Anaerobic Enrichment Cultures Research Articles

Description and genome analysis of a novel archaeon isolated from a syntrophic pyrite-forming enrichment culture and reclassification of Methanospirillum hungatei strains GP1 and SK as Methanospirillum purgamenti sp. nov.

The archaeal isolate J.3.6.1-F.2.7.3T was obtained from an anaerobic enrichment culture, where it may play an important role in methane production during pyrite formation. The new isolate formed a species-level clade with Methanospirillum hungatei strains GP1 and SK, which is separate from the type strain JF-1T. Cultivation-independent surveys indicate the occurrence of this phylogenetic group in sediments and anaerobic digesters. The abundance of this clade appears to be negatively affected by high nitrogen loads, indicating a sensitivity to certain nitrogen compounds that is not known in M. hungatei JF-1T. The relatively large core genome of this Methanospirillum clade is indicative of niche specialization and efficient control of horizontal gene transfer. Genes for nitrogenase and F420-dependent secondary alcohol dehydrogenase contribute to the metabolic versatility of this lineage. Characteristics of the new isolate such as the ability to utilize 2-propanol as an electron donor or the requirement for acetate as a carbon source are found also in the strains GP1 and SK, but not in the type strain M. hungatei JF-1T. Based on the genomic differences to related species, a new species within the genus Methanospirillum is proposed with the name M. purgamenti sp. nov. The determined phenotypic characteristics support this proposal and indicate a metabolic adaptation to a separate ecological niche.

Enrichment of anaerobic and facultative anaerobic bacteria from a graphite mine sample on graphene oxide

Graphene oxide (GO) is progressively synthesized and applied in various industrial fields, such as healthcare, medicine, gas transport, electric power industry, etc. Thus, its distribution in the environment increases, which leads to changes in various microbial communities. Mechanisms of interaction between bacterial communities and novel carbon-based nanomaterials, such as GO, are still to be elucidated. We developed stable consortia in order to identify bacteria with the ability to thrive in the presence of GO. Here, we show the results of metagenomic analyses of the graphite mine sample, a consortium with GO as a sole source of carbon and a consortium with fumarate as an additional carbon source. The aim of this study was to establish anaerobic enrichment cultures from a graphite mine sample and GO, and to identify their diversity/community composition.

Genome-Centric Metatranscriptomic Characterization of a Humin-Facilitated Anaerobic Tetrabromobisphenol A-Dehalogenating Consortium.

Tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant in electronics manufacturing, has caused global contamination due to improper e-waste disposal. Its persistence, bioaccumulation, and potential carcinogenicity drive studies of its transformation and underlying (a)biotic interactions. This study achieved an anaerobic enrichment culture capable of reductively dehalogenating TBBPA to the more bioavailable bisphenol A. 16S rRNA gene amplicon sequencing and quantitative PCR confirmed that successive dehalogenation of four bromide ions from TBBPA was coupled with the growth of both Dehalobacter sp. and Dehalococcoides sp. with growth yields of 5.0 ± 0.4 × 108 and 8.6 ± 4.6 × 108 cells per μmol Br- released (N = 3), respectively. TBBPA dehalogenation was facilitated by solid humin and reduced humin, which possessed the highest organic radical signal intensity and reducing groups -NH2, and maintained the highest dehalogenation rate and dehalogenator copies. Genome-centric metatranscriptomic analyses revealed upregulated putative TBBPA-dehalogenating rdhA (reductive dehalogenase) genes with humin amendment, cprA-like Dhb_rdhA1 gene in Dehalobacter species, and Dhc_rdhA1/Dhc_rdhA2 genes in Dehalococcoides species. The upregulated genes of lactate fermentation, de novo corrinoid biosynthesis, and extracellular electron transport in the humin amended treatment also stimulated TBBPA dehalogenation. This study provided a comprehensive understanding of humin-facilitated organohalide respiration.

Identification of acacia gum fermenting bacteria from pooled human feces using anaerobic enrichment culture.

