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To upcycle the nutrients from kitchen waste (KW), an integrated system consisting of anaerobic digestion (AD) reactor and microbial protein (MP) production reactor was established in this study. The subsystem I (AD system) demonstrated an efficient bio-energy production (282.37 mL CH4/g VS), with 553.54 mg/L of NH4+-N remained in the digestate. The subsystem II (MP production system) utilized the nitrogenous constituents of the digestate, with 2.04 g/L MP production. In order to further enhance the recovery efficiency, C/N ratio in the subsystem II was studied. NH4+-N recovery efficiency was 23.08% higher after C/N ratio optimization along with 0.24 g/L increment on MP production. Over 0.7 g/L of essential amino acids was obtained, according with the qualitative necessary for the feeds. Also, the key enzyme abundance of CO2 releasing and amino acid biosynthesis was obviously increased with max. 55.21%. Meanwhile, the integrated system was profitable via a simplified economic assessment.

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Nitrate pollution in groundwater is a serious problem worldwide, as its concentration in many areas exceeds the WHO-defined drinking water standard (50 mg/L). Hydrogen-oxidizing bacteria (HOB) are a group of microorganisms capable of producing single-cell protein (SCP) using hydrogen and oxygen. Furthermore, HOB can utilize various nitrogen sources, including nitrate. This study developed a novel hybrid biological-inorganic (HBI) system that coupled a new submersible water electrolysis system driven by renewable electricity with HOB fermentation for in-situ nitrate recovery from polluted groundwater and simultaneously upcycling it together with CO2 into single-cell protein. The performance of the novel HBI system was first evaluated in terms of bacterial growth and nitrate removal efficiency. With 5 V voltage applied and the initial nitrate concentration of 100 mg/L, the nitrate removal efficiency of 85.52 % and raw of 47.71 % (with a broad amino acid spectrum) were obtained. Besides, the HBI system was affected by the applied voltages and initial nitrogen concentrations. The water electrolysis with 3 and 4 V cannot provide sufficient H2 for HOB and the removal of nitrate was 57.12 % and 59.22 % at 180 h, while it reached 65.14 % and 65.42 % at 5 and 6 V, respectively. The nitrate removal efficiency reached 58.40 % and 50.72 % within 180 h with 200 and 300 mg/L initial nitrate concentrations, respectively. Moreover, a larger anion exchange membrane area promoted nitrate removal. The monitored of the determination of different forms of nitrogen indicated that around 60 % of the recovered nitrate was assimilated into cells, and 40 % was bio-converted to N2. The results demonstrate a potentially sustainable method for remediating nitrate contaminant in groundwater, upcycling waste nitrogen, CO2 sequestration and valorization of renewable electricity into food or feed.

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Xanthobacter sp. SoF1 (SoF1) is an autotrophic hydrogen-oxidizing bacteria that produces protein-rich biomass and has potential to be an alternative protein source that is more environmentally sustainable than animal and plant derived proteins. A protein-rich powder derived from SoF1 was the test material in a 90-day repeated-dose oral toxicity study to explore major toxic effects, demonstrate target organs, and provide an estimate of a no-observed-adverse-effect level (NOAEL). Daily doses of 0 (vehicle only), 375, 750, and 1500 mg/kg bw/day of the test material were administered by gavage to 10 Han:WIST rats/sex/group. An additional group was administered 1290 mg/kg bw/day whey protein concentrate as positive control. No treatment-related adverse effects were observed, and no target organs were determined after 90/91 days of consecutive administration of the test item. A NOAEL of 1500 mg/kg bw/day was determined.

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One way of limiting the environmental impact of food production and improving food security is to replace part of the animal- or plant-based protein in the human diet with protein sourced from microorganisms. The recently discovered bacterium Xanthobacter sp. SoF1 (VTT-E-193585) grows autotrophically using carbon dioxide gas as the only carbon source, yielding protein-rich biomass that can be processed further into a powder and incorporated into various food products. Since the safety of this microbial protein powder for human consumption had not been previously assessed, its genotoxic potential was evaluated employing three internationally recognized and standardized studies: a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes. No biologically relevant evidence of genotoxicity or mutagenicity was found.

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Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.

