Enhancement of hydrogen production rate by high biomass concentrations of Thermotoga neapolitana

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 H2/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

A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures

The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag® and the Halotag®. These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H5) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag® for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H5, proposing them as alternatives for in vivo studies in these extreme model organisms

Capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana: an unexpected deviation from dark fermentation

The heterotrophic bacterium Thermotoga neapolitana produces hydrogen by fermentation of organic substrates. The process is referred to as dark fermentation and is typically complemented by production of acetic acid. Here we show that synthesis of products derived by reductive metabolism of pyruvate, mainly lactic acid, occurs to the detriment of acetic acid fermentation when the cultures of the thermophilic bacterium are flushed by saturating level of CO2. Sodium bicarbonate in a very narrow range of concentrations (∼14 mM) also causes the same metabolic shift. The capnophilic (CO2-requiring) re-orientation of the fermentative process toward lactic acid does not affect hydrogen productivity thus challenging the currently accepted dark fermentation model that predicts reduction of this gas when glucose is converted into organic products different from acetat

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

Electro-Stimulation of Thermotoga Neapolitana Metabolism

Thermotoga neapolitana (DSM 4359, ATCC 49049), more than others Thermotogales is able to accumulate biohydrogen from waste organic matter, in a selective environment (>80ºC). Thanks to the high temperature, this bacterium is able to use a peculiar anaplerotic biochemical pathway named Capnophilic Lactic Fermentation (CLF) that accomplishes chain elongation of acetate with direct reduction of CO2, producing lactic acid (Pradhan et al. 2015). This is a carbon fixation mechanism which, differently from autotrophism, leads to direct elimination of fixed carbon into excreted metabolites, such as lactic acid, instead of converting and accumulating it into biomass. T. neapolitana is therefore a promising microorganism for recovering added-value chemicals from organic wastes (Fontana, EP14711847.5). In this work, the possibility of bioelectrostimulate the production of lactic acid by Thermotoga Neapolitana supplying additional electric energy in polarized and unpolarized bioreactors is investigated. For this, an alternating polarization up to ±1.2V was imposed between two identical electrodes of carbon cloth, immersed in the electrochemical bioreactors. Bioreactor without electrodes were also tested, as control. Glass reactors of 250 mL were operated at temperature of 85°C with a culture media of T. neapolitana containing 5 g/L (~30mM) of glucose, in triplicates. Three successive cycles of chemical measurements was carried out every 24 hours for three measurement cycles, to estimate the glucose consume and the acetic/lactic acid production, as well the hydrogen yieldin each tested bioreactor. The solution Optical density (OD) and micrographs produced by scanning electrode microscopy (SEM) showed a strong affinity of bacteria to form biofilm on the polarized and unpolarized electrodes. Evidence of the lactate/acetate ratio modification with respect the control bioreactors was achieved, especially under higher polarized condition, but also in the bioreactors with unpolarized electrodes. Glucose fermentation was strongly inhibited in electrochemical bioreactors in the first 24-48 hours test and during the whole three days test in some cases. Only in the control bioreactor glucose was oxidized to acetic acid in the molar rate similar to dark fermentation (glucose:acetic 1:2) and only the first day. Lactic acid in a relative high ratio versus the glucose consume, although at lower concentration with respect acetate, was found especially in some polarized bioereactors. These results globally confirm the affinity of Thermotoga neapolitana to carbon cloth electrodes and the possibility of electrostimultate their metabolism, opening future perspectives on electro-fermentation with this hyperthermophilic strain