Sporanaerobacter Acetigenes Research Articles

Description of a moderately acidotolerant and aerotolerant anaerobic bacterium Acidilutibacter cellobiosedens gen. nov., sp. nov. within the family Acidilutibacteraceae fam. nov., and proposal of Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov.

A Gram-stain positive, moderately thermophilic, acidotolerant and aerotolerant anaerobic bacterium, designated JN-28 T, was isolated from the pit mud of Chinese strong-flavor liquor. Growth was observed at 25–50 °C and pH 5.5–8.0 in the presence of 0–25 g l−1 NaCl (optimally at 45 °C, pH 6.0, without NaCl). Strain JN-28 T was heterotrophic, requiring yeast extract for growth. The major cellular fatty acids were iso-C15:0 and C14:0. The DNA G + C content of genomic DNA was 33.54 mol%. The strain was resistant to vancomycin (10 mg l−1). Genome analysis revealed the presence of genes involved in the response to mild acid stress and oxidative stress, and resistance to vancomycin. 16S rRNA gene-based phylogenetic analysis showed that strain JN-28 T shares ≤ 89.3 % sequence similarity with its closest relatives Sporanaerobacter acetigenes DSM 13106 T and other members in the order Tissierellales. Based on phenotypic and phylogenetic characteristics, Acidilutibacter cellobiosedens gen. nov., sp. nov. is proposed for the new genus and novel species with the type strain JN-28 T (=CCAM 418 T = JCM 39087 T). Further phylogenetic and phylogenomic analyses suggested strain JN-28 T represents a novel family within the order Tissierellales, for which Acidilutibacteraceae fam. nov. is proposed. In addition, the family Tissierellaceae was reclassified, Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov. were formally proposed. Emended description of the family Tissierellaceae is also provided.

Composition and profiles of volatile organic compounds during waste decomposition by the anaerobic bacteria purified from landfill

Volatile organic compounds (VOCs) become concerned pollutants in landfill gases, and their composition and concentration varied significantly during waste decomposition. Many environmental factors are known to affect VOC emissions, while the effect of indigenous bacteria in wastes on VOC production remains elusive. In this study, a simplified anaerobic degradation experiment, with the single substrate and the purified bacteria from a landfill, was set up to measure the degradation process and the dynamic changes of VOCs. The experiment excluded the abiotic factors for VOC variation. The two isolated bacteria, identified as Sporanaerobacter acetigenes and Clostridium sporogenes, could anaerobically ferment amino acids by Stickland reaction. They produced 51 and 57 species of VOCs in the experiment, respectively. The concentration changes of VOCs over bacterial growth and fermentation were clustered into four types by principal component analysis: three profiles were regular, similar to the variation of nitrate, hydrogen sulfide, and the major fermentation products (carbon dioxide, ammonium, and volatile organic acids), respectively; while one profile was unique to any degradation indicator. The various concentration profiles indicated different origins for VOCs, possibly from the extracellular environment, fermentation, and secondary reactions. The findings provide insights into the understanding of VOC diversity and variability during waste decomposition.

Draft Genome Sequence and Annotation of Sporanaerobacter acetigenes Strain F-12, Isolated from a Cattle Rumen.

We report the genome sequence of Sporanaerobacter acetigenes strain F-12, isolated from the rumen of a steer grazing on Rhodes grass in Townsville (Lansdown Research Station), Queensland, Australia. This draft genome consists of 2,866,191 bp, with 31.23% G+C content and 2,889 predicted coding sequences.

Mesophilic Acidogenesis of Food Waste-Recycling Wastewater: Effects of Hydraulic Retention Time, pH, and Temperature.

The effects of hydraulic retention time (HRT), pH, and operating temperature (T OP) on the degradation of food waste-recycling wastewater (FRW) were investigated in laboratory-scale hydrolysis/acidogenesis reactors. Response surface analysis was used to approximate the production of volatile organic acids and degradation of volatile suspended solids (VSS), carbohydrate, protein, and lipid with regard to the independent variables (1 ≤ HRT ≤ 3days, 4 ≤ pH ≤ 6, 25 ≤ T OP ≤ 45°C). Partial cubic models adequately approximated the corresponding response surfaces at α < 5%. The physiological conditions for maximum acidification (0.4g TVFA + EtOH/gVSadded) and the maximal degradation of VSS (47.5%), carbohydrate (92.0%), protein (17.7%), and lipid (73.7%) were different. Analysis of variance suggested that pH had a great effect on the responses in most cases, while T OP and HRT, and their interaction, were significant in some cases. Denaturing gradient gel electrophoresis analysis revealed that Sporanaerobacter acetigenes, Lactobacillus sp., and Eubacterium pyruvivorans-like microorganisms might be main contributors to the hydrolysis and acidogenesis of FRW. Biochemical methane potential test confirmed higher methane yield (538.2mLCH4/g VSadded) from an acidogenic effluent than from raw FRW.

