Hydrogen-oxidizing Bacterium Research Articles

Gas Fermentation Enhancement for Chemolithotrophic Growth of Cupriavidus necator on Carbon Dioxide

Cupriavidus necator, a facultative hydrogen-oxidizing bacterium, was grown on carbon dioxide, hydrogen, and oxygen for value-added products. High cell density and productivity were the goal of gas fermentation, but limited by gas substrates because of their low solubility in the aqueous medium solution. Enhancement of gas fermentation was investigated by (i) adding n-hexadecane as a gas vector to increase the volumetric mass transfer coefficient (kLa) and gas solubility, (ii) growing C. necator under a raised gas pressure, and (iii) using cell mass hydrolysates as the nutrients of chemolithotrophic growth. In contrast to previous studies, little positive but negative effects of the gas vector were observed on gas mass transfer and cell growth. The gas fermentation could be significantly enhanced under a raised pressure, resulting in a higher growth rate (0.12 h−1), cell density (18 g L−1), and gas uptake rate (200 mmole L−1 h−1) than a fermentation under atmospheric pressure. The gain, however, was not proportional to the pressure increase as predicted by Henry’s law. The hydrolysates of cell mass were found a good source of nutrients and the organic nitrogen was equivalent to or better than ammonium nitrogen for chemolithotrophic growth of C. necator on carbon dioxide.

Autotrophic, Heterotrophic, and Mixotrophic Nitrogen Assimilation for Single-Cell Protein Production by Two Hydrogen-Oxidizing Bacterial Strains.

To recover a nitrogen resource from high-ammonia-nitrogen wastewater, two amphitrophic hydrogen-oxidizing bacteria (HOB), Paracoccus denitrificans Y5 and P. versutus D6, capable of nitrogen assimilation for single-cell protein (SCP) production were isolated. These two HOB strains could grow autotrophically with H2 as an electron donor, O2 as an electron acceptor, CO2 as a carbon source, and ammonia nitrogen (NH4+-N) as a nitrogen source. The cell molecular formulas of strains Y5 and D6 determined by autotrophic cultivation were C3.33H6.83O2.58N0.77 and C2.87H5.34O3.17N0.57, respectively. The isolated strains could synchronously remove NH4+-N and organic carbon and produce SCP via heterotrophic cultivation. The rates of removal of NH4+-N and soluble chemical oxygen demand reached 35.47 and 49.04%, respectively, for Y5 under mixotrophic cultivation conditions with biogas slurry as a substrate. SCP content of strains Y5 and D6 was 67.34-73.73% based on cell dry weight. Compared with soybean meal, the SCP of Y5 contained a variety of amino acids.

Fixation of carbon dioxide by a hydrogen-oxidizing bacterium for value-added products.

With rapid technology progress and cost reduction, clean hydrogen from water electrolysis driven by renewable powers becomes a potential feedstock for CO2 fixation by hydrogen-oxidizing bacteria. Cupriavidus necator (formally Ralstonia eutropha), a representative member of the lithoautotrophic prokaryotes, is a promising producer of polyhydroxyalkanoates and single cell proteins. This paper reviews the fundamental properties of the hydrogen-oxidizing bacterium, the metabolic activities under limitation of individual gases and nutrients, and the value-added products from CO2, including the products with large potential markets. Gas fermentation and bioreactor safety are discussed for achieving high cell density and high productivity of desired products under chemolithotrophic conditions. The review also updates the recent research activities in metabolic engineering of C. necator to produce novel metabolites from CO2.

Complete Genome Sequence of Kyrpidia sp. Strain EA-1, a Thermophilic Knallgas Bacterium, Isolated from the Azores.

ABSTRACTKyrpidia sp. strain EA-1 is a thermophilic hydrogen-oxidizing bacterium isolated from hydrothermal systems at São Miguel Island, Portugal. Here, we present the complete genome sequence of the strain assembled to a single circular chromosome. The genome spans 3,352,175 bp, with a GC content of 58.7%.