Commercial acacia gum (AG) used in this study is a premium-grade free-flowing powder. It is a gummy exudate composed of arabinogalactan branched polysaccharide, a biopolymer of arabinose and galactose. Also known as food additive, acacia gum (E414), which is presently marketed as a functional dietary fiber to improve overall human gut health. The health effects may be related to the luminal pH regulation from the short-chain fatty acids (SCFA) production. Studies suggested that amylolytic and butyrogenic pathways are the major factors determining the SCFA outcome of AG in the lower gut. However, the primary bacteria involved in the fermentation have not been studied. This study aimed to investigate the putative primary degraders of acacia gum in the gut ecosystem. Isolation and identification of gum-fermenting bacteria were performed through enrichment culture fermentation. The experiment was conducted in an anaerobic chamber for 144 h in three stages. The study was conducted in triplicate using an anaerobic chamber system. This culture system allows specific responses to support only bacteria that are responsible for gum fermentation among the gut microbiota. Five bacterial strains were isolated and found to be gum-fermenting bacteria. Based on the 16s RNA sequence, the isolates matched to butyrate-producing Escherichia fergusonii, ATCC 35469.

New dechlorination products and mechanisms of tris(2-chloroethyl) phosphate by an anaerobic enrichment culture from a vehicle dismantling site

End-of-life vehicles (ELVs) dismantling sites are the notorious hotspots of chlorinated organophosphate esters (Cl-OPEs). However, the microbial-mediated dechlorination of Cl-OPEs at such sites has not yet been explored. Herein, the dechlorination products, pathways and mechanisms of tris(2-chloroethyl) phosphate (TCEP, a representative Cl-OPE) by an anaerobic enrichment culture (ZNE) from an ELVs dismantling plant were investigated. Our results showed that dechlorination of TCEP can be triggered by reductive transformation to form bis(2-chloroethyl) phosphate (BCEP), mono-chloroethyl phosphate (MCEP) and by hydrolytic dechlorination to form bis(2-chloroethyl) 2-hydroxyethyl phosphate (TCEP-OH), 2-chloroethyl bis(2-hydroxyethyl) phosphate (TCEP-2OH), 2-chloroethyl (2-hydroxyethyl) hydrogen phosphate (BCEP-OH). The combination of 16S rRNA gene amplicon sequencing, quantitative real-time PCR (qPCR) and metagenomics revealed that the Dehalococcoides played an important role in the reductive transformation of TCEP to BCEP and MCEP. A high-quality metagenome-assembled genome (completeness >99% and contamination <1%) of Dehalococcoides was obtained. The sulfate-reducing bacteria harboring haloacid dehalogenase genes (had) may be responsible for the hydrolytic dechlorination of TCEP. These findings provide insights into microbial-mediated anaerobic transformation products and mechanisms of TCEP at ELVs dismantling sites, having implications for the environmental fate and risk assessment of Cl-OPEs at those sites.

Biodegradation of the emerging contaminant 3-nitro-1,2,4-triazol-5-one and its product 3-amino-1,2,4-triazol-5-one in perlite/soil columns