Comprehensive study on volatile fatty acid production from Ettlia sp. residue with molecular analysis of the microbial community

The conversion of lipid extracted microalgal residues (LEμARs) into renewable energy provides a promising route for microalgal biodiesel production for various industries. In this study, volatile fatty acid (VFA) production and soluble chemical oxygen demand (SCOD) from the LEμAR of the green algae Ettlia sp. was optimized with the response surface methodology (RSM) varying the temperature from 25 to 65°C and the food to microorganism ratio (F/M ratio) from 0.5 to 2.5 as the independent variables. A F/M ratio of 1.76 (g COD/g VSS) and temperature of 47.97°C was found to be the optimal condition. Though the fit was rather lacking, the p-value was sufficiently significant at 1% α level validating the optimization model. Additionally, next generation sequencing (NGS) indicated significant correlations between VFA and its microbial community, revealing that an anaerobic bacterium species, Sporanaerobacter acetigenes, was a dominant VFA producer. This study shows that VFA production could indeed be a viable direction that would make the microalgae-based biofuel production competitive.

Characterization of Nitrate-Reducing and Amino Acid–Using Bacteria Prominent in Nitrotoxin-Enriched Equine Cecal Populations

Horses can be at risk for nitropoisoning by consuming plants containing 3-nitro-1-propionic acid or 3-nitro-1-propanol and to a lesser extent by plants containing nitrate. Populations of equine cecal microbes enriched for enhanced rates of 3-nitro-1-propionic acid (NPA) or nitrate metabolism were cultured for NPA- or nitrate-metabolizing bacteria on basal enrichment medium or tryptose soy agar supplemented with either 5-mM NPA or nitrate and under H2:CO2 (20:80) as the energy source. After 72 hours, separated colonies picked from plates, or roll tubes were cultured in fresh broth medium for 72 hours and then identified by 16S rRNA gene sequencing. Isolates from the NPA-enriched populations were identified as Streptococcus lutetiensis (five strains), Escherichia coli (two strains), and Sporanaerobacter acetigens (one strain). Strains isolated from nitrate-enriched populations were identified as Escherichia coli (one strain) and Wolinella succinogenes (three strains). None of these strains degraded NPA. Enriched populations of equine cecal microbes, the isolated pure strains and the type strain of Denitrobacterium detoxificans, a competent NPA-metabolizing microbe, were examined using denaturing gradient gel electrophoresis (DGGE). The DGGE analysis indicated that none of the strains in the enriched population of equine cecal microbes were similar to D. detoxificans. However, we report for the first time the isolation of the anaerobic amino acid–using Sporanaerobacter acetigenes from the equine cecum.

English

This work was done to study the bacterial diversity of mithun (Bos frontalis) fed on mixed tree leaves and rice straw based diet. Genomic DNA was extracted from the rumen liquor of mithun, 16S rDNA sequences were amplified, cloned and randomly selected for sequencing. The nearest neighbors were retrieved from the NCBI through a BLAST search and a phylogenetic tree was constructed. In our findings, 12% of clones showed similarity with known bacterial species (Prevotella ruminicola, Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Succinivibrio dextrinosolvens and &nbsp;Ruminococcus &nbsp;flavefaciens) and 6% of clones showed similarity with known bacterial genus (Butyrivibrio species, Streptococcus species) of 97-100% similarities. Twenty-two percent of clones showed similarity with known bacteria (P. ruminicola, Prevotella species, Sporanaerobacter acetigenes, Clostridiales bacterium, Bacteroidetes bacterium) of 90-97% similarities. Sequences of all the clones were also classified by using taxonomic classifier software available at Ribosomal Database Project and classification showed that, all the clones were under four phyla, namely Bacteroidetes (54%), Firmicutes (36%), Proteobacteria (4%) and Tenericutes (2%). The experiment showed that, bacterial population in the rumen of mithun fed on mixed tree leaves and rice straw based diet harbor diversified species of bacteria responsible for lignocellulosic feedstuffs. &nbsp;&nbsp;&nbsp;&nbsp; &nbsp; Key words: Tree leaves, bacteria, clones, sequences.

Effects of changes in microbial community on the fermentative production of hydrogen and soluble metabolites from Chlorella pyrenoidosa biomass in semi-continuous operation

16s rRNA gene sequence analysis is applied in the semi-continuous fermentation of Chlorella pyrenoidosa to monitor changes in the microbial community of HPB (hydrogen-producing bacteria). The microbial community of HPB on the 4th day is mainly composed of one genus (Clostridium) including one primary species (Clostridium butyricum). The microbial community of HPB on the 22nd day is mainly composed of three genera (Clostridium, Sporanaerobacter and Eubacterium) including three primary species (Clostridium acetireducens, Clostridium cochlearium and Sporanaerobacter acetigenes). Fermentation results show that production of hydrogen and soluble metabolites by the simple microbial community is unstable with fluctuating pH values at earlier stages (days 1–4). By contrast, the production of hydrogen (51.7–62.0 mL/g-total volatile solids) and soluble metabolites (acetate/butyrate: 0.4–0.5 mol/mol) by complex microbial communities is fairly stable with a steady pH value of approximately 5.9 at later stages of fermentation (days 11–22).

