Determination of the optimal culture conditions for detecting thermophilic campylobacters in environmental water

This study evaluated alternative protocols for culturing thermophilic campylobacters in environmental water. All samples were filtered through a sterile 0.45 μm pore-size membrane, which was then incubated in Preston enrichment broth. Four variables were compared: water sample volume (2000 mL vs. 500 mL), enrichment broth volume (25 mL vs. 100 mL), enrichment incubation duration (24 h vs. 48 h), and number of enrichment passages (one vs. two). In addition, DNA extracts were prepared from all final broths and analyzed using three rRNA PCR assays. River water was collected at 3 sampling sites weekly for 9 weeks. Among these 27 collections, 25 (93%) yielded Campylobacter spp. under at least one of the 16 culture conditions. By univariate analysis, yields were significantly better for the 2000 mL sample volume (68.5% vs. 43.0%, p < 0.0001) and the 25 mL enrichment broth volume (64.5% vs. 47.0%, p < 0.0004). Neither of the enrichment period had a significant effect, although there was a trend in favor of 48 h incubation (59.5% vs. 52.0%, p = 0.13). The three PCR methods gave concordant results for 66 (33%) of the culture-negative samples and 103 (50%) of the culture-positive samples. Compared with culture results, Lubeck's 16S PCR assay had the best performance characteristics, with a sensitivity of 82% and a specificity of 94%. Of the 12 culture-negative samples positive by Lubeck's PCR assay, 11 (92%) samples were also positive by Denis' 16S PCR assay, suggesting that in these cases the culture might have been falsely negative. Based on our results, we conclude that the optimal conditions for detecting Campylobacter spp. in natural waters include 2000 mL sample volume and a single enrichment broth of 25 mL PB incubated for 48 h.     

Isolation of a solventogenic Clostridium sp. strain: Fermentation of glycerol to n-butanol,analysis of the bcs operon region and its potential regulatory elements

A new solventogenic bacterium, strain GT6, was isolated from standing water sediment. 16S-rRNA gene analysis revealed that GT6 belongs to the heterogeneous Clostridium tetanomorphum group of bacteria exhibiting 99% sequence identity with C. tetanomorphum 4474^T. GT6 can utilize a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose and glycerol to produce mainly n-butanol without any acetone. Additional products of GT6 metabolism were ethanol, butyric acid, acetic acid, and trace amounts of 1,3-propanediol. Medium and substrate composition, and culture conditions such as pH and temperature influenced product formation. The major fermentation product from glycerol was n-butanol with a final concentration of up to 11.5 g/L. 3% (v/v) glycerol lead to a total solvent concentration of 14 g/L within 72 h. Growth was not inhibited by glycerol concentrations as high as 15% (v/v).     

Effective degradation of tannic acid by immobilized rumen microbes of a sika deer (Cervus nippon yesoensis) in winter

In winter seasons, wild sika deer (Cervus nippon yesoensis) inhabiting the Shiretoko Peninsula of Hokkaido Island, Japan, mainly graze woody materials (bark and twigs, etc.) as their feed source. Most of the tree species that they feed upon contain a high level of hydrolysable tannins within the inner bark. Tannins generally lead to low protein digestion and nutrient loss to these herbivorous mammals due to tannization of proteins. In winter months, it is speculated that wild sika deer develop a mechanism to degrade the tannins which are contained in their feed sources, but rumen fluid obtained from sika deer in winter months did not exhibit any ability to degrade tannins in liquid culture medium. However, constant degradation of hydrolysable tannin was observed when Ca-alginate gel beads were used for microbial immobilization and culturing. The gel beads that had been impregnated with 0.6×10⁴ fold-diluted rumen fluid of sika deer in winter and pre-incubated for 24 h under anaerobic conditions supplemented with a 1.5 g/L sugar were reacted with 5 g/L tannic acid solution. Under these conditions, the immobilized rumen bacteria grown in the macrogel beads effectively hydrolyzed tannic acid to release gallic acid monomers. Major bacterial colonies emerging in the Ca-alginate gel beads were identified as Streptococcus macedonicus and this bacterium (EC-D140) was regarded as the most likely candidate as the tannin-degrading bacterium.     