3-Nitro-1,2,4-triazol-5-one (NTO) is an ingredient of new safer-to-handle military insensitive munitions formulations. NTO can be microbially reduced to 3-amino-1,2,4-triazol-5-one (ATO) under anaerobic conditions if an electron donor is available. Conversely, ATO can undergo aerobic biodegradation. Previously, our research group developed an anaerobic enrichment culture that reduces NTO to ATO. A second culture could aerobically mineralize ATO. This study aimed to combine anaerobic/aerobic conditions within a down-flow perlite/soil column for simultaneous NTO reduction and ATO oxidation. Acetate biostimulation was investigated to promote oxygen depletion and create anaerobic micro-niches for NTO reduction, whereas perlite increased soil porosity and oxygen convection, allowing ATO oxidation. Two columns packed with a perlite/soil mixture (70:30, wet wt.%) or 100% perlite were operated aerobically and inoculated with the NTO- and ATO-degrading cultures. Initially, the influent consisted of ∼280 μM ATO, and after 30 days, the feeding was switched to ∼260 μM NTO and ∼250 μM acetate. By progressively increasing acetate from 250 to 4000 μM, the NTO removal gradually improved in both columns. The perlite/soil column reached a 100% NTO removal after 4000 μM acetate was supplemented. Additionally, there was no ATO accumulation, and inorganic nitrogen was produced, indicating ATO mineralization. Although NH4+ was produced following ATO oxidation, most nitrogen was recovered as NO3− likely via nitrification reactions. Microbial community analysis revealed that phylotypes hosted in the enrichment cultures specialized in NTO reduction (e.g., Geobacter) and ATO oxidation (e.g., Hydrogenophaga, Ramlibacter, Terrimonas, and Pseudomonas) were established in the columns. Besides, the predominant genera (Azohydromonas, Zoogloea, and Azospirillum) are linked to nitrogen cycling by performing nitrogen fixation, NO3− reduction, and nitroaromatics degradation. This study applied a bulking agent (perlite) and acetate biostimulation to achieve simultaneous NTO reduction and ATO oxidation in a single column. Such a strategy can assist with real-world applications of NTO and ATO biodegradation mechanisms.

Dual C-Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures.

Brominated organic compounds such as 1,2-dibromoethane (1,2-DBA) are highly toxic groundwater contaminants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C-Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide-respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εbulkC values (-1.8 ± 0.2 and -19.2 ± 3.5‰, respectively) were obtained, whereas their respective εbulkBr values were lower and similar to each other (-1.22 ± 0.08 and -1.2 ± 0.5‰), leading to distinctly different trends (ΛC-Br = Δδ13C/Δδ81Br ≈ εbulkC/εbulkBr) in a dual C-Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2-reaction). The strongly pathway-dependent ΛC-Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εbulkC values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites.

Phylogeny and Physiological Diversity of Cold-adapted Anaerobic Bacteria Isolated from Rice Field Soil in Japan.

Cold-adapted or psychrotrophic fermentative anaerobic bacteria were isolated from rice field soil in a temperate area in Japan using anaerobic enrichment cultures incubated at 5°C. Most isolates were obligately anaerobic, spore-forming rods and affiliated with different lineages of the genus Clostridium based on 16S rRNA gene sequences. The growth temperature ranges and physiological properties of three representative clostridial isolates (C5S7, C5S11T, and C5S18) were examined. Strain C5S7 grew at 0°C, but not at 20°C, and was identified as Clostridium estertheticum, a psychrophile isolated from spoiled, vacuum-packed, chilled meat (blown pack spoilage, BPS). Strain C5S7 produced butyrate, n-butanol, and abundant gases (H2 and CO2) as major fermentation products from the carbohydrates utilized. Strain C5S11T, which was recently described as Clostridium gelidum sp. nov., possessed psychrotrophic properties and grew at temperatures between 0 and 25°C. Strain C5S11T was saccharolytic, decomposed polysaccharides, such as inulin, pectin, and xylan, and produced acetate, butyrate, and gases. Strain C5S18 also grew at 0°C and the optimum growth temperature was 15°C. Strain C5S18 did not ferment carbohydrates and grew in a manner that was dependent on proteinaceous substrates. This strain was identified as the psychrotolerant species, Clostridium tagluense, originally isolated from a permafrost sample. Collectively, the present results indicate that psychrotrophic anaerobic bacteria with different physiological properties actively degrade organic matter in rice field soil, even in midwinter, in a cooperative manner using different substrates. Furthermore, different psychrotrophic species of the genus Clostridium with the ability to cause BPS inhabit cultivated soil in Japan.

