Thermoanaerobacter Species Research Articles

Biotechnological Prospects of Thermoanerobacter AK15: End-Product Formation from Carbohydrates, Amino Acids, and Lignocellulosic and Macroalgae Hydrolysates.

The conversion of lignocellulosic and algal biomass by thermophilic bacteria has been an area of active investigation. Thermoanaerobacter species have proven to be particularly capable in the production of bioethanol and biohydrogen from lignocellulosic biomass, although detailed studies of their abilities to utilize the full gamut of carbohydrate, amino acids, and proteins encountered in biomass hydrolysates are seldom comprehensively examined. Here, we re-evaluate the ability of Thermoanaerobacter strain AK15, a highly ethanologenic strain previously isolated from a hot spring in Iceland. Similar to other Thermoanaerobacter species, the strain degraded a wide range of mono- and di-saccharides and produced a maximum of 1.57 mol ethanol per mol of glucose degraded at high liquid-gas phase ratios. The ability of strain AK15 to utilize amino acids in the presence of thiosulfate is limited to the branched-chain amino acids as well as serine and threonine. Similar to other Thermoanaerobacter species, strain AK15 produces a mixture of branched-chain fatty acids and alcohols, making the strain of interest as a potential source of longer-chain alcohols. Finally, the strain was also shown to use butyrate as an electron sink during glucose degradation resulting in the reduced product butanol, in addition to end-products produced from glucose. Thus, strain AK15 is a promising candidate for ethanol and higher-order alcohols from a range of lignocellulosic and algal biomass.

Complete Genome Sequences of Two Thermophilic Indigenous Bacteria Isolated from Wheat Straw, Thermoclostridium stercorarium subsp. Strain RKWS1 and Thermoanaerobacter sp. Strain RKWS2.

Reported here are complete genome sequences for two anaerobic, thermophilic bacteria isolated from wheat straw, i.e., the (hemi)cellulolytic Thermoclostridium stercorarium subspecies strain RKWS1 (3,029,933 bp) and the hemicellulolytic Thermoanaerobacter species strain RKWS2 (2,827,640 bp). Discovery of indigenous thermophiles in plant biomass suggests that high-temperature microorganisms are more ubiquitous than previously thought.

Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review

The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.

Alcohol dehydrogenases AdhE and AdhB with broad substrate ranges are important enzymes for organic acid reduction in Thermoanaerobacter sp. strain X514

BackgroundThe industrial production of various alcohols from organic carbon compounds may be performed at high rates and with a low risk of contamination using thermophilic microorganisms as whole-cell catalysts. Thermoanaerobacter species that thrive around 50–75 °C not only perform fermentation of sugars to alcohols, but some also utilize different organic acids as electron acceptors, reducing them to their corresponding alcohols.ResultsWe purified AdhE as the major NADH- and AdhB as the major NADPH-dependent alcohol dehydrogenase (ADH) from the cell extract of the organic acid-reducing Thermoanaerobacter sp. strain X514. Both enzymes were present in high amounts during growth on glucose with and without isobutyrate, had broad substrate spectra including different aldehydes, with high affinities (< 1 mM) for acetaldehyde and for NADH (AdhE) or NADPH (AdhB). Both enzymes were highly thermostable at the physiological temperature of alcohol production. In addition to AdhE and AdhB, we identified two abundant AdhA-type ADHs based on their genes, which were recombinantly produced and biochemically characterized. The other five ADHs encoded in the genome were only expressed at low levels.ConclusionsAccording to their biochemical and kinetic properties, AdhE and AdhB are most important for ethanol formation from sugar and reduction of organic acids to alcohols, while the role of the two AdhA-type enzymes is less clear. AdhE is the only abundant aldehyde dehydrogenase for the acetyl-CoA reduction to aldehydes, however, acid reduction may also proceed directly by aldehyde:ferredoxin oxidoreductase. The role of the latter in bio-alcohol formation from sugar and in organic acid reduction needs to be elucidated in future studies.

