Thermophilic Anaerobic Bacterium Research Articles

Expression and characterization of monofunctional alcohol dehydrogenase enzymes in Clostridium thermocellum

Clostridium thermocellum is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native adhE gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase (adh) genes. We were able to express all of them successfully in C. thermocellum. Surprisingly, however, none of them increased ethanol production, and several actually decreased it. Our findings contribute to understanding the correlation between C. thermocellum ethanol production and Adh enzyme cofactor preferences. The identification of a set of adh genes that can be successfully expressed in this organism provides a foundation for future investigations into how the properties of Adh enzymes affect ethanol production.

Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45–75 °C (optimum 70–75 °C) and pH 6.5–8.5 (optimum pH 7.5–8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C16:0, iso-C15:0, and C18:0. The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Crassaminicella indica sp. nov., a novel thermophilic anaerobic bacterium isolated from a deep-sea hydrothermal vent.

A novel moderately thermophilic heterotrophic bacterium, designated strain 143-21T, was isolated from a deep-sea hydrothermal chimney sample collected from the Central Indian Ridge at a depth of 2 440 m. Phylogenetic analysis indicated that strain 143-21T belongs to the genus Crassaminicella. It was most closely related to Crassaminicella thermophila SY095T (96.79 % 16S rRNA gene sequence similarity) and Crassaminicella profunda Ra1766HT (96.52 %). Genomic analysis showed that strain 143-21T shares 79.79-84.45 % average nucleotide identity and 23.50-29.20 % digital DNA-DNA hybridization with the species of the genus Crassaminicella, respectively. Cells were rod-shaped, non-motile, Gram-positive-staining. Terminal endospores were observed in stationary-phase cells when strain 143-21T was grown on Thermococcales rich medium. Strain 143-21T was able to grow at 30-60 °C (optimum, 50 °C), pH 6.5-8.5 (optimum, pH 7.0) and in 1.0-7.0 % NaCl (w/v; optimum 2.0 %, w/v). Strain 143-21T utilized fructose, glucose, maltose, mannose, ribose, N-acetyl-d-(+)-glucosamine and casamino acids, as well as amino acids including glutamate, lysine, histidine and cysteine. The main fermentation products from glucose were acetate (2.07 mM), H2 and CO2. It did not reduce elemental sulphur, sulphate, thiosulphate, sulphite, fumarate, nitrate, nitrite and Fe (III). The predominant cellular fatty acids were C14 : 0 (48.8 %), C16 : 0 (12.9 %), and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 10.2 %). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, as well as two unidentified phospholipids and four unidentified aminolipids. No respiratory quinones were detected. Based on its phylogenetic analysis and physiological characteristics, strain 143-21T is considered to represent a novel species of the genus Crassaminicella, for which the name Crassaminicella indica sp. nov. is proposed. The type strain is strain 143-21T (=DSM 114408T= MCCC 1K06400T).

Extracellular Fe(III) reductase structure reveals a modular organization enabling S-layer insertion and electron transfer to insoluble substrates

The thermophilic anaerobic Gram-positive bacterium Carboxydothermus ferrireducens utilizes insoluble Fe(III) oxides as electron acceptors in respiratory processes using an extracellular 11-heme cytochrome cOmhA as a terminal reductase. OmhA is able to transfer electrons to soluble and insoluble Fe(III) compounds, substrates of multiheme oxidoreductases, and soluble electron shuttles. The crystal structure of OmhA at 2.5 Å resolution shows that it consists of two functionally distinct parts: the cytochrome с electron transfer and the S-layer binding domains. Nonaheme C-terminal subdomain of the cytochrome с domain isstructurally similar to the extracellular multiheme cytochrome OcwA from the metal-reducing Gram-positive bacterium "Thermincola potens." S-layer binding domain of OmhA is responsible for interaction with the S-layer that surrounds the Carboxydothermus ferrireducens cell envelope. The structural foundations enabling the embedding of extracellular multiheme cytochromes to the S-layer of a Gram-positive-type cell wall and putative electron transfer pathways to insoluble minerals are discussed.

