The effects of synthesis gas feedstocks and oxygen perturbation on hydrogen production by Parageobacillus thermoglucosidasius.

BACKGROUND: The facultatively anaerobic thermophile Parageobacillus thermoglucosidasius is able to produce hydrogen gas (H2) through the water-gas shift (WGS) reaction. To date this process has been evaluated under controlled conditions, with gas feedstocks comprising carbon monoxide and variable proportions of air, nitrogen and hydrogen. Ultimately, an economically viable hydrogenogenic system would make use of industrial waste/synthesis gases that contain high levels of carbon monoxide, but which may also contain contaminants such as H2, oxygen (O2) and other impurities, which may be toxic to P. thermoglucosidasius. RESULTS: We evaluated the effects of synthesis gas (syngas) mimetic feedstocks on WGS reaction-driven H2 gas production by P. thermoglucosidasius DSM 6285 in small-scale fermentations. Improved H2 gas production yields and faster onset towards hydrogen production were observed when anaerobic synthetic syngas feedstocks were used, at the expense of biomass accumulation. Furthermore, as the WGS reaction is an anoxygenic process, we evaluated the influence of O2 perturbation on P. thermoglucosidasius hydrogenogenesis. O2 supplementation improved biomass accumulation, but reduced hydrogen yields in accordance with the level of oxygen supplied. However, H2 gas production was observed at low O2 levels. Supplementation also induced rapid acetate consumption, likely to sustain growth. CONCLUSION: The utilisation of anaerobic syngas mimetic gas feedstocks to produce H2 and the relative flexibility of the P. thermoglucosidasius WGS reaction system following O2 perturbation further supports its applicability towards more robust and continuous hydrogenogenic operation.

Physiological basis for atmospheric methane oxidation and methanotrophic growth on air.

Atmospheric methane oxidizing bacteria (atmMOB) constitute the sole biological sink for atmospheric methane. Still, the physiological basis allowing atmMOB to grow on air is not well understood. Here we assess the ability and strategies of seven methanotrophic species to grow with air as sole energy, carbon, and nitrogen source. Four species, including three outside the canonical atmMOB group USCα, enduringly oxidized atmospheric methane, carbon monoxide, and hydrogen during 12 months of growth on air. These four species exhibited distinct substrate preferences implying the existence of multiple metabolic strategies to grow on air. The estimated energy yields of the atmMOB were substantially lower than previously assumed necessary for cellular maintenance in atmMOB and other aerobic microorganisms. Moreover, the atmMOB also covered their nitrogen requirements from air. During growth on air, the atmMOB decreased investments in biosynthesis while increasing investments in trace gas oxidation. Furthermore, we confirm that a high apparent specific affinity for methane is a key characteristic of atmMOB. Our work shows that atmMOB grow on the trace concentrations of methane, carbon monoxide, and hydrogen present in air and outlines the metabolic strategies that enable atmMOB to mitigate greenhouse gases.

Integration of biocompatible hydrogen evolution catalyst developed from metal-mix solutions with microbial electrosynthesis.

Microbial conversion of CO2 to multi-carbon compounds such as acetate and butyrate is a promising valorisation technique. For those reactions, the electrochemical supply of hydrogen to the biocatalyst is a viable approach. Earlier we have shown that trace metals from microbial growth media spontaneously form in situ electro-catalysts for hydrogen evolution. Here, we show biocompatibility with the successful integration of such metal mix-based HER catalyst for immediate start-up of microbial acetogenesis (CO2 to acetate). Also, n-butyrate formation started fast (after twenty days). Hydrogen was always produced in excess, although productivity decreased over the 36 to 50 days, possibly due to metal leaching from the cathode. The HER catalyst boosted microbial productivity in a two-step microbial community bioprocess: acetogenesis by a BRH-c20a strain and acetate elongation to n-butyrate by Clostridium sensu stricto 12 (related) species. These findings provide new routes to integrate electro-catalysts and micro-organisms showing respectively bio and electrochemical compatibility.

Biological fermentation pilot-scale systems and evaluation for commercial viability towards sustainable biohydrogen production.

Featuring high caloric value, clean-burning, and renewability, hydrogen is a fuel believed to be able to change energy structure worldwide. Biohydrogen production technologies effectively utilize waste biomass resources and produce high-purity hydrogen. Improvements have been made in the biohydrogen production process in recent years. However, there is a lack of operational data and sustainability analysis from pilot plants to provide a reference for commercial operations. In this report, based on spectrum coupling, thermal effect, and multiphase flow properties of hydrogen production, continuous pilot-scale biohydrogen production systems (dark and photo-fermentation) are established as a research subject. Then, pilot-scale hydrogen production systems are assessed in terms of sustainability. The system being evaluated, consumes 171,530 MJ of energy and emits 9.37 t of CO2 eq when producing 1 t H2, and has a payback period of 6.86 years. Our analysis also suggests future pathways towards effective biohydrogen production technology development and real-world implementation.

