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Methanogenesis allows methanogenic archaea (methanogens) to generate cellular energy for their growth while producing methane. Hydrogenotrophic methanogens thrive on carbon dioxide and molecular hydrogen as sole carbon and energy sources. Thermophilic and hydrogenotrophicMethanothermobacterspp. have been recognized as robust biocatalysts for a circular carbon economy and are now applied in power-to-gas technology. Here, we generated the first manually curated genome-scale metabolic reconstruction for threeMethanothermobacterspp‥ We investigated differences in the growth performance of three wild-type strains and one genetically engineered strain in two independent chemostat bioreactor experiments. In the first experiment, with molecular hydrogen and carbon dioxide, we found the highest methane production rate forMethanothermobacter thermautotrophicusΔH, whileMethanothermobacter marburgensisMarburg reached the highest biomass growth rate. Systems biology investigations, including implementing a pan-model that contains combined reactions from all three microbes, allowed us to perform an interspecies comparison. This comparison enabled us to identify crucial differences in formate anabolism. In the second experiment, with sodium formate, we found stable growth with anM. thermautotrophicusΔH plasmid-carrying strain with similar performance parameters compared to wild-typeMethanothermobacter thermautotrophicusZ-245. Our findings reveal that formate anabolism influences the diversion of carbon to biomass and methane with implications for biotechnological applications ofMethanothermobacterspp. in power-to-gas technology and for chemical production.Graphical AbstractBroader contextRenewable energy sources (e.g., wind and solar) provide carbon-free electric power. However, their intermittency and offset between peak production and demand generate the need to store this electric power. Furthermore, these technologies alone do not satisfy the demand for carbon-based commodities. Power-to-gas technology provides a means to store intermittent renewable electric power with concomitant carbon dioxide recycling into a chemical energy carrier, such as methane, on a centralized and decentralized scale. This is particularly important to establish equitable energy strategies forallcountries, as is highlighted by the United Nations Sustainable Development Goals. With this work, we provide an integrated systems-biology platform forMethanothermobacterspp. to optimize biological power-to-gas technology and formulate strategies to produce other value-added products besides methane.

Rice paddies are major contributors to anthropogenic greenhouse gas emissions via methane (CH4) flux. The accurate quantification of CH4 emissions from rice paddies remains problematic, in part due to uncertainties and omissions in the contribution of microbial aggregates on the soil surface to carbon fluxes. Herein, we comprehensively evaluated the contribution of one form of microbial aggregates, periphytic biofilm (PB), to carbon dioxide (CO2) and CH4 emissions from paddies distributed across three climatic zones, and quantified the pathways that drive net CH4 production as well as CO2 fixation. We found that PB accounted for 7.1%–38.5% of CH4 emissions and 7.2%–12.7% of CO2 fixation in the rice paddies. During their growth phase, PB fixed CO2 and increased the redox potential, which promoted aerobic CH4 oxidation. During the decay phase, PB degradation reduced redox potential and increased soil organic carbon availability, which promoted methanogenic microbial community growth and metabolism and increased CH4 emissions. Overall, PB acted as a biotic converter of atmospheric CO2 to CH4, and aggravated carbon emissions by up to 2,318 kg CO2 equiv ha−1 season−1. Our results provide proof-of-concept evidence for the discrimination of the contributions of surface microbial aggregates (i.e., PB) from soil microbes, and a profound foundation for the estimation and simulation of carbon fluxes in a potential novel approach to the mitigation of CH4 emissions by manipulating PB growth., Graphical abstract, Public summary • Field experiments were conducted in tropical, subtropical, and temperate rice paddies • Periphytic biofilm promoted CO2 fixation and CH4 emission • Periphytic biofilm acted as a biotic converter of atmospheric CO2 to CH4

Contribution to the International Chain Elongation Conference 2020 | ICEC 2020. An abstract can be found in the right column.