Influence of Culture Conditions on the Bioreduction of Organic Acids to Alcohols by Thermoanaerobacter pseudoethanolicus.

Thermoanaerobacter species have recently been observed to reduce carboxylic acids to their corresponding alcohols. The present investigation shows that Thermoanaerobacter pseudoethanolicus converts C2–C6 short-chain fatty acids (SCFAs) to their corresponding alcohols in the presence of glucose. The conversion yields varied from 21% of 3-methyl-1-butyrate to 57.9% of 1-pentanoate being converted to their corresponding alcohols. Slightly acidic culture conditions (pH 6.5) was optimal for the reduction. By increasing the initial glucose concentration, an increase in the conversion of SCFAs reduced to their corresponding alcohols was observed. Inhibitory experiments on C2–C8 alcohols showed that C4 and higher alcohols are inhibitory to T. pseudoethanolicus suggesting that other culture modes may be necessary to improve the amount of fatty acids reduced to the analogous alcohol. The reduction of SCFAs to their corresponding alcohols was further demonstrated using 13C-labelled fatty acids and the conversion was followed kinetically. Finally, increased activity of alcohol dehydrogenase (ADH) and aldehyde oxidation activity was observed in cultures of T. pseudoethanolicus grown on glucose as compared to glucose supplemented with either 3-methyl-1-butyrate or pentanoate, using both NADH and NADPH as cofactors, although the presence of the latter showed higher ADH and aldehyde oxidoreductase (ALDH) activity.

Fermentation of Mannitol Extracts From Brown Macro Algae by Thermophilic Clostridia.

Mannitol-containing macro algae biomass, such as Ascophyllum nodosum and Laminaria digitata, are a potential feedstock for the production of biofuels such as bioethanol. The purpose of this work was to evaluate the ability of thermophilic anaerobes within Class Clostridia to ferment mannitol and mannitol-containing algal extracts. Screening of the type strains of six genera, Caldanaerobius, Caldanaerobacter, Caldicellulosiruptor, Thermoanaerobacter, Thermobrachium, and Thermoanaerobacterium) was conducted on 20 mM mannitol and revealed that 11 of 41 strains could utilize mannitol with ethanol being the dominant end-product. Mannitol utilization seems to be most common within the genus of Thermoanaerobacter (7 of 16 strains) with yields up to 88% of the theoretical yield in the case of Thermoanaerobacter pseudoethanolicus. Six selected mannitol-degrading strains (all Thermoanaerobacter species) were grown on mannitol extracts prepared from A. nodosum and L. digitata. Five of the strains produced similar amounts of ethanol as compared with ethanol yields from mannitol only. Finally, T. pseudoethanolicus was kinetically monitored using mannitol and mannitol extracts made from two macro algae species, A. nodosum and L. digitata for end-product formation.

Thermoanaerobacter species differ in their potential to reduce organic acids to their corresponding alcohols.

The reduction of organic acids to their corresponding alcohols has been shown for some bacterial species within the Firmicutes super-phylum and a genetically modified strain of the hyperthermophilic archaeon Pyrococcus furiosus. In the latter strain, an aldehyde:ferredoxin oxidoreductase (AOR) catalyzed the reduction of a variety of organic acids to their corresponding aldehydes, as shown by the deletion of the corresponding aor gene. Here, we found that the genomes of a few thermophilic bacterial species within the genus Thermoanaerobacter which have been described to efficiently ferment sugars to ethanol harbor a copy of aor, while others do not. Specific AOR activity was only found in strains with aor, and the gene was highly expressed in Thermoanaerobacter sp. strain X514. The reduction of a variety of organic acids was observed for several Thermoanaerobacter sp.; however, strains with aor reduced, e.g., isobutyrate at much higher rates of up to 5.1mMh-1g-1. Organic acid reduction also led to increased growth rates in Thermoanaerobacter sp. strain X514 and in Thermoanaerobacter pseudethanolicus. Organic acid activation may proceed via acyl-CoA with subsequent NADH-dependent reduction by an aldehyde dehydrogenase (ALDH), or via direct reduction by AOR. Cell-free extracts of Thermoanaerobacter sp. strain X514 exhibited both enzyme activities at comparable rates. Therefore, the biochemistry of organic acid reduction to alcohols in Thermoanaerobacter sp. remains to be elucidated; however, relatively high specific activities and the correlation of AOR specific activities with alcohol production rates suggest a role for AOR.

