A hot topic: thermophilic plastic biodegradation.

Biological degradation of plastic waste is an environmentally and economically friendlier alternative to current recycling practices and enables the cycling of plastic monomers back into virgin-quality plastics. However, due to slow reaction rates, there is a lack of an industrially viable biodegradation strategy for most plastics. Here, we highlight the applicability of a thermophilic biodegradation strategy over a mesophilic approach, to enhance enzyme accessibility and catalyze plastic biodegradation. Thus, at reactions closer to the melting temperature or glass transition temperature of plastics, thermophilic reactions can offer an alternative direction to conventional plastic biodegradation strategies.

Draft Genome Sequence of Microbacterium sp. TNHR37B Isolated from a Heated Aquifer Bore Well of the Great Artesian Basin, Australia.

Microbacterium sp. strain TNHR37B was isolated from a geothermal bore well sample (50°C) collected from a region of coal seam gas extraction activities. The 3.5-Mb genome with a G+C content of 69.9% contained unique genes, and a low similarity value for average nucleotide identity using BLAST was observed with the available 73 Microbacterium sp. genomes.

Draft Genome Sequence of the 1,2-Dichloroethane-Utilizing Micrococcus sp. Strain NDB3Y10, Isolated from an Australian Bore Well Producing Coal Seam Gas.

Micrococcus luteus strain NDB3Y10, which utilizes 1,2-dichloroethane as a carbon source, was isolated from a bore well that produces coal seam gas. The draft genome size of the strain was 2.49 Mb with a G+C content of 72.97%. Genes involved in the metabolism of halogenated substrates, including halogenated hydrocarbons, were identified.

Draft Genome Sequence of Cellulosilyticum sp. I15G10I2, a Novel Bacterium Isolated from a Coal Seam Gas Water Treatment Pond.

Cellulosilyticum sp. strain I15G10I2 was isolated from a coal seam gas water treatment pond at the Spring Gully water treatment facility, Roma, Queensland, Australia. Analysis of the genome of 4,489,861 bp and G+C content of 35.23% revealed that strain I15G10I2 shared limited similarity to members of the genus Cellulosilyticum, family Lachnospiraceae.

Draft Genome Sequence of Caloramator mitchellensis, a Thermoanaerobe Isolated from the Waters of the Great Artesian Basin.

The genome sequence of Caloramator mitchellensis strain VF08, a rod-shaped, heterotrophic, strictly anaerobic bacterium isolated from the free-flowing waters of a Great Artesian Basin (GAB) bore well located in Mitchell, an outback Queensland town in Australia, is reported here. The analysis of the 2.42-Mb genome sequence indicates that the attributes of the genome are consistent with its physiological and phenotypic traits.

Engineering a thermostable Halothermothrix orenii ß-glucosidase for improved galacto-oligosaccharide synthesis.

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining ß-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a ß-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k cat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k cat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

A molecular phylogenetic framework for Bacillus subtilis using genome sequences and its application to Bacillus subtilis subspecies stecoris strain D7XPN1, an isolate from a commercial food-waste degrading bioreactor.

A thermophilic, heterotrophic and facultatively anaerobic bacterium designated strain D7XPN1 was isolated from Baku BakuKing™, a commercial food-waste degrading bioreactor (composter). The strain grew optimally at 45 °C (growth range between 24 and 50 °C) and pH 7 (growth pH range between pH 5 and 9) in Luria Broth supplemented with 0.3 % glucose. Strain D7XPN1 tolerated up to 7 % NaCl and showed amylolytic and xylanolytic activities. 16S rRNA gene analysis placed strain D7XPN1 in the cluster represented by Bacillus subtilis and the genome analysis of the 4.1 Mb genome sequence determined using RAST (Rapid Annotation using Subsystem Technology) indicated a total of 5116 genomic features were present of which 2320 features could be grouped into several subsystem categories. Of these, 615 features were related to carbohydrate metabolism which included a range of enzymes with potential in the biodegradation of food wastes, a property consistent with the ecological habitat of the isolate. ANIb (Average Nucleotide Identity based on BLAST) analysis with 49 Bacillus subtilis genomes indicated that it was distantly related to the three currently taxonomically validated B. subtilis subspecies namely B. subtilis subsp. subtilis (95.6 %), B. subtilis subsp. spizizenii (93 %) and B. subtilis subsp. inaquosorum (92 %) and based on our current knowledge warranted that it be included as a separate cluster together with strain JS which it was closely related (98.69 %). The close relationship of strains D7XPN1 and JS is also supported from our results from electronic DNA-DNA Hybridization (e-DDH) studies. Furthermore, our additional in-depth phylogenomic analyses using three different datasets unequivocally supported the creation of a fourth B. subtilis subspecies to include strains D7XPN1 and JS for which we propose strain D7XPN1T (=KCTC 33554T, JCM 30051T) as the type strain, and designate it as B. subtilis subsp. stecoris.