BackgroundBulk production of medium-chain carboxylates (MCCs) with 6-12 carbon atoms is of great interest to biotechnology. Open cultures (e.g., reactor microbiomes) have been utilized to generate MCCs in bioreactors. When in-line MCC extraction and prevention of product inhibition is required, the bioreactors have been operated at mildly acidic pH (5.0-5.5). However, model chain-elongating bacteria grow optimally at neutral pH values. Here, we isolated a chain-elongating bacterium (strain 7D4C2) that thrives at mildly acidic pH. We studied its metabolism and compared its whole genome and the reverse β-oxidation (rBOX) genes to other bacteria.ResultsStrain 7D4C2 produces lactate, acetate,n-butyrate,n-caproate, biomass, and H2/CO2from hexoses. With only fructose as substrate (pH 5.5), the maximumn-caproate specificity (i.e., productsperother carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose andn-butyrate (electron acceptor) were combined as a substrate. A comparison of serum bottles with fructose andn-butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasingn-caproate specificity from ∼92% at mildly acidic pH (pH 4.5-5.0) to ∼24% at alkaline pH (pH 9.0). Moreover, when carboxylates were extracted from the broth (undissociatedn-caproic acid was ∼0.3 mM), then-caproate selectivity (i.e., productpersubstrate fed) was 42.6 ± 19.0% higher compared to serum bottles without extraction. Based on the 16S rRNA gene sequence, strain 7D4C2 is most closely related to the isolatesCaproicibacter fermentans(99.5%) andCaproiciproducens galactitolivorans(94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2,C. fermentans, andC. galactitolivoransbelong to the same genus ofCaproiciproducens. Their rBOX genes are conserved and located next to each other, forming a gene cluster, which is different than for other chain-elongating bacteria such asMegasphaeraspp.ConclusionsCaproiciproducensspp., comprising strain 7D4C2,C. fermentans, C. galactitolivorans, and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster.Caproiciproducenssp. 7D4C2 (DSM 110548) was studied here to understandn-caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars inton-caproate.

The increasing use and associated loss of phosphorus to the environment pose risks to aquatic ecosystems. Technology for phosphorus removal based on microbial aggregates is a natural, ecologically widespread, and sustainable reclamation strategy. Two main processes dominate phosphorus removal by microbial aggregates: extra- and intra-cellular entrapment. Extracellular phosphorus entrapment relies on extracellular polymeric substances, while intracellular entrapment uses a wider variety of phosphorus-entrapping mechanisms. In microbial aggregates, microalgae–bacteria interactions, quorum sensing, and acclimation can enhance phosphorus removal. Based on these insights, we propose novel avenues for entrapping phosphorus using ecological and genetic engineering, manipulated interactions, and integrated processes to create phosphorus removal technology mediated by microbial aggregates.

Across human populations, 16S rRNA gene-based surveys of gut microbiomes have revealed that the bacterial familyChristensenellaceae and the archaeal familyMethanobacteriaceaeco-occur and are enriched in individuals with a lean, compared to an obese, BMI. Whether these association patterns reflect interactions between metabolic partners remains to be ascertained, as well as whether these associations play a role in the lean host phenotype with which they associate. Here, we validated previously reported co-occurrence patterns of the two families, and their association with a lean BMI, with a meta-analysis of 1,821 metagenomes derived from 10 independent studies. Furthermore, we report positive associations at the genus and species level betweenChristensenellaspp. andMethanobrevibacter smithii,the most abundant methanogen of the human gut. By co-culturing threeChristensenellaspp. WithM. smithii,we show thatChristensenellaspp. efficiently support the ofM. smithiivia H2production, far better thanBacteroides thetaiotaomicron.C. minutaforms flocs colonized byM. smithiieven when H2is in excess. In culture withC. minuta, H2consumption byM. smithiishifts the metabolic output ofC. minuta’s fermentation towards acetate rather than butyrate. Together, these results indicate that the widespread co-occurrence of these microbiota is underpinned by both physical and metabolic interactions. Their combined metabolic activity may provide insights into their association with a lean host BMI.ImportanceThe human gut microbiome is made of trillions of microbial cells, most of which areBacteria, with a subset ofArchaea. The bacterial familyChristensenellaceaeand the archaeal familyMethanobacteriaceaeare widespread in human guts. They correlate with each other and with a lean body type. Whether species of these two families interact, and how they affect the body type, are unanswered questions. Here, we showed that species within these families correlate with each other across people. We also demonstrated that particular species of these two families grow together in dense flocs, wherein the bacteria provide hydrogen gas to the archaea, which then make methane. When the archaea are present, the ratio of bacterial products (which are nutrients for humans) is changed. These observations indicate when these species grow together, their products have the potential to affect the physiology of their human host.