Elucidating the mechanisms associated with the anaerobic biotransformation of the emerging contaminant nitroguanidine

Nitroguanidine (NQ) is a constituent of gas generators for automobile airbags, smokeless pyrotechnics, insecticides, propellants, and new insensitive munitions formulations applied by the military. During its manufacture and use, NQ can easily spread in soils, ground, and surface waters due to its high aqueous solubility. Very little is known about the microbial biotransformation of NQ. This study aimed to elucidate important mechanisms operating during NQ anaerobic biotransformation. To achieve this goal, we developed an anaerobic enrichment culture able to reduce NQ to nitrosoguanidine (NsoQ), which was further abiotically transformed to cyanamide. Effective electron donors for NQ biotransformation were lactate and, to a lesser extent, pyruvate. The results demonstrate that the enrichment process selected a sulfate-reducing culture that utilized lactate as its electron donor and sulfate as its electron acceptor while competing with NQ as an electron sink. A unique property of the culture was its requirement for exogenous nitrogen (e.g., from yeast extract or NH4Cl) for NQ biotransformation since NQ itself did not serve as a nitrogen source. The main phylogenetic groups associated with the NQ-reducing culture were sulfate-reducing and fermentative bacteria, namely Cupidesulfovibrio oxamicus (63.1% relative abundance), Dendrosporobacter spp. (12.0%), and Raoultibacter massiliens (10.9%). The molecular ecology results corresponded to measurable physiological properties of the most abundant members. The results establish the conditions for NQ anaerobic biotransformation and the microbial community associated with the process, improving our present comprehension of NQ environmental fate and assisting the development of NQ remediation strategies.

Composition of Prokaryotic Communities in Anaerobic Enrichment Cultures of Crude Oil from Romashkino Oilfield (Russia)

Despite the many papers published on oilfield ecosystems, there is a lack of data about microbial community structure and functioning in many crude oils or oilfields and wells. One of such poorly studied oilfields is supergiant Romashkino situated in Tatarstan (Russia). In the present study, prokaryotic community composition and abundance of functional genes encoding sulfate reduction and rhamnolipid production, was investigated in enrichment cultures of four samples from Romashkino oilfield production wells with different oil/water ratios. The enrichment was conducted under anaerobic conditions, sulfate and nitrate were used as electron acceptors, and acetate – as an electron donor. In enrichment culture A and enrichment culture B, bacteria of the Pseudomonadaceae family predominated with 99% and 93% relative abundance, respectively. In enrichment culture C (obtained on the basis of initial sample contained oil with medium water content) and D (obtained on the basis of initial sample contained oil with very high water content), Pseudomonadaceae were one of the two predominating families, along with Micrococcaceae (enrichment culture C) and Dietziaceae (enrichment culture D). The proportion of dsrA and apsA genes encoding sulfate reduction to 16S rRNA gene copy numbers was quite low and ranged between 0.003% (enrichment cultures A, B) and 0.027% (enrichment culture D). The proportion of rhlB gene encoding rhamnolipid production from the total 16S rRNA gene copy number was 1.10% in sample A and 0.06% in sample B. In enrichment cultures C and D the rhlB gene was not found.

Organohalide Respiration with Diclofenac by Dehalogenimonas.

Diclofenac (DCF) is a pharmaceutically active contaminant frequently found in aquatic ecosystems. The transformation pathways and microbiology involved in the biodegradation of DCF, particularly under anoxic conditions, remain poorly understood. Here, we demonstrated microbially mediated reductive dechlorination of DCF in anaerobic enrichment culture derived from contaminated river sediment. Over 90% of the initial 76.7 ± 3.6 μM DCF was dechlorinated at a maximum rate of 1.8 ± 0.3 μM day-1 during a 160 days' incubation. Mass spectrometric analysis confirmed that 2-(2-((2-chlorophenyl)amino)phenyl)acetic acid (2-CPA) and 2-anilinophenylacetic acid (2-APA) were formed as the monochlorinated and nonchlorinated DCF transformation products, respectively. A survey of microbial composition and Sanger sequencing revealed the enrichment and dominance of a new Dehalogenimonas population, designated as Dehalogenimonas sp. strain DCF, in the DCF-dechlorinating community. Following the stoichiometric conversion of DCF to 2-CPA (76.0 ± 2.1 μM) and 2-APA (3.7 ± 0.8 μM), strain DCF cell densities increased by 24.4 ± 4.4-fold with a growth yield of 9.0 ± 0.1 × 108 cells per μmol chloride released. Our findings expand the metabolic capability in the genus Dehalogenimonas and highlight the relevant roles of organohalide-respiring bacteria for the natural attenuation of halogenated contaminants of emerging concerns (e.g., DCF).

