An integrated systems biology approach reveals differences in formate metabolism in the genus Methanothermobacter.

Methanogenesis allows methanogenic archaea to generate cellular energy for their growth while producing methane. Thermophilic hydrogenotrophic species of the genus Methanothermobacter have been recognized as robust biocatalysts for a circular carbon economy and are already applied in power-to-gas technology with biomethanation, which is a platform to store renewable energy and utilize captured carbon dioxide. Here, we generated curated genome-scale metabolic reconstructions for three Methanothermobacter strains and investigated differences in the growth performance of these same strains in chemostat bioreactor experiments with hydrogen and carbon dioxide or formate as substrates. Using an integrated systems biology approach, we identified differences in formate anabolism between the strains and revealed that formate anabolism influences the diversion of carbon between biomass and methane. This finding, together with the omics datasets and the metabolic models we generated, can be implemented for biotechnological applications of Methanothermobacter in power-to-gas technology, and as a perspective, for value-added chemical production.

Contribution of periphytic biofilm of paddy soils to carbon dioxide fixation and methane emissions.

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.

Functional sustainability of nutrient accumulation by periphytic biofilm under temperature fluctuations.

Temperature can fluctuate widely between different seasons, and this may greatly impact many biological processes. However, little is known about its influence on the functioning of benthic microbial communities. Here we investigated the nutrient accumulation capability of periphytic biofilm under temperature fluctuations (17-35°C). Periphytic biofilm maintained the same nutrient accumulation capacity after experiencing the 'warming-hot-cooling' temperature fluctuation under both lab and outdoor conditions as those without temperature disturbance. In response to temperature increase, both community composition and species richness changed greatly and the increase in biodiversity was identified as being the underlying mechanism boosting the sustainable function in nutrient accumulation, indicating zero net effects of community changes. These findings provide insights into the underlying mechanisms of how benthic microbial communities adapt to temperature fluctuations to maintain nutrient accumulation capacity and elucidate that periphytic biofilm plays important roles in influencing nutrient cycling in aquatic ecosystems under temperature changes such as seasonal fluctuations.

Using Microbial Aggregates to Entrap Aqueous Phosphorus.

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.

Syntrophy via Interspecies H2 Transfer between Christensenella and Methanobrevibacter Underlies Their Global Cooccurrence in the Human Gut.

Across human populations, 16S rRNA gene-based surveys of gut microbiomes have revealed that the bacterial family Christensenellaceae and the archaeal family Methanobacteriaceae cooccur and are enriched in individuals with a lean, compared to an obese, body mass index (BMI). Whether these association patterns reflect interactions between metabolic partners, as well as whether these associations play a role in the lean host phenotype with which they associate, remains to be ascertained. Here, we validated previously reported cooccurrence 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 levels between Christensenella spp. and Methanobrevibacter smithii, the most abundant methanogen of the human gut. By coculturing three Christensenella spp. with M. smithii, we show that Christensenella spp. efficiently support the metabolism of M. smithii via H2 production far better than Bacteroides thetaiotaomicron does. Christensenella minuta forms flocs colonized by M. smithii even when H2 is in excess. In culture with C. minuta, H2 consumption by M. smithii shifts the metabolic output of C. minuta's fermentation toward acetate rather than butyrate. Together, these results indicate that the widespread cooccurrence of these microorganisms is underpinned by both physical and metabolic interactions. Their combined metabolic activity may provide insights into their association with a lean host BMI.IMPORTANCE The human gut microbiome is made of trillions of microbial cells, most of which are Bacteria, with a subset of Archaea The bacterial family Christensenellaceae and the archaeal family Methanobacteriaceae are 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 show that species within these families correlate with each other across people. We also demonstrate 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 that when these species grow together, their products have the potential to affect the physiology of their human host.

The Isolate Caproiciproducens sp. 7D4C2 Produces n-Caproate at Mildly Acidic Conditions From Hexoses: Genome and rBOX Comparison With Related Strains and Chain-Elongating Bacteria.