Harmful algal blooms (HABs) are a worldwide problem with numerous negative effects on water systems, which have prompted researchers to study applicable measures to inhibit and control them. This review summarized the current microorganisms-based methods or technologies aimed at controlling HABs. Based on their characteristics, these methods can be divided into two categories: methods based on single-species microorganisms and methods based on microbial aggregates, and four types: methods for rapid decrease of algal cells density (e.g., alga-bacterium and alga-fungus bioflocculation), inhibition of harmful algal growth, lysis of harmful algae (e.g. algicidal bacteria, fungi, and actinomycete), and methods based on microbial aggregates (periphytons and biofilms). An integrative process of "flocculation-lysis-degradation-nutrients regulation" is proposed to control HABs. This review not only offers a systematic understanding of HABs control technologies based on microorganisms but also elicits a re-thinking of HABs control based on microbial aggregates.

Continuous bioreactors operated at low hydraulic retention times have rarely been explored for reductive dehalogenation of chlorinated ethenes. The inability to consistently develop such bioreactors affects the way growth approaches for Dehalococcoides mccartyi bioaugmentation cultures are envisioned. It also affects interpretation of results from in situ continuous treatment processes. We report bioreactor performance and dehalogenation kinetics of a D. mccartyi-containing consortium in an upflow bioreactor. When fed synthetic groundwater at 11–3.6 h HRT, the upflow bioreactor removed >99.7% of the influent trichloroethene (1.5–2.8 mM) and produced ethene as the main product. A trichloroethene removal rate of 98.51 ± 0.05 me– equiv L–1 d–1 was achieved at 3.6 h HRT. D. mccartyi cell densities were 1013 and 1012 16S rRNA gene copies L–1 in the bioflocs and planktonic culture, respectively. When challenged with a feed of natural groundwater containing various competing electron acceptors and 0.3–0.4 mM tri...

The increasing use and discharge of nanoparticles (NPs) pose risks to microorganisms that maintain the health of aquatic ecosystems. Although NPs are toxic to microorganisms, they tend to form microbial aggregates to protect themselves. Two main mechanisms account for the reduced toxicity: the dense physical structure acts as a barrier to NP exposure in the interior of the aggregate, and aggregation stabilizes a complex microbial ecosystem that enhances the ability of the community to adapt to prolonged NP exposure. We highlight the opportunities and challenges for managing microbial aggregates in wastewater treatment to remove or control NPs. For example, understanding the resistance mechanisms can help to design smart NPs that are less toxic to useful microorganisms or more toxic towards pathogenic microorganisms.

Carbon monoxide (CO)-metabolism and phenotypic and phylogenetic characterization of a novel anaerobic, mesophilic and hydrogenogenic carboxydotroph are reported. Strain SVCO-16 was isolated from anaerobic sludge and grows autotrophically and mixotrophically with CO. The genes cooS and cooF, coding for a CO dehydrogenase complex, and genes similar to hycE2, encoding a CO-induced hydrogenase, were present in its genome. The isolate produces H2 and CO2 from CO, and acetate and formate from organic substrates. Based on the 16S rRNA sequence, it is an Alphaproteobacterium most closely related to the genus Pleomorphomonas (98.9%-99.2% sequence identity). Comparison with other previously characterized Pleomorphomonas showed that P. diazotrophica and P. oryzae do not metabolize CO, and P. diazotrophica does not grow anaerobically with organic substrates. Average nucleotide identity values between strain SVCO-16 and P. diazotrophica, P. oryzae or P. koreensis were 86.66 ± 0.21%. These values are below the boundary to define species (95%-96%). Digital DNA-DNA hybridization estimates between strain SVCO-16 and reference strains were also below the 70% threshold for species delineation: 29.1%-34.5%. Based on the differences in CO metabolism, genome analyses and cellular fatty acid composition, the isolate should be classified into the genus Pleomorphomonas as a representative of a novel species, Pleomorphomonas carboxyditropha. The type strain of Pleomorphomonas carboxyditropha is SVCO-16T (strain deposit numbers, DSM 106132T and TSD-119T).