Nutritional characterization of Mucuna pruiriens: 2. In vitro ruminal fluid fermentability of Mucuna pruriens,Mucunal-dopa and soybean meal incubated with or without l-dopa

Three in vitro experiments were conducted to determine the rumen fermentability of Mucuna (M) pruriens (24 g 3,4-dihydroxy-l-phenylalanine (l-dopa)/kg dry matter (DM) and soybean meal treated with (SBD) or without (SB) 138 g l-dopa/kg DM). Additional objectives were to determine if l-dopa inhibits rumen fermentation, and if ruminal microbes can adapt to l-dopa or M. In Experiment 1, ground (1 mm) substrates were incubated in triplicate at 38 °C in 9 ml nutrient media and 1 ml rumen fluid in a series of six, 48 h, consecutive batch cultures. The first culture was inoculated with rumen fluid from two donor cows. Subsequent cultures were inoculated with fluid (1 ml) from the previous culture. The DM digestibility (DMD, 616 g/kg vs. 540 g/kg; P<0.01) and gas production (51.7 ml/g vs. 44.2 ml/g DM; P<0.05) were higher from fermentation of M versus SB but similar for SB and SBD (540 g/kg vs. 554 g/kg and 44.2 ml/g DM vs. 43.5 ml/g DM, respectively). The slopes of the relationships between DMD (g/kg) or gas production (ml/g DM) and fermentation period were not reduced by fermenting M (−0.014 DMD slope; 2.28 gas production slope) or SBD (−0.014 DMD slope; 0.459 gas production slope), instead of SB (−0.002 DMD slope; 1.039 gas production slope), indicating microbial adaptation to M and SBD. Total volatile fatty acid concentration (VFA; 53.7, 54.9 and 54.9 mmol/l) and molar proportions of VFA were similar among substrates. Gas production kinetics of M versus SB (Experiment 2), and SB versus SBD (Experiment 3) were also measured after substrates were incubated in triplicate in buffered rumen fluid for 24 h using a non-linear exponential model to fit the data. Residual l-dopa was measured after separate fermentation of substrates in triplicate for 0, 4, 8, 16 and 24 h. Fermentation of M versus SB produced more (P<0.05) gas (250 ml/g vs. 100 ml/g DM) and total VFA (203 mmol/l vs. 180 mmol/l) and a lower (P<0.05) acetate:propionate ratio (1.35 vs. 1.87; P<0.05). Adding l-dopa to SB increased (P<0.01) gas production (92 ml/g DM vs. 200 ml/g DM), and total VFA concentration (132 mmol/l vs. 188 mmol/l), but reduced (P<0.05) gas production rate (0.08 ml/h vs. 0.05 ml/h). The concentration of l-dopa in fermented M and SBD decreased by 53 and 47%, respectively during fermentation. In conclusion, M was more fermented than SB and degradation of l-dopa during ruminal fermentation and microbial adaptation to l-dopa were confirmed. Adding l-dopa to SB did not impair ruminal fermentation.     

Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15μm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99-100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.     

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.     

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.     

Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00° N, 25.30° E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17 × 10⁹, 5.17 × 10¹¹ and 4.02 × 10⁷, respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.     

Development and use of quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems

This paper describes the use of a quantitative competitive polymerase chain reaction (QC-PCR) assay; using PCR primers to the rRNA locus of rumen fungi and a standard-control DNA including design and validation. In order to test the efficiency of this method for quantifying anaerobic rumen fungi, it has been attempted to evaluate this method in in vitro conditions by comparing with an assay based on measuring cell wall chitin. The changes in fungal growth have been studied when they are grown in in vitro on either untreated (US) or sodium hydroxide treated wheat straw (TS). Results showed that rumen fungi growth was significantly higher in treated samples compared with untreated during the 12 d incubation (P < 0.05) and plotting the chitin assay's results against the competitive PCR's showed high positive correlation (R² ≥ 0.87). The low mean values of the coefficients of variance in repeatability in the QC-PCR method against the chitin assay demonstrated more reliability of this new approach. And finally, the efficiency of this method was investigated in in vivo conditions. Samples of rumen fluid were collected from four fistulated Holstein steers which were fed four different diets (basal diet, high starch, high sucrose and starch plus sucrose) in rotation. The results of QC-PCR showed that addition of these non-structural carbohydrates to the basal diets caused a significant decrease in rumen anaerobic fungi biomass. The QC-PCR method appears to be a reliable and can be used for rumen samples.     