Moorella sulfitireducens sp. nov., a thermophilic anaerobic bacterium isolated from a terrestrial thermal spring.

In hydrothermal ecosystems, the dissolution of sulfur dioxide in water results in the formation of sulfite, which can be used in microbial metabolism. A limited number of thermophiles have been isolated using sulfite as an electron acceptor. From a terrestrial thermal spring, Sakhalin Island, Russia, we isolated a thermophilic anaerobic bacterium (strain SLA38T). Cells of strain SLA38T were spore-forming straight rods. Growth was observed at temperatures 45-65°C (optimum at 60°C) and pH 5.5-9.0 (optimum at pH 6.5-7.0). The novel isolate was capable of anaerobic respiration with sulfite, thiosulfate, fumarate and perchlorate or fermentative growth. Strain SLA38T utilized glycerol, lactate, pyruvate and yeast extract. It grew lithoautotrophically on carbon monoxide with thiosulfate as electron acceptor, producing acetate. The genome size of the isolate was 2.9 Mbp and genomic DNA G + C content was 53.6mol%. Analysis of the 16S rRNA gene sequences revealed that strain SLA38T belongs to the genus Moorella. Based on the physiological features and phylogenetic analysis, we propose to assign strain SLA38T to a new species of the genus Moorella, as Moorella sulfitireducens sp. nov. The type strain is SLA38T (= DSM 111068T = VKM B-3584T).

Description of a moderately acidotolerant and aerotolerant anaerobic bacterium Acidilutibacter cellobiosedens gen. nov., sp. nov. within the family Acidilutibacteraceae fam. nov., and proposal of Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov.

A Gram-stain positive, moderately thermophilic, acidotolerant and aerotolerant anaerobic bacterium, designated JN-28 T, was isolated from the pit mud of Chinese strong-flavor liquor. Growth was observed at 25–50 °C and pH 5.5–8.0 in the presence of 0–25 g l−1 NaCl (optimally at 45 °C, pH 6.0, without NaCl). Strain JN-28 T was heterotrophic, requiring yeast extract for growth. The major cellular fatty acids were iso-C15:0 and C14:0. The DNA G + C content of genomic DNA was 33.54 mol%. The strain was resistant to vancomycin (10 mg l−1). Genome analysis revealed the presence of genes involved in the response to mild acid stress and oxidative stress, and resistance to vancomycin. 16S rRNA gene-based phylogenetic analysis showed that strain JN-28 T shares ≤ 89.3 % sequence similarity with its closest relatives Sporanaerobacter acetigenes DSM 13106 T and other members in the order Tissierellales. Based on phenotypic and phylogenetic characteristics, Acidilutibacter cellobiosedens gen. nov., sp. nov. is proposed for the new genus and novel species with the type strain JN-28 T (=CCAM 418 T = JCM 39087 T). Further phylogenetic and phylogenomic analyses suggested strain JN-28 T represents a novel family within the order Tissierellales, for which Acidilutibacteraceae fam. nov. is proposed. In addition, the family Tissierellaceae was reclassified, Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov. were formally proposed. Emended description of the family Tissierellaceae is also provided.

Identification, molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus.

We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family 15 of glycosylhydrolases (GH15). The expression of this enzyme was optimized in E. coli (BL21) cells in order to have the highest amount of soluble protein (around 3mg/l of culture medium). TeGA showed a high optimum temperature of 75°C. It also showed one of the highest specific activities reported for a bacterial glucoamylase (75.3 U/mg) and was also stable in a wide pH range (3.0-10.0). Although the enzyme was preferentially active with maltose, it was also able to hydrolyze different soluble starches such as those from potato, corn or rice. TeGA showed a high thermostability up to around 70°C, which was increased in the presence of PEG8000, and also showed to be stable in the presence of moderate concentrations of ethanol. We propose that TeGA could be suitable for use in different industrial processes such as biofuel production and food processing.

