Process conditions affect microbial diversity and activity in a haloalkaline biodesulfurization system.

Biodesulfurization (BD) systems that treat sour gas employ mixtures of haloalkaliphilic sulfur-oxidizing bacteria to convert sulfide to elemental sulfur. In the past years, these systems have seen major technical innovations that have led to changes in microbial community composition. Different studies have identified and discussed the microbial communities in both traditional and improved systems. However, these studies do not identify metabolically active community members and merely focus on members' presence/absence. Therefore, their results cannot confirm the activity and role of certain bacteria in the BD system. To investigate the active community members, we determined the microbial communities of six different runs of a pilot-scale BD system. 16S rRNA gene-based amplicon sequencing was performed using both DNA and RNA. A comparison of the DNA- and RNA-based sequencing results identified the active microbes in the BD system. Statistical analyses indicated that not all the existing microbes were actively involved in the system and that microbial communities continuously evolved during the operation. At the end of the run, strains affiliated with Alkalilimnicola ehrlichii and Thioalkalivibrio sulfidiphilus were confirmed as the most active key bacteria in the BD system. This study determined that microbial communities were shaped predominantly by the combination of hydraulic retention time (HRT) and sulfide concentration in the anoxic reactor and, to a lesser extent, by other operational parameters.IMPORTANCEHaloalkaliphilic sulfur-oxidizing bacteria are integral to biodesulfurization (BD) systems and are responsible for converting sulfide to sulfur. To understand the cause of conversions occurring in the BD systems, knowing which bacteria are present and active in the systems is essential. So far, only a few studies have investigated the BD system's microbial composition, but none have identified the active microbial community. Here, we reveal the metabolically active community, their succession, and their influence on product formation.

How microbial glycosyl hydrolase activity in the gut mucosa initiates microbial cross-feeding.

The intestinal epithelium is protected from direct contact with gut microbes by a mucus layer. This mucus layer consists of secreted mucin glycoproteins. The outer mucus layer in the large intestine forms a niche that attracts specific gut microbiota members of which several gut commensals can degrade mucin. Mucin glycan degradation is a complex process that requires a broad range of glycan degrading enzymes, as mucin glycans are intricate and diverse molecules. Consequently, it is hypothesized that microbial mucin breakdown requires concerted action of various enzymes in a network of multiple resident microbes in the gut mucosa. This review investigates the evolutionary relationships of microbial carbohydrate-active enzymes that are potentially involved in mucin glycan degradation and focuses on the role that microbial enzymes play in the degradation of gut mucin glycans in microbial cross-feeding and syntrophic interactions.

Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes.

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na+). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as Nε-acetyl-ß-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments. IMPORTANCE Using genome-centric metagenomics, we discovered a new Methanothrix harundinacea subspecies that appears to be a halotolerant acetoclastic methanogen with the flexibility for adaptation in the anaerobic digestion process both at low (5 g/L Na+) and high salinity conditions (20 g/L Na+). Annotation of the recovered M. harundinacea genome revealed salinity stress-related functions, including the modification of EPS glycoconjugates and the production of compatible solutes. This is the first study reporting these genomic features within a Methanothrix sp., a milestone further supporting previous studies that identified M. harundinacea as a key-driver in anaerobic granulation under high salinity stress.

Comparative analysis of microbial communities from different full-scale haloalkaline biodesulfurization systems.

In biodesulfurization (BD) at haloalkaline and dO2-limited conditions, sulfide-oxidizing bacteria (SOB) effectively convert sulfide into elemental sulfur that can be used in agriculture as a fertilizer and fungicide. Here we show which bacteria are present in this biotechnological process. 16S rRNA gene amplicon sequencing of biomass from ten reactors sampled in 2018 indicated the presence of 444 bacterial Amplicon Sequence Variants (ASVs). A core microbiome represented by 30 ASVs was found in all ten reactors, with Thioalkalivibrio sulfidiphilus as the most dominant species. The majority of these ASVs are phylogenetically related to bacteria previously identified in haloalkaline BD processes and in natural haloalkaline ecosystems. The source and composition of the feed gas had a great impact on the microbial community composition followed by alkalinity, sulfate, and thiosulfate concentrations. The halophilic SOB of the genus Guyparkeria (formerly known as Halothiobacillus) and heterotrophic SOB of the genus Halomonas were identified as potential indicator organisms of sulfate and thiosulfate accumulation in the BD process. KEY POINTS: • Biodesulfurization (BD) reactors share a core microbiome • The source and composition of the feed gas affects the microbial composition in the BD reactors • Guyparkeria and Halomonas indicate high concentrations of sulfate and thiosulfate in the BD process.

Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate, formate and/or hydrogen as electron donor.

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO2 fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO2 , and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.

Concurrent use of methanol and ethanol for chain-elongating short chain fatty acids into caproate and isobutyrate.

Microbial chain elongation (MCE) is a bioprocess that could utilise a mixed-culture fermentation to valorise organic waste. MCE converting ethanol and short chain fatty acids (SCFA; derived from organic waste) to caproate has been studied extensively and implemented. Recent studies demonstrated the conversion of SCFAs and methanol or ethanol into isomerised fatty acids as novel products, which may expand the MCE application and market. Integrating caproate and isomerised fatty acid production in one reactor system is theoretically feasible given the employment of a mixed culture and may increase the economic competence of MCE; however, the feasibility of such has never been demonstrated. This study investigated the feasibility of using two electron donors, i.e. methanol and ethanol, for upgrading SCFAs into isobutyrate and caproate concurrently in MCE Results show that supplying methanol and ethanol in MCE simultaneously converted acetate and/or butyrate into caproate and isobutyrate, by a mixed-culture microbiome. The butyrate supplement stimulated the caproate production rate from 1.5 to 2.6 g/L.day and induced isobutyrate production (1.5 g/L.day). Further increasing ethanol feeding rate from 140 to 280 mmol carbon per litre per day enhanced the direct use of butyrate for caproate production, which improved the caproate production rate to 5.9 g/L.day. Overall, the integration of two electron donors, i.e. ethanol and methanol, in one chain-elongation reactor system for upgrading SCFAs was demonstrated. As such, MCE could be applied to valorise organic waste (water) streams into a wider variety of value-added biochemical.

Propionibacterium ruminifibrarum sp. nov., isolated from cow rumen fibrous content.

A novel propionate producing bacterium, strain JV5T, was isolated from the rumen fibrous content of a Holstein Friesian dairy cow. Cells of strain JV5T were Gram-stain-positive, non-motile and aerotolerant. Growth occurred between 35 and 45 °C, with an optimum at 39 °C. The pH range for growth was 6.5-8, with an optimum at pH 7. The 16S rRNA gene sequence of strain JV5T was 98.4 and 96.5 % identical to those of Propionibacterium australiense DSM 15818T and Propionibacterium acidifaciens DSM 21887T, respectively. Genome wide average nucleotide identity and digital DNA-DNA hybridization values were 88.3 and 35.5 %, respectively, against P. australiense DSM 15818T. The G+C content of strain JV5T was 68.9  mol%. Strain JV5T did not produce urease and was able to metabolize glutamate, but not aspartate and glycine. Strain JV5T was able to ferment a range of substrates including certain simple and complex carbohydrates, sugar alcohols and amino acids. Chemotaxonomic analysis of strain JV5T revealed the presence of meso-diamino pimelic acid isomers similar those found in P. australiense, but different from P. acidifaciens. The observed major (>10 %) cellular fatty acids in strain JV5T (C18 : 1 ω9c, anteiso-C15 : 1, C16 : 0, C17 : 0 and C16 : 0 alcohol) were also different from those observed in P. australiense and P. acidifaciens. Based on these findings, a novel species is proposed within the genus Propionibacterium, Propionibacterium ruminifibrarum sp. nov. (type strain JV5T=DSM 106771T=TISTR 2629T).

Microbial Diversity and Organic Acid Production of Guinea Pig Faecal Samples.

The guinea pig (Cavia porcellus) or cavy is a grass-eating rodent. Its main diet consists of grass or hay, which comprises cellulose, hemicellulose, lignin and their derivatives. Here, the microbial diversity of faecal samples of two guinea pigs and microbial enrichments made with substrates, including starch waste and dried grass, were investigated along with organic acid production profiles. The microbial communities of the faecal samples were dominated by the phyla Bacteroidetes (40%) and Firmicutes (36%). Bacteroidales S24-7 (11% in Cavy 1 and 21% in Cavy 2) was the most abundant order. At genus level, many microorganisms remained unclassified. Different carbon sources were used for organic acid production in faecal enrichments. The dominant bacterial groups in the secondary enrichments with dried grass, starch waste and xylose were closely related to Prevotella and Blautia. Acetate was the predominant organic acid from all enrichments. The organic acid production profiles corresponded to a mixed acid fermentation but differed depending on the substrate. Eight phylogenetically different isolates were obtained, including a novel Streptococcus species, strain Cavy grass 6. This strain had a low abundance (1%) in one of the faecal samples but was enriched in the dried grass enrichment (3%). Cavy grass 6, a fast-growing heterolactic bacterium, ferments cellobiose to lactate, acetate, formate and ethanol. Our results show that cavy faecal samples can be applied as microbial source for organic acid production from complex organic substrates. The cavy gut contains many as-yet-uncultivated bacteria which may be appropriate targets for future studies.