Trickle-Bed Bioreactors for Acetogenic H2/CO2 Conversion

Acetic acid is an essential industrial building block and can be produced by acetogenic bacteria from molecular hydrogen (H2) and carbon dioxide (CO2). When gasses are supplied as substrates, bioreactor design plays an important role for their availability. Trickle-bed bioreactors (TBs) have an enhanced gas-to-liquid mass transfer and cells remain in the system by forming a biofilm on the carriers. So far, TBs have been investigated extensively for bio-methanation processes, whereas studies for their use in acetic acid production are rare. In this study, we evaluated the reproducibility of two parallel TBs for acetic acid production from H2:CO2(= 70:30) by a mixed culture with a gas flow rate of 3.8 mL min−1and a medium flow rate of 10 mL min−1. Additionally, the effect of glucose addition during the starting phase on the resulting products and microbial composition was investigated by setting up a third TB2. Partial medium exchanges to decrease the internal acetic acid concentration (AAC) combined with recycling of withdrawn cells had a positive impact on acetic acid production rates with maxima of around 1 g L−1d−1even at high AACs of 19–25 g L−1. Initial glucose addition resulted in the accumulation of unwanted butyric acid up to concentrations of 2.60 ± 0.64 g L−1. The maximum AAC of 40.84 g L−1was obtained without initial glucose addition. The main families identified in the acetogenic TBs were Peptococcaceae, Ruminococcaceae, Planococcaceae, Enterobacteriaceae, Clostridiaceae, Lachnospiraceae, Dysgonomonadaceae and Tannerellaceae. We conclude that a TB is a viable solution for conversion of H2/CO2to acetate using an anaerobic enrichment culture.

Metagenomic Investigation of a Low Diversity, High Salinity Offshore Oil Reservoir.

Oil reservoirs can represent extreme environments for microbial life due to low water availability, high salinity, high pressure and naturally occurring radionuclides. This study investigated the microbiome of saline formation water samples from a Gulf of Mexico oil reservoir. Metagenomic analysis and associated anaerobic enrichment cultures enabled investigations into metabolic potential for microbial activity and persistence in this environment given its high salinity (4.5%) and low nutrient availability. Preliminary 16S rRNA gene amplicon sequencing revealed very low microbial diversity. Accordingly, deep shotgun sequencing resulted in nine metagenome-assembled genomes (MAGs), including members of novel lineages QPJE01 (genus level) within the Halanaerobiaceae, and BM520 (family level) within the Bacteroidales. Genomes of the nine organisms included respiratory pathways such as nitrate reduction (in Arhodomonas, Flexistipes, Geotoga and Marinobacter MAGs) and thiosulfate reduction (in Arhodomonas, Flexistipes and Geotoga MAGs). Genomic evidence for adaptation to high salinity, withstanding radioactivity, and metal acquisition was also observed in different MAGs, possibly explaining their occurrence in this extreme habitat. Other metabolic features included the potential for quorum sensing and biofilm formation, and genes for forming endospores in some cases. Understanding the microbiomes of deep biosphere environments sheds light on the capabilities of uncultivated subsurface microorganisms and their potential roles in subsurface settings, including during oil recovery operations.