Genome sequence and Carbohydrate Active Enzymes (CAZymes) repertoire of the thermophilic Caldicoprobacter algeriensis TH7C1T

BackgroundOmics approaches are widely applied in the field of biology for the discovery of potential CAZymes including whole genome sequencing. The aim of this study was to identify protein encoding genes including CAZymes in order to understand glycans-degrading machinery in the thermophilic Caldicoprobacter algeriensis TH7C1T strain.ResultsCaldicoprobacter algeriensis TH7C1T is a thermophilic anaerobic bacterium belonging to the Firmicutes phylum, which grows between the temperatures of 55 °C and 75 °C. Next generation sequencing using Illumina technology was performed on the C. algeriensis strain resulting in 45 contigs with an average GC content of 44.9% and a total length of 2,535,023 bp. Genome annotation reveals 2425 protein-coding genes with 97 ORFs coding CAZymes. Many glycoside hydrolases, carbohydrate esterases and glycosyltransferases genes were found linked to genes encoding oligosaccharide transporters and transcriptional regulators; suggesting that CAZyme encoding genes are organized in clusters involved in polysaccharides degradation and transport. In depth analysis of CAZomes content in C. algeriensis genome unveiled 33 CAZyme gene clusters uncovering new enzyme combinations targeting specific substrates.ConclusionsThis study is the first targeting CAZymes repertoire of C. algeriensis, it provides insight to the high potential of identified enzymes for plant biomass degradation and their biotechnological applications.

Direct conversion of cellulose to l-lactic acid by a novel thermophilic Caldicellulosiruptor strain

BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to l-lactic acid using thermophilic cellulolytic/hemicellulolytic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic/hemicellulolytic enzymes. Most studies on the mesophilic and thermophilic CBP of lignocellulose to lactic acid concentrate on cultivation of non-cellulolytic mesophilic and thermophilic bacteria at temperatures of 30–55 °C with external addition of cellulases/hemicellulases for saccharification of substrates.Resultsl-Lactic acid was generated by fermenting microcrystalline cellulose or lignocellulosic substrates with a novel thermophilic anaerobic bacterium Caldicellulosiruptor sp. DIB 104C without adding externally produced cellulolytic/hemicellulolytic enzymes. Selection of this novel bacterium strain for lactic acid production is described as well as the adaptive evolution towards increasing the l-lactic acid concentration from 6 to 70 g/l on microcrystalline cellulose. The evolved strains grown on microcrystalline cellulose show a maximum lactic acid production rate of 1.0 g/l*h and a lactic acid ratio in the total organic fermentation products of 96 wt%. The enantiomeric purity of the l-lactic acid generated is 99.4%. In addition, the lactic acid production by these strains on several other types of cellulose and lignocellulosic feedstocks is also reported.ConclusionsThe evolved strains originating from Caldicellulosiruptor sp. DIB 104C were capable of producing unexpectedly large amounts of l-lactic acid from microcrystalline cellulose in fermenters. These strains produce l-lactic acid also from lignocellulosic feedstocks and thus represent an ideal starting point for development of a highly integrated commercial l-lactic acid production process from such feedstocks.

Deferrivibrio essentukiensis sp. nov., gen. nov., a Representative of Deferrivibrionaceae fam. nov., Isolated from the Subsurface Aquifer of Caucasian Mineral Drinking Waters