Comparative Analysis of Carbon Monoxide Tolerance among Thermoanaerobacter Species.

An anaerobic thermophilic strain (strain PCO) was isolated from a syngas-converting enrichment culture. Syngas components cannot be used by strain PCO, but the new strain is very tolerant to carbon monoxide (pCO = 1.7 × 105 Pa, 100% CO). 16S rRNA gene analysis and DNA-DNA hybridization revealed that strain PCO is a strain of Thermoanaerobacter thermohydrosulfuricus. The physiology of strain PCO and other Thermoanaerobacter species was compared, focusing on their tolerance to carbon monoxide. T. thermohydrosulfuricus, T. brockii subsp. finnii, T. pseudethanolicus, and T. wiegelii were exposed to increased CO concentrations in the headspace, while growth, glucose consumption and product formation were monitored. Remarkably, glucose conversion rates by Thermoanaerobacter species were not affected by CO. All the tested strains fermented glucose to mainly lactate, ethanol, acetate, and hydrogen, but final product concentrations differed. In the presence of CO, ethanol production was generally less affected, but H2 production decreased with increasing CO partial pressure. This study highlights the CO resistance of Thermoanaerobacter species.

Growth and metabolic profiling of the novel thermophilic bacterium Thermoanaerobacter sp. strain YS13.

A strictly anaerobic, thermophilic bacterium, designated strain YS13, was isolated from a geothermal hot spring. Phylogenetic analysis using the 16S rRNA genes and cpn60 UT genes suggested strain YS13 as a species of Thermoanaerobacter. Using cellobiose or xylose as carbon source, YS13 was able to grow over a wide range of temperatures (45-70 °C), and pHs (pH 5.0-9.0), with optimum growth at 65 °C and pH 7.0. Metabolic profiling on cellobiose, glucose, or xylose in 1191 medium showed that H2, CO2, ethanol, acetate, and lactate were the major metabolites. Lactate was the predominant end product from glucose or cellobiose fermentations, whereas H2 and acetate were the dominant end products from xylose fermentation. The metabolic balance shifted away from ethanol to H2, acetate, and lactate when YS13 was grown on cellobiose as temperatures increased from 45 to 70 °C. When YS13 was grown on xylose, a metabolic shift from lactate to H2, CO2, and acetate was observed in cultures as the temperature of incubation increased from 45 to 65 °C, whereas a shift from ethanol and CO2 to H2, acetate, and lactate was observed in cultures incubated at 70 °C.

Investigating the potential of thermophilic species for ethanol production from industrial spent sulfite liquor