Accelerated adsorption of tetracyclines and microbes with FeOn(OH)m modified oyster shell: Its application on biotransformation of oxytetracycline in anaerobic enrichment culture

Due to the persistence in residue and the emission to environment, antibiotics have become the emerging contaminants. Tetracyclines, as the largest amount of antibiotics in the breeding industry, were investigated in this study. For eliminating the tetracyclines, anaerobic culture was enriched from livestock waste after four generations and FeOn(OH)m modified oyster shell powders (OSPs) were amended into the enrichment culture as auxiliary method. Tetracycline and oxytetracycline were selected as the model compounds. The adsorption models (Langmuir and Freundlich isotherms) of tetracycline and microbes on OSPs were simulated respectively. With the addition of FeOn(OH)m modified OSPs, the transformation efficiency of oxytetracycline achieved 81.5% after 22 days’ incubation, approximately 1.85 times higher than the 4th generation (G-IV) enrichment culture without modified OSPs. Dehydration, demethylation, dehydroxylation and decarboxylation took place under anaerobic condition, inferred from the detected metabolites transformed from oxytetracycline. Genus Methanosarcina and two operational taxonomic units (OTUs) assigned to order Clostridiales, showed an increasing trend on abundance, indicating the contribution to transformation of oxytetracycline. Therefore, the FeOn(OH)m modified OSPs and enrichment culture can accelerate the transformation of oxytetracycline simultaneously, indicating a promising utilization not only for waste water system but also for antibiotic contaminated biomass treatment.

Halo(natrono)archaea from hypersaline lakes can utilize sulfoxides other than DMSO as electron acceptors for anaerobic respiration.

Dimethylsulfoxide (DMSO) has long been known to support anaerobic respiration in a few species of basically aerobic extremely halophilic euryarchaea living in hypersaline lakes. Recently, it has also been shown to be utilized as an additional electron acceptor in basically anaerobic sulfur-reducing haloarchaea. Here we investigated whether haloarchaea would be capable of anaerobic respiration with other two sulfoxides, methionine sulfoxide (MSO) and tetramethylene sulfoxide (TMSO). For this, anaerobic enrichment cultures were inoculated with sediments from hypersaline salt and soda lakes in southwestern Siberia and southern Russia. Positive enrichments were obtained for both MSO and TMSO with yeast extract but not with formate or acetate as the electron donor. Two pure cultures obtained from salt lakes, either with MSO or TMSO, were obligate anaerobes closely related to sulfur-reducing Halanaeroarchaeum sulfurireducens, although the type strain of this genus was unable to utilize any sulfoxides. Two pure cultures isolated from soda lakes were facultatively anaerobic alkaliphilic haloarchaea using O2, sulfur and sulfoxides as the electron acceptors. One isolate was identical to the previously described sulfur-reducing Natrarchaeobaculum sulfurireducens, while another one, enriched at lower alkalinity, is forming a new species in the genus Halobiforma. Since all isolates enriched with either MSO or TMSO were able to respire all three sulfoxides including DMSO and the corresponding activities were cross-induced, it suggest that a single enzyme of the DMSO-reductase family with a broad substrate specificity is responsible for various sulfoxide-dependent respiration in haloarchaea.

Reductive Dechlorination of 1,2-dichloroethane to Ethylene by Anaerobic Enrichment Culture Containing Vulcanibacillus spp.

Bioremediation has been widely used for clean-up of 1,2-dichloroethane (DCA) at contaminated sites. Only a small number of specific anaerobes, halorespiring bacteria (HRB), have been reported to degrade DCA. The goals of this research were the screening and isolation of HRB with capable dechlorination of DCA. HRB were screened and isolated from 7 enrichment cultures (ES1-ES7), from DCA-contaminated soils, on bicarbonate-buffered basal salts medium containing 10 mM acetate and 250 µM DCA under anaerobic conditions and analyzed by gas chromatography. The results showed that the mixed cultures of ES3 and ES5 could reductively dechlorinate DCA to ethylene via a direct reductive dihaloelimination pathway. In particular, ES5 showed rapid transformation of 250 µM DCA to ethylene within 6 days at 30 °C. Specific microbial populations of Vulcanibacillus spp., elucidated by polymerase chain reaction-denaturing gradient gel electrophoresis, were found only in ES3 and ES5, which was related to reductive dihaloelimination activities on DCA. A 16S rRNA gene analysis of isolate es5d8 from mixed culture ES5 revealed 2 strains of Vulcanibacillus spp. (KKU-DCA1 and KKU-DCA2) concerned with dechlorinating DCA. These findings suggested that Vulcanibacillus spp. were HRB that have the potential for detoxifying DCA.

