Biogeography of Lentibacter algarum, description of a new strain isolated from the North Sea and emended genus and species descriptions.

A new heterotrophic, aerobic alphaproteobacterium, designated strain SH36 (=DSM 23330=LMG 25292), was obtained from a seawater sample collected in the open North Sea during a phytoplankton bloom. Analysis of the 16S rRNA gene sequence revealed affiliation of strain SH36 to the species Lentibacter algarum (family Roseobacteraceae), showing 100 and 99.9 % sequence similarity to the 16S rRNA genes of the strains L. algarum ZXM098 and ZXM100T. Digital DNA-DNA hybridization of strain SH36 with the type strain of L. algarum showed 98.0 % relatedness, confirming that strain SH36 can be classified within the same species. All three L. algarum strains were compared by physiological, morphological, chemotaxonomic, and genotypic characteristics. The strains showed only minor differences in the composition of fatty acids and polar lipids, but considerable physiological differences. Comparison of the 16S rRNA gene sequence of SH36 with sequences present in GenBank revealed that phylotypes with ≥98.65 % sequence identity to the type strain of L. algarum were found at different marine and estuarine locations of temperate and subtropic regions. Furthermore, by using a specific PCR approach L. algarum was detected throughout annual cycles at the offshore station at Helgoland Roads in the German Bight, indicating that this species is a permanent member of the microbial community in the North Sea.

Roseobacter fucihabitans sp. nov., isolated from the brown alga Fucus spiralis.

A Gram-negative, aerobic, pink-pigmented, and bacteriochlorophyll a-containing bacterial strain, designated B14T, was isolated from the macroalga Fucus spiralis sampled from the southern North Sea, Germany. Based on 16S rRNA gene sequences, species of the genera Roseobacter and Sulfitobacter were most closely related to strain B14T with sequence identities ranging from 98.15 % (Roseobacter denitrificans Och 114T) to 99.11 % (Roseobacter litoralis Och 149T), whereas Sulfitobacter mediterraneus CH-B427T exhibited 98.52 % sequence identity. Digital DNA-DNA hybridization and average nucleotide identity values between the genome of the novel strain and that of closely related Roseobacter and Sulfitobacter type strains were <20 % and <77 %, respectively. The novel strain contained ubiquinone-10 as the only respiratory quinone and C18 : 1 ω7c, C16 : 0, C18 : 0, C12 : 1 ω7c, C18 : 2 ω7,13c, and C10 : 0 3-OH as the major cellular fatty acids. The predominant polar lipids of strain B14T were phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. The genome of strain B14T comprises a chromosome with a size of 4.5 Mbp, one chromid, and four plasmids. The genome contains the complete gene cluster for aerobic anoxygenic photosynthesis required for a photoheterotrophic lifestyle. The results of this study indicate that strain B14T (=DSM 116946T=LMG 33352T) represents a novel species of the genus Roseobacter for which the name Roseobacter fucihabitans sp. nov. is proposed.

Anaeropeptidivorans aminofermentans gen. nov., sp. nov., a mesophilic proteolytic salt-tolerant bacterium isolated from a laboratory-scale biogas fermenter, and emended description of Clostridium colinum.

An anaerobic bacterial strain, designated strain M3/9T, was isolated from a laboratory-scale biogas fermenter fed with maize silage supplemented with 5 % wheat straw. Cells were straight, non-motile rods, which stained Gram-negative. Optimal growth occurred between 30 and 40°C, at pH 7.5-8.5, and up to 3.9 % (w/v) NaCl was tolerated. When grown on peptone from casein and soymeal, strain M3/9T produced mainly acetic acid, ethanol, and isobutyric acid. The major cellular fatty acids of the novel strain were C16 : 0 and C16 : 0 DMA. The genome of strain M3/9T is 3757  330 bp in size with a G+C content of 38.45 mol%. Phylogenetic analysis allocated strain M3/9T within the family Lachnospiraceae with Clostridium colinum DSM 6011T and Anaerotignum lactatifermentans DSM 14214T being the most closely related species sharing 57.86 and 56.99% average amino acid identity and 16S rRNA gene sequence similarities of 91.58 and 91.26 %, respectively. Based on physiological, chemotaxonomic and genetic data, we propose the description of a novel species and genus Anaeropeptidivorans aminofermentans gen. nov., sp. nov., represented by the type strain M3/9T (=DSM 100058T=LMG 29527T). In addition, an emended description of Clostridium colinum is provided.

Proteiniborus indolifex sp. nov., isolated from a thermophilic industrial-scale biogas plant.