Sulfate-reducing bacteria decreases fractional pressure of H2 to accelerate short-chain fatty acids production from waste activated sludge fermentation assisted with zero-valent iron activated sulfite pretreatment.

The production of short-chain fatty acids (SCFAs) is constrained by substrate availability and the increased fractional pressure of H2 emitted by acidogenic/fermentative bacteria during anaerobic fermentation of waste activated sludge (WAS). This study introduced a novel approach employing zero-valent iron (ZVI)-activated sulfite pretreatment combined with H2-consuming sulfate-reducing bacteria (SRB) mediation to improve SCFAs, especially acetate production from WAS fermentation. Experimental results showed that the combined ZVI-activated sulfite and incomplete-oxidative SRB (io-SRB) process achieved a peak SCFAs production of 868.11 mg COD/L, with acetate accounting for 80.55 %, which was 7.90- and 2.18-fold higher than that obtained from raw WAS fermentation, respectively. This could be firstly attributed to the SO4- and OH generated by ZVI-activated sulfite, which significantly promoted WAS decomposition, e.g., soluble proteins and carbohydrates increased 14.3- and 10.8-fold, respectively, over those in raw WAS. The biodegradation of dissolved organic matter was subsequently enhanced by the synergistic interaction and H2 transfer between anaerobic fermentation bacteria (AFB) and io-SRB. The positive and negative correlations among AFB, nitrate-reducing bacteria (NRB) and the io-SRB consortia were revealed by molecular ecological network (MEN) and Mantel test. Moreover, the expression of functional genes was also improved, for instance, in relation to acetate formation, the relative abundances of phosphate acetyltransferase and acetate kinase was 0.002 % and 0.005 % higher than that in the control test, respectively. These findings emphasized the importance of sulfate radicals-based oxidation pretreatment and the collaborative relationships of multifunctional microbes on the value-added chemicals and energy recovery from sludge fermentation.

Archaeal community composition as key driver of H2 consumption rates at the start-up of the biomethanation process.

The performance of hydrogen consumption by various inocula derived from mesophilic anaerobic digestion plants was evaluated under ex situ biomethanation. A panel of 11 mesophilic inocula was operated at a concentration of 15 gVS.L-1 at a temperature of 35 °C in batch system with two successive injections of H2:CO2 (4:1 mol:mol). Hydrogen consumption and methane production rates were monitored from 44 h to 72 h. Hydrogen consumption kinetics varies significantly based on the inoculum origin, with no accumulation of volatile fatty acids. Microbial community analyses revealed that microbial indicators such as the increase in Methanosarcina sp. abundance and the increase of the Archaea/Bacteria ratio were associated to high initial hydrogen consumption rates. The improvement in the hydrogen consumption rate between the two injections was correlated with the enrichment in hydrogenotrophic methanogens. This work provides new insights into the early response of microbial communities to hydrogen injection and on the microbial structures that may favor their adaptation to the biomethanation process.

The formate-hydrogen axis and its impact on the physiology of enterobacterial fermentation.

Formic acid (HCOOH) and dihydrogen (H2) are characteristic products of enterobacterial mixed-acid fermentation, with H2 generation increasing in conjunction with a decrease in extracellular pH. Formate and acetyl-CoA are generated by radical-based and coenzyme A-dependent cleavage of pyruvate catalysed by pyruvate formate-lyase (PflB). Formate is also the source of H2, which is generated along with carbon dioxide through the action of the membrane-associated, cytoplasmically-oriented formate hydrogenlyase (FHL-1) complex. Synthesis of the FHL-1 complex is completely dependent on the cytoplasmic accumulation of formate. Consequently, formate determines its own disproportionation into H2 and CO2 by the FHL-1 complex. Cytoplasmic formate levels are controlled by FocA, a pentameric channel that translocates formic acid/formate bidirectionally between the cytoplasm and periplasm. Each protomer of FocA has a narrow hydrophobic pore through which neutral formic acid can pass. Two conserved amino acid residues, a histidine and a threonine, at the center of the pore control directionality of translocation. The histidine residue is essential for pH-dependent influx of formic acid. Studies with the formate analogue hypophosphite and amino acid variants of FocA suggest that the mechanisms of formic acid efflux and influx differ. Indeed, current data suggest, depending on extracellular formate levels, two separate uptake mechanisms exist, both likely contributing to maintain pH homeostasis. Bidirectional formate/formic acid translocation is dependent on PflB and influx requires an active FHL-1 complex. This review describes the coupling of formate and H2 production in enterobacteria.