Validation of a novel extraction method for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation by ruminal microbiota

A simple, fast liquid-liquid extraction method was developed for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation using small sample volumes. The method was tested in vitro with anaerobic incubations of RDX with whole rumen fluid (WRF) and a commercial Sporanaerobacter acetigenes strain in methanogenic media for RDX. Additionally, validation experiments were conducted in deionized water in order to show applicability toward various aqueous matrices. Conditions for extraction were as follows: 300 μL of sample were mixed with an equal volume of a 0.34 M ammonium hydroxide solution to reach a basic pH, extracted with a hexane/ethyl acetate 1:1 (v/v) solution (1 mL) and shaken vigorously for 10 s. The resulting organic phase was transferred, then dried under a constant flow of N2 and reconstituted with acetonitrile (300 μL) for HPLC-UV and LC-MS/MS analysis. Percent recovery values were obtained (83-101%) in all matrices for RDX. In WRF (n=3 animals), RDX degradation was observed with almost 100% elimination of RDX after 4 h. The five nitroso and ring cleavage metabolites were observed by mass spectrometry. Liquid cultures of S. acetigenes did not show significant RDX biodegradation activity. RDX extractions from deionized water samples indicated acceptable recoveries with low variability, suggesting suitability of the method for aqueous matrices. Overall, the new method demonstrated acceptable efficiency and reproducibility across three matrices, providing an advantageous alternative for studies where complex matrices and small volume samples are in use.

Characterization of Microbial Community in the Leachate Associated with the Decomposition of Entombed Pigs

Foot and mouth disease (FMD) is one of the acute infectious diseases in hoofed and even-toed mammals, including pigs, and it occurs via acute infection by Aphthovirus. When FMD is suspected, animals around the location of origin are typically slaughtered and buried. Other methods such as rendering, composting, and incineration have not been verified in practice in Korea. After the FMD incident, the regular monitoring of the microbial community is required, as microorganisms greatly modify the characteristics of the ecosystem in which they live. This is the result of their metabolic activities causing chemical changes to take place in the surrounding environment. In this study, we investigated changes in the microbial community during a 24 week period with DNA extracts from leachate, formed by the decomposition of buried pigs at a laboratory test site, using denaturing gradient gel electrophoresis (DGGE) with a genomic DNA. Our results revealed that Bacteroides coprosuis, which is common in pig excreta, and Sporanaerobacter acetigenes, which is a sulfur-reduced microbe, were continuously observed. During the early stages (0~2 weeks) of tissue decomposition, Clostridium cochlearium, Fusobacterium ulcerans, and Fusobacterium sp., which are involved in skin decomposition, were also observed. In addition, various microbes such as Turicibacter sanguinis, Clostridium haemolyticum, Bacteroides propionicifaciens, and Comamonas sp. were seen during the later stages (16~24 weeks). In particular, the number of existing microbial species gradually increased during the early stages, including the exponential phase, decreased during the middle stages, and then increased again during the later stages. Therefore, these results indicate that the decomposition of pigs continues for a long period of time and leachate is created continuously during this process. It is known that leachate can easily flow into the neighboring environment, so a long-term management plan is needed in burial locations for FMD-infected animals.

Microbial community analysis during continuous fermentation of thermally hydrolysed waste activated sludge

Acidogenic fermentation of thermally hydrolysed waste activated sludge was carried out at laboratory scale in two reactors operated under different hydraulic retention times (HRT). Process performance was assessed in terms of volatile fatty acid (VFA) composition and yield. The diversity of the microbial population was investigated by constructing a 16S rRNA gene library and subsequent phylogenetic analysis of clones. Fluorescence in situ hybridization (FISH) was used to assess the relative abundance of different bacterial groups. Bacteroidetes and Firmicutes were the dominant taxonomic groups representing 93% of the total sequences obtained in the reactor with 4 d HRT. A similar VFA yield (0.4-0.5 g VFA(COD) g SCOD(-1)) was obtained for the HRTs tested (1-4 d), indicating that extended retention times were not useful. Within Firmicutes, Clostridia was the major group detected in the clone sequences. These had close affiliation to Sporanaerobacter acetigenes, suggesting organisms of this group were important for hydrolysis of the protein fraction of the substrate. However, FISH analysis failed to detect the major portion of the bacteria, and this is most likely due to the lack of appropriate probes. This work emphasizes the diversity of fermentative communities, and indicates that more work is needed to identify and detect the important members.