Comparative proteome analysis of propionate degradation by Syntrophobacter fumaroxidans in pure culture and in coculture with methanogens.

Syntrophobacter fumaroxidans is a sulfate-reducing bacterium able to grow on propionate axenically or in syntrophic interaction with methanogens or other sulfate-reducing bacteria. We performed a proteome analysis of S. fumaroxidans growing with propionate axenically with sulfate or fumarate, and in syntrophy with Methanospirillum hungatei, Methanobacterium formicicum or Desulfovibrio desulfuricans. Special attention was put on the role of hydrogen and formate in interspecies electron transfer (IET) and energy conservation. Formate dehydrogenase Fdh1 and hydrogenase Hox were the main confurcating enzymes used for energy conservation. In the periplasm, Fdh2 and hydrogenase Hyn play an important role in reverse electron transport associated with succinate oxidation. Periplasmic Fdh3 and Fdh5 were involved in IET. The sulfate reduction pathway was poorly regulated and many enzymes associated with sulfate reduction (Sat, HppA, AprAB, DsrAB and DsrC) were abundant even at conditions where sulfate was not present. Proteins similar to heterodisulfide reductases (Hdr) were abundant. Hdr/Flox was detected in all conditions while HdrABC/HdrL was exclusively detected when sulfate was available; these complexes most likely confurcate electrons. Our results suggest that S. fumaroxidans mainly used formate for electron release and that different confurcating mechanisms were used in its sulfidogenic metabolism.

Controlling Ethanol Use in Chain Elongation by CO2 Loading Rate.

Chain elongation is an open-culture biotechnological process which converts volatile fatty acids (VFAs) into medium chain fatty acids (MCFAs) using ethanol and other reduced substrates. The objective of this study was to investigate the quantitative effect of CO2 loading rate on ethanol usages in a chain elongation process. We supplied different rates of CO2 to a continuously stirred anaerobic reactor, fed with ethanol and propionate. Ethanol was used to upgrade ethanol itself into caproate and to upgrade the supplied VFA (propionate) into heptanoate. A high CO2 loading rate (2.5 LCO2·L-1·d-1) stimulated excessive ethanol oxidation (EEO; up to 29%) which resulted in a high caproate production (10.8 g·L-1·d-1). A low CO2 loading rate (0.5 LCO2·L-1·d-1) reduced EEO (16%) and caproate production (2.9 g·L-1·d-1). Heptanoate production by VFA upgrading remained constant (∼1.8 g·L-1·d-1) at CO2 loading rates higher than or equal to 1 LCO2·L-1·d-1. CO2 was likely essential for growth of chain elongating microorganisms while it also stimulated syntrophic ethanol oxidation. A high CO2 loading rate must be selected to upgrade ethanol (e.g., from lignocellulosic bioethanol) into MCFAs whereas lower CO2 loading rates must be selected to upgrade VFAs (e.g., from acidified organic residues) into MCFAs while minimizing use of costly ethanol.

Reverse Methanogenesis and Respiration in Methanotrophic Archaea.

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., "trace methane oxidation"). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.

Inhibitory Effect of Coumarin on Syntrophic Fatty Acid-Oxidizing and Methanogenic Cultures and Biogas Reactor Microbiomes.