Thermophilic microorganisms hold a great potential for bioethanol production on waste biomass, due to their ability to utilize pentoses and hexoses alike. However, to date hardly any data on thermophiles growing directly on industrial substrates like spent sulfite liquor (SSL) are available. This contribution investigates the ability of Thermoanaerobacter species to utilize the main sugars in the used SSL (mannose, glucose and xylose) and the effect of process parameters (pH, temperature and sugar concentration) on their growth. Based on these results the strain T. mathranii was chosen for further studies. The ability of T. mathranii to grow directly on SSL was investigated and the effect of several inhibiting substances on growth was elucidated. Furthermore it was tested whether pretreatment with activated charcoal can increase the fermentability of SSL. The fermentations were evaluated based on yields and specific rates. It could be shown that T. mathranii was able to ferment all sugars in the investigated softwood SSL and fermented diluted, untreated SSL (up to 2.7% (w/w) dry matter). Pretreatment with activated charcoal could slightly reduce the amount of phenols in the substrate and thus facilitate growth and ethanol production on higher SSL concentrations (up to 4.7% (w/v) dry matter). Ethanol yields of 0.29-0.44 Cmmol of ethanol per Cmmol sugar were obtained on untreated and pretreated spent sulfite liquor, respectively. These results on an industrial substrate strengthen the claim that thermophilic microorganisms might be the optimal candidates for forest biorefinery.

A Computational Framework for Tracing the Origins of Genomic Islands in Prokaryotes.

Genomic islands (GIs) are chunks of genomic fragments that are acquired from nongenealogical organisms through horizontal gene transfer (HGT). Current researches on studying donor-recipient relationships for HGT are limited at a gene level. As more GIs have been identified and verified, the way of studying donor-recipient relationships can be better modeled by using GIs rather than individual genes. In this paper, we report the development of a computational framework for detecting origins of GIs. The main idea of our computational framework is to identify GIs in a query genome, search candidate genomes that contain genomic regions similar to those GIs in the query genome by BLAST search, and then filter out some candidate genomes if those similar genomic regions are also alien (detected by GI detection tools). We have applied our framework in finding the GI origins for Mycobacterium tuberculosis H37Rv, Herminiimonas arsenicoxydans, and three Thermoanaerobacter species. The predicted results were used to establish the donor-recipient network relationships and visualized by Gephi. Our studies have shown that donor genomes detected by our computational approach were mainly consistent with previous studies. Our framework was implemented with Perl and executed on Windows operating system.

Branched-chain alcohol formation from branched-chain amino acids by Thermoanaerobacter brockii and Thermoanaerobacter yonseiensis

Thermoanaerobacter species degrade branched-chain amino acids to a mixture of their corresponding branched-chain fatty acids and alcohols in the presence of thiosulfate; only acid formation occurred when Thermoanaerobacter strains were cultivated in co-culture with a hydrogenotrophic methanogen. Increased pH2 at high liquid–gas phase ratios increases the relative concentration of branched-chain alcohol.

Scalable production of microbially mediated zinc sulfide nanoparticles and application to functional thin films

A series of semiconducting zinc sulfide (ZnS) nanoparticles were scalably, reproducibly, controllably and economically synthesized with anaerobic metal-reducing Thermoanaerobacter species. These bacteria reduced partially oxidized sulfur sources to sulfides that extracellularly and thermodynamically incorporated with zinc ions to produce sparingly soluble ZnS nanoparticles with ∼5nm crystallites at yields of ∼5gl−1month−1. A predominant sphalerite formation was facilitated by rapid precipitation kinetics, a low cation/anion ratio and a higher zinc concentration compared to background to produce a naturally occurring hexagonal form at the low temperature, and/or water adsorption in aqueous conditions. The sphalerite ZnS nanoparticles exhibited narrow size distribution, high emission intensity and few native defects. Scale-up and emission tunability using copper doping were confirmed spectroscopically. Surface characterization was determined using Fourier transform infrared and X-ray photoelectron spectroscopies, which confirmed amino acid as proteins and bacterial fermentation end products not only maintaining a nano-dimensional average crystallite size, but also increasing aggregation. The application of ZnS nanoparticle ink to a functional thin film was successfully tested for potential future applications.

