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Cereal distillers grains, a by-product from bioethanol industry, proved to be a suitable feedstock for biogas production in laboratory scale anaerobic digesters. Five continuously stirred tank reactors were run under constant conditions and monitored for biogas production and composition along with other process parameters. Iron additives for sulfide precipitation significantly improved the process stability and efficiency, whereas aerobic pretreatment of the grains had no effect. The microbial communities in the reactors were investigated for their phylogenetic composition by terminal restriction fragment length polymorphism analysis and sequencing of 16S rRNA genes. The bacterial subcommunities were highly diverse, and their composition did not show any correlation with reactor performance. The dominant phylotypes were affiliated to the Bacteroidetes. The archaeal subcommunities were less diverse and correlated with the reactor performance. The well-performing reactors operated at lower organic loading rates and amended with iron chloride were dominated by aceticlastic methanogens of the genus Methanosaeta. The well-performing reactor operated at a high organic loading rate and supplemented with iron hydroxide was dominated by Methanosarcina ssp. The reactor without iron additives was characterized by propionate and acetate accumulation and high hydrogen sulfide content and was dominated by hydrogenotrophic methanogens of the genus Methanoculleus.

A gram-negative prototrophic bacterial species, strain MC1, was isolated from the vicinity of herbicide-contaminated building rubble and identified by 16S rDNA sequence analysis, its physiological properties, GC content, and fatty acid composition as Comamonas acidovorans. This strain displays activity for the productive degradation of the two enantiomers of dichlorprop [(RS)-2-(2,4-dichlorophenoxy-)propionate; (RS)-2,4-DP] and mecoprop [(RS)-2-(4-chloro-2-methyl-) phenoxypropionate ; (RS)-MCPP] in addition phenoxyacetate herbicides, i.e. 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), and various chlorophenols were utilized. Rates amounted to 1.2 mmoles/h g dry mass (2,4-D) and 2.7 mmoles/h g dry mass [(RS)-2,4-DP]. Degradation of (RS)-2,4-DP was not inhibited up to concentrations of 500 mg/l, nor of 2,4-D up to 200 mg/l. The optimum pH value of (RS)-2,4-DP degradation was around 8. The application of respective primers for PCR amplification revealed the presence of tfdB and tfdC genes.

Aims: To investigate the effect of the forage source and feed particle size (FPS) in ruminant rations on the composition of the ruminal Firmicutes community in vitro.Methods and Results: Three diets, varying in maize silage to grass silage ratio and FPS, were incubated in a rumen simulation system. Microbial samples were taken from the liquid fermenter effluents. Microbial community analysis was performed by 16S rRNA-based techniques. Clostridia-specific single-strand conformation polymorphism profiles revealed changes of the community structure in dependence on both factors tested. The coarse grass silage-containing diets seemed to enhance the occurrence of different Roseburia species. As detected by real-time quantitative PCR, Ruminococcus albus showed a higher abundance with decreasing FPS. A slightly lower proportion of Bacilli was found with increasing grass silage to maize silage ratio by fluorescence in situ hybridization (FISH). In contrast, a slightly higher proportion of bacterial species belonging to the Clostridium-clusters XIV a and b was detected by FISH with increasing grass silage contents in the diet.Conclusions: The ruminal Firmicutes community is affected by the choice of the forage source and FPS.Significance and Impact of the Study: This study supplies fundamental knowledge about the response of ruminal microbial communities to changing diets. Moreover, the data suggest a standardization of grinding of feeds for in vitro studies to facilitate the comparison of results of different laboratories

Analysis of ISCR8 (ISPps1) revealed that this group of insertion elements has to be subdivided into three subgroups: ISCR8, ISCR22, and ISCR23. The distinction of three subgroups is supported by phylogenetic analysis of the transposase open reading frames (ORFs). Comparison of over 20 complete and partial ISCR8/22/23 elements identified oriIS candidate sequences for all groups and a terIS candidate sequence for ISCR8. The oriIS sequences, their distance to the transposase ORFs, and the sequence of this intervening region are group specific, further supporting the definition of two new ISCR elements. ISCR8/22/23 have a very broad host range, including Gram-positive and Gram-negative bacteria, among which are several (opportunistic) pathogens. The IS often resides on plasmids or in the vicinity of other mobile elements and is mostly associated with genes for the degradation of halo- or nitro-aromatics. However, in one case ISCR8 was found in the neighborhood of an antibiotic resistance determinant in Klebsiella pneumoniae. ISCR8 resembles other IS91 family elements in mediating genetic rearrangements by homologous recombination between two copies. In Delftia acidovorans this led to the loss of the genes encoding dichlorprop cleavage. In conclusion, this study shows that ISCR8 could be a fully functional and active member of the IS91 family of insertion elements.