Influence of urea calcium mixture supplementation on ruminal fermentation characteristics of beef cattle fed on concentrates containing high levels of cassava chips and rice straw

The purpose of this study was to evaluate the effects of various N sources in concentrates containing high levels of cassava chips, with rice straw as the basal forage, on rumen ecology, rumen microbial counts, microbial crude (CP) protein synthesis, and digestibility of nutrients. Four ruminally fistulated crossbred (Brahman × native) beef steers with initial body weight (BW) of 400 ± 40.2 kg were randomly assigned according to a 4 × 4 Latin square design. The dietary treatments were different sources of N in the concentrates and were: T1 = urea (control; urea); T2 = soybean meal (SBM); T3 = urea CaCl₂ mixture (U-Cal); T4 = urea CaSO₄ mixture (U-Cas). All steers were kept in individual pens and supplemented with concentrate at 5 g/kg of BW daily. The experiment was 4 periods, and each lasted 21 d. During the first 14 d, all steers were fed their respective diets ad libitum and for during the last 7 d, they were moved to metabolism crates for total urine and fecal collection. Dry matter intake ranged from 9.8 to 10.5 kg daily and was not altered by diet, while digestibility of NDF differed among treatments and was highest with U-Cas supplementation (P<0.05). Ruminal NH₃ N and plasma urea N with U-Cal, U-Cas, and SBM diets were lower compared with the urea supplemented group (P<0.05). Ruminal volatile fatty acid concentrations were not altered by treatments. Total viable, and cellulolytic bacteria, differed among treatments and were highest with U-Cas (9.1 × 10¹¹, and 4.0 × 10⁹ cfu/mL, respectively). In addition, efficiency of rumen microbial CP synthesis based on organic matter (OM) truly digested in the rumen was increased by SBM or U-Cal supplementation, and was highest with U-Cas supplementation (18.2 g of N/kg of OM truly digested in the rumen). Supplementation of U-Cas to a concentrate containing a high level of cassava chips improved rumen ecology and microbial CP synthesis in beef cattle, suggesting that urea calcium mixtures can replace soybean meal or urea in beef cattle diets without adverse affects on rumen fermentation and other rumen parameters.     

Bacteria associated with mucus and tissues of the coral Oculina patagonica in summer and winter

The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 x 10(7) and 8.3 x 10(8)/cm2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different alpha-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (> 99.5% identity). Of these, 295 were novel (< 99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.     

Neotabrizicola shimadae gen. nov., sp. nov., an aerobic anoxygenic phototrophic bacterium harbouring photosynthetic genes in the family Rhodobacteraceae,isolated from a terrestrial hot spring

