis strategy also increased the biomass yield by 81.2, 42.1, and 49.5% in the case of systems fed with glucose, cellulose, and corn stover, respectively, when compared with the systems without applied oxidation potential. This is the first work reporting the effects of applied oxidation potential on increasing the rates of degradation of lignocellulosic biomass and enhanced biofilm formation.

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 pH's, wide substrate specificity, and higher hydrolytic activity on recalcitrant lignocellulose, make this endo-xylanase a suitable candidate for biofuel and bioprocess industries.

A thermophilic naphthalene- and aliphatic hydrocarbon-degrading bacterium SH-1 was isolated from a deep oil well and identified as Geobacillus sp. n-alkanes from C12 to C33 in crude oil and naphthalene were effectively degraded by strain SH-1, and this strain could readily utilize these compounds as its sole carbon and energy resources. During the degradation of naphthalene, strain SH-1 initiated its attack on naphthalene by a monooxygenation at its C-1 to give 1-naphthol and further monooxygenation at C-2 to produce 1,2-dihydroxynaphthalene. The ring of 1,2-dihydroxynaphthalene was cleaved to form trans-o-hydroxybenzylidenepyruvate. Subsequently, trans-o-hydroxybenzylidenepyruvate was transformed to (2E)-3-(2-hydroxyphenyl)prop-2-enal by losing a carboxyl group. Additionally, benzoic acid was identified as an intermediate in the naphthalene degradation pathway of this Geobacillus strain. This study highlights an important potential use of the thermophilic degradative strain SH-1 in the cleanup of environmental contamination by naphthalene and crude oil and presents a mechanism for naphthalene metabolism.

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus . The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50 °C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24 h. This cellulase (∼54 kDa) was most active at pH 6.5 and 70 °C. The enzyme in citrate phosphate buffer (10 mM) was stable at 60 °C for at least 1 h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.

The composition of thermophilic (60 °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 °C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had K m values of 3.11 and 1.08 mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 °C for 1 day. 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.

A novel pretreatment method combining freezing/thawing with Geobacillus sp. G1 was employed to pretreat waste activated sludge (WAS) for enhancing the WAS hydrolysis and subsequent short-chain fatty acids (SCFAs) production. Results showed that freezing/thawing combined with Geobacillus sp. G1 pretreatment achieved the maximal concentrations of soluble protein from 40±6mg COD/L (non-pretreated) to 1226±24mg COD/L (pretreated), and accumulated SCFAs concentration increased from 248±81mg COD/L to 3032±53mg COD/L. Excitation emission matrix (EEM) fluorescence spectroscopy revealed the highest fluorescence intensity (FI) of protein-like substances, which was the dominant fluorescent organic matters, indicating the synergistic effect of freezing/thawing and Geobacillus sp. G1 pretreatment on organics hydrolysis. High-throughput pyrosequencing analysis investigated that the abundance of bacteria responsible for WAS hydrolysis (such as Clostridium and Caloramator) and SCFAs production (such as Parabacteroides and Bacterodies) was greatly enhanced due to the novel pretreatment method used.

The β-xylosidase encoding gene (XsidB) of the extremely thermophilic bacterium Geobacillus thermodenitrificans has been cloned and expressed in Escherichia coli. The homotrimeric recombinant XsidB is of 204.0 kDa, which is optimally active at 60 °C and pH 7.0 with T1/2 of 58 min at 70 °C. The β-xylosidase remains unaffected in the presence of most metal ions and organic solvents. The Km [p-nitrophenyl β-xyloside (pNPX)], Vmax and kcat values of the enzyme are 2 × 10−3 M, 1250 μmoles mg−1 min−1 and 13.20 × 105 min−1, respectively. The enzyme catalyzes transxylosylation reactions in the presence of alcohols as acceptors. The pharmaceutically important β-methyl- d -xylosides could be produced using pNPX as the donor and methanol as acceptor. The products of transxylosylation were identified by TLC and HPLC, and the structure was confirmed by 1H NMR analysis. The enzyme is also useful in synthesizing transxylosylation products from the wheat bran hydrolysate.

