Kinetic modeling of hydrogen and L-lactic acid production by Thermotoga neapolitana via capnophilic lactic fermentation of starch.

This study investigated the feasibility of hydrogen (H2) and L-lactic acid production from starch under capnophilic lactic fermentation (CLF) conditions by using Thermotoga neapolitana. Batch experiments were performed in 120 mL serum bottles and a 3 L pH-controlled continuous stirred-tank reactors (CSTR) system with potato and wheat starch as the substrates. A H2 yield of 3.34 (±0.17) and 2.79 (±0.17) mol H2/mol of glucose eq. was achieved with, respectively, potato and wheat starch. In the presence of CO2, L-lactic acid production by the acetyl-CoA carboxylation was significantly higher for the potato starch (0.88 ± 0.39 mol lactic acid/mol glucose eq.) than wheat starch (0.33 ± 0.11 mol lactic acid/mol glucose eq.). A kinetic model was applied to simulate and predict the T. neapolitana metabolic profile and bioreactor performance under CLF conditions. The CLF-based starch fermentation suggests a new direction to biotransform agri-food waste into biofuels and valuable biochemicals.

CO2-Induced Transcriptional Reorganization: Molecular Basis of Capnophillic Lactic Fermentation in Thermotoga neapolitana.

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway able to convert sugars to lactic acid (LA) and hydrogen using CO2 as carbon enhancer in the hyperthermophilic bacterium Thermotoga neapolitana. In order to give further insights into CLF metabolic networks, we investigated the transcriptional modification induced by CO2 using a RNA-seq approach. Transcriptomic analysis revealed 1601 differentially expressed genes (DEGs) in an enriched CO2 atmosphere over a total of 1938 genes of the T. neapolitana genome. Transcription of PFOR and LDH genes belonging to the CLF pathway was up-regulated by CO2 together with 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydratase (EDD) of the Entner-Doudoroff (ED) pathway. The transcriptomic study also revealed up-regulation of genes coding for the flavin-based enzymes NADH-dependent reduced ferredoxin:NADP oxidoreductase (NFN) and NAD-ferredoxin oxidoreductase (RNF) that control supply of reduced ferredoxin and NADH and allow energy conservation-based sodium translocation through the cell membrane. These results support the hypothesis that CO2 induces rearrangement of the central carbon metabolism together with activation of mechanisms that increase availability of the reducing equivalents that are necessary to sustain CLF. In this view, this study reports a first rationale of the molecular basis of CLF in T. neapolitana and provides a list of target genes for the biotechnological implementation of this process.

Model development and experimental validation of capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana.

The aim of the present study was to develop a kinetic model for a recently proposed unique and novel metabolic process called capnophilic (CO2-requiring) lactic fermentation (CLF) pathway in Thermotoga neapolitana. The model was based on Monod kinetics and the mathematical expressions were developed to enable the simulation of biomass growth, substrate consumption and product formation. The calibrated kinetic parameters such as maximum specific uptake rate (k), semi-saturation constant (kS), biomass yield coefficient (Y) and endogenous decay rate (kd) were 1.30 h(-1), 1.42 g/L, 0.1195 and 0.0205 h(-1), respectively. A high correlation (>0.98) was obtained between the experimental data and model predictions for both model validation and cross validation processes. An increase of the lactate production in the range of 40-80% was obtained through CLF pathway compared to the classic dark fermentation model. The proposed kinetic model is the first mechanistically based model for the CLF pathway. This model provides useful information to improve the knowledge about how acetate and CO2 are recycled back by Thermotoga neapolitana to produce lactate without compromising the overall hydrogen yield.

Hydrogen Production by the Thermophilic Bacterium Thermotoga neapolitana.

As the only fuel that is not chemically bound to carbon, hydrogen has gained interest as an energy carrier to face the current environmental issues of greenhouse gas emissions and to substitute the depleting non-renewable reserves. In the last years, there has been a significant increase in the number of publications about the bacterium Thermotoga neapolitana that is responsible for production yields of H2 that are among the highest achievements reported in the literature. Here we present an extensive overview of the most recent studies on this hyperthermophilic bacterium together with a critical discussion of the potential of fermentative production by this bacterium. The review article is organized into sections focused on biochemical, microbiological and technical issues, including the effect of substrate, reactor type, gas sparging, temperature, pH, hydraulic retention time and organic loading parameters on rate and yield of gas production.

Recycling of carbon dioxide and acetate as lactic acid by the hydrogen-producing bacterium Thermotoga neapolitana.