A bacteriochlorophyll-containing bacterium, designated as strain N10^T, was isolated from a terrestrial hot spring in Nagano Prefecture, Japan. Gram-stain-negative, oxidase- and catalase-positive and ovoid to rod-shaped cells showed the features of aerobic anoxygenic phototrophic bacteria, i.e., strain N10^T synthesised bacteriochlorophylls under aerobic conditions and could not grow anaerobically even under illumination. Genome analysis found genes for bacteriochlorophyll and carotenoid biosynthesis, light-harvesting complexes and type-2 photosynthetic reaction centre in the chromosome. Phylogenetic analyses based on the 16S rRNA gene sequence and 92 core proteins revealed that strain N10^T was located in a distinct lineage near the type species of the genera Tabrizicola and Xinfangfangia and some species in the genus Rhodobacter (e.g., Rhodobacter blasticus). Strain N10^T shared?<?97.1% 16S rRNA gene sequence identity with those species in the family Rhodobacteraceae. The digital DNA–DNA hybridisation, average nucleotide identity and average amino acid identity values with the relatives, Tabrizicola aquatica RCRI19^T (an aerobic anoxygenic phototrophic bacterium), Xinfangfangia soli ZQBW^T and R. blasticus ATCC 33485^T were 19.9–20.7%, 78.2–79.1% and 69.1–70.1%, respectively. Based on the phenotypic features, major fatty acid and polar lipid compositions, genome sequence and phylogenetic position, a novel genus and species are proposed for strain N10^T, to be named Neotabrizicola shimadae (=?JCM 34381^T?=?DSM 112087T). Strain N10^T which is phylogenetically located among aerobic anoxygenic phototrophic bacteria (Tabrizicola), bacteriochlorophyll-deficient bacteria (Xinfangfangia) and anaerobic anoxygenic phototrophic bacteria (Rhodobacter) has great potential to promote studies on the evolution of photosynthesis in Rhodobacteraceae.

     

Enhanced start-up of anaerobic facultatively autotrophic biocathodes in bioelectrochemical systems

Biocathodes in bioelectrochemical systems (BESs) can be used to convert CO₂ into diverse organic compounds through a process called microbial electrosynthesis. Unfortunately, start-up of anaerobic biocathodes in BESs is a difficult and time consuming process. Here, a pre-enrichment method was developed to improve start-up of anaerobic facultatively autotrophic biocathodes capable of using cathodes as the electron donor (electrotrophs) and CO₂ as the electron acceptor. Anaerobic enrichment of bacteria from freshwater bog sediment samples was first performed in batch cultures fed with glucose and then used to inoculate BES cathode chambers set at −0.4 V (versus a standard hydrogen electrode; SHE). After two weeks of heterotrophic operation of BESs, CO₂ was provided as the sole electron acceptor and carbon source. Consumption of electrons from cathodes increased gradually and was sustained for about two months in concert with a significant decrease in cathode chamber headspace CO₂. The maximum current density consumed was −34 ± 4 mA/m². Biosynthesis resulted in organic compounds that included butanol, ethanol, acetate, propionate, butyrate, and hydrogen gas. Bacterial community analyses based on 16S rRNA gene clone libraries revealed Trichococcus palustris DSM 9172 (99% sequence identity) as the prevailing species in biocathode communities, followed by Oscillibacter sp. and Clostridium sp. Isolates from autotrophic cultivation were most closely related to Clostridium propionicum (99% sequence identity; ZZ16), Clostridium celerecrescens (98–99%; ZZ22, ZZ23), Desulfotomaculum sp. (97%; ZZ21), and Tissierella sp. (98%; ZZ25). This pre-enrichment procedure enables simplified start-up of anaerobic biocathodes for applications such as electrofuel production by facultatively autotrophic electrotrophs.     

Analysis of Community Composition during Moderately Thermophilic Bioleaching of Pyrite,Arsenical Pyrite,and Chalcopyrite.

An analysis of the community composition of three previously undefined mixed cultures of moderately thermophilic bioleaching bacteria grown at 45°C on pyrite, arsenical pyrite, and chalcopyrite has been carried out. The bacterial species present were identified by comparative sequence analysis of the 16S rRNA gene isolated from the bioleaching vessels and analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The mixed cultures leached all three minerals, as shown by the increase in iron released from the mineral concentrates. The species identified from the mixed cultures during bioleaching of pyrite, arsenical pyrite, and chalcopyrite were clones closely related to Acidithiobacillus caldus C-SH12, Sulfobacillus thermosulfidooxidans AT-1, Sulfobacillus montserratensis L15, and an uncultured thermal soil bacterium YNP. It was also found that the same mixed culture maintained for over a year on chalcopyrite mineral selected approximately the same consortia of bacteria as the original mixed culture grown on chalcopyrite.     

Sulfidogenesis in Low pH (3.8–4.2) Media by a Mixed Population of Acidophilic Bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8–4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8–4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.     