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To investigate the effects of nitrogen source supply on microbial protein (MP) production by hydrogen-oxidizing bacteria (HOB) under continuous feed gas provision, a sequencing batch culture comparison (N2 fixation versus ammonium assimilation) was performed. The results confirmed that even under basic cultivation conditions, N2-fixing HOB (NF-HOB) communities showed higher levels of CO2 and N2 fixation (190.45 mg/L Δ CODt and 11.75 mg/L Δ TNbiomass) than previously known, with the highest biomass yield being 0.153 g CDW/g COD-H2. Rich ammonium stimulated MP synthesis and the biomass accumulation of communities (increased by 7.4 ～ 14.3 times), presumably through the enhancement of H2 and CO2 absorption. The micro mechanism may involve encouraging the enrichment of species like Xanthobacter and Acinetobacter then raising the abundance of nitrogenase and glutamate synthase to facilitate the nitrogen assimilation. This would provide NF-HOB with ideas for optimizing their MP synthesis activity.

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We report the complete genome of Mycolicibacterium mageritense strain H4_3_1, which was isolated from a reactor of a hybrid biological-inorganic system. This genome will provide useful information about hydrogen-oxidizing bacteria as well as mycolicibacteria in non-host environments.

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We report the complete genome of Achromobacter xylosoxidans strain H1_3_1, which was isolated from a reactor of a hybrid biological-inorganic system. The complete genome comprised 7,071,873 bp, including 6,428 codings, 10 rRNA, and 70 tRNA, with 67.4% G + C content.

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Hybrid biological-inorganic (HBI) systems show great promise as CO2 conversion platforms combining CO2 fixation by hydrogen-oxidizing bacteria (HOB) with water splitting. Herein, halotolerant HOB were enriched using an HBI system with a high-ionic-strength medium containing 180 mM phosphate buffer to identify new biocatalysts. The reactors were inoculated with samples from saline environments and applied with a voltage of 2.0 V. Once an increase in biomass was observed with CO2 consumption, an aliquot of the medium was transferred to a new reactor. After two successive subcultures, Achromobacter xylosoxidans strain H1_3_1 and Mycolicibacterium mageritense strain H4_3_1 were isolated from the reactor media. Genome sequencing indicated the presence of genes for aerobic hydrogen-oxidizing chemolithoautotrophy and synthesis of the compatible solute hydroxyectoine in both strains. Furthermore, both strains produced hydroxyectoine in the reactors under the high-ionic-strength condition, suggesting the potential for new HBI systems using halotolerant HOB to produce high-value-added chemicals.

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Upgrading of waste nitrogen sources is considered as an important approach to promote sustainable development. In this study, a multifunctional bio-electrochemical system with three chambers was established, innovatively achieving 2.02 g/L in-situ microbial protein (MP) production via hydrogen-oxidizing bacteria (HOB) in the protein chamber (middle chamber), along with over 2.9 L CO2/(L·d) consumption rate. Also, 69% chemical oxygen demand was degraded by electrogenic bacteria in the anode chamber, resulting in the 394.67 J/L electricity generation. Focusing on the NH4+-N migration in the system, the current intensity contributed 4%-9% in the anode and protein chamber, whereas, the negative effect of -6.69% on contribution was shown in the cathode chamber. On the view of kinetics, NH4+-N migration in anode and cathode chambers was fitted well with Levenberg-Marquardt equation (R2 > 0.92), along with the well-matched results of HOB growth in the protein chamber based on Gompertz model (R2 > 0.99). Further evaluating MPs produced by HOB, 0.45 g/L essential amino acids was detected, showing the better amino acid profile than fish and soybean. Multifunctional bio-electrochemical system revealed the economic potential of producing 6.69 €/m3 wastewater according to a simplified economic evaluation.

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With the utilization of pesticides and fertilizers (e.g. urea), the presence of nitrogen and heavy metals (e.g. copper) can enter and pollute the environment. Biofertilizers can be used to replace chemical fertilizers to increase crop yields and reduce environmental stress. The utilization of hydrogen-oxidizing bacteria (HOB) to be biofertilizers has recently attracted more attention. However, the enrichment of HOB on urea and the effect of copper are undetermined. HOB were successfully enriched using urea in this investigation. The average urea conversion rate (AUCR) was 180.08 mgN/L/d with a hydraulic retention time of 10 h. Microbial community (R1) was dominated by Hydrogenophaga (83.92%), a biofertilizer-type HOB. After addition of 5.47 mg/L Cu2+, the AUCR was decreased by 16%-151.18 mgN/L/d, and the growth of HOB was inhibited by 48%. Meanwhile, inhibition was also reflected by the increase of polysaccharide content (20.27 ± 0.57 to 33.45 ± 2.53 mg/gVSS) and protein content (106.19 ± 19.39 to 125.14 ± 24.73 mg/gVSS) of extracellular polymeric substances in the HOB. The resulting microbial community (R2) was changed to Azospiralium-dominated flora (91.33%). Both enriched microbial communities (R1 and R2) exhibited the abilities of ACC degradation and phosphate solubilization. This study demonstrates that employing urea can directly enrich biofertilizer-type HOB and copper-tolerant HOB can be obtained in a 5.47 mg/L Cu2+ environment. The results provide potential methods to obtain biofertilizer from copper-containing urea wastewater via HOB.