The Yessentukskoye deposit of Caucasian mineral waters contains balneologically valuable drinking mineral water, which is extracted from the Upper Cretaceous 1 km subsurface aquifer and is almost unexplored by microbiologists. We have sampled this water via continuously operating production wells, characterized the phylogenetic diversity of its microbial community, and obtained enrichments of thermophilic iron reducers from the source aquifer. From the enrichments, a novel anaerobic thermophilic bacterium, reducing Fe(III) in the mineral ferrihydrite with acetate as the electron donor, was isolated into a pure culture. The novel isolate, designated as strain Es71-Z0220T belonging to Deferribacterales order, is thermophilic, neutrophilic, halotolerant, motile vibrio. It utilizes synthesized ferrihydrite, fumarate, nitrate or elemental sulfur as the electron acceptors with organic acids as the electron donors. The strain is incapable of soluble Fe(III) complexes reduction and fermentative growth. The draft genome assembly of strain Es71-Z0220T resulted in 65 contigs with a total size of ca. 2.3 Mb. On the basis of whole-genome phylogenetic reconstruction and physiological characterization, the novel isolate was considered to represent a novel family, genus and species for which the name Deferrivibrio essentukiensis gen. nov., sp. nov. is proposed. Genome analysis revealed key determinants of anaerobic respiration and carbon substrate utilization pathways in the organism with peculiarities related to putative Fe(III)-reducing electron transfer chain. Considering the revealed metabolic features of Deferrivibrio essentukiensis, the involvement of the organism in its subsurface environment in biogeochemical by carbon cycling by coupling the organic matter oxidation with Fe(III) minerals reduction is discussed.

دراسة تأثير المعاملة بمستخلص قشور الرّمان على الحمولة الميكروبية لعينات لحم حملان العواس المغلّفة عند حفظها بالتجميد

تم في هذا البحث دراسة التأثير على تعداد الكائنات الحية الدقيقة وتغيرات هذه الحمولة الميكروبية على لحم حملان العواس بعد تغليفها وتخزينها لمدة 6 أشهر تحت ظروف التجميد (-18م) من خلال المعاملة بمستخلص قشور الرّمان. وقد أجريت اختبارات البحث في مخابر قسم علوم الأغذية في كلية الهندسة الزراعية بدير الزور، إذ أظهرت نتائج اختبارات تعداد الحمولة الميكروبية وجود فروق معنوية في أعداد البكتريا اللاهوائية المحبة للحرارة المتوسطة بين العينات المعاملة بالمستخلص وعينات الشاهد خلال فترة التخزين. كما لوحظ وجود فروق معنوية في أعداد بكتريا E.coli وكانت العينات المعاملة بالتركيز (1.5%) الأقل تعداداً مقارنة بالعينات المعاملة بالتراكيز الأخرى، فقد انخفض متوسط لوغاريتم أعداد بكتريا E.coli في عينات لحم حملان العواس من (7.74، 7.45، 6.64) في بداية فترة التخزين إلى (3.72، 2.54، 1.21) في نهايتها وذلك عند استخدام تراكيز مستخلص قشور الرّمان (0.5، 1، 1.5%) على التوالي. وانخفض متوسط لوغاريتم أعداد البكتريا Pseudomonas وفقاً لتركيز المستخلص (0.5%، 1%، 1.5%) من (11.07، 11.02، 10.46) في بداية فترة التخزين إلى (7.12، 6.23، 2.98) في نهاية فترة التخزين على التوالي، أما بالنسبة لعينات الشاهد (غير المعاملة بالمستخلص) فقد ازدادت فيها أعداد البكتريا بشكل غير معنوي.

Structural Modelling and in-silico characterization of a Novel Thermophilic β-amylase from Clostridium thermosulfuregenes