Enterobacter cloacae strain JD, which produces water-insoluble biopolymers at optimal temperature of 30°C, and a thermophilic Geobacillus strain were used to construct an engineered strain for exopolysaccharide production at high temperatures by protoplast fusion. The obtained fusant strain ZR3 produced exopolysaccharides at up to 45°C with optimal growth temperature at 35°C. The fusant produced exopolysaccharides of approximately 7.5 g/L or more at pH between 7.0 and 9.0. The feasibility of the enhancement of crude oil recovery with the fusant was tested in a sand-packed column at 40°C. The results demonstrated that bioaugmentation of the fusant was promising approach for MEOR. Mass growth of the fusant was confirmed in fermentor tests.

A xylanase gene (xyl-gt) of 1.224 kbp was cloned from the extremely thermophilic bacterium Geobacillus thermoleovorans that encodes a protein containing 408 amino acid residues. Eight conserved regions (signature sequences) of GH family 10 xylanases have been found in the xylanase. When the xylanase gene was cloned and expressed in Escherichia coli BL21 (DE3), the recombinant strain produced xylanase titer of 270 U mg(-1) which is 27-fold higher than the wild strain. It is optimally active at 80°C and pH 8.5 with a high thermostability over broad range of pH (6-12) and temperature (40-100°C). The end products of the hydrolysis of birch wood xylan and agro-residues included xylobiose, xylotriose, xylotetraose and xylopentaose. The xylanase of G. thermoleovorans is one of the rare xylanases that exhibits thermo-alkali-stability, and thus, it is a suitable candidate for pre-bleaching of paper pulps and generating xylooligosaccharides from agro-residues for use as prebiotics.

Geobacillus pallidus XS2 and XS3 were isolated from oil contaminated soil samples in Yumen oilfield, China, and were able to produce bioemulsifiers on different hydrocarbons. Biodegradation assays exhibited that approximately 70% of PAH (250 mg/L) or 85% of crude oil (500 mg/L) was removed by the thermophilic bacteria after 20 days. The bioemulsifiers of the two strains were isolated and obtained a productive yield of 4.24 ± 0.08 and 3.82 ± 0.11 g/L, respectively. GPC analysis revealed that the number-average molecular weights (Mn) of the two bioemulsifiers were 271,785 Da and 526,369 Da, with PDI values of 1.104 and 1.027, respectively. Chemical composition studies exhibited that the bioemulsifier XS2 consisted of carbohydrates (68.6%), lipids (22.7%) and proteins (8.7%) while the bioemulsifier XS3 was composed by carbohydrates (41.1%), lipids (47.6%) and proteins (11.3%). Emulsification assays approved the effectiveness of bioemulsifiers over a wide range of temperature, pH and salinity. Highlights ► Isolation and identification of thermophilic hydrocarbon-degrading bacteria. ► Investigation of hydrocarbon degradation pattern. ► Bioemulsifier: chemical characterization and emulsifying properties

The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.

An inoculum containing two amylolytic and three cellulolytic thermophilic bacteria, isolated from a preceding compost pile and identified as Geobacillus species by 16S rRNA gene sequencing, was applied to a mixture of market waste, rice straw and cow dung (5:1:0.2) so that the initial cell density was 2 x 10(8) colony forming unit (CFU) per gram dry weight at 55 degrees Celsius. The inoculation increased the total cell count particularly in the thermophilic stage as determined by flow cytometry. Concomitantly, there was a significant rise in microbial metabolism in the compost pile as reflected by the dehydrogenase activity. As a result, the C/N ratio dropped more rapidly in the inoculated mixture than that in the control without inoculum. The study, therefore, suggested that inoculation by thermophilic bacteria would be effective in the composting process at least in the thermophilic stage.