Coumarins are widely found in plants as natural constituents having antimicrobial activity. When considering plants that are rich in coumarins for biogas production, adverse effects on microorganisms driving the anaerobic digestion process are expected. Furthermore, coumarin derivatives, like warfarin, which are used as anticoagulating medicines, are found in wastewater, affecting its treatment. Coumarin, the structure common to all coumarins, inhibits the anaerobic digestion process. However, the details of this inhibition are still elusive. Here, we studied the impact of coumarin on acetogenesis and methanogenesis. First, coumarin was applied at four concentrations between 0.25 and 1 g · liter-1 to pure cultures of the methanogens Methanosarcina barkeri and Methanospirillum hungatei, which resulted in up to 25% less methane production. Acetate production of syntrophic propionate- and butyrate-degrading cultures of Syntrophobacter fumaroxidans and Syntrophomonas wolfei was inhibited by 72% at a coumarin concentration of 1 g · liter-1 Coumarin also inhibited acetogenesis and acetoclastic methanogenesis in a complex biogas reactor microbiome. When a coumarin-adapted microbiome was used, acetogenesis and methanogenesis were not inhibited. According to amplicon sequencing of bacterial 16S rRNA genes and mcrA genes, the communities of the two microbiomes were similar, although Methanoculleus was more abundant and Methanobacterium less abundant in the coumarin-adapted than in the nonadapted microbiome. Our results suggest that well-dosed feeding with coumarin-rich feedstocks to full-scale biogas reactors while keeping the coumarin concentrations below 0.5 g · liter-1 will allow adaptation to coumarins by structural and functional community reorganization and coumarin degradation.IMPORTANCE Coumarins from natural and anthropogenic sources have an inhibitory impact on the anaerobic digestion process. Here, we studied in detail the adverse effects of the model compound coumarin on acetogenesis and methanogenesis, which are two important steps of the anaerobic digestion process. Coumarin concentrations lower than 0.5 g · liter-1 had only a minor impact. Even though similar inhibitory effects can be assumed for coumarin derivatives, little effects on the anaerobic treatment of wastewater are expected where concentrations of coumarin derivatives are lower than 0.5 g · liter-1 However, when full-scale reactors are fed with coumarin-rich feedstocks, the biogas processes might be inhibited. Hence, these feedstocks should be utilized in a well-dosed manner or after adaptation of the microbial community.

Streptococcus caviae sp. nov., isolated from guinea pig faecal samples.

A novel cellobiose-degrading and lactate-producing bacterium, strain Cavy grass 6T, was isolated from faecal samples of guinea pigs (Cavia porcellus). Cells of the strain were ovalshaped, non-motile, non-spore-forming, Gram-stain-positive and facultatively anaerobic. The strain gr at 25-40 °C (optimum 37 °C) and pH 4.5-9.5 (optimum 8.0). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Cavy grass 6T belongs to the genus Streptococcus with its closest relative being Streptococcus devriesei CCUG 47155T with only 96.5 % similarity. Comparing strain Cavy grass 6T and Streptococcus devriesei CCUG 47155T, average nucleotide identity and level of digital DNA-DNA hybridization dDDH were only 86.9 and 33.3 %, respectively. Housekeeping genes groEL and gyrA were different between strain Cavy grass 6T and other streptococci. The G+C content of strain Cavy grass 6T was 42.6±0.3 mol%. The major (>10 %) cellular fatty acids of strain Cavy grass 6T were C16:0, C20 : 1ω9c and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Strain Cavy grass 6T ferment a range of plant mono- and disaccharides as well as polymeric carbohydrates, including cellobiose, dulcitol, d-glucose, maltose, raffinose, sucrose, l-sorbose, trehalose, inulin and dried grass extract, to lactate, formate, acetate and ethanol. Based on phylogenetic and physiological characteristics, Cavy grass 6T can be distinguished from other members of the genus Streptococcus. Therefore, a novel species of the genus Streptococcus, family Streptococcaceae, order Lactobacillales is proposed, Streptococcuscaviae sp. nov. (type strain Cavy grass 6T=TISTR 2371T=DSM 102819T).

Thiosulfate Conversion to Sulfide by a Haloalkaliphilic Microbial Community in a Bioreactor Fed with H2 Gas.

In industrial gas biodesulfurization systems, where haloalkaline conditions prevail, a thiosulfate containing bleed stream is produced. This bleed stream can be treated in a separate bioreactor by reducing thiosulfate to sulfide and recycling it. By performing treatment and recycling of the bleed stream, its disposal decreases and less caustics are required to maintain the high pH. In this study, anaerobic microbial thiosulfate conversion to sulfide in a H2/CO2 fed bioreactor operated at haloalkaline conditions was investigated. Thiosulfate was converted by reduction to sulfide as well as disproportionation to sulfide and sulfate. Formate production from H2/CO2 was observed as an important reaction in the bioreactor. Formate, rather than H2, might have been used as the main electron donor by thiosulfate/sulfate-reducing bacteria. The microbial community was dominated by bacteria belonging to the family Clostridiaceae most closely related to Tindallia texcoconensis. Bacteria phylogenetically related to known haloalkaline sulfate and thiosulfate reducers, thiosulfate-disproportionating bacteria, and remarkably sulfur-oxidizing bacteria were also detected. On the basis of the results, two approaches to treat the biodesulfurization waste stream are proposed: (i) addition of electron donor to reduce thiosulfate to sulfide and (ii) thiosulfate disproportionation without the need for an electron donor. The concept of application of solely thiosulfate disproportionation is discussed.

Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation.

Since the discovery that anaerobic methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane coupled to sulfate reduction in marine sediments, different primers and probes specifically targeting the 16S rRNA gene of these archaea have been developed. Microbial investigation of the different ANME subtypes (ANME-1; ANME-2a, b, and c; and ANME-3) was mainly done in sediments where specific subtypes of ANME were highly enriched and methanogenic cell numbers were low. In different sediments with higher archaeal diversity and abundance, it is important that primers and probes targeting different ANME subtypes are very specific and do not detect other ANME subtypes or methanogens that are also present. In this study, primers and probes that were regularly used in AOM studies were tested in silico on coverage and specificity. Most of the previously developed primers and probes were not specific for the ANME subtypes, thereby not reflecting the actual ANME population in complex samples. Selected primers that showed good coverage and high specificity for the subclades ANME-1, ANME-2a/b, and ANME-2c were thoroughly validated using quantitative polymerase chain reaction (qPCR). From these qPCR tests, only certain combinations seemed suitable for selective amplification. After optimization of these primer sets, we obtained valid primer combinations for the selective detection and quantification of ANME-1, ANME-2a/b, and ANME-2c in samples where different ANME subtypes and possibly methanogens could be present. As a result of this work, we propose a standard workflow to facilitate selection of suitable primers for qPCR experiments on novel environmental samples.

Effect of humic acid on anaerobic digestion of cellulose and xylan in completely stirred tank reactors: inhibitory effect, mitigation of the inhibition and the dynamics of the microbial communities.

Inhibition effect of humic acid (HA) on anaerobic digestion of cellulose and xylan and the mitigation potential of the inhibition were evaluated in controlled fed batch reactors at 30 °C and a hydraulic retention time (HRT) of 20 days. Reactor performances were evaluated by biogas production and metabolite measurements for 220 days. Microbial population dynamics of the reactors were monitored with next-generation 16S rRNA gene sequencing at nine different sampling times. Our results showed that increasing levels of HA inhibited the hydrolysis efficiency of the digestion by 40% and concomitantly reduced the methane yield. Addition of hydrolytic enzymes helped to reverse the negative effects of HA, whereas calcium addition did not reverse HA inhibition. Microbiological analyses showed that the relative abundance of hydrolytic/fermentative bacterial groups such as Clostridiales, Bacteroidales and Anaerolineales was significantly lowered by the presence of HA. HA also affected the archaeal populations. Mostly hydrogenotrophic methanogens were negatively affected by HA. The relative abundance of Methanobacteriaceae, Methanomicrobiales-WCHA208 and Unassigned Thermoplasmata WCHA1-57 were negatively affected by the presence of HA, whereas Methanosaetacea was not affected.

Sodium lauryl ether sulfate (SLES) degradation by nitrate-reducing bacteria.

The surfactant sodium lauryl ether sulfate (SLES) is widely used in the composition of detergents and frequently ends up in wastewater treatment plants (WWTPs). While aerobic SLES degradation is well studied, little is known about the fate of this compound in anoxic environments, such as denitrification tanks of WWTPs, nor about the bacteria involved in the anoxic biodegradation. Here, we used SLES as sole carbon and energy source, at concentrations ranging from 50 to 1000 mg L-1, to enrich and isolate nitrate-reducing bacteria from activated sludge of a WWTP with the anaerobic-anoxic-oxic (A2/O) concept. In the 50 mg L-1 enrichment, Comamonas (50%), Pseudomonas (24%), and Alicycliphilus (12%) were present at higher relative abundance, while Pseudomonas (53%) became dominant in the 1000 mg L-1 enrichment. Aeromonas hydrophila strain S7, Pseudomonas stutzeri strain S8, and Pseudomonas nitroreducens strain S11 were isolated from the enriched cultures. Under denitrifying conditions, strains S8 and S11 degraded 500 mg L-1 SLES in less than 1 day, while strain S7 required more than 6 days. Strains S8 and S11 also showed a remarkable resistance to SLES, being able to grow and reduce nitrate with SLES concentrations up to 40 g L-1. Strain S11 turned out to be the best anoxic SLES degrader, degrading up to 41% of 500 mg L-1. The comparison between SLES anoxic and oxic degradation by strain S11 revealed differences in SLES cleavage, degradation, and sulfate accumulation; both ester and ether cleavage were probably employed in SLES anoxic degradation by strain S11.