β-amylase is a hydrolytic enzyme that is involved in breaking down starch and producing energy. Since the discovery of β-amylase, it has been applied in various applications especially in the food industry. In this study, a novel β-amylase from Clostridium thermosuluregen, a thermophilic anaerobic bacterium that ferments its extracellular emulsion to ethanol at 62 ℃ was modelled and studied using bioinformatics tools and compared with B. cereus β-amylases that functions at mesophilic conditions. The results showed that the overall structural conformations, secondary structures, and important residues involved in active and binding sites were identified in both proteins. The results revealed that the modelled β-amylase of C. thermosulfuregen is very similar with respect to the global conformation, location of active and binding sites. Both proteins showed identical structural domains with the thermophilic variant possessing a high percentage of hydrophobic amino acid residues, polar amino acid residues, and differences in secondary composition such as loops and beta sheets as the potential evolutionary thermal adaptations that make it stable enzyme that functions up to 70 ℃. The results suggest that the thermal stability are not dependent on one single unique mechanism and may use one or a combination of the mechanisms to sustain its structural conformation at a higher operating temperature. Overall, considering the common properties of this modelled protein with the β-amylase of B. cereus, it can be assumed that if the β-amylase of C. thermosulfuregen were expressed in-vitro, it would produce a stable protein that possesses the hydrolysis function for C. thermosulfuregen to break down the starch and sugar formation.

Bioethanol production from pretreated whole slurry rice straw by thermophilic co-culture

In this study, an innovative approach is proposed for the valorization of all sugars (cellulosic and hemicellulosic) contained in a lignocellulosic feedstock, as is rice straw biomass (RSB), for the production of second-generation bioethanol. For this purpose, the whole slurry obtained after dilute sulfuric acid pretreatment at a biomass loading of 5% (w/v) was subjected to consolidated bioprocessing without previous solid-liquid separation and/or detoxification and the concentrated solution of carbohydrates recovered was fermented to bioethanol by a co-culture of thermophilic anaerobic bacteria. To bypass the inhibitory effect of undetoxified whole slurry on the lower fermentability of the co-culture, the concentration of different additives was optimized for improved buffering capacity, enhanced substrate-microbe interaction, and by-product elimination. The individual supplementation of 3 selected additives at optimum concentration; 20 mM calcium carbonate, 0.4% (v/v) polyethylene glycol, and 1% (w/v) sodium acetate resulted in enhanced ethanol yield of 130.83 mM, 106.37 mM, and 99.10 mM, respectively, compared to 75.03 mM ethanol yield in control. The combined effect of optimal dosage of these additives was able to improve the ethanol concentration and yield to 142 mM and 48% of the theoretical maximum respectively, suggesting about 88.58% increase compared to control medium without additives supplementation. The combined supplementation of additives proved to be an advantageous strategy for bioethanol production using undetoxified whole slurry biomass.

Engineering Thermoanaerobacterium aotearoense SCUT27/Δldh with pyruvate formate lyase-activating protein (PflA) knockout for enhanced ethanol tolerance and production

Thermophilic anaerobic bacteria have received increasing interest as a promising bioprocessing platform for bioethanol production due to their broad substrate range and higher operating temperature. However, ethanol concentration is still lower than the commercial requirements on account of the high by-product formation and low ethanol tolerance. Our previous transcriptome analysis showed the gene expression level of pyruvate formate lyase-activating protein (PflA) was significantly down-regulated when Thermoanaerobacterium aotearoense SCUT27/Δldh was stimulated by various concentrations of ethanol. Based on this, we constructed the mutant strain T. aotearoense SCUT27/Δldh/ΔpflA, which not only possesses higher ethanol tolerance and production but also has enhanced ethanol/acetate ratio that is important for economical ethanol production. Compared to T. aotearoense SCUT27/Δldh, the mutant strain could resist to 5% (v/v) exogenous ethanol and produce 49 % more ethanol (38.82 g/L vs. 26.05 g/L) in 5-L bioreactor. In addition, the final concentration of ethanol reached 45.01 g/L by immobilized cell fermentation, which is the highest value obtained in T. aotearoense SCUT27. Simultaneously, the ethanol/acetate ratio of the mutant strain achieved 6.82, which was 2.16-fold higher than that of the parental strain.