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Aerobic hydrogen-oxidizing 'Knallgas' bacteria are promising candidates for microbial cell factories due to their ability to use hydrogen and carbon dioxide as the sole energy and carbon sources, respectively. These bacteria can convert atmospheric CO2 to chemicals which could help to mitigate climate change by replacing fossil fuel-based chemicals. A known method to enhance the product yield is to disrupt competing metabolic pathways in the host organism. One such pathway in many 'Knallgas' bacteria is polyhydroxybutyrate (PHB) biosynthesis. In this study, the PHB biosynthesis genes of a non-model 'Knallgas' bacterium Xanthobacter sp. SoF1 were identified. Consequently, the phaA, phaB and phaC genes were individually deleted and the resulting knockouts were evaluated for their ability to produce PHB in autotrophic shake flask and small-scale bioreactor cultivations. The results demonstrate that PHB production was inactivated in the phaC1 knockout strain, which advances the development of Xanthobacter sp. SoF1 as a production host.

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Hybrid biological-inorganic (HBI) systems comprising inorganic water-splitting catalysts and aerobic hydrogen-oxidizing bacteria (HOB) have previously been used for CO2 conversion. In order to identify new biocatalysts for CO2 conversion, the present study used an HBI system to enrich HOB directly from environmental samples. Three sediment samples (from a brackish water pond, a beach, and a tide pool) and two activated sludge samples (from two separate sewage plants) were inoculated into HBI systems using a cobalt phosphorus (Co-P) alloy and cobalt phosphate (CoPi) as inorganic catalysts with a fixed voltage of 2.0 V. The gas composition of the reactor headspaces and electric current were monitored. An aliquot of the reactor medium was transferred to a new reactor when significant consumption of H2 and CO2 was detected. This process was repeated twice (with three reactors in operation for each sample) to enrich HOB. Increased biomass concomitant with increased H2 and CO2 consumption was observed in the third reactor, indicating enrichment of HOB. 16S rRNA gene amplicon sequencing demonstrated enrichment of sequences related to HOB (including bacteria from Mycobacterium, Hydrogenophaga, and Xanthobacter genera) over successive sub-cultures. Finally, four different HOB belonging to the Mycobacterium, Hydrogenophaga, Xanthobacter, and Acidovorax genera were isolated from reactor media, representing potential candidates as HBI system biocatalysts.

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It is of great significance to utilize CO2 as feedstock to synthesize biobased products, particularly single cell protein (SCP) as the alternative food and feed. Bioelectrochemical system (BES) driven by clean electric energy has been regarded as a promising way for Cupriavidus necator to produce SCP from CO2 directly. At present, the key problem of culturing C. necator in BES is that reactive oxygen species (ROS) generated in cathode chamber are harmful to bacterial growth. Therefore, it is necessary to find a solution to mitigate the negative effect of ROS. In this study, we constructed a number of C. necator strains displayed with superoxide dismutase (SOD), which allowed the decomposition of superoxide anion radical. The effects of promoters and signal peptides on the cell surface displayed SOD were analyzed. The proteins displayed on the surface were further verified by the fluorescence experiment. Finally, the growth of C. necator CMS incorporating a pBAD-SOD-E-tag-IgAß plasmid could achieve 4.9 ± 1.0 of OD600 by 7 days, equivalent to 1.7 ± 0.3 g/L dry cell weight (DCW), and the production rate was 0.24 ± 0.04 g/L/d DCW, around 2.7-fold increase than the original C. necator CMS (1.8 ± 0.3 of OD600). This study can provide an effective and novel strategy of cultivating strains for the production of CO2-derived SCP or other chemicals in BES.