A novel strictly anaerobic bacterium, designated strain BA2-13T, was isolated from a thermophilic industrial-scale biogas plant. Cells were rod-shaped and Gram-stain-positive. Growth occurred at temperatures of 25 to 50 °C and between pH 6.3 and 9.5. Strain BA2-13T produced indole. Cell growth was stimulated by yeast extract, peptone, meat extract, a mixture of 20 amino acids, glucose, pyruvate and ribose. When grown on peptone and yeast extract, the main fermentation products were acetic acid, H2 and CO2. The predominant cellular fatty acids were iso-C15 : 0 and iso-C14 : 0 3-OH. Major polar lipids were diphosphatidylglycerol, glycolipids, phospholipids and phosphatidylgycerol. Phylogenetic analysis based on 16S rRNA gene nucleotide sequence analysis placed strain BA2-13T within the order Clostridiales showing closest affiliation with Proteiniborusethanoligenes with 95.9 % sequence identity. Physiological, genotypic and chemotaxonomic differences of strain BA2-13T from P. ethanoligenes support the description of a new species within the genus Proteiniborus for which we suggest the name Proteiniborusindolifex sp. nov. (type strain BA2-13T=DSM 103060T=LMG 29818T).

Herbinix luporum sp. nov., a thermophilic cellulose-degrading bacterium isolated from a thermophilic biogas reactor.

Phylogenetic studies were performed on a group of novel Gram-stain-positive, anaerobic, non-sporulating rod-shaped bacteria isolated from a thermophilic biogas plant. The novel organisms were able to degrade crystalline cellulose. 16S rRNA gene sequence comparison indicated that the isolates SD1DT, SD1G, SD1I and RK1P share 100 % sequence identity, and are most closely related to Herbinix hemicellulosilytica T3/55T with which they share a 16S rRNA gene sequence similarity of 96.4 %. As a representative of the whole group of isolates, strain SD1DT was further characterized. Strain SD1DT was catalase-negative, indole-negative, and produced acetate, ethanol, butyric acid and hydrogen as major end-products during fermentative cellobiose utilization. Cells are rod-shaped, growing optimally at 40-65 °C and pH 6.5-8.5. The major cellular fatty acids (>10 %) were C19 : 0cyc 9,10 dimethyl acetal, C16 : 0 and C14 : 0. The DNA G+C content was 35.1 mol%. Due to the genetic and phenotypic differences to the most closely affiliated species, the isolates represent a novel species of the genus Herbinix within the family Lachnospiraceae, for which the name Herbinix luporum sp. nov. is proposed. The type strain is SD1DT(=DSM 100831T=CECT 8959T).

Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov., isolated from mesophilic laboratory-scale biogas reactors, and emended description of the genus Proteiniphilum.

Three novel, facultatively anaerobic bacteria of the family Porphyromonadaceae (phylum Bacteroidetes) were isolated from mesophilic laboratory-scale biogas reactors. The strains were Gram-negative rods. Optimal growth occurred between 35 and 45 °C and at pH 7.1-7.8. The main fermentation products were acetic and propionic acids. The predominant fatty acid in all strains was anteiso-C15 : 0, and the only respiratory quinone detected was menaquinone MK-8. 16S rRNA gene sequence comparison indicated that strains M3/6T and ING2-E5BT were most closely related to the type strain of Proteiniphilum acetatigenes, with sequence similarities of 97.3 and 94.5 %. Strain ING2-E5AT showed the closest affiliation to the type strain of Petrimonas sulfuriphila, with 97 % sequence identity. DNA-DNA hybridization of strain M3/6T and ING2-E5AT with the most closely related type strains showed 43.3-45.6 and 23.8-25.7 % relatedness, respectively, which supports the conclusion that both isolates represent novel species. Phylogenetic analysis and comparison of cellular fatty acid patterns indicated that strain ING2-E5BT cannot be classified as a member of any previously described genus. Therefore, because of the physiological, genotypic and chemotaxonomic differences, it is proposed to designate novel species within the genera Proteiniphilum and Petrimonas, Proteiniphilum saccharofermentans sp. nov. (type strain M3/6T = DSM 28694T = CECT 8610T = LMG 28299T) and Petrimonas mucosa sp. nov. (type strain ING2-E5AT = DSM 28695T = CECT 8611T), and a novel species of a new genus, Fermentimonas caenicola gen. nov., sp. nov. (type strain of Fermentimonas caenicola is ING2-E5BT = DSM 28696T = CECT 8609T = LMG 28429T). In addition, an emended description of the genus Proteiniphilum is provided.