This study examined whether different corn silage to grass silage ratios in ruminant rations and different grinding levels of the feed affect the composition of the ruminal Bacteroides-Prevotella community in vitro. Three diets, composed of 10% soybean meal as well as of different corn silage and grass silage proportions, were ground through 1 mm or 4 mm screened sieves and incubated in a semi-continuous rumen simulation system. On day 14 of the incubation microbes were harvested by centrifugation from the liquid effluent of fermenter vessels. Microbial DNA was extracted for single strand conformation polymorphism (SSCP) analysis of 16S rRNA genes followed by sequencing of single SSCP bands. Fluorescence in situ hybridization (FISH) and real-time quantitative (q) PCR were used to quantify differences in the relative abundance of Bacteroides-Prevotella and Prevotella bryantii. SSCP profiles revealed a significant influence of the forage source as well as of the feed particle size on the community structure of the Bacteroides-Prevotella group. Different, phylogenetically distinct, so far uncultured Prevotella species were detected by sequence analysis of several treatment-dependent occurring SSCP bands indicating different nutritional requirements of these organisms for growth. No quantitative differences in the occurrence of Bacteroides-Prevotella-related species were detected between diets by FISH with probe BAC303. However, real-time qPCR data revealed a higher abundance of P. bryantii with increasing grass silage to corn silage ratio, thus again indicating changes within the community composition of the Bacteroides-Prevotella group. As P. bryantii possesses high proteolytic activity its higher abundance may have been caused by the higher contents of crude protein in the grass silage containing diets. To conclude, results of this study show an influence of the forage source on the ruminal community of Bacteroides-Prevotella. Furthermore, they suggest an effect of t

Electrokinetically stimulated bioremediation of soils (electro-bioremediation) requires that the application of weak electric fields has no negative effect on the contaminant degrading microbial communities. This study evaluated the hypothesis that weak direct electric current (DC) fields per se do not negatively influence the physiology and composition of soil microbial communities given that secondary electrokinetic phenomena such as soil pH changes and temperatures are minimized. Mildly buffered, water-saturated laboratory mesocosms with agricultural soil were subjected for 34 days to a constant electric field (X = 1.4 V cm- 1; J 1.0 mA cm- 2) and the spatiotemporal changes of soil microbial communities assessed by fingerprints of phospholipids fatty acids (PLFA) and terminal restriction fragment length polymorphisms (T-RFLP) of bacterial 16S rRNA genes. DC-induced electrolysis of the pore water led to pH changes (< 1.5 pH units) in the immediate vicinity of the electrodes and concomitant distinct soil microbial community changes. By contrast, DC-treated bulk soil distant to the electrodes showed no pH changes and developed similar PLFA- and T-RFLP-fingerprints as control soil in the absence of DC. Our data suggest that the presence of an electric field, if suitably applied, will not influence the composition and physiology of soil microbial communities and hence not affect their potential to biodegrade contaminants.

Sulfate-reducing bioreactors are a promising option for the treatment of acid mine drainage. We studied the structure and function of a biofilm in a methanol-fed fixed-bed in-lake reactor for the treatment of an acidic pit lake by a combination of laboratory incubations, chemical and molecular analyses and confocal laser scanning microscopy to determine whether competition by different groups of microorganisms as well as the precipitation of minerals affect reactor performance negatively. The biofilm growing on the surface of a synthetic carrier material consisted of dense microbial colonies covered by iron-sulfide precipitates. The microorganisms continuously had to overgrow this mineral coating, resulting in a high biomass turnover. About one third of the added methanol was used by sulfate reduction, and the rest by competing reactions. Sulfate-reducing bacteria as well as methanogens and acetogens were involved in methanol consumption. Six different groups of Deltaproteobacteria, dominated by the genera Desulfomonile, Desulfobacterium and a phylotype related to Geobacter, Gram-positive sulfate reducers of the genus Desulfosporosinus, acetogenic Acetobacteria, different fermenting bacteria as well as methylotrophic methanogens were identified. The versatility of the microbial food web is probably an important factor stabilizing the biofilm function under fluctuating and partly oxidizing conditions in the reactor.

