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Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF. [ABSTRACT FROM AUTHOR]

In this study, the morphological characteristics of the T. neapolitana biofilms on a ceramic carrier, stainless steel, graphite foil, carbon paper, carbon felt and carbon cloth using 3D reconstruction technology was investigated. This was based on the micrographs available in Squadrito et al. (Data Brief 33: 106–403, 2020). Besides the ceramic carrier, the other surfaces were conductive and slightly positively polarised (0.8 and 1.2 V). A simple drying technique was used to show the biofilm and avoid its detachment while chemical fixing with glutaraldehyde was used to better highlight the bacterial morphology within the biofilm. The latter was more suitable for investigating biofilm morphology while the former for bacteria morphology. For the ceramic carrier and stainless steel electrode surfaces, a regular undulating pattern of the biofilm was highlighted by the 3D rendering whilst the glutaraldehyde fixed sample showed a rod-like bacteria morphology. For the other surfaces, a regular undulating pattern of the biofilm and a mixture of a rod-like and a coccoid form of settled bacteria were evidenced also. Carbon cloth was the more suitable electrode for the current application due to its richer filamentous network of bacteria biofilm suggesting a better prevention of bacteria detachment from the electrode surface. Indeed, a preserved biofilm was highlighted on the surfaces of the polarised carbon cloth. [ABSTRACT FROM AUTHOR]

Hyperthermophile bacteria were seldom investigated in bioelectrochemical systems although they allow more effective control of the inoculum in comparison with mesophilic bacteria. Biofilm formed in hyperthermophilic conditions (>60 °C) also rarely was documented (d'Ippolito et al., 2020; Belkin et al., 1986, Pysz et al., 2004).Scanning Electron Microscopy (SEM) micrographs documenting biofilms formed by the Hyperthermophile bacterium Thermotoga neapolitana on different solid materials (ceramic carrier, stainless steel mesh, carbon felt, carbon paper, expanse graphite, and carbon cloth) are shown in this report. Also, micrographs of the biofilm formed on electrodes of carbon cloth under a dynamic polarization oscillating around ±1 V (±0.8 V and ±1.2 V) are reported.Two procedures of sample preparation for SEM analyses are described and used: 1) a fast drying of samples, which is enough to underline the biofilm shape that covers solids, and 2) a chemical treating of the samples with glutaraldehyde, which better preserves the shape of bacterial cell components in the biofilm, although this treatment might cause the detachment of pieces of the biofilm.The different effect of potentiostatic and potentiodynamic polarizations on the glucose metabolism of T. neapolitana has been screened and discussed in the associated article [1]. Here, data of Optical Densities (O.D.) of culture media are provided, indicating the presence or absence of bacteria growth in the bulk of the media. Data have been collected every 24 h from the differently polarized bioreactors. The electrodes set-up of small bioreactors is also illustrated.Chemical data, optical data and SEM images, accordingly, document a retard in the glucose fermentation process due to a settlement of T. Neapolitana in a stationary phase. The polarization of electrodes can modify the stationary condition, inducing a possible change of the bacteria metabolism.

• Mutant C6 of Cel12A obtained after four rounds of ep PCR showed nearly 5-fold increase in activity. • The mutation E57 K in C6 produced conformational changes promoting binding affinity with the ligand and thus enhancing activity. • Analysis of the hydrolytic products showed that Cel12A should be a cellobiohydrolase instead of an endoglucanase as previously reported. Using multi-step error prone PCR (ep-PCR) of the gene encoding endoglucanase Cel12A (27 kDa) from Thermotoga neapolitana, mutants were obtained with many fold increase in the enzyme activity. The best mutant (C6, N47S/E57 K/ V88A/S157 P/K165 H) obtained after four rounds of ep-PCR showed 2.7−, 5− and 4.8−fold increase in activity against CMC, RAC and Avicel, respectively, compared with the wild type enzyme. The other characteristics of the mutated enzyme with respect to stability, optimum working pH and temperature were comparable to the wild type enzyme.C6 mutant showed higher binding efficiency towards the rice straw (∼50%) than the wild type (∼41%). The structural information obtained from the protein docking of the wild type Cel12A and its mutant showed that E57 K improved the binding affinity between enzyme and ligand by producing conformational changes in the catalytic cavity. The other mutations can facilitate the enzyme-substrate binding interactions to enhance catalytic activity although they are not directly involved in catalysis. The wild type and mutant enzyme produce cellobiose as the major products for both soluble and insoluble substrates, suggesting that this enzyme should be a cellobiohydrolase instead of endoglucanase as previously reported. [ABSTRACT FROM AUTHOR]