Comparison analysis on the energy efficiencies and biomass yields in microbial CO 2 fixation

Abstract CO 2 fixation by a hydrogen-oxidizing bacterium, Cupriavidus necator, was evaluated in a packed bed bioreactor under a constant flow rate of gas mixtures (H 2 , O 2 , CO 2 ). The overall energy efficiency depends on the efficiencies of CO 2 fixation into carbohydrate and the reduced carbon into biomass and bioproducts, respectively. The efficiencies varied with the limiting gas substrate. Under O 2 limitation, the efficiency (20–30%) of CO 2 fixation increased with time and was higher than the overall efficiency (12–18%). Under H 2 limitation, the efficiency of CO 2 fixation declined with time while the biomass yield was quite similar to that under O 2 limitation. A cellular metabolic model was suggested for the lithoautotrophic growth of C. necator , including CO 2 fixation into carbohydrate followed by the main metabolic pathway of reduced carbon. Under CO 2 limitation, most H 2 energy was wasted, resulting in a very low biomass yield. Under a dual limitation of O 2 and nitrogen, biosynthesis of poly(3-hydroxybutyrate) was triggered, and the energy efficiency or yield of biopolyester was lower than those of microbial cell mass. Compared with a green microalga Neochloris oleoabundans that produces lipid under nutrient limitation, C. necator exhibited a much higher (3–6 times) energy efficiency in producing biomass and bioproducts from CO 2 .

The NiFe Hydrogenases of the Tetrachloroethene-Respiring Epsilonproteobacterium Sulfurospirillum multivorans: Biochemical Studies and Transcription Analysis.

The organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans is able to grow with hydrogen as electron donor and with tetrachloroethene (PCE) as electron acceptor; PCE is reductively dechlorinated to cis-1,2-dichloroethene. Recently, a genomic survey revealed the presence of four gene clusters encoding NiFe hydrogenases in its genome, one of which is presumably periplasmic and membrane-bound (MBH), whereas the remaining three are cytoplasmic. To explore the role and regulation of the four hydrogenases, quantitative real-time PCR and biochemical studies were performed with S. multivorans cells grown under different growth conditions. The large subunit genes of the MBH and of a cytoplasmic group 4 hydrogenase, which is assumed to be membrane-associated, show high transcript levels under nearly all growth conditions tested, pointing toward a constitutive expression in S. multivorans. The gene transcripts encoding the large subunits of the other two hydrogenases were either not detected at all or only present at very low amounts. The presence of MBH under all growth conditions tested, even with oxygen as electron acceptor under microoxic conditions, indicates that MBH gene transcription is not regulated in contrast to other facultative hydrogen-oxidizing bacteria. The MBH showed quinone-reactivity and a characteristic UV/VIS spectrum implying a cytochrome b as membrane-integral subunit. Cell extracts of S. multivorans were subjected to native polyacrylamide gel electrophoresis (PAGE) and hydrogen oxidizing activity was tested by native staining. Only one band was detected at about 270 kDa in the particulate fraction of the extracts, indicating that there is only one hydrogen-oxidizing enzyme present in S. multivorans. An enrichment of this enzyme and SDS PAGE revealed a subunit composition corresponding to that of the MBH. From these findings we conclude that the MBH is the electron-donating enzyme system in the PCE respiratory chain. The roles for the other three hydrogenases remain unproven. The group 4 hydrogenase might be involved in hydrogen production upon fermentative growth.

Gas mass transfer with microbial CO2 fixation and poly(3-hydroxybutyrate) synthesis in a packed bed bioreactor

Gas mass transfer is the rate-limiting step in CO2 fixation by a hydrogen-oxidizing bacterium Cupriavidus necator under chemolithoautotrophic conditions. The mass transfer rates (i.e. the gas uptake rates) were measured in a packed bed bioreactor under the conditions of microbial growth on CO2. The kLa values of O2 and H2 were determined under either O2 or H2 limitation, respectively, and the ratio was proportional to a square root ratio of the molecular diffusivities in water, indicating that a surface renew model is suitable for the gas mass transfer in the packed bed. At a low gassing rate, the O2 mass transfer coefficient was to different extent affected by packing materials, bed geometry, liquid velocity, and liquid distribution. The liquid distributor and/or bed void space exhibited a high effect on the mass transfer coefficient. With microbial growth, the gas fermentation was becoming limited either by dissolved O2 or H2, depending on gas composition, but neither O2 nor H2 limitation could trigger the formation of a substantial amount of poly(3-hydroxybutyrate) (PHB) under nutrient-rich conditions. Instead, when a nitrogen nutrient control strategy was applied, PHB synthesis was significantly improved and the PHB content reached up to 67wt% of cell mass.

