Low-Cost Clamp-On Photometers (ClampOD) and Tube Photometers (TubeOD) for Online Cell Density Determination

Optical density (OD) measurement is the gold standard to estimate microbial cell density in aqueous systems. Recording microbial growth curves is essential to assess substrate utilization, gauge sensitivity to inhibitors or toxins, or determine the perfect sampling point. Manual sampling for cuvette-photometer-based measurements can cause disturbances and impact growth, especially for strictly anaerobic or thermophilic microbes. For slow growing microbes, manual sampling can cause data gaps that complicate analysis. Online OD measurement systems provide a solution, but are often expensive and ill-suited for applications such as monitoring microbial growth in custom or larger anaerobic vessels. Furthermore, growth measurements of thermophilic cultures are limited by the heat sensitivity of complex electronics. Here, we present two simple, low-cost, self-assembled photometers—a “TubeOD” for online measurement of anaerobic and thermophilic cultures in Hungate tubes and a “ClampOD” that can be attached to virtually any transparent growth vessel. Both OD-meters can be calibrated in minutes. We detail the manufacturing and calibration procedure and demonstrate continuous acquisition of high quality cell density data of a variety of microbes, including strict anaerobes, a thermophile, and gas-utilizing strains in various glassware. When calibrated and operated within their detection limits (ca. 0.3–90% of the photosensor voltage range), these self-build OD-meters can be used for continuous measurement of microbial growth in a variety of applications, thereby, simplifying and enhancing everyday lab operations.

Phosphitispora fastidiosa gen. nov. sp. nov., a new dissimilatory phosphite-oxidizing anaerobic bacterium isolated from anaerobic sewage sludge

A new strictly anaerobic bacterium, strain DYL19T, was enriched and isolated with phosphite as the sole electron donor and CO2 as a single carbon source and electron acceptor from anaerobic sewage sludge sampled at a sewage treatment plant in Constance, Germany. It is a Gram-positive, spore-forming, slightly curved, rod-shaped bacterium which oxidizes phosphite to phosphate while reducing CO2 to biomass and small amounts of acetate. Optimal growth is observed at 30 °C, pH 7.2, with a doubling time of 3 days. Beyond phosphite, no further inorganic or organic electron donor can be used, and no other electron acceptor than CO2 is reduced. Sulphate inhibits growth with phosphite and CO2. The G+C content is 45.95 mol%, and dimethylmenaquinone-7 is the only quinone detectable in the cells. On the basis of 16S rRNA gene sequence analysis and other chemotaxonomic properties, strain DYL19T is described as the type strain of a new genus and species, Phosphitispora fastidiosa gen. nov., sp. nov.

Mariniplasma anaerobium gen. nov., sp. nov., a novel anaerobic marine mollicute, and proposal of three novel genera to reclassify members of Acholeplasma clusters II–IV

A novel strictly anaerobic chemoorganotrophic bacterium, designated Mahy22T, was isolated from sulfidic bottom water of a shallow brackish meromictic lake in Japan. Cells of the strain were Gram-stain-negative, non-motile and coccoid in shape with diameters of about 600–800 nm. The temperature range for growth was 15–37 °C, with optimum growth at 30–32 °C. The pH range for growth was pH 6.2–8.9, with optimum growth at pH 7.2–7.4. The strain grew with NaCl concentrations of 5% or below (optimum, 2–3%). Growth of the strain was enhanced by the addition of thiosulfate. The major cellular fatty acids were C16:0 and anteiso-C15:0. Respiratory quinones were not detected. The complete genome sequence of strain Mahy22T possessed a 1 885 846 bp circular chromosome and a 12 782 bp circular genetic element. The G+C content of the genome sequence was 30.1 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the family Acholeplasmataceae , class Mollicutes . The closest relative of strain Mahy22T with a validly published name was Acholeplasma palmae J233T with a 16S rRNA gene sequence similarity of 90.5%. Based on the results of polyphasic analysis, the name Mariniplasma anaerobium gen. nov., sp. nov. is proposed to accommodate strain Mahy22T, along with reclassification of some Acholeplasma species into Alteracholeplasma gen. nov., Haploplasma gen. nov. and Paracholeplasma gen. nov.