Hydrogen is a strong inhibitor of dark fermentation. We aimed at directly correlating the hydrogen production by Thermotoga neapolitana with the supersaturation of hydrogen in the liquid phase (H 2aq), which is often disregarded. Different agitation speeds, biogas recirculation and bubble induction by AnoxK™ K1 carrier were tested to prevent the supersaturation of H 2aq. At 100 rpm agitation, the H 2aq was 29.7 (±1.4) mL/L, which is 3-times higher than 9.7 mL/L, i.e. the equilibrium concentration given by Henry's law. Increasing the agitation speed up to 600 rpm reduced the H 2aq until 8.5 (±0.1) mL/L in 2 h and increased the hydrogen production rate (HPR) from 39 (±2) mL/L/h at 0 rpm to 198 (±4) mL/L/h at 600 rpm. Similar to 600 rpm, biogas recirculation and the presence of K1 carrier at 200 rpm maintained the H 2aq below the equilibrium concentration. This study demonstrates the reciprocal influence of HPR and H 2aq and revealed an inverse nonlinear correlation between the two parameters. Therefore, we conclude that an adequate gas-liquid mass transfer, efficiently provided by biogas recirculation or the presence of solid materials (e.g. a biomass carrier), is essential to remove H 2 from the liquid phase and prevent H 2 supersaturation. • An inverse nonlinear correlation was found between HPR and H 2aq. • The equilibrium between H 2aq and HPR depends on the gas-liquid mass transfer. • Agitation at 100 rpm resulted in a supersaturation of H 2aq up to 29.7 (±1.4) mL/L. • Gas recirculation and K1 carrier led to the lowest H 2aq , i.e. 5.8 (±0.5) mL/L. • K1 carrier induced H 2 bubble formation reducing H 2aq by about 50% in the first 15 h. [ABSTRACT FROM AUTHOR]

This study focused on the supersaturation of hydrogen in the liquid phase (H 2aq) and its inhibitory effect on dark fermentation by Thermotoga neapolitana cf. capnolactica by increasing the agitation (from 100 to 500 rpm) and recirculating H 2 -rich biogas (GaR). At low cell concentrations, both 500 rpm and GaR reduced the H 2aq from 30.1 (±4.4) mL/L to the lowest values of 7.4 (±0.7) mL/L and 7.2 (±1.2) mL/L, respectively. However, at high cell concentrations (0.79 g CDW/L), the addition of GaR at 300 rpm was more efficient and increased the hydrogen production rate by 271%, compared to a 136% increase when raising the agitation to 500 rpm instead. While H 2aq primarily affected the dark fermentation rate, GaR concomitantly increased the hydrogen yield up to 3.5 mol H 2 /mol glucose. Hence, H 2aq supersaturation highly depends on the systems gas-liquid mass transfer and strongly inhibits dark fermentation. • H 2 supersaturates in the liquid phase at 100 rpm despite hyperthermophilic conditions. • High H 2aq concentrations directly inhibit the specific rates of dark fermentation. • H 2 supersaturation and H 2 production rate depend on the gas-liquid mass transfer. • H 2 -rich biogas recirculation is an effective method to prevent H 2 supersaturation. • Gas recirculation increased the hydrogen production rate by 271%. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Identification of two novel, thermostable hemicellulolytic enzymes from T. neapolitana. • Design of an enzyme cocktail for the deconstruction of lignocellulose in bioenergy feedstock at high temperatures. • Treatment of complex substrates with the hemicellulolytic cocktail increased the enzymatic degradation of cellulose up to 65%. Abstract The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-β-galactanase and an α- l -arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (k cat values of ˜177 and ˜133 s−1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases – endo-1,4-β-xylanase (XynA), endo-1,4-β-mannanase (ManB/Man5A) and β-glucosidase (GghA) – to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Hydrogen production rate increased with increasing feed glucose until 27.8 mM. • Hydrogen yield was negatively correlated with feed glucose concentration. • Process performance was unaffected by continuously-fed acetic acid up to 240 mM. • Acetic acid reduced the hydrogen yield and production rate in batch bioassays. • Hydrogen yield increased by 47% in 110 d of continuous operation. Abstract This study focused on the effect of feed glucose and acetic acid on biohydrogen production by Thermotoga neapolitana under continuous-flow conditions. Increasing the feed glucose concentration from 11.1 to 41.6 mM decreased the hydrogen yield from 3.6 (±0.1) to 1.4 (±0.1) mol H 2 /mol glucose. The hydrogen production rate concomitantly increased until 27.8 mM of feed glucose but remained unaffected when feed glucose was further raised to 41.6 mM. Increasing the acetic acid concentration from 0 to 240 mM hampered dark fermentation in batch bioassays, diminishing the cumulative hydrogen production by 45% and the hydrogen production rate by 57%, but induced no negative effect during continuous operation. Indeed, throughout the continuous flow operation the process performance improved considerably, as indicated by the 47% increase of hydrogen yield up to 3.1 (±0.1) mol H 2 /mol glucose on day 110 at 27.8 mM feed glucose. [ABSTRACT FROM AUTHOR]