Novel dichloromethane-fermenting bacteria in the Peptococcaceae family

Dichloromethane (DCM; CH2Cl2) is a toxic groundwater pollutant that also has a detrimental effect on atmospheric ozone levels. As a dense non-aqueous phase liquid, DCM migrates vertically through groundwater to low redox zones, yet information on anaerobic microbial DCM transformation remains scarce due to a lack of cultured organisms. We report here the characterisation of DCMF, the dominant organism in an anaerobic enrichment culture (DFE) capable of fermenting DCM to the environmentally benign product acetate. Stable carbon isotope experiments demonstrated that the organism assimilated carbon from DCM and bicarbonate via the Wood–Ljungdahl pathway. DCMF is the first anaerobic DCM-degrading population also shown to metabolise non-chlorinated substrates. It appears to be a methylotroph utilising the Wood–Ljungdahl pathway for metabolism of methyl groups from methanol, choline, and glycine betaine. The flux of these substrates from subsurface environments may either directly (DCM, methanol) or indirectly (choline, glycine betaine) affect the climate. Community profiling and cultivation of cohabiting taxa in culture DFE without DCMF suggest that DCMF is the sole organism in this culture responsible for substrate metabolism, while the cohabitants persist via necromass recycling. Genomic and physiological evidence support placement of DCMF in a novel genus within the Peptococcaceae family, ‘Candidatus Formimonas warabiya’.

A multi-omic screening approach for the discovery of thermoactive glycoside hydrolases.

Next-generation sequencing and computational biology have facilitated the implementation of new combinatorial screening approaches to discover novel enzymes of biotechnological interest. In this study, we describe the successful establishment of a multi-omic approach for the identification of thermostable hydrolase-encoding genes by determination of gene expression levels. We applied this combinatorial approach using an anaerobic enrichment culture from an Azorean hot spring sample grown on green coffee beans as recalcitrant substrate. An in-depth analysis of the microbial community resulted in microorganisms capable of metabolizing the selected substrate, such as the genera Caloramator,Dictyoglomus and Thermoanaerobacter as active and abundant microorganisms. To discover glycoside hydrolases, 90,342 annotated genes were screened for specific reaction types. A total number of 106 genes encoding cellulases (EC 3.2.1.4), beta-glucosidases (EC 3.2.1.21) and endo-1,4-beta-mannosidases (EC 3.2.1.78) were selected. Mapping of RNA-Seq reads to the related metagenome led to expression levels for each gene. Amongst those, 14 genes, encoding glycoside hydrolases, showed highest expression values, and were used for further cloning. Four proteins were biochemically characterized and were identified as thermoactive glycoside hydrolases with a broad substrate range. This work demonstrated that a combinatory omic approach is a suitable strategy identifying unique thermoactive enzymes from environmental samples.

Selecting for lactic acid producing and utilising bacteria in anaerobic enrichment cultures.

Lactic acid‐producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology‐based process design and can aid the development of sustainable and energy‐efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose‐based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid‐producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.

"Candidatus Galacturonibacter soehngenii" Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex.

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of “Candidatus Galacturonibacter soehngenii,” from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of “Ca. G. soehngenii” was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the “Ca. G. soehngenii” MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of “Ca. G. soehngenii” enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of “Ca. G. soehngenii.”