Anaerostipes hominis sp. nov., a novel butyrate-producing bacteria isolated from faeces of a patient with Crohn's disease

Cultivation and isolation of gut bacteria are necessary for understanding their role in the intestinal ecosystem. We isolated a novel bacterium, designated strain BG01T, from the faeces of a patient with Crohn's disease. Strain BG01T was a strictly anaerobic, rod-shaped, Gram-variable and endospore-forming bacterium. Strain BG01T possessed C12 : 0, C18 : 0 dimethyl aldehyde (DMA) and C18 : 1 ω9c DMA as predominant cellular fatty acids and meso-diaminopimelic acid as a diagnostic diamino acid. Strain BG01T grew at 15–45 °C (optimum, 37 °C), with 0–4 % (w/v) NaCl (optimum, 0–1 %), at pH 6–10 (optimum, pH 7) and was resistant to bile salt, but not to ampicillin, metronidazole, vancomycin and cefoperazone. Butyrate, propionate, oxalacetate and fumarate were produced as fermentation end products from Gifu anaerobic medium broth. Strain BG01T showed 97.7 % 16S rRNA gene sequence similarity, and 92.0 and 48.5 % of average nucleotide identity and digital DNA–DNA hybridization values, respectively, with Anaerostipes caccae KCTC 15019T. Genomic analysis indicated that strain BG01T had a butyrate-producing pathway. The genomic G+C content of the strain was 43.5 mol%. Results of the phenotypic, phylogenetic and genotypic analyses indicated that strain BG01T represents a novel butyrate-producing species of the genus Anaerostipes , for which the name Anaerostipes hominis sp. nov. is proposed. The type strain is BG01T (=KCTC 15617T=JCM 32275T).

Bacterial microcompartments for isethionate desulfonation in the taurine-degrading human-gut bacterium Bilophila wadsworthia

Background Bilophila wadsworthia, a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2-aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H2S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves the glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslAB), which cleaves isethionate (2-hydroxyethanesulfonate) into acetaldehyde and sulfite. Results We demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential iron-sulfur (FeS) cluster-containing shell protein identified by sequence analysis. Conclusions We characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H2S production in the human gut.

Iocasia fonsfrigidae NS-1 gen. nov., sp. nov., a Novel Deep-Sea Bacterium Possessing Diverse Carbohydrate Metabolic Pathways

Resolving metabolisms of deep-sea microorganisms is crucial for understanding ocean energy cycling. Here, a strictly anaerobic, Gram-negative strain NS-1 was isolated from the deep-sea cold seep in the South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain NS-1 was most closely related to the type strain Halocella cellulosilytica DSM 7362T (with 92.52% similarity). A combination of phylogenetic, genomic, and physiological traits with strain NS-1, was proposed to be representative of a novel genus in the family Halanaerobiaceae, for which Iocasia fonsfrigidae NS-1 was named. It is noteworthy that I. fonsfrigidae NS-1 could metabolize multiple carbohydrates including xylan, alginate, starch, and lignin, and thereby produce diverse fermentation products such as hydrogen, lactate, butyrate, and ethanol. The expressions of the key genes responsible for carbohydrate degradation as well as the production of the above small molecular substrates when strain NS-1 cultured under different conditions, were further analyzed by transcriptomic methods. We thus predicted that part of the ecological role of Iocasia sp. is likely in the fermentation of products from the degradation of diverse carbohydrates to produce hydrogen as well as other small molecules, which are in turn utilized by other members of cold seep microbes.