Clostridium bornimense sp. nov., isolated from a mesophilic, two-phase, laboratory-scale biogas reactor.

A novel anaerobic, mesophilic, hydrogen-producing bacterium, designated strain M2/40(T), was isolated from a mesophilic, two-phase, laboratory-scale biogas reactor fed continuously with maize silage supplemented with 5% wheat straw. 16S rRNA gene sequence comparison revealed an affiliation to the genus Clostridium sensu stricto (cluster I of the clostridia), with Clostridium cellulovorans as the closest characterized species, showing 93.8% sequence similarity to the type strain. Cells of strain M2/40(T) were rods to elongated filamentous rods that showed variable Gram staining. Optimal growth occurred at 35 °C and at pH 7. Grown on glucose, the main fermentation products were H2, CO2, formate, lactate and propionate. The DNA G+C content was 29.6 mol%. The major fatty acids (>10 %) were C(16 : 0), summed feature 10 (C(18 : 1)ω11c/ω9t/ω6t and/or unknown ECL 17.834) and C(18 : 1)ω11c dimethylacetal. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain M2/40(T) represents a novel species within the genus Clostridium, for which we propose the name Clostridium bornimense sp. nov. The type strain is M2/40(T) ( = DSM 25664(T) = CECT 8097(T)).

Pelagimonas varians gen. nov., sp. nov., isolated from the southern North Sea.

A heterotrophic, Gram-stain-negative, aerobic bacterium, designated strain SH4-1(T), was obtained from a seawater sample collected from the southern North Sea during a phytoplankton bloom. The 16S rRNA gene sequence comparison revealed affiliation to the Roseobacter clade (class Alphaproteobacteria) with Sulfitobacter marinus SW-265(T) as the most closely related characterized strain, showing 97.2 % 16S rRNA gene sequence similarity. Calculation of phylogenetic trees based on 16S rRNA gene sequences indicated, however, that members of the genus Roseobacter, Roseobacter denitrificans Och 114(T) and Roseobacter litoralis Och 149(T) (95 % and 96 % sequence similarity, respectively) fall between strain SH4-1(T) and the Sulfitobacter cluster including Oceanibulbus indolifex HEL-45(T) (≥95.4 % sequence similarity). Cells of strain SH4-1(T) are irregular rods with at least one flagellum. Optimal growth occurred between 28 and 32 °C and at a pH between 7.0 and 8.5. Cells require the vitamin nicotinic acid amide as well as sodium ions for growth. The DNA G+C content was 55.1 mol%. The fatty acids (>1 %) comprised C10 : 0 3-OH, C12 : 1, C14 : 1 3-OH, C16 : 0, C18 : 0, C18 : 2, C18 : 1ω7c and 11-methyl C18 : 1ω7c. The polar lipid pattern indicated the presence of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylmonomethylethanolamine, an unidentified aminolipid, one unidentified phospholipid and one other unidentified lipid. On the basis of phenotypic, chemotaxonomic and phylogenetic differences, strain SH4-1(T) represents a novel species in a new genus within the family Rhodobacteraceae, for which we propose the name Pelagimonas varians gen. nov., sp. nov. The type strain of the type species is SH4-1(T) ( = DSM 23678(T) = LMG 26343(T) = CIP 110297(T)).

Planktotalea frisia gen. nov., sp. nov., isolated from the southern North Sea.

A heterotrophic, aerobic bacterium, designated strain SH6-1(T), was obtained from a seawater sample collected from the open North Sea during a phytoplankton bloom. Strain SH6-1(T) was isolated from a 10(-6) dilution culture, which indicated a high abundance of this organism in the environmental sample. 16S rRNA gene sequence comparison revealed that strain SH6-1(T) belonged to the marine Roseobacter clade (order Rhodobacterales) within the class Alphaproteobacteria. Pelagicola litoralis CL-ES2(T) was the closest phylogenetic neighbour (96.4% 16S rRNA gene sequence similarity). Cells of strain SH6-1(T) were small or elongated irregular rods. Optimal growth occurred between 20 and 25 °C and between pH 7.5 and 9.0 with peptone and yeast extract. On marine agar, the isolate formed non-pigmented, small, circular, convex colonies. For growth, cells required sodium ions and the vitamins pantothenic acid and nicotinic acid amide. The DNA G+C content was 53.8 mol%. The fatty acids (>1%) were C(10:0) 3-OH, C(16:0), C(12:1), C(12:1) 3-OH, C(18:0), C(18:1)ω7c, C(18:2) and 11-methyl C(18:1)ω7c. The polar lipid pattern indicated the presence of phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and one unidentified phospholipid. The major respiratory lipoquinone was ubiquinone Q-10. Strain SH6-1(T) contained the genes pufLM, which code for the bacterial photosynthesis reaction centre; however, no bacteriochlorophyll a could be detected. Physiological, genotypic and phenotypic differences from P. litoralis support the description of a novel genus and species, for which we suggest the name Planktotalea frisia gen. nov., sp. nov; the type strain of the type species is SH6-1(T) (=DSM 23709(T)=LMG 25294(T)).