The objective of this study was to enhance the hydrogen production rate of dark fermentation in batch operation. For the first time, the hyperthermophilic pure culture of Thermotoga neapolitana cf. Capnolactica was applied at elevated biomass concentrations. The increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight/L led to a general acceleration of the fermentation process, reducing the fermentation time of 5 g glucose/L down to 3 h with a lag phase of 0.4 h. The volumetric hydrogen production rate increased from 323 (±11) to 654 (±30) mL/L/h with a concomitant enhancement of the biomass growth and glucose consumption rate. The hydrogen yield of 2.45 (±0.09) mol H 2 /mol glucose, the hydrogen concentration of 68% in the produced gas and the composition of the end products in the digestate, i.e. 62.3 (±2.5)% acetic acid, 23.5 (±2.9)% lactic acid and 2.3 (±0.1)% alanine, remained unaffected at increasing biomass concentrations. [ABSTRACT FROM AUTHOR]

This study demonstrates the potential for biohydrogen production in a co-culture of two ecologically distant species, Thermatoga neapolitana and Caldicellulosiruptor saccharolyticus , and the development of a quantitative real-time PCR (qPCR) method for quantifying the hyperthermophilic bacterium of the genus Thermotoga. Substrate utilization and H 2 production performance was compared to those of their individual cultures. The highest H 2 yields obtained were 2.7 ± 0.05, 2.5 ± 0.07 and 2.8 ± 0.09 mol H 2 /mol glucose for C. saccharolyticus , T. neapolitana, and their co-culture respectively. Statistical analysis comparing the H 2 production rate of the co-culture to either C. saccahrolyticus or T. neapolitana pure cultures indicated a significant difference in the H 2 production rate (p < 0.05: t -test), with the highest rate of H 2 production (36.02 mL L −1 h −1 ) observed from the co-culture fermentations. In order to monitor the presence of T. neapolitana in the bioprocess, we developed a qPCR method using 16S rRNA gene and hydrogenase (hydA) gene targets. The qPCR data using hydA primers specific to T. neapolitana showed an increase in hydA gene copies from 3.32 × 10 7 to 4.4 × 10 8 hydA gene copies per mL confirming the influence of T. neapolitana in the synthetic consortium. [ABSTRACT FROM AUTHOR]

Thermotoga neapolitana is a hyperthermophilic eubacterium that produces hydrogen by sugar fermentation. A lab strain of T. neapolitana DSMZ 4359 T maintained in a CO 2 -enriched atmosphere showed a stable increase of lactic acid production under capnophilic lactic fermentation (CLF) conditions. The genotypic comparison between the putative mutant (TN-CMut) and the original strain DSMZ 4359 T (WT4359) revealed 88.1 (±2.4)% DNA homology. RiboPrint ® and MALDI-TOF mass analyses support a genetic differentiation beyond subspecies level. The phenotypic characterization indicated a high correlation between the two strains, except for the lactic acid production. Under identical operating conditions, the lab mutant produced significantly more lactic acid than the parent strain without impairing the hydrogen yield. The highest divergence between TN-CMut and WT4359 was observed for fermentation of glucose or lactose at 80 °C. Based on these results, we propose that the lab strain is a new subspecies of the genus Thermotoga that is named T. neapolitana subsp. capnolactica with regards to its improved feature to produce lactic acid under capnophilic conditions. [ABSTRACT FROM AUTHOR]