Conjugated Polymer/Recombinant Escherichia coli Biohybrid Systems for Photobiocatalytic Hydrogen Production.

Biohybrid photocatalysts are composite materials that combine the efficient light-absorbing properties of synthetic materials with the highly evolved metabolic pathways and self-repair mechanisms of biological systems. Here, we show the potential of conjugated polymers as photosensitizers in biohybrid systems by combining a series of polymer nanoparticles with engineered Escherichia coli cells. Under simulated solar light irradiation, the biohybrid system consisting of fluorene/dibenzo [b,d]thiophene sulfone copolymer (LP41) and recombinant E. coli (i.e., a LP41/HydA BL21 biohybrid) shows a sacrificial hydrogen evolution rate of 3.442 mmol g-1 h-1 (normalized to polymer amount). It is over 30 times higher than the polymer photocatalyst alone (0.105 mmol g-1 h-1), while no detectable hydrogen was generated from the E. coli cells alone, demonstrating the strong synergy between the polymer nanoparticles and bacterial cells. The differences in the physical interactions between synthetic materials and microorganisms, as well as redox energy level alignment, elucidate the trends in photochemical activity. Our results suggest that organic semiconductors may offer advantages, such as solution processability, low toxicity, and more tunable surface interactions with the biological components over inorganic materials.

The delayed senescence in harvested blueberry by hydrogen-based irrigation is functionally linked to metabolic reprogramming and antioxidant machinery.

Molecular hydrogen is beneficial for fruits quality improvement. However, the mechanism involved, especially cellular metabolic responses, has not been well established. Here, the integrated widely targeted metabolomics analysis (UPLC-MS/MS) and biochemical evidence revealed that hydrogen-based irrigation could orchestrate, either directly or indirectly, an array of physiological responses in blueberry (Vaccinium spp.) during harvesting stage, especially for the delayed senescence in harvested stage (4 °C for 12 d). The hubs to these changes are wide-ranging metabolic reprogramming and antioxidant machinery. A total of 1208 distinct annotated metabolites were identified, and the characterization of differential accumulated metabolites (DAMs) revealed that the reprogramming, particularly, involves phenolic acids and flavonoids accumulation. These changes were positively matched with the transcriptional profiles of representative genes for their synthesis during the growth stage. Together, our findings open a new window for development of hydrogen-based agriculture that increases the shelf-life of fruits in a smart and sustainable manner.

The role of hydrogen-rich water in delaying the pulp breakdown of litchi fruit during postharvest storage.

Previous studies have indicated that hydrogen-rich water (HW) treatment can delay fruit ripening and senescence. However, little is known about the HW-delaying pulp breakdown. In this study, eight physiological characteristics revealed that HW treatment delayed both pericarp browning and pulp breakdown of litchi fruit. To gain a comprehensive understanding of the changes in litchi pulp, a combination of multiple metabolomics and gene expression analyses was conducted, assessing 67 primary metabolites, 103 volatiles, 31 amino acids, and 13 crucial metabolite-related genes. Results showed that HW treatment promoted starch degradation, decelerated cell wall degradation and glycolysis, and maintained the flavor and quality of litchi fruit. Furthermore, HW treatment stimulated the production of volatile alcohols, aldehydes, ketones, olefins, and amino acids, which might play a vital role in HW-delaying pulp breakdown. This study sheds light on the mechanism by which HW delayed pulp breakdown by investigating small molecule metabolites and metabolic pathways.

Hydrogen production pathways in Clostridia and their improvement by metabolic engineering.

Biological production of hydrogen has a tremendous potential as an environmentally sustainable technology to generate a clean fuel. Among the different available methods to produce biohydrogen, dark fermentation features the highest productivity and can be used as a means to dispose of organic waste biomass. Within this approach, Clostridia have the highest theoretical H2 production yield. Nonetheless, most strains show actual yields far lower than the theoretical maximum: improving their efficiency becomes necessary for achieving cost-effective fermentation processes. This review aims at providing a survey of the metabolic network involved in H2 generation in Clostridia and strategies used to improve it through metabolic engineering. Together with current achievements, a number of future perspectives to implement these results will be illustrated.