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox

Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H2-driven production of biodegradable polymers and hydrocarbons. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H2 oxidation with the reduction of NAD+ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD+ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD+] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD+] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD+] ratios represents a novel and sensitive tool to determine the redox state of cells.

Autotrophic Growth of Paracoccus denitrificans in Aerobic Condition and the Accumulation of Biodegradable Plastics from CO<sub>2</sub>

The cell growth on H2 and O2 as the energy source and CO2 as the sole carbon source in the autotrophic culture condition was tested for the gram-negative bacteria Paracoccus spp. The aerobic growth in the autotrophic condition was only observed in Paracoccus denitrificans NBRC13301 and P.pantotrophus NBRC102493. Both strains were sensitive to O2 in particular the growth of P.pantotrophus was completely inhibited at the concentrations above 2% O2. P.denitrificans grew until 15% O2 however the optimum O2 concentration was 5%. The growth characteristic of P.denitrificans was investigated by pH-controlled batch culture with supplying the gas mixture of H2, O2 and CO2. The specific growth rate was 0.12 h-1 at 5% O2, 30℃ and pH7.0. The growth was much slower than other hydrogen-oxidizing bacteria however P.denitrificans accumulated biodegradable plastic, polyhydroxybutyrate, PHB with the content of 57.3% w/w in the cell under nitrogen limitation. Under DO limitation, the cell concentration increased to 25 g/L without accumulating PHB if NH3 solution as the nitrogen source was fed sufficiently.

Artificial photosynthesis steps up

Bioenergy Photosynthesis fixes CO2 from the air by using sunlight. Industrial mimics of photosynthesis seek to convert CO2 directly into biomass, fuels, or other useful products. Improving on a previous artificial photosynthesis design, Liu et al. combined the hydrogen-oxidizing bacterium Raistonia eutropha with a cobalt-phosphorus water-splitting catalyst. This biocompatible self-healing electrode circumvented the toxicity challenges of previous designs and allowed it to operate aerobically. When combined with solar photovoltaic cells, solar-to-chemical conversion rates should become nearly an order of magnitude more efficient than natural photosynthesis. Science , this issue p. [1210][1] [1]: /lookup/doi/10.1126/science.aaf5039

Autotrophic nitrogen assimilation and carbon capture for microbial protein production by a novel enrichment of hydrogen-oxidizing bacteria

Domestic used water treatment systems are currently predominantly based on conventional resource inefficient treatment processes. While resource recovery is gaining momentum it lacks high value end-products which can be efficiently marketed. Microbial protein production offers a valid and promising alternative by upgrading low value recovered resources into high quality feed and also food. In the present study, we evaluated the potential of hydrogen-oxidizing bacteria to upgrade ammonium and carbon dioxide under autotrophic growth conditions. The enrichment of a generic microbial community and the implementation of different culture conditions (sequenced batch resp. continuous reactor) revealed surprising features. At low selection pressure (i.e. under sequenced batch culture at high solid retention time), a very diverse microbiome with an important presence of predatory Bdellovibrio spp. was observed. The microbial culture which evolved under high rate selection pressure (i.e. dilution rate D = 0.1 h−1) under continuous reactor conditions was dominated by Sulfuricurvum spp. and a highly stable and efficient process in terms of N and C uptake, biomass yield and volumetric productivity was attained. Under continuous culture conditions the maximum yield obtained was 0.29 g cell dry weight per gram chemical oxygen demand equivalent of hydrogen, whereas the maximum volumetric loading rate peaked 0.41 g cell dry weight per litre per hour at a protein content of 71%. Finally, the microbial protein produced was of high nutritive quality in terms of essential amino acids content and can be a suitable substitute for conventional feed sources such as fishmeal or soybean meal.