The thermostable β-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric threedomain representative from glycoside hydrolase family 3. By using chemical reactivation with exogenous nucleophiles in previous studies with TnBg13B, the catalytic nucleophile (D242) and corresponding acid/base residue (E458) were determined. Identifying these residues led to the attempt of converting TnBgl3B into a β-glucosynthase, where three nucleophilic variants were created (TnBgl3B_D242G, TnBgl3B_D242A, TnBgl3B_D242S) and all of them failed to exhibit glucosynthase activity. A deeper analysis of the TnBgl3B active site led to the generation of three additional variants, each of which received a single-point mutation. Two of these variants were altered at the -1 subsite (Y210F, W243F) and the third received a substitution near the binding site's aglycone region (N248R). Kinetic evaluation of these three variants revealed that W243F substitution reduced hydrolytic turnover while maintaining KM. This key W243F mutation was then introduced into the original nucleophile variants and the resulting double mutants were successfully converted into β-glucosynthases that were assayed using two separate biosynthetic methods. The first reaction used an a-glucosyl fluoride donor with a 4-nitrophenyl-β-D-glucopyranoside (4NPGlc) acceptor, and the second used 4NPGlc as both the donor and acceptor in the presence of the exogenous nucleophile formate. The primary specificity observed was a β-1,3-linked disaccharide product, while a secondary β-1,4-linked disaccharide product was observed with increased incubation times. Additional analysis revealed that substituting quercetin-3-glycoside for the second reaction's acceptor molecule resulted in the successful production of quercetin-3,4'-diglycosides with yields up to 40%. [ABSTRACT FROM AUTHOR]

Competing Interests: Declaration of interests No interests are declared.

Two-stage process based on photofermentation of dark fermentation effluents is widely recognized as the most effective method for biological production of hydrogen from organic substrates. Recently, it was described an alternative mechanism, named capnophilic lactic fermentation, for sugar fermentation by the hyperthermophilic bacterium Thermotoga neapolitana in CO-rich atmosphere. Here, we report the first application of this novel process to two-stage biological production of hydrogen. The microbial system based on T. neapolitana DSM 4359 and Rhodopseudomonas palustris 42OL gave 9.4 mol of hydrogen per mole of glucose consumed during the anaerobic process, which is the best production yield so far reported for conventional two-stage batch cultivations. The improvement of hydrogen yield correlates with the increase in lactic production during capnophilic lactic fermentation and takes also advantage of the introduction of original conditions for culturing both microorganisms in minimal media based on diluted sea water. The use of CO during the first step of the combined process establishes a novel strategy for biohydrogen technology. Moreover, this study opens the way to cost reduction and use of salt-rich waste as feedstock. [ABSTRACT FROM AUTHOR]

The biochemical properties of a putative β-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein ( TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against β-1,3-xylan and released β-1,3-xylobiose and β-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3-10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble β-1,3-xylan K value was 10.35 mg/ml and the k value was 588.24 s. The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction. [ABSTRACT FROM AUTHOR]

Highlights: [•] Total quercetin content was determined in different onion varieties. [•] Hot water extraction was combined with hydrolysis using thermostable β-glucosidase. [•] Enzyme activity was highest in water containing 5% ethanol at 84°C and pH 5.5. [•] The immobilized enzyme showed good stability over 72h at the optimized conditions. [•] The developed method gave comparable yield of quercetin compared to conventional methods. [ABSTRACT FROM AUTHOR]