Improvement of 1,3-Propanediol Production From Crude Glycerol by Co-cultivation of Anaerobic and Facultative Microbes Under Non-strictly Anaerobic Conditions

Background: Natural microbial consortia could efficiently produce 1,3-propanediol, the most promising bulk biochemical derived from glycerol that can be used as a monomer in the synthesis of polytrimethylene terephthalate (PTT). While natural microbial communities are made up of a diverse range of microbes with frequently unknown functions, the construction of synthetic microbial consortia allows for creating more defined systems with lower complexity.Results: In this study, the synthetic microbial consortia were constructed by combing facultative microbes of Klebsiella pneumoniae DUT2 (KP) and/or Escherichia coli DUT3 (EC) cultures with the strict anaerobic microbe of Clostridium butyricum DUT1 (CB) cultures under micro-aerobic conditions. The function of EC and KP during the fermentation process was to deplete oxygen and provide an anaerobic environment for CB. Furthermore, KP competes with CB to consume crude glycerol and produce 1,3-PDO. The interaction of commensalism and competition resulted in synthetic microbial consortia that could efficiently convert crude glycerol to 1,3-PDO even under micro-aerobic conditions. In a batch fermentation, the synthetic CB:KP co-culture at an initial abundance ratio of 92.5:7.5 yielded a maximum 1,3-PDO concentration of 52.08 g/L, with a yield of 0.49 g/g and a productivity of 1.80 g/(L.h), which increased by 10%, 9%, and 12%, respectively, when compared to the CB mono-culture under strictly anaerobic conditions. Compared to the KP mono-culture, the final 1,3-PDO concentration, yield, and productivity by the synthetic CB:KP consortia increased by 16%, 19%, and 84%, respectively. The synthetic CB:KP:EC co-culture achieved the highest 1,3-PDO flux of 49.17% at an initial abundance ratio of 85:7.5:7.5, while 7.43%, 5.77%, 3.15% 4.24%, and 2.13% of flux was distributed to butyric acid, acetic acid, lactic acid, ethanol, and succinic acid pathways. In a fed-batch fermentation, synthetic CB:KP:EC co-culture demonstrated a maximum 1,3-PDO concentration of 77.68 g/L with a yield of 0.51 g/g which is 30% and 13% higher than the production by the CB mono-culture at 0.02 vvm N2 supply. The initial abundance of CB guaranteed to be at least 85% facilitates 1,3-PDO production from crude glycerol efficiently by the development of synthetic microbial consortia. Conclusion: Under micro-aerobic conditions, the synthetic microbial consortia demonstrated excellent performance on 1,3-propanediol production via the interaction of commensalism and competition. The experimental results demonstrated the potential benefit of using the synthetic microbial consortia to produce 1,3-propanediol from crude glycerol.

Metabolism of Daidzein and Genistein by Gut Bacteria of the Class Coriobacteriia

The intake of isoflavones is presumed to be associated with health benefits in humans, but also potential adverse effects of isoflavones are controversially discussed. Isoflavones can be metabolized by gut bacteria leading to modulation of the bioactivity, such as estrogenic effects. Especially bacterial strains of the Eggerthellaceae, a well-known bacterial family of the human gut microbiota, are able to convert the isoflavone daidzein into equol. In addition, metabolization of genistein is also described for strains of the Eggerthellaceae. The aim of this study was to identify and investigate gut bacterial strains of the family Eggerthellaceae as well as the narrowly related family Coriobacteriaceae which are able to metabolize daidzein and genistein. This study provides a comprehensive, polyphasic approach comprising in silico analysis of the equol gene cluster, detection of genes associated with the daidzein, and genistein metabolism via PCR and fermentation of these isoflavones. The in silico search for protein sequences that are associated with daidzein metabolism identified sequences with high similarity values in already well-known equol-producing strains. Furthermore, protein sequences that are presumed to be associated with daidzein and genistein metabolism were detected in the two type strains ‘Hugonella massiliensis’ and Senegalimassilia faecalis which were not yet described to metabolize these isoflavones. An alignment of these protein sequences showed that the equol gene cluster is highly conserved. In addition, PCR amplification supported the presence of genes associated with daidzein and genistein metabolism. Furthermore, the metabolism of daidzein and genistein was investigated in fermentations of pure bacterial cultures under strictly anaerobic conditions and proofed the metabolism of daidzein and genistein by the strains ‘Hugonella massiliensis’ DSM 101782T and Senegalimassilia faecalis KGMB04484T.