Experimental investigation on feasible bioreactor using mechanism of hydrogen oxidation of natural soil for detritiation system

A passive reactor for tritium oxidation at room temperature has been widely studied in nuclear engineering especially for a detritiation system (DS) of a tritium process facility taking possible extraordinary situation severely into consideration. We have focused on bacterial oxidation of tritium by hydrogen-oxidizing bacteria in natural soil to realize the passive oxidation reactor. The purpose of this study was to examine the feasibility of a bioreactor with hydrogen-oxidizing bacteria in soil from a point of view of engineering. The efficiency of the bioreactor was evaluated by kinetics. The bioreactor packed with natural soil shows a relative high conversion rate of tritium under the saturated moisture condition at room temperature, which is obviously superior to that of a Pt/Al2O3 catalyst generally used for tritium oxidation in the existing tritium handling facilities. The order of reaction for tritium oxidation with soil was the pseudo-first order as assessed with Michaelis-Menten kinetics model. Our engineering suggestion to increase the reaction rate is the intentional addition of hydrogen at a small concentration in the feed gas on condition that the oxidation of tritium with soil is expressed by the Michaelis-Menten kinetics model.

Sodium-Driven Energy Conversion for Flagellar Rotation of the Earliest Divergent Hyperthermophilic Bacterium

Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here, we cultured this hyperthermophilic bacterium and observed motile behavior with a variable–temperature chamber for optical microscopy [1, 2]. The swimming of the cells was about 90 µm s−1 at 85 °C, and decreased with decreasing temperature of the chamber [3]. Next, we expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na+. As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na+ for energy coupling of the flagellar motor. The Na+-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.[1] Nishiyama, M. and Sowa Y. 2012. Microscopic Analysis of Bacterial Motility at High Pressure. Biophys. J. 102, 1872-1880.[2] Nishiyama, M. et al. 2013. High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor. J. Bactetiol. 195, 1809-1814.[3] Takekawa, N. et al. 2015. Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium. Sci. Rep. 5, 12711.

Application of elevated pressures to cultures of the hydrogen-oxidizing bacteria, Cupriavidus necator

Application of elevated pressures to cultures of the hydrogen-oxidizing bacteria, Cupriavidus necator

Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage.

Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha.

Characterization of bacterial diversity associated with deep sea ferromanganese nodules from the South China Sea.

Deep sea ferromanganese (FeMn) nodules contain metallic mineral resources and have great economic potential. In this study, a combination of culture-dependent and culture-independent (16S rRNA genes clone library and pyrosequencing) methods was used to investigate the bacterial diversity in FeMn nodules from Jiaolong Seamount, the South China Sea. Eleven bacterial strains including some moderate thermophiles were isolated. The majority of strains belonged to the phylum Proteobacteria; one isolate belonged to the phylum Firmicutes. A total of 259 near full-length bacterial 16S rRNA gene sequences in a clone library and 67,079 valid reads obtained using pyrosequencing indicated that members of the Gammaproteobacteria dominated, with the most abundant bacterial genera being Pseudomonas and Alteromonas. Sequence analysis indicated the presence of many organisms whose closest relatives are known manganese oxidizers, iron reducers, hydrogen-oxidizing bacteria and methylotrophs. This is the first reported investigation of bacterial diversity associated with deep sea FeMn nodules from the South China Sea.

Analysis of Microbial Communities in Biofilms from CSTR-Type Hollow Fiber Membrane Biofilm Reactors for Autotrophic Nitrification and Hydrogenotrophic Denitrification.

Two hollow fiber membrane biofilm reactors (HF-MBfRs) were operated for autotrophic nitrification and hydrogenotrophic denitrification for over 300 days. Oxygen and hydrogen were supplied through the hollow fiber membrane for nitrification and denitrification, respectively. During the period, the nitrogen was removed with the efficiency of 82-97% for ammonium and 87-97% for nitrate and with the nitrogen removal load of 0.09-0.26 kg NH4(+)-N/m(3)/d and 0.10-0.21 kg NO3(-)-N/m(3)/d, depending on hydraulic retention time variation by the two HF-MBfRs for autotrophic nitrification and hydrogenotrophic denitrification, respectively. Biofilms were collected from diverse topological positions in the reactors, each at different nitrogen loading rates, and the microbial communities were analyzed with partial 16S rRNA gene sequences in denaturing gradient gel electrophoresis (DGGE). Detected DGGE band sequences in the reactors were correlated with nitrification or denitrification. The profile of the DGGE bands depended on the NH4(+) or NO3(-) loading rate, but it was hard to find a major strain affecting the nitrogen removal efficiency. Nitrospira-related phylum was detected in all biofilm samples from the nitrification reactors. Paracoccus sp. and Aquaspirillum sp., which are an autohydrogenotrophic bacterium and an oligotrophic denitrifier, respectively, were observed in the denitrification reactors. The distribution of microbial communities was relatively stable at different nitrogen loading rates, and DGGE analysis based on 16S rRNA (341f /534r) could successfully detect nitrate-oxidizing and hydrogen-oxidizing bacteria but not ammonium-oxidizing bacteria in the HF-MBfRs.