An intracellular α-amylase, AmyB, has been cloned from the hyperthermophilic bacterium Thermotoga neapolitana. AmyB belongs to glycoside hydrolase family 13 and liberates maltose from diverse substrates, including starch, amylose, amylopectin and glycogen. The final product of AmyB is similar to that of typical maltogenic amylases, but AmyB cleaves maltose units from the nonreducing end, which is a unique property of this α-amylase. In this study, the crystal structure of AmyB from T. neapolitana has been determined at 2.4 Å resolution, revealing that the monomeric AmyB comprises domains A, B and C like other α-amylases, but with structural variations. In the structure, a wider active site and a putative extra sugar-binding site at the top of the active site were found. Subsequent biochemical results suggest that the extra sugar-binding site is suitable for recognizing the nonreducing end of the substrates, explaining the unique activity of this enzyme. These findings provide a structural basis for the ability of an α-amylase that has the common α-amylase structure to show a diverse substrate specificity. [ABSTRACT FROM AUTHOR]

The gene encoding a putative mannose-6-phosphate isomerase (TnMPI) from Thermotoga neapolitana DSM4359 was cloned and expressed in Escherichia coli. TnMPI showed the highest isomerization activity between d-fructose and d-mannose at 75°oC in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM of Cu. TnMPI can be activated by some divalent metal ions, such as Cu, Mn, and Co. In the presence of 1 mM Cu, TnMPI activity on conversion from d-fructose to d-mannose was significantly enhanced up to 271% of that without Cu. In addition, its isomerization equilibrium between d-fructose and Dmannose was strongly affected by reaction temperature and pH. As reaction temperature decreased from 95 to 55°C, the equilibrium ratio of d-fructose to d-mannose was gradually shifted from 73:27 to 55:45. As reaction pH decreased from pH 8.5 to 5.5, the equilibrium ratio of Dfructose to d-mannose was shifted from 68:32 to 49:51. [ABSTRACT FROM AUTHOR]

Constraint-based models of biochemical reaction networks require experimental validation to test model-derived hypotheses and iteratively improve the model. Physiological and proteomic analysis of Thermotoga neapolitana growth on cellotetraose was conducted to identify gene products related to growth on cellotetraose to improve a constraint-based model of T. neapolitana central carbon metabolism with incomplete cellotetraose pathways. In physiological experiments comparing cellotetraose to cellobiose and glucose as growth substrates, product formation yields on cellotetraose, cellobiose, and glucose were similar; however cell yields per mol carbon consumed were higher on cellotetraose than on cellobiose or glucose. Proteomic analysis showed increased expression of several proteins from cells grown on cellotetraose compared with glucose cell cultures, including cellobiose phosphorylase (CTN_0783), endo-1,4-β-glucosidase (CTN_1106), and an ATP-binding protein (CTN_1296). The CTN_1296 gene product should be evaluated further for participation in cellotetraose metabolism and is included as one of two hypothetical gene-protein-reaction associations in the T. neapolitana constraint-based model to reinstate cellotetraose metabolism in model simulations. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012 [ABSTRACT FROM AUTHOR]

Abstract: Biohydrogen production from xylose by Thermotoga neapolitana was investigated in batch culture using serum bottles and a continuously stirred anaerobic bioreactor (CSABR). The effect of various xylose concentrations on growth and H2 production were studied in small batch culture for highly efficient H2 production. The highest hydrogen production of 32.1 ± 1.6 mmol-H2/L and maximum biomass concentration of 959.63 ± 47.9 mg/L were obtained at initial xylose concentration of 5.0 g/L. To develop a large-scale biohydrogen production system as well as overcome the problems in small batch culture, a continuously stirred anaerobic bioreactor was tested on T. neapolitana in both pH-uncontrolled batch culture and pH-controlled batch culture. The results showed that the production level of H2 from fermentation in a pH-controlled batch culture was much higher than those from a pH-uncontrolled batch culture for H2 production from xylose. The H2 yield in a pH-controlled batch culture on xylose substrate was 2.22 ± 0.11 mol-H2 mol−1 xyloseconsumed, which was nearly 1.2-fold higher than pH-uncontrolled batch cultures. In order to study the precise effect of a stable pH on hydrogen production, and metabolite pathway involved, cultures was conducted with pH-controlled at different levels ranging from 6.5 to 7.5. The maximum H2 yield of 2.8 ± 0.14 mol-H2 mol−1 xyloseconsumed was measured while the pH was maintained at 7.0. The acetic acid and lactic acid production were 2.98 ± 0.15 g/L and 0.36 ± 0.02 g/L, respectively. [Copyright &y& Elsevier]