Ovine Ruminal Microbes Are Capable of Biotransforming Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX)

Bioremediation is of great interest in the detoxification of soil contaminated with residues from explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although there are numerous forms of in situ and ex situ bioremediation, ruminants would provide the option of an in situ bioreactor that could be transported to the site of contamination. Bovine rumen fluid has been previously shown to transform 2,4,6-trinitrotoluene (TNT), a similar compound, in 4 h. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within 4 h. Whole ovine rumen fluid was then inoculated into five different types of media to select for archaeal and bacterial organisms capable of RDX biotransformation. Cultures containing 30 μg mL(-1) RDX were transferred each time the RDX concentration decreased to 5 μg mL(-1) or less. Time point samples were analyzed for RDX biotransformation by HPLC. The two fastest transforming enrichments were in methanogenic and low nitrogen basal media. After 21 days, DNA was extracted from all enrichments able to partially or completely transform RDX in 7 days or less. To understand microbial diversity, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted. Cloning and sequencing of partial 16S rRNA fragments were performed on both low nitrogen basal and methanogenic media enrichments. Phylogenetic analysis revealed similar homologies to eight different bacterial and one archaeal genera classified under the phyla Firmicutes, Actinobacteria, and Euryarchaeota. After continuing enrichment for RDX degraders for 1 year, two consortia remained: one that transformed RDX in 4 days and one which had slowed after 2 months of transfers without RDX. DGGE comparison of the slower transforming consortium to the faster one showed identical banding patterns except one band. Homology matches to clones from the two consortia identified the same uncultured Clostridia genus in both; Sporanaerobacter acetigenes was identified only in the consortia able to completely transform RDX. This is the first study to examine the rumen as a potential bioremediation tool for soils contaminated with RDX, as well as to discover S. acetigenes in the rumen and its potential ability to metabolize this energetic compound.

Diversity of the resident microbiota in a thermophilic municipal biogas plant

Biogas plants continuously convert biological wastes mainly into a mixture of methane, CO2 and H2O-a conversion that is carried out by a consortium of bacteria and archaea. Especially in the municipal plants dedicated towards waste treatment, the reactor feed may vary considerably, exposing the resident microbiota to a changing variety of substrates. To evaluate how and if such changes influence the microbiology, an established biogas plant (6,600 m3, up to 600 m3 biogas per h) was followed over the course of more than 2 years via polymerase chain reaction-denaturing gradient gel electrophoresis of 16S rRNA genes and subsequent sequencing. Both the bacterial and the archaeal community remained stable over the investigation. Of the bacterial consortium, about half of the sequences were in decreasing order of occurrence: Thermoacetogenium sp., Anaerobaculum mobile, Clostridium ultunense, Petrotoga sp., Lactobacillus hammesii, Butyrivibrio sp., Syntrophococcus sucromutans, Olsenella sp., Tepidanaerobacter sp., Sporanaerobacter acetigenes, Pseudoramibacter alactolyticus, Lactobacillus fuchuensis or Lactobacillus sakei, Lactobacillus parabrevis or Lactobacillus spicheri and Enterococcus faecalis. The other half matched closely to ones from similar habitats (thermophilic anaerobic methanogenic digestion). The archaea consisted of Methanobrevibacter sp., Methanoculleus bourgensis, Methanosphaera stadtmanae, Methanimicrococcus blatticola and uncultured Methanomicrobiales. The role of these species in methane production is discussed.

Sporanaerobacter acetigenes gen. nov., sp. nov., a novel acetogenic, facultatively sulfur-reducing bacterium.

A strictly anaerobic, moderately thermophilic, sporulating rod, designated strain Lup 33T, was isolated from an upflow anaerobic sludge blanket (UASB) reactor in Mexico. Strain Lup 33T possessed a few laterally inserted flagella, had a DNA G+C content of 32.2 mol % and grew optimally at pH 7.4 and 40 degrees C. Growth was observed at temperatures of up to 50 degrees C and was inhibited in the presence of 5% NaCl. Strain Lup 33T is heterotrophic and utilized some sugars, peptides and various single amino acids. Gelatin and casein were not used as energy sources. It performed the Stickland reaction and reduced elemental sulfur to sulfide. Acetate was the only fatty acid detected from glucose fermentation, whereas acetate together with isobutyrate and isovalerate were found as end products from peptone fermentation. Phylogenetically, strain Lup 33T branched with members of cluster XII of the order Clostridiales, with Clostridium hastiforme as the closest relative (similarity of 93%). On the basis of the phenotypic, genotypic and phylogenetic characteristics of the isolate, it is proposed as a novel species of a new genus, Sporanaerobacter acetigenes gen. nov., sp. nov. The type strain is strain Lup 33T (= DSM 13106T = CIP 106730T).