Development of an oligonucleotide probe targeting 16S rRNA and its application for detection and quantitation of the ruminal bacterium Synergistes jonesii in a mixed-population chemostat.

Radiolabelled and fluorescent-dye-conjugated oligonucleotide probes which targeted rRNA sequences were developed for the enumeration of the ruminal bacterium Synergistes jonesii 78-1 in mixed culture. Two probes were tested, and both were highly specific for the respective complementary sequences of the target organism. Individual cells of S. jonesii in pure and mixed cultures were clearly visualized in situ by hybridization with the fluorescent-dye-conjugated probe but could not be detected in natural samples. Therefore the radiolabelled probe was used to monitor the population of S. jonesii introduced into a chemostat which simulated the rumen ecosystem. The S. jonesii probe did not hybridize to RNA extracted from the culture prior to inoculation with the target organism. After inoculation, S. jonesii rRNA represented 4.5% of the total bacterial rRNA and then rapidly declined to < 0.2% before increasing to about 1% of the total bacterial rRNA during the following 3 weeks. This study demonstrates that rRNA-targeted probes could be used for tracking organisms introduced into the rumen ecosystem.     

Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia)

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.     

Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food

Ting is a spontaneously fermented sorghum food that is popular for its sour taste and unique flavour. Insight of the microbial diversity and population dynamics during sorghum fermentations is an essential component of the development of starter cultures for commercial production of ting. In this study, bacterial populations associated with spontaneous sorghum fermentations were examined using a culture-independent strategy based on denaturing gradient gel electrophoresis and sequence analysis of V3-16S rRNA gene amplicons, and a culture-dependent strategy using conventional isolation based on culturing followed by 16S rRNA and/or pheS gene sequence analysis. The entire fermentation process was monitored over a 54 h period and two phases were observed with respect to pH evolution and microbial succession. The first phase of the process (0-6 h) was characterized by relatively high pH conditions and the presence of Enterococcus mundtii, albeit that this species was only detected with the culture-dependent approach. The second phase of the fermentation process (12-54 h) was characterized by increased acidity and the predominance of a broader range of lactic acid bacteria, including Lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Weissella cibaria, Enterococcus faecalis, and a close relative of Lactobacillus curvatus, as well as some members of the Enterobacteriaceae family. The Lb. curvatus-like species was only detected with PCR-DGGE, while the majority of the other species was only detected using the culture-dependent approach. These findings highlighted the fact that a combination of both approaches was essential in revealing the microbial diversity and dynamics during spontaneous sorghum fermentations.     

Arthrospiribacter ruber gen. nov., sp. nov., a novel bacterium isolated from Arthrospira cultures

Polyphasic analysis of ten isolates of the red-pigmented bacteria isolated from ten Arthrospira cultures originating from different parts of the world is described. The 16S rRNA analysis showed <95 % identity with the known bacteria on public databases, therefore, additional analyses of fatty acids profiles, MALDI-TOF/MS, genome sequencing of the chosen isolate and following phylogenomic analyses were performed. Gram-stain-negative, strictly aerobic rods were positive for catalase, negative for oxidase, proteolytic and urease activity. Major fatty acids were 15 : 0 iso, 17:0 iso 3 OH and 17:1 iso w9c/16:0 10-methyl. The whole phylogenomic analyses revealed that the genomic sequence of newly isolated strain DPMB0001 was most closely related to members of Cyclobacteriaceae family and clearly indicated distinctiveness of newly isolated bacteria. The average nucleotide identity and in silico DNA–DNA hybridisation values were calculated between representative of the novel strains DPMB0001 and its phylogenetically closest species, Indibacter alkaliphilus CCUG57479 (LW1)^T (ANI 69.2 % is DDH 17.2 %) and Mariniradius saccharolyticus AK6^T (ANI 80.02 % isDDH 26.1 %), and were significantly below the established cut-off <94 % (ANI) and <70 % (isDDH) for species and genus delineation.The obtained results showed that the analysed isolates represent novel genus and species, for which names Arthrospiribacter gen nov. and Arthrospiribacter ruber sp. nov. (type strain DPMB0001 = LMG 31078 = PCM 3008) is proposed.