Background Syngas fermentation, the bioconversion of CO, CO2, and H2 to biofuels and chemicals, has undergone considerable optimization for industrial applications. Even more, full-scale plants for ethanol production from syngas fermentation by pure cultures are being built worldwide. The composition of syngas depends on the feedstock gasified and the gasification conditions. However, it remains unclear how different syngas mixtures affect the metabolism of carboxidotrophs, including the ethanol/acetate ratios. In addition, the potential application of mixed cultures in syngas fermentation and their advantages over pure cultures have not been deeply explored. In this work, the effects of CO2 and H2 on the CO metabolism by pure and mixed cultures were studied and compared. For this, a CO-enriched mixed culture and two isolated carboxidotrophs were grown with different combinations of syngas components (CO, CO:H2, CO:CO2, or CO:CO2:H2). Results The CO metabolism of the mixed culture was somehow affected by the addition of CO2 and/or H2, but the pure cultures were more sensitive to changes in gas composition than the mixed culture. CO2 inhibited CO oxidation by the Pleomorphomonas-like isolate and decreased the ethanol/acetate ratio by the Acetobacterium-like isolate. H2 did not inhibit ethanol or H2 production by the Acetobacterium and Pleomorphomonas isolates, respectively, but decreased their CO consumption rates. As part of the mixed culture, these isolates, together with other microorganisms, consumed H2 and CO2 (along with CO) for all conditions tested and at similar CO consumption rates (2.6 ± 0.6 mmol CO L−1 day−1), while maintaining overall function (acetate production). Providing a continuous supply of CO by membrane diffusion caused the mixed culture to switch from acetate to ethanol production, presumably due to the increased supply of electron donor. In parallel with this change in metabolic function, the structure of the microbial community became dominated by Geosporobacter phylotypes, instead of Acetobacterium and Pleomorphomonas phylotypes. Conclusions These results provide evidence for the potential of mixed-culture syngas fermentation, since the CO-enriched mixed culture showed high functional redundancy, was resilient to changes in syngas composition, and was capable of producing acetate or ethanol as main products of CO metabolism. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0910-1) contains supplementary material, which is available to authorized users.

Medium-chain fatty acids (MCFA) are important biofuel precursors. Carbon monoxide (CO) is a sustainable electron and carbon donor for fatty acid elongation, since it is metabolized to MCFA precursors, it is toxic to most methanogens, and it is a waste product generated in the gasification of waste biomass. The main objective of this work was to determine if the inhibition of methanogenesis through the continuous addition of CO would lead to increased acetate or MCFA production during fermentation of ethanol. The effects of CO partial pressures (PCO ; 0.08-0.3 atm) on methanogenesis, fatty acids production, and the associated microbial communities were studied in batch cultures fed with CO and ethanol. Methanogenesis was partially inhibited at PCO ≥ 0.11 atm. This inhibition led to increased acetate production during the first phase of fermentation (0-19 days). However, a second addition of ethanol (day 19) triggered MCFA production only at PCO ≥ 0.11 atm, which probably occurred through the elongation of acetate with CO-derived ethanol and H2 :CO2 . Accordingly, during the second phase of fermentation (days 20-36), the distribution of electrons to acetate decreased at higher PCO , while electrons channeled to MCFA increased. Most probably, Acetobacterium, Clostridium, Pleomorphomonas, Oscillospira, and Blautia metabolized CO to H2 :CO2 , ethanol and/or fatty acids, while Peptostreptococcaceae, Lachnospiraceae, and other Clostridiales utilized these metabolites, along with the provided ethanol, for MCFA production. These results are important for biotechnological systems where fatty acids production are preferred over methanogenesis, such as in chain elongation systems and microbial fuel cells.

The aim of this work was to study the characteristics and flocculating properties of extracellular polymeric substances (EPS) extracted from periphytic biofilms. The periphytic EPS, with an extracted yield of 491.8 mg/g, were mainly composed of hetero-polysaccharides and proteins, and the elements C1s, N1s, and O1s. Polysaccharides represented 53.28% of the periphytic EPS. Proteins constituted 20.26% of the EPS, and contributed to at least 34.65% of the total flocculating activity. The periphytic EPS showed high turbidity removal capacity (86.76 ± 1.52%, 10 min) and efficient aniline blue (AB) removal capacity (56.46 ± 1.41%, 30 min). The mechanism of AB removal by the periphytic EPS seemed to be a combined technique of “adsorption-flocculation”. This study reveals the flocculating capability of periphytic EPS, and suggests that periphytic biofilms are novel sources for bioflocculants preparation.