Abstract: Biohydrogen fermentation by the hyperthermophile Thermotoga neapolitana was conducted in a continuously stirred anaerobic bioreactor (CSABR). The production level of H2 from fermentation in a batch culture with pH control was much higher than without pH control from pentose (xylose) and hexose (glucose and sucrose) substrates. The respective H2 yield in the batch culture with pH control from xylose and glucose was 2.22 ± 0.11 mol-H2 mol−1 xyloseconsumed and 3.2 ± 0.16 mol-H2 mol−1 glucoseconsumed, which was nearly 1.2-fold greater for xylose and 1.6-fold greater for glucose than without pH control. In the case of sucrose, the H2 yield from fermentation increased by 40.63%, compared with fermentation in batch cultures without pH control, from 3.52 ± 0.171 to 4.95 ± 0.25 mol-H2 mol−1 sucroseconsumed. The effects of stirring speed and different pH levels on growth and H2 production were studied in the CSABR for highly efficient H2 production. Growth and H2 production of this bacterial strain in a batch culture with pH control or without pH control using a 3 L bioreactor was limited within 24 h due to substrate exhaustion and a decrease in the culture’s pH. The pH-controlled fed-batch culture with a xylose substrate added in doses was studied for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The highest H2 production rates were approximately 4.6, 4.1, 3.9, and 4.3 mmol-H2 L−1 h−1 at 32, 52, 67, and 86 h, respectively. [Copyright &y& Elsevier]

We cloned the gene for an extracellular α-amylase, AmyE, from the hyperthermophilic bacterium Thermotoga neapolitana and expressed it in Escherichia coli. The molecular mass of the enzyme was 92 kDa as a monomer. Maximum activity was observed at pH 6.5 and temperature 75°C and the enzyme was highly thermostable. AmyE hydrolyzed the typical substrates for α-amylase, including soluble starch, amylopectin, and maltooli-gosaccharides. The hydrolytic pattern of AmyE was similar to that of a typical α-amylase; however, unlike most of the calcium (Ca)-dependent α-amylases, the activity of AmyE was unaffected by Ca. The specific activities of AmyE towards various substrates indicated that the enzyme preferred maltooligosaccharides which have more than four glucose residues. AmyE could not hydrolyze maltose and maltotriose. When maltoheptaose was incubated with AmyE at the various time courses, the products consisting of maltose through maltopentaose was evenly formed indicating that the enzyme acts in an endo-fashion. The specific activity of AmyE (7.4 U/mg at 75° C, pH 6.5, with starch as the substrate) was extremely lower than that of other extracellular α-amylases, which indicates that AmyE may cooperate with other highly active extracellular α-amylases for the breakdown of the starch or α-glucans into maltose and maltotriose before transport into the cell in the members of Thermotoga sp. [ABSTRACT FROM AUTHOR]

Abstract: Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H2) production than untreated waste. Nitrogen (N2) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H2 partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H2 mol−1 glycerolconsumed by optimising N2 sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N2 sparging, the H2 yield increased to 2.73 ± 0.14 mol-H2 mol−1 glycerolconsumed, which was 2.22-fold higher than the non-N2 sparged H2 yield (1.23 ± 0.06 mol-H2 mol−1 glycerolconsumed). [Copyright &y& Elsevier]

Abstract: The gene encoding a type I pullulanase from the hyperthermophilic anaerobic bacterium Thermotoga neapolitana (pulA) was cloned in Escherichia coli and sequenced. The pulA gene from T. neapolitana showed 91.5% pairwise amino acid identity with pulA from Thermotoga maritima and contained the four regions conserved in all amylolytic enzymes. pulA encodes a protein of 843 amino acids with a 19-residue signal peptide. The pulA gene was subcloned and overexpressed in E. coli under the control of the T7 promoter. The purified recombinant enzyme (rPulA) produced a 93-kDa protein with pullulanase activity. rPulA was optimally active at pH 5–7 and 80°C and had a half-life of 88min at 80°C. rPulA hydrolyzed pullulan, producing maltotriose, and hydrolytic activities were also detected with amylopectin, starch, and glycogen, but not with amylose. This substrate specificity is typical of a type I pullulanase. Thin layer chromatography of the reaction products in the reaction with pullulan and aesculin showed that the enzyme had transglycosylation activity. Analysis of the transfer product using NMR and isoamylase treatment revealed it to be α-maltotriosyl-(1,6)-aesculin, suggesting that the enzyme transferred the maltotriosyl residue of pullulan to aesculin by forming α-1,6-glucosidic linkages. Our findings suggest that the pullulanase from T. neapolitana is the first thermostable type I pullulanase which has α-1,6-transferring activity. [Copyright &y& Elsevier]