Enhancing proton transport in polyvinylidenedifluoride membranes and reducing biofouling for improved hydrogen production in microbial electrolysis cells.

Hydrophilic porous membranes, exemplified by polyvinylidene fluoride (PVDF) membranes, have demonstrated significant potential for replacing ion exchange membranes in microbial electrolysis cells (MECs). Membrane fouling remains a major challenge in MECs, impeding proton transport and consequently limiting hydrogen production. This study aims to investigate a synergistic antifouling strategy for PVDF membrane through the incorporation of a coating composed of polydopamine (PDA), polyethyleneimine (PEI), and silver nanoparticles (AgNPs). The PDA-PEI-Ag@PVDF membrane not only effectively mitigates fouling through steric and electrostatic repulsion forces, but also amplifies ion transport by facilitating water diffusion and electromigration. The PDA-PEI-Ag@PVDF membrane exhibited a reduced membrane resistance of 1.01 mΩ m2 and PDA-PEI-Ag modifying PVDF membrane was found to be effective in enhancing the proton transportation of PVDF membrane. Therefore, the enhanced hydrogen production rate of 2.65 ± 0.02 m3/m3/d was achieved in PDA-PEI-Ag@PVDF-MECs.

Design of a sustainable system for wastewater treatment and generation of biofuels based on the biomass of the aquatic plant Eichhornia Crassipes.

Colombia's continuous contamination of water resources and the low alternatives to produce biofuels have affected the fulfillment of the objectives of sustainable development, deteriorating the environment and affecting the economic productivity of this country. Due to this reality, projects on environmental and economic sustainability, phytoremediation, and the production of biofuels such as ethanol and hydrogen were combined. The objective of this article was to design and develop a sustainable system for wastewater treatment and the generation of biofuels based on the biomass of the aquatic plant Eichhornia crassipes. A system that simulates an artificial wetland with live E. crassipes plants was designed and developed, removing organic matter contaminants; subsequently, and continuing the sustainability project, bioreactors were designed, adapted, and started up to produce bioethanol and biohydrogen with the hydrolyzed biomass used in the phytoremediation process, generating around 12 g/L of bioethanol and around 81 ml H2/g. The proposed research strategy suggests combining two sustainable methods, bioremediation and biofuel production, to preserve the natural beauty of water systems and their surroundings.

Efficient phosphate and hydrogen recovery from sludge fermentation liquid by sacrificial iron anode in electro-fermentation system.

Electro-fermentation (EF) has been extensively studied for recovering hydrogen and phosphorus from waste activated sludge (WAS), while was limited for the further application due to the low hydrogen yield and phosphorus recovery efficiency. This study proposed an efficient strategy for hydrogen and vivianite recovery from the simulated sludge fermentation liquid by sacrificial iron anode in EF. The optimum hydrogen productivity and the utilization efficiency of short chain fatty acids (SCFAs) reached 45.2 mmol/g COD and 77.6% at 5 d in pH 8. Phosphate removal efficiency achieved at 90.8% at 2 d and the high crystallinity and weight percentage of vivianite (84.8%) was obtained. The functional microbes, i.e., anaerobic fermentative bacteria, electrochemical active bacteria, homo-acetogens and iron-reducing bacteria were highly enriched and the inherent interaction between the microbial consortia and environmental variables was thoroughly explored. This work may provide a theoretical basis for energy/resource recovery from WAS in the further implementation.

Nitrous oxide respiration in acidophilic methanotrophs.

Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.

Incorporation of hydrogen-producing magnesium into minced beef meat protects the quality attributes and safety of the product during cold storage.

The impact of hydrogen (H2) producing magnesium (Mg) incorporation into minced beef meat (MBM) on the quality and safety of the product was investigated. The H2-producing Mg (H2-P-Mg)-incorporated MBMs were vacuumed (VP) and stored at 4 °C for 12 days. Other MBMs were vacuumed and gassed with H2 or N2. At the end of storage, the lowest browning index values were for H2 and H2-P-Mg samples. H2- PMg and VP methods generally decreased the counts of mesophilic and psychrotrophic bacteria and yeast molds and restricted the formation of thiobarbituric acid reactive substances and biogenic amines. Heat mapping, PCA, and multivariate analysis methods confirmed chemical analysis results. The volatile compounds were at their highest levels in the control samples at the end of storage, followed by H2, N2, H2-P-Mg, and VP samples. Using the H2-P-Mg method in MBM preparation could protect the quality characteristics and safety of the product during cold storage.

Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.

Bioenergy production from chicken feather waste by anaerobic digestion and bioelectrochemical systems.

BACKGROUND: Poultry feather waste has a potential for bioenergy production because of its high protein content. This research explored the use of chicken feather hydrolysate for methane and hydrogen production via anaerobic digestion and bioelectrochemical systems, respectively. Solid state fermentation of chicken waste was conducted using a recombinant strain of Bacillus subtilis DB100 (p5.2). RESULTS: In the anaerobic digestion, feather hydrolysate produced maximally 0.67 Nm3 CH4/kg feathers and 0.85 mmol H2/day.L concomitant to COD removal of 86% and 93%, respectively. The bioelectrochemical systems used were microbial fuel and electrolysis cells. In the first using a microbial fuel cell, feather hydrolysate produced electricity with a maximum cell potential of 375 mV and a current of 0.52 mA. In the microbial electrolysis cell, the hydrolysate enhanced the hydrogen production rate to 7.5 mmol/day.L, with a current density of 11.5 A/m2 and a power density of 9.26 W/m2. CONCLUSIONS: The data indicated that the sustainable utilization of keratin hydrolysate to produce electricity and biohydrogen via bioelectrical chemical systems is feasible. Keratin hydrolysate can produce electricity and biofuels through an integrated aerobic-anaerobic fermentation system.

Biomass acid pretreatment impacts on metabolic routes and bacterial composition of dark fermentation process.

Complex organic matter represents a suitable substrate to produce hydrogen through dark fermentation (DF) process. To increase H2 yields, pretreatment technology is often required. The main objective of the present work was to investigate thermo-acid pretreatment impact on sugar solubilization and biotic parameters of DF of sorghum or organic fraction of municipal solid waste (OFMSW). Biochemical hydrogen potential tests were carried out without inoculum using raw or thermo-acid pretreated substrates. Results showed an improvement in sugar solubilization after thermo-acid pretreatments. Pretreatments led to similar DF performances (H2 and total metabolite production) compared to raw biomasses. Nevertheless, they were responsible for bacterial shifts from Enterobacteriales towards Clostridiales and Bacillales as well as metabolic changes from acetate towards butyrate or ethanol. The metabolic changes were attributed to the biomass pretreatment impact on indigenous bacteria as no change in the metabolic profile was observed after performing thermo-acid pretreatments on irradiated OFMSW (inactivated indigenous bacteria and inoculum addition). Consequently, acid pretreatments were inefficient to improve DF performances but led to metabolic and bacterial community changes due to their impact on indigenous bacteria.

Ammonia-dependent reducing power redistribution for purple phototrophic bacteria culture-based biohydrogen production.

The global energy crisis has intensified the search for sustainable and clean alternatives, with biohydrogen emerging as a promising solution to address environmental challenges. Leveraging photo fermentation (PF) process, purple phototrophic bacteria (PPB) can harness reducing power derived from organic substrates to facilitate hydrogen production. However, existing studies report much lower H2 yields than theoretical value when using acetate as carbon source and ammonia as nitrogen source, primarily attributed to the widely employed pulse-feeding mode which suffers from ammonia inhibition effect on nitrogenase. To address this issue, a continuous feeding mode was applied to avoid ammonia accumulation in this study. On the other hand, other pathways like carbon fixation and polyhydroxyalkanoate (PHA) formation could compete reducing power with H2 production. However, the reducing power allocation under continuous feeding mode is not yet clear. In this study, the reducing power allocation and hydrogen production performance were evaluated under various ammonia loading, using acetate as carbon source and infrared LED at around 50 W·m-2 as light source. The results show that (a) The absence of ammonia resulted in the best performance for hydrogen production, with 44 % of the reducing power distributed to H2 and the highest H2 volumetric productivity, while the allocation of reducing power to hydrogen production stopped when ammonia loading was above 7.6 mg NH4-N·L-1·d-1; (b) when PPB required to eliminate reducing power under ammonia limited conditions, PHA production was the preferred pathway followed by the hydrogen production pathway, but once PHA accumulation reached saturation, hydrogen generation pathway dominated; (c) under ammonia limited conditions, the TCA cycle was more activated rendering higher NADH (i.e. reducing power) production compared with that under ammonia sufficient conditions which was verified by metagenomics analysis, and all the hydrogen production, PHA accumulation and carbon fixation pathways were highly active to dissipate reducing power. This work provides the insight of reducing power distribution and PPB biohydrogen production variated by ammonia loading under continuous feeding mode.