Genome and proteome analysis of Pseudomonas chloritidismutans†AW-1T that grows on n-decane with chlorate or oxygen as electron acceptor.

Growth of Pseudomonas chloritidismutans AW-1T on C7 to C12 n-alkanes with oxygen or chlorate as electron acceptor was studied by genome and proteome analysis. Whole genome shotgun sequencing resulted in a 5 Mbp assembled sequence with a G + C content of 62.5%. The automatic annotation identified 4767 protein-encoding genes and a putative function could be assigned to almost 80% of the predicted proteins. The distinct phylogenetic position of P. chloritidismutans AW-1T within the Pseudomonas stutzeri cluster became clear by comparison of average nucleotide identity values of sequenced genomes. Analysis of the proteome of P. chloritidismutans AW-1T showed the versatility of this bacterium to adapt to aerobic and anaerobic growth conditions with acetate or n-decane as substrates. All enzymes involved in the alkane oxidation pathway were identified. An alkane monooxygenase was detected in n-decane-grown cells, but not in acetate-grown cells. The enzyme was found when grown in the presence of oxygen or chlorate, indicating that under both conditions an oxygenase-mediated pathway is employed for alkane degradation. Proteomic and biochemical data also showed that both chlorate reductase and chlorite dismutase are constitutively present, but most abundant under chlorate-reducing conditions.

Lachnotalea glycerini gen. nov., sp. nov., an anaerobe isolated from a nanofiltration unit treating anoxic groundwater.

A strictly anaerobic bacterium, strain DLD10T, was isolated from a biofilm that developed on a nanofiltration membrane treating anoxic groundwater using glycerol as substrate. Cells were straight to slightly curved rods 0.2-0.5 µm in diameter and 1-3 µm in length, non-motile and non-spore-forming. The optimum temperature and pH for growth were 30 °C and pH 7.0. Strain DLD10T was able to grow in the presence of 0.03-4.5 % (w/v) NaCl. Substrates utilized by strain DLD10T included glycerol and various carbohydrates (glucose, sucrose, fructose, mannose, arabinose, pectin, starch, xylan), which were mainly converted to ethanol, acetate, H2 and formate. Thiosulphate, sulphur and Fe(III) were used as electron acceptors, but sulphate, fumarate and nitrate were not. The predominant membrane fatty acids were C16 : 0, iso-C17 : 1 and C17 : 1ω8c. The DNA G+C content was 36.4 mol%. Strain DLD10T belongs to the family Lachnospiraceae and is distantly related to Clostridium populeti DSM 5832T, Hespellia porcina DSM 15481T and Robinsoniella peoriensis CCUG 48729T (93 % 16S rRNA gene sequence similarity). Physiological characteristics and phylogenetic analysis indicated that strain DLD10T is a representative of a novel species of a new genus, for which the name Lachnotalea glycerini gen. nov., sp. nov. is proposed. The type strain of Lachnotalea glycerini is DLD10T ( = DSM 28816T = JCM 30818T).

Chain Elongation with Reactor Microbiomes: Open-Culture Biotechnology To Produce Biochemicals.

Chain elongation into medium-chain carboxylates, such as n-caproate and n-caprylate, with ethanol as an electron donor and with open cultures of microbial consortia (i.e., reactor microbiomes) under anaerobic conditions is being developed as a biotechnological production platform. The goal is to use the high thermodynamic efficiency of anaerobic fermentation to convert organic biomass or organic wastes into valuable biochemicals that can be extracted. Several liter-scale studies have been completed and a first pilot-plant study is underway. However, the underlying microbial pathways are not always well understood. In addition, an interdisciplinary approach with knowledge from fields ranging from microbiology and chemical separations to biochemistry and environmental engineering is required. To bring together research from different fields, we reviewed the literature starting with the microbiology and ending with the bioprocess engineering studies that already have been performed. Because understanding the microbial pathways is so important to predict and steer performance, we delved into a stoichiometric and thermodynamic model that sheds light on the effect of substrate ratios and environmental conditions on product formation. Finally, we ended with an outlook.