Microbial anaerobic conversion of carbon monoxide (CO) and syngas (mainly composed of CO, CO2 and H2) leads to the production of important industrial products, such as acetate and ethanol. The composition of CO- and syngas-converting microbial communities and the microbial interactions involved are still largely unknown. The main objectives of this study were (i) to understand the effects of CO, CO2, and H2 on the structure and function of a CO-consuming microbial community, and (ii) to identify key carboxidotrophs in the mixed culture. For this, sludge was anaerobically enriched with CO as the sole carbon/energy source at incrementally increasing CO partial pressures (PCO). Phylotypes of Methanobacteriaceae and methane production were detected at PCO ≤ 44.1 kPa. At higher PCO, enriched phylotypes were Acetobacterium, Oscillospira and Pleomorphomonas, and acetate was the main end product. The addition of CO2/HCO3- or H2 to CO fermentation increased the acetate/ethanol ratio and species diversity, compared to growth with CO as sole substrate. Phylotypes associated with Pleomorphomonas and Acetobacterium increased in relative abundance during exponential CO utilization. The Pleomorphomonas-like isolate produced H2:CO2, whereas the Acetobacterium-like isolate produced ethanol, when CO was the only electron/carbon source. These findings shed light on the interplay between syngas components and microbial communities.

Inhibition by ammonium at concentrations above 1000 mgN/L is known to harm the methanogenesis phase of anaerobic digestion. We anaerobically digested swine waste and achieved steady state COD-removal efficiency of around 52% with no fatty-acid or H2accumulation. As the anaerobic microbial community adapted to the gradual increase of total ammonia-N (NH3-N) from890±295to2040±30 mg/L, the Bacterial and Archaeal communities became less diverse. Phylotypes most closely related to hydrogenotrophicMethanoculleus(36.4%) andMethanobrevibacter(11.6%), along with acetoclasticMethanosaeta(29.3%), became the most abundant Archaeal sequences during acclimation. This was accompanied by a sharp increase in the relative abundances of phylotypes most closely related to acetogens and fatty-acid producers (Clostridium,Coprococcus, andSphaerochaeta) and syntrophic fatty-acid Bacteria (Syntrophomonas,Clostridium, Clostridiaceae species, and Cloacamonaceae species) that have metabolic capabilities for butyrate and propionate fermentation, as well as for reverse acetogenesis. Our results provide evidence countering a prevailing theory that acetoclastic methanogens are selectively inhibited when the total ammonia-N concentration is greater than ~1000 mgN/L. Instead, acetoclastic and hydrogenotrophic methanogens coexisted in the presence of total ammonia-N of ~2000 mgN/L by establishing syntrophic relationships with fatty-acid fermenters, as well as homoacetogens able to carry out forward and reverse acetogenesis.

Dark fermentation for bio-hydrogen (bio-H2) production is an easily operated and environmentally friendly technology. However, low bio-H2 production yield has been reported as its main drawback. Two strategies have been followed in the past to improve this fact: genetic modifications and adjusting the reaction conditions. In this paper, the second one is followed to regulate the bio-H2 release from the reactor. This operating condition alters the metabolic pathways and increased the bio-H2 production twice. Gas release was forced in the continuous culture to study the equilibrium in the mass transfer between the gaseous and liquid phases. This equilibrium depends on the H2, CO2, and volatile fatty acids production. The effect of reducing the bio-H2 partial pressure (bio-H2 pp) to enhance bio-H2 production was evaluated in a 30 L continuous stirred tank reactor. Three bio-H2 release strategies were followed: uncontrolled, intermittent, and constant. In the so called uncontrolled fermentation, without bio-H2 pp control, a bio-H2 molar yield of 1.2 mol/mol glucose was obtained. A sustained low bio-H2 pp of 0.06 atm increased the bio-H2 production rate from 16.1 to 108 mL/L/h with a stable bio-H2 percentage of 55 % (v/v) and a molar yield of 1.9 mol/mol glucose. Biogas release enhanced bio-H2 production because lower bio-H2 pp, CO2 concentration, and reduced volatile fatty acids accumulation prevented the associated inhibitions and bio-H2 consumption.