Symbiotic chitin degradation by a novel anaerobic thermophilic bacterium Hydrogenispora sp. UUS1-1 and the bacterium Tepidanaerobacter sp. GT38

Chitin is the second most abundant organic compound in nature. Although mesophilic bacteria degrade insoluble chitin, there is a paucity of data describing degradation of insoluble chitin by anaerobic thermophilic bacteria. In this report, we screened cow manure compost for new chitin degradation systems, and identified a chitinolytic bacterial community (CBC) that showed high chitin degradation activity under thermophilic conditions, i.e., 1% (w/v) chitin powder degraded completely within 7 days at 60 °C. Metagenomic analysis revealed that the CBC was dominated by two bacterial genera from Hydrogenispora, an uncultured taxonomic group, and Tepidanaerobacter. Hydrogenispora were abundant in the early-to-mid stages of culturing with chitin, whereas the population of Tepidanaerobacter increased during the later stages of culturing. Strains UUS1-1 and GT38, which were isolated as pure cultures using the roll-tube method with colloidal chitin, N-acetyl-d-glucosamine, and glucose as carbon sources, were found to be closely related to H. ethanolica and T. acetatoxydans, respectively. Strain UUS1−1 readily degraded chitin and is the first anaerobic thermophilic chitinolytic bacterium reported, whereas strain GT38 showed no chitinolytic activity. Based on phylogenetic analysis, UUS1−1 and GT38 should be classified as novel genera and species. Zymogram analysis revealed that UUS1−1 produces at least two chitinases with molecular weights of 150 and 40 kDa. A coculture of UUS1−1 and GT38 degraded crystalline chitin faster with lower accumulation of lactate compared with UUS1−1 alone, indicating that the strains maintained a symbiotic association through assimilation of organic acids in chitin degradation and that strain GT38 consumed end-products to reduce end-product inhibition and enhance the degradation of crystalline chitin.

Genomic Characterization and Environmental Distribution of a Thermophilic Anaerobe Dissulfurirhabdus thermomarina SH388T Involved in Disproportionation of Sulfur Compounds in Shallow Sea Hydrothermal Vents

Marine hydrothermal systems are characterized by a pronounced biogeochemical sulfur cycle with the participation of sulfur-oxidizing, sulfate-reducing and sulfur-disproportionating microorganisms. The diversity and metabolism of sulfur disproportionators are studied to a much lesser extent compared with other microbial groups. Dissulfurirhabdus thermomarina SH388T is an anaerobic thermophilic bacterium isolated from a shallow sea hydrothermal vent. D. thermomarina is an obligate chemolithoautotroph able to grow by the disproportionation of sulfite and elemental sulfur. Here, we present the results of the sequencing and analysis of the high-quality draft genome of strain SH388T. The genome consists of a one circular chromosome of 2,461,642 base pairs, has a G + C content of 71.1 mol% and 2267 protein-coding sequences. The genome analysis revealed a complete set of genes essential to CO2 fixation via the reductive acetyl-CoA (Wood-Ljungdahl) pathway and gluconeogenesis. The genome of D. thermomarina encodes a complete set of genes necessary for the dissimilatory reduction of sulfates, which are probably involved in the disproportionation of sulfur. Data on the occurrences of Dissulfurirhabdus 16S rRNA gene sequences in gene libraries and metagenome datasets showed the worldwide distribution of the members of this genus. This study expands our knowledge of the microbial contribution into carbon and sulfur cycles in the marine hydrothermal environments.

Isolation and characterization of thermophilic cellulose and hemicellulose degrading bacterium, Thermoanaerobacterium sp. R63 from tropical dry deciduous forest soil.

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0–9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes.

Genome analysis of Thermosulfuriphilus ammonigenes ST65T, an anaerobic thermophilic chemolithoautotrophic bacterium isolated from a deep-sea hydrothermal vent.