High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with α-L-arabinofuranosidase activity.

A gene from the heterotrophic, halothermophilic marine bacterium Halothermothrix orenii has been cloned and overexpressed in Escherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by H. orenii. The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10 Šresolution. As for other GH43 members, the enzyme folds as a five-bladed ß-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, the H. orenii glycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4 M NaCl and Mn(2+) at pH 6.5. The gene encoding the H. orenii GH43 enzyme has previously been annotated as a putative α-L-arabinofuranosidase. Activity was detected with p-nitrophenyl-α-L-arabinofuranoside as a substrate, and therefore the name HoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4 M NaCl and Mn(2+) at pH 6.5, giving a specific activity of 20-36 µmol min(-1) mg(-1). The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution of HoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.

Draft Genome Sequence of Anoxybacillus Strain BCO1, Isolated from a Thermophilic Microbial Mat Colonizing the Outflow of a Bore Well of the Great Artesian Basin of Australia.

Anoxybacillus strain BCO1, isolated from a thermophilic (50°C) microbial mat colonizing an outflow of a Great Artesian bore well of Australia, possessed a genome of ~2.8 Mb, with a G+C content of 41.7 mol%, and encoded 3,205 genes.

Draft Genome Sequence of Paenibacillus Strain P1XP2, a Polysaccharide-Degrading, Thermophilic, Facultative Anaerobic Bacterium Isolated from a Commercial Bioreactor Degrading Food Waste.

The analysis of the ~5.8-Mb draft genome sequence of a moderately thermophilic, heterotrophic, facultative anaerobic bacterium, Paenibacillus strain P1XP2, identified genes for enzymes with the potential for degrading complex food wastes, a property consistent with the ecological habitat of the isolate.

Biochemical and structural characterization of a thermostable ß-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial ß-glucosidases (BGL) with significant ß-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific ß-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal ß-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are ß-D-Galp-(1→6)-D-Lac (6GALA) and ß-D-Galp-(1→3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

Draft Genome Sequence of Bacillus subtilis Strain D7XPN1, Isolated from Commercial Bioreactor-Degrading Food Waste.

The analysis of the 4.1-Mb draft genome sequence of a moderately thermophilic, heterotrophic, and facultatively anaerobic bacterium, Bacillus subtilis strain D7XPN1, identified genes for a range of enzymes with potential in the biodegradation of food waste, a property consistent with the ecological habitat of the isolate.

Draft Genome Sequence of Fervidicella metallireducens Strain AeBT, an Iron-Reducing Thermoanaerobe from the Great Artesian Basin.

The genome sequence of Fervidicella metallireducens strain AeB(T), a curved, heterotrophic, thermoanaerobic, and iron-reducing bacterium isolated from a gray microbial mat colonizing the free-flowing waters of a Great Artesian Basin (GAB) bore well located in outback Queensland, Australia, is reported here. The analysis of the 2.9-Mb sequence indicates that the attributes of the genome are consistent with its physiological and phenotypic traits.

Caloramator quimbayensis sp. nov., an anaerobic, moderately thermophilic bacterium isolated from a terrestrial hot spring.