Clostridium zeae sp. nov., isolated from corn silage

A rod-shaped, Gram-stain-negative, strictly anaerobic, catalase-negative and endospore-forming bacterial strain CSC2T was isolated from corn silage preserved in Tochigi, Japan. The strain CSC2T grew at 15–40 °C, at pH 5.0–7.7 and with up to 0.5 % (w/v) NaCl. The main cellular fatty acids were C14 : 0, C16 : 0 and C16 : 0 dimethyl acetal. The cellular polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, lysophosphatidylethanolamine, phosphatidylserine, lysophosphatidylcholine and two unidentified polar lipids. Phylogenetic analysis of the 16S rRNA gene showed that strain CSC2T was a member of the genus Clostridium and closely related to Clostridium polyendosporum DSM 57272T (95.6 % gene sequence similarity) and Clostridium fallax ATCC 19400T (95.3 %). The genomic DNA G+C content of strain CSC2T was 31.1 mol% (whole genome analysis). The average nucleotide identity based on blast and digital DNA–DNA hybridization values between strain CSC2T and the type strains of phylogenetically related species were below 71 and 24 %, respectively. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, it is proposed to designate strain CSC2T as representing Clostridium zeae sp. nov. The type strain is CSC2T (=MAFF212476T=JCM 33766T=DSM 111242T).

Blautia liquoris sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese strong-flavour liquor

A novel Gram-positive, non-motile, non-flagellated, strictly anaerobic, non-spore-forming and dumbbell-shaped, coccoid- or chain-shaped bacterium, designated strain LZLJ-3T, was isolated from a mud fermentation cellar which has been used for the production of Chinese strong-flavour liquor for over 100 years. Strain LZLJ-3T grew at 20–40 °C (optimum, 37 °C), at pH 6.0–8.0 (optimum, pH 8.0) and with NaCl concentrations up to 1 % (w/v; optimum, 0 %). Phylogenetic trees established based on 16S rRNA gene sequences showed that strain LZLJ-3T belonged to the genus Blautia of the family Lachnospiraceae, with the highest sequence similarity to Blautia stercoris GAM6-1T (91.7 %) and Blautia faecicola KGMB01111T (91.7 %). Comparative genome analysis showed that the orthologous average nucleotide identity (OrthoANI) and genome-to-genome distance (GGD) values between strain LZLJ-3T and B. stercoris GAM6-1T were respectively 69.1 and 22.9 %; the OrthoANI and GGD values between strain LZLJ-3T and B. faecicola KGMB01111T were respectively 70.86 and 36 % . The DNA G+C content of strain LZLJ-3T genome was 42.1 mol%. The predominant celluar fatty acids (>10 %) of strain LZLJ-3T were C16 : 0 FAME (27.9 %), C14 : 0 FAME (17.6 %) and C16 : 0 DMA (13.0 %). Arabinose, glucose and maltose could be utilized by strain LZLJ-3T as sole carbon sources for growth, with weak utilization of raffinose and l-fucose. API ZYM analysis gave positive reactions with α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase. The major end product of glucose fermentation was acetic acid. Based on the results of phenotypic, genotypic and phylogenetic analyses, strain LZLJ-3T is considered to represent a novel species of Blautia , for which the name Blautia liquoris sp. nov. is proposed. The type strain is LZLJ-3T (=KCTC 25163T=CGMCC 1.5299T=JCM 34225T).