Hydrogen production by Pseudomonas stutzeri JX442762 isolated from thermal soil at Mettur power station, Salem district, Tamil Nadu, India

BackgroundCarbohydrate based substrates presents a promising route of biological hydrogen production compared with chemical routes. Pure substrates including starch, sucrose as well as different organic waste materials can be used for hydrogen production. Among a large number of microbial species, strict anaerobes and facultative anaerobic chemoheterotrophs, thermoanaerobacter species are efficient producers of hydrogen. Since hydrogen is a high-energy fuel than hydrocarbon fuel, it is essential to find an alternative source. ObjectivesTo isolate hydrogen producing organism from thermal soil sample, to identify the organism as it is a cost effective way for biological hydrogen production. The hydrogen production was assessed in pure starch, sucrose and also in mango juice effluent obtained from Krishnagiri dist., to find a better substrate. MethodologyMorphological, biochemical characterization of the isolate was evaluated and finally confirmed by 16S rRNA gene sequencing. Hydrogen production was measured by simple water displacement method for the selected substrates at 70 °C, initial pH 4.0 as well as for the effluent. Results16S rRNA gene sequencing confirms the organism as Pseudomonas stutzeri which was deposited in gene bank under the accession number JX442762 and the identified strain was named as SSKVM 2012. The G + C content of the strain P. stutzeri was found to be 53 mol%. The obtained results showed that the maximum hydrogen production was observed with pure starch (255.98 ± 0.76 ml) but in sucrose it was found to be 212.82 ± 0.57 ml. The mango juice effluent showed 190.03 ± 0.81 ml hydrogen. Even though it was less when compared to hydrogen production by starch and sucrose, mango juice effluent could be a better substrate for the identified organism. ConclusionThe identified strain confirms that it can use effluent as a good source for the hydrogen production as its initial pH is 6.0 and also freely available in the environment.

Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.

Microbial synthesis of iron-based nanomaterials—A review

Nanoparticles are the materials having dimensions of the order of 100 nm or less. They exhibit a high surface/volume ratio leading to different properties far different from those of the bulk materials. The development of uniform nanoparticles has been intensively pursued because of their technological and fundamental scientific importance. A number of chemical methods are available and are extensively used, but these are often energy intensive and employ toxic chemicals. An alternative approach for the synthesis of uniform nanoparticles is the biological route that occurs at ambient temperature, pressure and at neutral pH. The main aim of this review is to enlist and compare various methods of synthesis of iron-based nanoparticles with emphasis on the biological method. Biologically induced and controlled mineralization mechanisms are the two modes through which the micro-organisms synthesize iron oxide nanoparticles. In biologically induced mineralization (BIM) mode, the environmental factors like pH, pO2, pCO2, redox potential, temperature etc govern the synthesis of iron oxide nanoparticles. In contrast, biologically controlled mineralization (BCM) process initiates the micro-organism itself to control the synthesis. BIM can be observed in the Fe(III) reducing bacterial species of Shewanella, Geobacter, Thermoanaerobacter, and sulphate reducing bacterial species of Archaeoglobus fulgidus, Desulfuromonas acetoxidans, whereas BCM mode can be observed in the magnetotactic bacteria (MTB) like Magnetospirillum magnetotacticum, M. gryphiswaldense and sulphate-reducing magnetic bacteria (Desulfovibrio magneticus). Magnetite crystals formed by Fe(III)-reducing bacteria are epicellular, poorly crystalline, irregular in shapes, having a size range of 10–50 nm super-paramagnetic particles, with a saturation magnetization value ranging from 75–77 emu/g and are not aligned in chains. Magnetite crystals produced by MTB have uniform species-specific morphologies and sizes, which are mostly unknown from inorganic systems. The unusual characteristics of magnetosome particles have attracted a great interdisciplinary interest and inspired numerous ideas for their biotechnological applications. The nanoparticles synthesized through biological method are uniform with size ranging from 5 to 100 nm, which can potentially be used for various applications.