Synthesis of nickel-iron hydrogenase in Cupriavidus metallidurans is controlled by metal-dependent silencing and un-silencing of genomic islands.

Cupriavidus metallidurans CH34 is able to grow autotrophically as a hydrogen-oxidizing bacterium and produces nickel-dependent hydrogenases, even under heterotrophic conditions. Loss of its two native plasmids resulted in inability of the resulting strain AE104 to synthesize the hydrogenases and to grow autotrophically in phosphate-poor, Tris-buffered mineral salts medium (TMM). Three of eleven previously identified catabolic genomic islands (CMGIs; Van Houdt et al., 2009), two of which harbor the genes for the membrane-bound (CMGI-2) and the soluble hydrogenase (CMGI-3), were silenced in strain AE104 when cultivated in phosphate-poor TMM, explaining its inability to produce hydrogenases. Production of the soluble hydrogenase from the aut region 1 of CMGI-3, and concomitant autotrophic growth, was recovered when the gene for the zinc importer ZupT was deleted in strain AE104. The transcriptome of the ΔzupT mutant exhibited two up-regulated gene regions compared to its parent strain AE104. Expression of the genes in the aut region 1 increased independently of the presence of added zinc. A second gene region was expressed only under metal starvation conditions. This region encoded a TonB-dependent outer membrane protein, a putative metal chaperone plus paralogs of essential zinc-dependent proteins, indicating the presence of a zinc allocation pathway in C. metallidurans. Thus, expression of the genes for the soluble hydrogenase and the Calvin cycle enzymes on aut region 1 of CMGI-3 of C. metallidurans is under global control and needs efficient ZupT-dependent zinc allocation for a regulatory role, which might be discrimination of nickel.

A comparison of different pretreatments on hydrogen fermentation from waste sludge by fluorescence excitation-emission matrix with regional integration analysis

To gain a better understanding of the influence on hydrogen fermentation with different pretreatments, the compositional and structural characteristics of extracellular polymeric substances (EPS) and dissolved organic matters (DOM) were analyzed during hydrogen fermentation using excitation-emission matrix (EEM) with fluorescence regional integration (FRI). In order to accelerate the hydrogen production with waste sludge, multi-enzyme, thermophilic bacteria, heat and microwave were performed for sludge pretreatments. The best method was found to be heating with the maximum hydrogen yield of 15.3 ml H2/g VSS (Volatile suspended solid). EPS and DOM component of soluble chemical oxygen demand (SCOD), carbohydrate and protein, the microbial community structure evolutions, soluble metabolite of volatile fatty acids (VFAs) and ethanol distribution characteristics and pH were also evaluated. Different pretreatments could change the microbial community structure in waste sludge, which led to the diversity of substrates degradation and metabolite with hydrogen bacteria.

Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent

Hydrogen production (HP) from dark fermentation effluent of starch wastewater via vertical tubular photo-bioreactor was investigated. The reactor was inoculated with mixed culture of bacteria and operated at light intensity of 190W/m2. Hydraulic retention time (HRT) and organic loading rate (OLR) was varied between 0.9 to 4.0h and 3.2 to 16gCOD/l.d., respectively. Increasing the HRT from 0.9 to 2.5h, significantly (P<0.05) increased HP from 1±0.04 to 3.05±0.19l/d, respectively. However, minimal increase in HP occurred when increasing the HRT up to 4.0h. The HP remained unaffected when increasing the OLR from 3.2 to 6.4gCOD/l.d. Further increase in the OLR up to 8.2 and 16gCOD/l.d., resulted in a drop in HP i.e. 0.96 and 0.19l/d, respectively. Microbial community analysis of the reactor samples showed the presence and dominance of hydrogen producing purple non-sulfur phototrophic (PNS) bacterium, Rhodopseudomonas palustris in the reactor.