Thermosulfuriphilus ammonigenes ST65T is an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent chimney. T. ammonigenes is an obligate chemolithoautotroph utilizing elemental sulfur as an electron donor and nitrate as an electron acceptor with sulfate and ammonium formation. It also is able to grow by disproportionation of elemental sulfur, thiosulfate and sulfite. Here, we present the complete genome sequence of strain ST65T. The genome consists of a single chromosome of 2,287,345 base pairs in size and has a G+C content of 51.9mol%. The genome encodes 2172 proteins, 48 tRNA genes, and 3 rRNA genes. Genome analysis revealed a complete set of genes essential to CO2 fixation and gluconeogenesis. Homologs of genes encoding known enzyme systems for nitrate ammonification are absent in the genome of T. ammonigenes assuming unique mechanism for this pathway. The genome of strain ST65T encodes a complete set of genes necessary for dissimilatory sulfate reduction, which are probably involved in sulfur disproportionation and anaerobic oxidation. This is the first reported genome of a bacterium from the genus Thermosulfuriphilus, providing insights into the microbial contribution into carbon, sulfur and nitrogen cycles in the deep-sea hydrothermal vent environment.

One-step biohydrogen production from cassava pulp using novel enrichment of anaerobic thermophilic bacteria community

The aim of this study is to produce biohydrogen from untreated cassava pulp (CP) using a novel selected consortium of anaerobic thermophilic bacteria and the associated microbial communities were elucidated by metagenomic analysis. CP was chosen as a sole carbon source to enrich efficient hydrogen-producers from a total of 100 different composts and soil sediments under anaerobic dark fermentation at 60 °C. The stable enrichment culture was selected, base on CP degradation and hydrogen production. The selected consortium, namely BK16 from the residue of soil sediment around mangroves at Bangkhuntiean province, Thailand shows the highest hydrogen production yield (35.08 ml H 2 /g substrate) and hydrogen production rate (64.47 mL/day) in one-step. Besides, polysaccharide-degrading enzyme such as amylase, cellulase and xylanase of the BK16 were detected in culture supernatant at 70, 73 and 100 U/mg protein, respectively. In particular, the dominant microbes from selected consortium BK16 comprised of Clostridium thermopalmarium (61.2%) and Clostridium isatidis (25.3%) which have ability to degrade biomass and co-existed with acidogenesis bacteria, belong to the genus Thermoanaerobacterium (6.7%) and Fonticella tunisiensis (5.06%) to produced hydrogen as a consolidate bioprocess (CBP) system. The optimization condition for the hydrogen production of BK16 were at pH 7.0 and 10% (w/v) cassava pulp, in which 760 mL of H 2 /L was produced. Thus, these results revealed that the selected consortia BK16 can be used as an attractive candidate for direct hydrogen production, using cassava pulp as potential raw material for future bio-energy production. • The BK16 has isolated and identified the novel associated microbial communities. • Key biomass-degrading enzyme e.g., amylase, cellulase and xylanase were investigated. • One-step hydrogen production using non-pretreated cassava pulp were optimized.

Thermophilic whole-cell degradation of polyethylene terephthalate using engineered Clostridium thermocellum.

SummaryPolyethylene terephthalate (PET) is a mass‐produced synthetic polyester contributing remarkably to the accumulation of solid plastics waste and plastics pollution in the natural environments. Recently, bioremediation of plastics waste using engineered enzymes has emerged as an eco‐friendly alternative approach for the future plastic circular economy. Here we genetically engineered a thermophilic anaerobic bacterium, Clostridium thermocellum, to enable the secretory expression of a thermophilic cutinase (LCC), which was originally isolated from a plant compost metagenome and can degrade PET at up to 70°C. This engineered whole‐cell biocatalyst allowed a simultaneous high‐level expression of LCC and conspicuous degradation of commercial PET films at 60°C. After 14 days incubation of a batch culture, more than 60% of the initial mass of a PET film (approximately 50 mg) was converted into soluble monomer feedstocks, indicating a markedly higher degradation performance than previously reported whole‐cell‐based PET biodegradation systems using mesophilic bacteria or microalgae. Our findings provide clear evidence that, compared to mesophilic species, thermophilic microbes are a more promising synthetic microbial chassis for developing future biodegradation processes of PET waste.