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Polyhydroxyalkanoates (PHAs) are eco-friendly plastics that are thermoplastic and biodegradable in nature. The hydrogen-oxidizing bacterium Ralstonia eutropha can biosynthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], the most common PHA, from carbon dioxide using hydrogen and oxygen as energy sources. In conventional autotrophic cultivation using R. eutropha, a gas mixture containing 75−80 vol% hydrogen is supplied; however, a gas mixture with such a high hydrogen content has a risk of explosion due to gas leakage. In this study, we aimed to develop an efficient cell culture system with a continuous supply of a non-combustible gas mixture (H2: O2: CO2: N2 = 3.8: 7.3: 13.0: 75.9) for safe autotrophic culture to produce P(3HB) by hydrogen-oxidizing bacteria, with a controlled hydrogen concentration under a lower explosive limit concentration. When the gas mixture was continuously supplied to the jar fermentor, the cell growth of R. eutropha H16 significantly improved compared to that in previous studies using flask cultures. Furthermore, an increased gas flow rate and agitation speed enhanced both cell growth and P(3HB) production. Nitrogen source deficiency promoted P(3HB) production, achieving up to 2.94 g/L P(3HB) and 89 wt% P(3HB) content in the cells after 144 h cultivation. R. eutropha NCIMB 11599, recombinant R. eutropha PHB-4, and Azohydromonas lata grew in a low-hydrogen-content gas mixture. R. eutropha H16 and recombinant R. eutropha PHB-4 expressing PHA synthase from Bacillus cereus YB-4 synthesized P(3HB) with a high weight-average molecular weight of 13.5−16.9 × 105. Thus, this autotrophic culture system is highly beneficial for PHA production from carbon dioxide using hydrogen-oxidizing bacteria as the risk of explosion is eliminated.

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Hydrogen oxidizing autotrophic bacteria are promising hosts for conversion of CO2 into chemicals. In this work, we engineered the metabolically versatile lithoautotrophic bacterium R. opacus strain DSM 43205 for synthesis of polymer precursors. Aspartate decarboxylase (panD) or lactate dehydrogenase (ldh) were expressed for beta-alanine or L-lactic acid production, respectively. The heterotrophic cultivations on glucose produced 25 mg L-1 beta-alanine and 742 mg L-1 L-lactic acid, while autotrophic cultivations with CO2, H2, and O2 resulted in the production of 1.8 mg L-1 beta-alanine and 146 mg L-1 L-lactic acid. Beta-alanine was also produced at 345 µg L-1 from CO2 in electrobioreactors, where H2 and O2 were provided by water electrolysis. This work demonstrates that R. opacus DSM 43205 can be engineered to produce chemicals from CO2 and provides a base for its further metabolic engineering.

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Bacteria are key denitrifiers in the reduction of nitrate (NO3 --N), which is a contaminant in wastewater treatment plants (WWTPs). They can also produce carbon dioxide (CO2) and nitrous oxide (N2O). In this study, the autotrophic hydrogen-oxidizing bacterium Rhodoblastus sp. TH20 was isolated for sustainable treatment of NO3 --N in wastewater. Efficient removal of NO3 --N and recovery of biomass nitrogen were achieved. Up to 99% of NO3 --N was removed without accumulation of nitrite and N2O, consuming CO2 of 3.25 mol for each mole of NO3 --N removed. The overall removal rate of NO3 --N reached 1.1 mg L-1 h-1 with a biomass content of approximately 0.71 g L-1 within 72 h. TH20 participated in NO3 --N assimilation and aerobic denitrification. Results from 15N-labeled-nitrate test indicated that removed NO3 --N was assimilated into organic nitrogen, showing an assimilation efficiency of 58%. Seventeen amino acids were detected, accounting for 43% of the biomass. Nitrogen loss through aerobic denitrification was only approximately 42% of total nitrogen. This study suggests that TH20 can be applied in WWTP facilities for water purification and production of valuable biomass to mitigate CO2 and N2O emissions.