Bulk 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 grows at mildly acidic pH. We studied its metabolism and compared its whole genome and the reverse ß-oxidation (rBOX) genes to other bacteria. Strain 7D4C2 produces lactate, acetate, n-butyrate, n-caproate, biomass, and H2/CO2 from hexoses. With only fructose as substrate (pH 5.5), the maximum n-caproate specificity (i.e., products per other carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose and n-butyrate (electron acceptor) were combined as a substrate. A comparison of 7D4C2 cultures with fructose and n-butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasing n-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 (undissociated n-caproic acid was ∼0.3 mM), the n-caproate selectivity (i.e., product per substrate fed) was 42.6 ± 19.0% higher compared to 7D4C2 cultures without extraction. Based on the 16S rRNA gene sequence, strain 7D4C2 is most closely related to the isolates Caproicibacter fermentans (99.5%) and Caproiciproducens galactitolivorans (94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2, C. fermentans, and C. galactitolivorans belong to the same genus of Caproiciproducens. 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 as Megasphaera spp. In conclusion, Caproiciproducens spp., comprising strain 7D4C2, C. fermentans, C. galactitolivorans, and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster. Caproiciproducens sp. 7D4C2 (DSM 110548) was studied here to understand n-caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars into n-caproate.

How Microbial Aggregates Protect against Nanoparticle Toxicity.

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.

Anaerobic carbon monoxide metabolism by Pleomorphomonas carboxyditropha sp. nov., a new mesophilic hydrogenogenic carboxydotroph.

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).

Microorganisms-based methods for harmful algal blooms control: A review.

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.

Uncovering the flocculating potential of extracellular polymeric substances produced by periphytic biofilms.

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 491.8mg/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%, 10min) and efficient aniline blue (AB) removal capacity (56.46±1.41%, 30min). 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.

Impact of carbon monoxide partial pressures on methanogenesis and medium chain fatty acids production during ethanol fermentation.

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 effects of CO2 and H2 on CO metabolism by pure and mixed microbial cultures.

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.

Coupling Bioflocculation of Dehalococcoides mccartyi to High-Rate Reductive Dehalogenation of Chlorinated Ethenes.

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 trichloroethene, trichloroethene removal was sustained at >99.6%. Electron micrographs revealed that D. mccartyi were abundant within the bioflocs, not only in multispecies structures, but also as self-aggregated microcolonies. This study provides fundamental evidence toward the feasibility of upflow bioreactors containing D. mccartyi as high-density culture production tools or as a high-rate, real-time remediation biotechnology.

Glucose triggers the cytotoxicity of Citrobacter sp. R1 against Microcystis aeruginosa.

Algicidal bacteria offer a promising option for killing Microcystis aeruginosa, one notorious cyanobacteria causing harmful algal blooms. In this study, Citrobacter sp. R1 presented high algicidal activity (81.6±2.2%, 72h) against M. aeruginosa when cultured using glucose, while it showed no algicidal activity (0±3.4%) when cultured using wheat bran, suggesting that appropriate carbon source is crucial for algicidal bacteria in killing M. aeruginosa. The underlying algicidal mechanism of strain R1 was explored by studying the effect of different carbon sources (glucose and wheat bran) on its key algicidal gene expression and total protein translation. While the glycogen synthase gene (glgA), cloned from strain R1 via transposon mutagenesis, was for the first time related to algicidal activity, its transcriptional level was not positively correlated with the algicidal activity of strain R1. We found that, the translation of total protein of strain R1 was relatively less when cultured with glucose, compared to growth with wheat bran. This indicated that the functional algicidal gene of strain R1 exerts its algicidal activity at protein translational level. These findings not only reveal the importance of appropriate carbon source for strain R1 for controlling M. aeruginosa, but also bring insights into its underlying algicidal mechanism.

Evolution of microbial communities growing with carbon monoxide, hydrogen, and carbon dioxide.

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.

Archaea and Bacteria Acclimate to High Total Ammonia in a Methanogenic Reactor Treating Swine Waste.

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 H2 accumulation. As the anaerobic microbial community adapted to the gradual increase of total ammonia-N (NH3-N) from 890 ± 295 to 2040 ± 30 mg/L, the Bacterial and Archaeal communities became less diverse. Phylotypes most closely related to hydrogenotrophic Methanoculleus (36.4%) and Methanobrevibacter (11.6%), along with acetoclastic Methanosaeta (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, and Sphaerochaeta) 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.