The heterotrophic bacterium Thermotoga neapolitana produces hydrogen by fermentation of sugars. Under capnophilic (carbon dioxide requiring) conditions, the process is preferentially associated with the production of lactic acid, which, as shown herein, is synthesized by reductive carboxylation of acetyl coenzyme A. The enzymatic coupling is dependent on the carbon dioxide stimulated activity of heterotetrameric pyruvate:ferredoxin oxidoreductase. Under the same culture conditions, T. neapolitana also operates the unfavorable synthesis of lactic acid from an exogenous acetate supply. This process, which requires carbon dioxide (or carbonate) and an unknown electron donor, allows for the conversion of carbon dioxide into added-value chemicals without biomass deconstruction.

Salinivibrio sharmensis sp. nov., a novel haloalkaliphilic bacterium from a saline lake in Ras Mohammed Park (Egypt).

A novel haloalkaliphilic, facultative anaerobic and Gram-negative Salinivibrio-like microorganism (designated strain BAG(T)) was recovered from a saline lake in Ras Mohammed Park (Egypt). Cells were motile, curved rods, not spore-forming and occurred singly. Strain BAG(T) grew optimally at 35°C (temperature growth range 25-40°C) with 10.0% (w/v) NaCl [NaCl growth range 6.0-16.0% (w/v)] and at pH 9.0 (pH growth range 6.0-10.0). Strain BAG(T) had phosphatidylethanolamine (PEA) and phosphatidylglycerol (PG) as the main polar lipids, C16:0 (54.0%) and C16:1 (26.0%) as the predominant cellular fatty acids and Q-8 as the major respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BAG(T) was a member of Salinivibrio genus, with the highest sequence similarities of 99.1, 98.4 and 98.1% to Salinivibrio siamensis JCM 14472(T), Salinivibrio proteolyticus DSM 19052(T) and Salinivibrio costicola subsp. alcaliphilus DSM 16359(T), respectively. DNA-DNA hybridization values of strain BAG(T) with members of Salinivibrio genus were lower than 55.0%. DNA G + C content was 51.0 mol%. On the basis of the polyphasic taxonomic results revealed in this study, strain BAG(T) should be classified as a novel species of Salinivibrio genus, for which the name Salinivibrio sharmensis sp. nov. is proposed, with the type strain BAG(T) (=ATCC BAA-1319(T) = DSM 18182(T)).

The effect of the surface charge of hydrogel supports on thermophilic biohydrogen production.

The effect of the surface charge of different immobilizing hydrogels on biohydrogen production in batch cultures was investigated using a novel isolate associated to the genus Thermoanaerobacterium. Two crosslinked polysaccharide-based hydrogels and two acrylic hydrogels were tested as polymeric carriers for cell adsorption. Immobilization improved both substrate conversion and hydrogen cumulative production compared to the suspended culture, and a yield of 1.9 mol H(2)/mol glucose was observed after 24h for alginate-supported cultures. Cationic carriers dramatically increased cell immobilization, leading to markedly faster kinetics of substrate degradation and hydrogen production in batch operation, with a peak of 3.6 mol H(2)/mol glucose for the acrylic hydrogel HM92. Accumulation of gaseous and acidic metabolites inhibited further H(2) production, shifting the carbon flow to reduced end-products and biomass synthesis. Preliminary tests showed that all the tested hydrogels had good durability and allowed hydrogen production on repeated batch runs.

Thermoanaerobacterium thermostercus sp. nov., a new anaerobic thermophilic hydrogen-producing bacterium from buffalo-dung.

A novel thermophilic, anaerobic, rod-shaped bacterium strain, designated Buff, was isolated from buffalo-dung samples collected from a buffalo-farm located in Caserta (Campania, south of Italy). Strain Buff was Gram-positive, motile and no spore-forming. The growth temperature range was 40-65 degrees C with an optimum at 60 degrees C, while pH growth range at 60 degrees C was 5.5-8.0 with an optimum at about pH 6.5. NaCl growth concentration ranged from 0 to 2.0% with an optimum at 0.5% (w/v); no growth was observed with the presence of NaCl 3.0% (w/v). The strain produced ethanol, acetate, lactate, H(2), H(2)S and CO(2) by glucose fermentation. The DNA G + C content was 34.4 mol%. As determined by 16S rRNA sequence analysis, this organism belonged to the genus Thermoanaerobacterium. On the basis of the physiological and molecular properties, we propose for strain Buff the new species designation Thermoanaerobacterium thermostercus sp. nov. This novel organism represents the first species of the genus Thermoanaerobacterium isolated from buffalo-dung. The type strain is Buff (=DSM 22141 = ATCC BAA-1776).

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359).

This is the first report describing the purification and enzymatic properties of a native invertase (beta-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85 degrees C at pH 6.0. At temperatures of 80-85 degrees C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K(m) and k(cat)/K(m) values (at 85 degrees C and pH 6.0) of about 14 mM and 5.2 x 10(8) M(-1) s(-1), respectively.