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Growing concerns over the use of limited fossil fuels and their negative impacts on the ecological niches have facilitated the exploration of alternative routes. The use of conventional plastic material also negatively impacts the environment. One such green alternative is polyhydroxyalkanoates, which are biodegradable, biocompatible, and environmentally friendly. Recently, researchers have focused on the utilization of waste gases particularly those belonging to C1 sources derived directly from industries and anthropogenic activities, such as carbon dioxide, methane, and methanol as the substrate for polyhydroxyalkanoates production. Consequently, several microorganisms have been exploited to utilize waste gases for their growth and biopolymer accumulation. Methylotrophs such as Methylobacterium organophilum produced highest amount of PHA up to 88% using CH4 as the sole carbon source and 52-56% with CH3OH. On the other hand Cupriavidus necator, produced 71-81% of PHA by utilizing CO and CO2 as a substrate. The present review shows the potential of waste gas valorization as a promising solution for the sustainable production of polyhydroxyalkanoates. Key bottlenecks towards the usage of gaseous substrates obstructing their realization on a large scale and the possible technological solutions were also highlighted. Several strategies for PHA production using C1 gases through fermentation and metabolic engineering approaches are discussed. Microbes such as autotrophs, acetogens, and methanotrophs can produce PHA from CO2, CO, and CH4. Therefore, this article presents a vision of C1 gas into bioplastics are prospective strategies with promising potential application, and aspects related to the sustainability of the system.

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Increasing efforts are directed towards the development of sustainable alternative protein sources among which microbial protein (MP) is one of the most promising. Especially when waste streams are used as substrates, the case for MP could become environmentally favorable. The risks of using organic waste streams for MP production-the presence of pathogens or toxicants-can be mitigated by their anaerobic digestion and subsequent aerobic assimilation of the (filter-sterilized) biogas. Even though methane and hydrogen oxidizing bacteria (MOB and HOB) have been intensively studied for MP production, the potential benefits of their co-cultivation remain elusive. Here, we isolated a diverse group of novel HOB (that were capable of autotrophic metabolism), and co-cultured them with a defined set of MOB, which could be grown on a mixture of biogas and H2/O2. The combination of MOB and HOB, apart from the CH4 and CO2 contained in biogas, can also enable the valorization of the CO2 that results from the oxidation of methane by the MOB. Different MOB and HOB combinations were grown in serum vials to identify the best-performing ones. We observed synergistic effects on growth for several combinations, and in all combinations a co-culture consisting out of both HOB and MOB could be maintained during five days of cultivation. Relative to the axenic growth, five out of the ten co-cultures exhibited 1.1-3.8 times higher protein concentration and two combinations presented 2.4-6.1 times higher essential amino acid content. The MP produced in this study generally contained lower amounts of the essential amino acids histidine, lysine and threonine, compared to tofu and fishmeal. The most promising combination in terms of protein concentration and essential amino acid profile was Methyloparacoccus murrelli LMG 27482 with Cupriavidus necator LMG 1201. Microbial protein from M. murrelli and C. necator requires 27-67% less quantity than chicken, whole egg and tofu, while it only requires 15% more quantity than the amino acid-dense soybean to cover the needs of an average adult. In conclusion, while limitations still exist, the co-cultivation of MOB and HOB creates an alternative route for MP production leveraging safe and sustainably-produced gaseous substrates.

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Novel food production technologies are being developed to address the challenges of securing sustainable and healthy nutrition for the growing global population. This study assessed the environmental impacts of microbial protein (MP) produced by autotrophic hydrogen-oxidizing bacteria (HOB). Data was collected from a company currently producing MP using HOB (hereafter simply referred to as MP) on a small-scale. Earlier studies have performed an environmental assessment of MP on a theoretical basis but no study yet has used empirical data. An attributional life cycle assessment (LCA) with a cradle-to-gate approach was used to quantify global warming potential (GWP), land use, freshwater and marine eutrophication potential, water scarcity, human (non-)carcinogenic toxicity, and the cumulative energy demand (CED) of MP production in Finland. A Monte Carlo analysis was performed to assess uncertainties while a sensitivity analysis was used to explore the impacts of alternative production options and locations. The results were compared with animal- and plant-based protein sources for human consumption as well as protein sources for feed. Electricity consumption had the highest contribution to environmental impacts. Therefore, the source of energy had a substantial impact on the results. MP production using hydropower as an energy source yielded 87.5% lower GWP compared to using the average Finnish electricity mix. In comparison with animal-based protein sources for food production, MP had 53-100% lower environmental impacts depending on the reference product and the source of energy assumed for MP production. When compared with plant-based protein sources for food production, MP had lower land and water use requirements, and eutrophication potential but GWP was reduced only if low-emission energy sources were used. Compared to protein sources for feed production, MP production often resulted in lower environmental impact for GWP (FHE), land use, and eutrophication and acidification potential, but generally caused high water scarcity and required more energy.

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