The novel oligopeptide utilizing species Anaeropeptidivorans aminofermentans M3/9T, its role in anaerobic digestion and occurrence as deduced from large-scale fragment recruitment analyses.

Research on biogas-producing microbial communities aims at elucidation of correlations and dependencies between the anaerobic digestion (AD) process and the corresponding microbiome composition in order to optimize the performance of the process and the biogas output. Previously, Lachnospiraceae species were frequently detected in mesophilic to moderately thermophilic biogas reactors. To analyze adaptive genome features of a representative Lachnospiraceae strain, Anaeropeptidivorans aminofermentans M3/9T was isolated from a mesophilic laboratory-scale biogas plant and its genome was sequenced and analyzed in detail. Strain M3/9T possesses a number of genes encoding enzymes for degradation of proteins, oligo- and dipeptides. Moreover, genes encoding enzymes participating in fermentation of amino acids released from peptide hydrolysis were also identified. Based on further findings obtained from metabolic pathway reconstruction, M3/9T was predicted to participate in acidogenesis within the AD process. To understand the genomic diversity between the biogas isolate M3/9T and closely related Anaerotignum type strains, genome sequence comparisons were performed. M3/9T harbors 1,693 strain-specific genes among others encoding different peptidases, a phosphotransferase system (PTS) for sugar uptake, but also proteins involved in extracellular solute binding and import, sporulation and flagellar biosynthesis. In order to determine the occurrence of M3/9T in other environments, large-scale fragment recruitments with the M3/9T genome as a template and publicly available metagenomes representing different environments was performed. The strain was detected in the intestine of mammals, being most abundant in goat feces, occasionally used as a substrate for biogas production.

Complete Genome Sequence of a New Clostridium sp. Isolated from Anaerobic Digestion and Biomethanation.

Here, we present the genome sequence and annotation of the bacterial strain HV4-5-A1G, a potentially new Clostridium species. Based on its genomic data, this strain may act as a keystone microorganism in the hydrolysis of complex polymers, as well as in the different acidogenesis and acetogenesis steps during anaerobic digestion.

Draft Genome Sequence of a New Oscillospiraceae Bacterium Isolated from Anaerobic Digestion of Biomass.

Here, we present the genome sequence and annotation of the novel bacterial strain HV4-5-C5C, which may represent a new genus within the family Oscillospiraceae (order Eubacteriales). This strain is a potential keystone species in the hydrolysis of complex polymers during anaerobic digestion of biomass.

Complete Genome Sequence of a New Bacteroidaceae Bacterium Isolated from Anaerobic Biomass Digestion.

Here, we present the genome sequence and annotation of HV4-6-C5C, a bacterial strain isolated from a mesophilic two-stage laboratory-scale leach bed biogas reactor system. Strain HV4-6-C5C may represent a new genus of the family Bacteroidaceae and may have a key role in acidogenesis and acetogenesis steps during anaerobic biomass digestion.

Proteiniphilum saccharofermentans str. M3/6T isolated from a laboratory biogas reactor is versatile in polysaccharide and oligopeptide utilization as deduced from genome-based metabolic reconstructions.

Proteiniphilum saccharofermentans str. M3/6T is a recently described species within the family Porphyromonadaceae (phylum Bacteroidetes), which was isolated from a mesophilic laboratory-scale biogas reactor. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding biomass degradation and fermentation pathways. The P. saccharofermentans str. M3/6T genome consists of a 4,414,963 bp chromosome featuring an average GC-content of 43.63%. Genome analyses revealed that the strain possesses 3396 protein-coding sequences. Among them are 158 genes assigned to the carbohydrate-active-enzyme families as defined by the CAZy database, including 116 genes encoding glycosyl hydrolases (GHs) involved in pectin, arabinogalactan, hemicellulose (arabinan, xylan, mannan, ß-glucans), starch, fructan and chitin degradation. The strain also features several transporter genes, some of which are located in polysaccharide utilization loci (PUL). PUL gene products are involved in glycan binding, transport and utilization at the cell surface. In the genome of strain M3/6T, 64 PUL are present and most of them in association with genes encoding carbohydrate-active enzymes. Accordingly, the strain was predicted to metabolize several sugars yielding carbon dioxide, hydrogen, acetate, formate, propionate and isovalerate as end-products of the fermentation process. Moreover, P. saccharofermentans str. M3/6T encodes extracellular and intracellular proteases and transporters predicted to be involved in protein and oligopeptide degradation. Comparative analyses between P. saccharofermentans str. M3/6T and its closest described relative P. acetatigenes str. DSM 18083T indicate that both strains share a similar metabolism regarding decomposition of complex carbohydrates and fermentation of sugars.