An anaerobic, moderately thermophilic, terminal-spore-forming bacterium, designated strain USBA A(T), was isolated from a terrestrial hot spring located at an altitude of 2683 m in the Andean region of Colombia (04° 50' 14.0″ N 75° 32' 53.4″ W). Cells of strain USBA A(T) were Gram-stain-positive, straight to slightly curved rods (0.9×2.5 µm), that were arranged singly or in pairs, and were motile by means of flagella. Growth occurred at 37-55 °C and pH 6.0-8.0, with a doubling time of 2 h under the optimal conditions (50 °C and pH 7.0). Glucose fermentation in strain USBA A(T) required yeast extract or peptone (each at 0.2 %, w/v). The novel strain fermented sugars, amino acids, Casamino acids, propanol, propionate, starch and dextrin, but no growth was observed on galactose, lactose, xylose, histidine, serine, threonine, benzoate, butyrate, lactate, pyruvate, succinate, methanol, ethanol, glycerol, casein, gelatin or xylan. The end products of glucose fermentation were formate, acetate, ethanol and lactate. Strain USBA A(T) did not grow autotrophically (with CO2 as carbon source and H2 as electron donor) and did not reduce thiosulfate, sulfate, elemental sulfur, sulfite, vanadium (V) or Fe (III) citrate. Growth of strain USBA A(T) was inhibited by ampicillin, chloramphenicol, kanamycin, penicillin and streptomycin (each at 10 µg ml(-1)). The predominant fatty acids were iso-C15 : 0, C16 : 0 and iso-C17 : 0 and the genomic DNA G+C content was 32.6 mol%. 16S rRNA gene sequence analysis indicated that strain USBA A(T) belonged in the phylum Firmicutes and that its closest relative was Caloramator viterbiensis JW/MS-VS5(T) (95.0 % sequence similarity). A DNA-DNA relatedness value of only 30 % was recorded in hybridization experiments between strain USBA A(T) and Caloramator viterbiensis DSM 13723(T). Based on the phenotypic, chemotaxonomic and phylogenetic evidence and the results of the DNA-DNA hybridization experiments, strain USBA A(T) represents a novel species of the genus Caloramator, for which the name Caloramator quimbayensis sp. nov. is proposed. The type strain is USBA A(T) ( = CMPUJ U833(T)  = DSM 22093(T)).

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophile Halothermothrix orenii.

A ribokinase gene (rbk) from the anaerobic halothermophilic bacterium Halothermothrix orenii was cloned and overexpressed in Escherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 45.6, b = 61.1, c = 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

Draft genome sequence of Caloramator australicus strain RC3T, a thermoanaerobe from the Great Artesian Basin of Australia.

Caloramator australicus strain RC3(T) (JCM 15081(T) = KCTC 5601(T)) is the type strain of a newly identified thermophilic species, which was isolated from red microbial mats that thrive at 66°C in the runoff channel of a Great Artesian Basin bore (New Lorne bore, registered number 17263) in outback Queensland, Australia. The ability of the C. australicus strain to use metals as terminal electron acceptors has led to concerns that it could colonize and enhance corrosion of the metal casing of Great Artesian Basin bore well pipes and that this could subsequently lead to bore failure and loss of water availability for the community which is so reliant on it. The genome of the C. australicus strain has been sequenced, and annotation of the ~2.65-Mb sequence indicates that the attributes are consistent with physiological and phenotypic traits.

Expression, purification and preliminary crystallographic analysis of the recombinant ß-glucosidase (BglA) from the halothermophile Halothermothrix orenii.

The ß-glucosidase A gene (bglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA; EC 3.2.1.21) was bacterially expressed, purified using metal ion-affinity chromatography and subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress.

Desulfosoma caldarium gen. nov., sp. nov., a thermophilic sulfate-reducing bacterium from a terrestrial hot spring.