Thermoanaerobacter spp. control ethanol pathway via transcriptional regulation and versatility of key enzymes

Ethanologenic Thermoanaerobacter species produce ethanol from lignocellulose derived substrates at temperatures above 70 °C. In the final steps of ethanol formation, two bifunctional acetaldehyde/alcohol dehydrogenases, AdhB and AdhE, and an alcohol dehydrogenase, AdhA, catalyze redox reactions between acetyl-CoA and ethanol via an acetaldehyde intermediate. DNA cloning and analysis revealed that the dehydrogenase genes and their transcriptional regulatory regions were highly conserved in these species . As determined by real-time PCR, the transcription of adhE was activated by ethanol, while adhB was transcribed without ethanol; however, all of their transcription was reduced at higher ethanol concentrations. Under imitating physiological conditions, AdhE played a crucial role in ethanol formation, and AdhB favored ethanol consumption when ethanol concentration was high e.g. 1%. Thus, the ethanol titer of fermentation is controlled via transcriptional regulation and the properties of specific enzymes in Thermoanaerobacter. These results provide evidence for an ethanol balance model and offer the possibility to raise the ethanol titer by metabolic engineering.

Chiral (R)- and (S)-allylic alcohols via a one-pot chemoenzymatic synthesis

Chiral (R)- and (S)-allylic alcohols with an enantiomeric excess exceeding 99% have been prepared in good to high overall isolated yields through a two-step one-pot chemoenzymatic process based on the palladium-catalyzed Heck reaction of aryl iodides with butenone followed by an enzymatic reduction of the resultant vinylic substitution products. Alcohol dehydrogenases from Lactobacillus brevis and Thermoanaerobacter species were used to attain (R)- and (S)-stereoselectivity, respectively.

Effect of additives on gas-phase catalysis with immobilised Thermoanaerobacter species alcohol dehydrogenase (ADH T)

This paper presents a strategy for preparing an efficient immobilised alcohol dehydrogenase preparation for a gas-phase reaction. The effects of additives such as buffers and sucrose on the immobilisation efficiency (residual activity and protein loading) and on the gas-phase reaction efficiency (initial reaction rate and half-life) of Thermoanaerobacter sp. alcohol dehydrogenase were studied. The reduction of acetophenone to 1-phenylethanol under in situ cofactor regeneration using isopropanol as co-substrate was used as a model reaction at fixed reaction conditions (temperature and thermodynamic activities). A strongly enhanced thermostability of the enzyme in the gas-phase reaction was achieved when the enzyme was immobilised with 50 mM phosphate buffer (pH 7) containing sucrose five times the protein amount (on weight/weight basis). This resulted in a remarkable productivity of 200 g L(-1) day(-1) even at non-optimised reaction conditions. The interaction of additives with the enzyme and water affects the immobilisation and gas-phase efficiencies of the enzyme. However, it was not possible to predict the effect of additives on the gas-phase reaction efficiency even after knowing their effect on the immobilisation efficiency.

Isolation from canned foods of a novel Thermoanaerobacter species phylogenetically related to Thermoanaerobacter mathranii (Larsen 1997): Emendation of the species description and proposal of Thermoanaerobacter mathranii subsp. Alimentarius subsp. Nov

Several anaerobic, thermophilic, Gram-positive bacteria were isolated from dairy products and canned meats. While some isolates were identified as Thermoanaerobacter thermohydrosulfuricus, comparisons of 16S rDNA genes indicated that others were phylogenetically closely related to Thermoanaerobacter mathranii, and more distantly related to Thermoanaerobacter thermocopriae and Thermoanaerobacter italicus. Biochemical characteristics, phylogenetic analysis, G+C content, and DNA–DNA hybridization experiments demonstrated that the strains AIP 504.99, AIP 505.99 T and AIP 431.03, notwithstanding their high sequence similarities differ from T. mathranii and represent a novel T. mathranii subspecies for which the name T. mathranii subsp. alimentarius is proposed. The type strain is strain AIP 505.99 T=CIP 108280 T=CCUG 49566 T. Emendation of the species description for T. mathranii is proposed to include this subspecies.