We have investigated H production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7-3.8 mol mol sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel. [ABSTRACT FROM AUTHOR]

Abstract: Rice straw, a low-cost lignocellulosic biomass was used as feedstock for thermophilic hydrogen fermentation by Thermotoga neapolitana. Hydrogen production, the growth and cellulose digestibility of the hyperthermophile in batch mode from untreated as well as chemically pretreated (ammonia and dilute sulfuric acid) Korean rice straws were investigated. Pretreatment method using combination of 10% ammonia and 1.0% dilute sulfuric acid was developed to increase the digestibility of rice straw for the hyperthermophilic H2 fermentation and to decrease the time consumption. In a typical fermentation using raw rice straw, 29% of the substrate was digested and 2.3 mmol H2/g straw of hydrogen yield was consistently obtained. Compared with the pretreatments using only ammonia or dilute sulfuric acid, the combined pretreatment method using both chemical agents significantly increases the digestibility of rice straw with 85.4% of substrate consumption. H2 production on rice straw from this combined pretreatment showed the highest yield (2.7 mmol H2/g straw) and the highest sugar conversions (72.9% of glucose and 95.7% of xylose). [ABSTRACT FROM AUTHOR]

Abstract: Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO2 and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H2) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H2 from algal starch with H2 yield of 1.8–2.2 mol H2/mol glucose and the total accumulated H2 level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H2 production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H2 fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H2 yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H2 productivity of the bacterium to 64% (v/v) of total accumulated H2 level and a H2 yield of 2.5 mol H2/mol glucose. Our results demonstrated that direct H2 fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H2 production. [ABSTRACT FROM AUTHOR]

Immobilization of a thermostable D-xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N-terminal fusion with a chitin-binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD-TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD-TNXI were greater than the wild-type enzyme: kcat (min-1) was ∼1,000, 3,800, and 5,800 at 80°C compared to 1,140, 10,350, and 7,000 at 90°C, for the wild-type, unbound, and immobilized enzymes, respectively. These kcat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi-phasic inactivation model, showed that the CBD-TNXI bound to chitin had a half-life approximately three times longer than the soluble wild-type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD-TNXI had a significantly longer half-life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N-terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD-TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate-binding affinities and generating higher turnover rates in the unbound fusion protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]

Abstract: A highly thermostable recombinant xylanase from Thermotoga neapolitana was used as a catalyst for single-step synthesis of n-octyl xylobioside and n-octyl xylotrioside from xylan and n-octanol. Effect of xylan concentration, enzyme dose, reaction water content, reaction temperature and initial pH on the yield of these surfactants has been studied. The optimal conditions for n-octyl xylobioside and n-octyl xylotrioside synthesis are found to be different. The maximum n-octyl xylobioside and n-octyl xylotrioside yield were 120 and 38mg/g of xylan, respectively. The n-octyl xylobioside yield achieved in this study was better than the yield achieved in all other enzymatic synthesis studies reported so far except what is achieved with the use of supercritical fluoroform under high pressure. The n-octyl xylotrioside yield is the highest ever achieved through enzymatic synthesis. An integrated system of production and recovery of n-octyl xylosides has improved the yield of n-octyl xylobioside and n-octyl xylotrioside by a factor of 1.7 and 2.4, respectively. [Copyright &y& Elsevier]

Abstract: Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized—that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable β-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 Å. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 Å resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (α/β)8 domain similar to a triose phosphate isomerase barrel, a five-stranded α/β sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second β-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the −1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the −1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability. [Copyright &y& Elsevier]