A thermophilic, sulfate-reducing bacterium, designated strain USBA-053(T), was isolated from a terrestrial hot spring located at a height of 2500 m in the Colombian Andes (5° 45' 33.29″ N 73° 6' 49.89″ W), Colombia. Cells of strain USBA-053(T) were oval- to rod-shaped, Gram-negative and motile by means of a single polar flagellum. The strain grew autotrophically with H(2) as the electron donor and heterotrophically on formate, propionate, butyrate, valerate, isovalerate, lactate, pyruvate, ethanol, glycerol, serine and hexadecanoic acid in the presence of sulfate as the terminal electron acceptor. The main end products from lactate degradation, in the presence of sulfate, were acetate, CO(2) and H(2)S. Strain USBA-053(T) fermented pyruvate in the absence of sulfate and grew optimally at 57 °C (growth temperature ranged from 50 °C to 62 °C) and pH 6.8 (growth pH ranged from 5.7 to 7.7). The novel strain was slightly halophilic and grew in NaCl concentrations ranging from 5 to 30 g l(-1), with an optimum at 25 g l(-1) NaCl. Sulfate, thiosulfate and sulfite were used as electron acceptors, but not elemental sulfur, nitrate or nitrite. The G+C content of the genomic DNA was 56±1 mol%. 16S rRNA gene sequence analysis indicated that strain USBA-053(T) was a member of the class Deltaproteobacteria, with Desulfacinum hydrothermale MT-96(T) as the closest relative (93 % gene sequence similarity). On the basis of physiological characteristics and phylogenetic analysis, it is suggested that strain USBA-053(T) represents a new genus and novel species for which the name Desulfosoma caldarium gen. nov., sp. nov. is proposed. The type strain of the type species is USBA-053(T) ( = KCTC 5670(T) = DSM 22027(T)).

Caloramator mitchellensis sp. nov., a thermoanaerobe isolated from the geothermal waters of the Great Artesian Basin of Australia, and emended description of the genus Caloramator.

A strictly thermophilic anaerobe, designated strain VF08(T), was isolated from a water sample collected from a Great Artesian Basin bore (registered bore number 22981) situated at Mitchell, QLD, Australia. Cells of isolate VF08(T) were slightly curved, non-sporulating rods (1.5-3.5 x 0.4-0.8 µm), which stained Gram-negative but possessed a Gram-positive cell-wall ultrastructure. The strain grew optimally in tryptone-yeast extract-glucose (TYEG) medium at 55 °C (temperature growth range between 37 and 60 °C) and a pH of 7 (pH growth range, 6.0-9.0). Yeast extract or tryptone was required for growth on glucose, fructose, xylose, maltose, sucrose, raffinose, cellobiose, ribose, pyruvate, tryptone, peptone, Casamino acids, amyl media and serine, but could also support growth as the sole carbon source. End products from glucose fermentation were acetate, ethanol, CO2 and H2. The strain reduced vanadium(V), but not iron(III), manganese(IV), elemental sulfur, sulfate, thiosulfate, sulfite, nitrate or nitrite in the presence of 0.2  % yeast extract, peptone, tryptone, glucose, sucrose and Casamino acids, but an increase in the growth rate or cell yield was not observed. Growth was inhibited by chloramphenicol, streptomycin, tetracycline, penicillin, ampicillin and ≥ 2  % NaCl (w/v). The G+C content of the DNA was 38.4 ± 0.8 mol% as determined by the thermal denaturation (T(m)) method. 16S rRNA gene sequence analysis revealed that isolate VF08(T) was a member of the genus Caloramator with Caloramator australicus and Caloramator fervidus (formerly Clostridium fervidus) being the closest relatives with similarity values of 85.0 and 86.1  %, respectively, when helix 6 nucleotides were included in the analysis, and 95.2  % and 94  %, respectively, when these nucleotides were masked from the analysis. Further analysis revealed that strain VF08(T) formed an individual cluster (cluster II) within the genus Caloramator and could be distinguished from other species within the genus Caloramator (clusters I, III and IV) on the basis of signature nucleotides and differences in phenotypic traits. These data suggest that strain VF08(T) is a novel species of the genus Caloramator, for which the name Caloramator mitchellensis sp. nov. is proposed. The type strain is VF08(T) (=JCM 15828(T)=KCTC 5735(T)). An emended description of the genus Caloramator is also provided.