Complete Genome Sequence of a New Ruminococcaceae Bacterium Isolated from Anaerobic Biomass Hydrolysis.

A new Ruminococcaceae bacterium, strain HV4-5-B5C, participating in the anaerobic digestion of grass, was isolated from a mesophilic two-stage laboratory-scale leach bed biogas system. The draft annotated genome sequence presented in this study and 16S rRNA gene sequence analysis indicated the affiliation of HV4-5-B5C with the family Ruminococcaceae outside recently described genera.

Microbial communities involved in biogas production exhibit high resilience to heat shocks.

We report here the impact of heat-shock treatments (55 and 70 °C) on the biogas production within the acidification stage of a two-stage reactor system for anaerobic digestion and biomethanation of grass. The microbiome proved both taxonomically and functionally very robust, since heat shocks caused minor community shifts compared to the controls, and biogas yield was not decreased. The strongest impact on the microbial profile was observed with a combination of heat shock and low pH. Since no transient reduction of microbial diversity occured after the shock, biogas keyplayers, but also potential pathogens, survived the treatment. All along the experiment, the heat-resistant bacterial profile consisted mainly of Firmicutes, Bacteroidetes and Proteobacteria. Bacteroides and Acholeplasma were reduced after heat shocks. An increase was observed for Aminobacterium. Our results prove the stability to thermal stresses of the microbial communities involved in acidification, and the resilience in biogas production irrespectively of the thermal treatment.

The completely annotated genome and comparative genomics of the Peptoniphilaceae bacterium str. ING2-D1G, a novel acidogenic bacterium isolated from a mesophilic biogas reactor.

The strictly anaerobic Peptoniphilaceae bacterium str. ING2-D1G (=DSM 28672=LMG 28300) was isolated from a mesophilic laboratory-scale completely stirred tank biogas reactor (CSTR) continuously co-digesting maize silage, pig and cattle manure. Based on 16S rRNA gene sequence comparison, the closest described relative to this strain is Peptoniphilus obesi ph1 showing 91.2% gene sequence identity. The most closely related species with a validly published name is Peptoniphilus indolicus DSM 20464T whose 16S rRNA gene sequence is 90.6% similar to the one of strain ING2-D1G. The genome of the novel strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding anaerobic digestion of biomass. The strain harbors a circular chromosome with a size of 1.6 Mb that contains 1466 coding sequences, 53 tRNA genes and 4 ribosomal RNA (rrn) operons. The genome carries a 28,261bp prophage insertion comprising 47 phage-related coding sequences. Reconstruction of fermentation pathways revealed that strain ING2-D1G encodes all enzymes for hydrogen, lactate and acetate production, corroborating that it is involved in the acido- and acetogenic phase of the biogas process. Comparative genome analyses of Peptoniphilaceae bacterium str. ING2-D1G and its closest relative Peptoniphilus obesi ph1 uncovered rearrangements, deletions and insertions within the chromosomes of both strains substantiating a divergent evolution. In addition to genomic analyses, a physiological and phenotypic characterization of the novel isolate was performed. Grown in Brain Heart Infusion Broth with added yeast extract, cells were spherical to ovoid, catalase- and oxidase-negative and stained Gram-positive. Optimal growth occurred between 35 and 37°C and at a pH value of 7.6. Fermentation products were acetate, butanoate and carbon dioxide.

Complete Genome Sequence of a New Firmicutes Species Isolated from Anaerobic Biomass Hydrolysis.

A new Firmicutes isolate, strain HV4-6-A5C, was obtained from the hydrolysis stage of a mesophilic and anaerobic two-stage lab-scale leach-bed system for biomethanation of fresh grass. It is assumed that the bacterial isolate contributes to plant biomass degradation. Here, we report a draft annotated genome sequence of this organism.

Genomics and prevalence of bacterial and archaeal isolates from biogas-producing microbiomes.

BACKGROUND: To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. RESULTS: Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. CONCLUSIONS: Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members.