Aims: This study intended to unravel the physiological interplay in an anaerobic microbial community that degrades toluene under sulfate-reducing conditions combining proteomic and genetic techniques.Methods and Results: An enriched toluene-degrading community (Zz5-7) growing in batch cultures was investigated by DNA- and protein-based analyses. The affiliation and diversity of the community were analysed using 16S ribosomal RNA (rRNA) genes as a phylogenetic marker as well as bssA and dsrAB genes as functional markers. Metaproteome analysis was carried out by a global protein extraction and a subsequent protein separation by two-dimensional gel electrophoresis (2-DE). About 85% of the proteins in the spots were identified by nano-liquid chromatography coupled with electrospray mass spectrometry (nano-LC-ESI-MS/MS) analysis. DNA sequencing of bssA and the most abundant dsrAB amplicons revealed high similarities to a member of the Desulfobulbaceae, which was also predominant according to 16S rRNA gene amplicons. Metaproteome analysis provided 202 unambiguous protein identifications derived from 236 unique protein spots. The proteins involved in anaerobic toluene activation, dissimilatory sulfate reduction, hydrogen production/consumption and autotrophic carbon fixation were mainly affiliated to members of the Desulfobulbaceae and several other Deltaproteobacteria.Conclusion: Phylogenetic and metaproteomic analyses revealed a member of the Desulfobulbaceae as the key player of anaerobic toluene degradation in a sulfate-reducing consortium.Significance and Impact of the Study: This is the first study that combines genetic and proteomic analyses to indicate the interactions in an anaerobic toluene-degrading microbial consortium.

Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

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A sulfate-reducing bacterial consortium was enriched from an anoxic aquifer contaminated with BTEX compounds, using toluene as a growth substrate. Total cell counts, protein contents and sulfide production were determined to follow growth at the in situ temperature (14 °C) and at 25 °C, respectively. Community members were identified by 16S rRNA gene cloning and sequencing. Phylogenetic analysis revealed 12 sequence types belonging to Deltaproteobacteria (several groups), Epsilonproteobacteria, Bacteroidetes, Spirochaetaceae and an unclassified bacterial clade. The most prominent phylotype comprising 34% of all clones was affiliated to the Desulfobulbaceae and closely related to environmental clones retrieved from hydrocarbon-contaminated aquifers. Flow-cytometric methods were applied to analyze the community dynamics and to identify key organisms involved in toluene assimilation. Flow-cytometric measurement of DNA contents and scatter behavior served to detect and quantify dominant and newly emerging clusters of subcommunities. Up to seven subcommunities, two of them dominant, were distinguished. Cell sorting was used to facilitate the analysis of conspicuous clusters for phylogenetic identity by terminal restriction fragment length polymorphism profiling of the 16S rRNA genes. The Desulfobulbaceae phylotype accounted for up to 87% in proliferating subcommunities, indicating that it represents the key organism of toluene degradation within this complex anaerobic consortium.

Information on anaerobic phenol metabolism by physiological groups of bacteria other than nitrate reducers is scarce. We investigated phenol degradation in the strictly anaerobic iron-reducing deltaproteobacterium Geobacter metallireducens GS-15 using metabolite, transcriptome, proteome, and enzyme analyses. The results showed that the initial steps of phenol degradation are accomplished by phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) as known from Thauera aromatica, but they also revealed some distinct differences. The pps-ppc gene cluster identified in the genome is functional, as shown by transcription analysis. In contrast to T. aromatica, transcription of the pps- and ppc-like genes was induced not only during growth on phenol, but also during growth on benzoate. In contrast, proteins were detected only during growth on phenol, suggesting the existence of a posttranscriptional regulation mechanism for these initial steps. Phenylphosphate synthase and phenylphosphate carboxylase activities were detected in cell extracts. The carboxylase does not catalyze an isotope exchange reaction between 14CO2 and 4-hydroxybenzoate, which is characteristic of the T. aromatica enzyme. Whereas the enzyme of T. aromatica is encoded by ppcABCD, the pps-ppc gene cluster of G. metallireducens contains only a ppcB homologue. Nearby, but oriented in the opposite direction, is a ppcD homologue that is transcribed during growth on phenol. Genome analysis did not reveal obvious homologues of ppcA and ppcC, leaving open the question of whether these genes are dispensable for phenylphosphate carboxylase activity in G. metallireducens or are quite different from the Thauera counterparts and located elsewhere in the genome.

Microcosms filled with different solids (sand, lava, Amberlite XAD-7) were exposed for 67 days in the sulfidic part of a groundwater monitoring well downstream of the source zone of a benzene-contaminated aquifer and subsequently incubated in the laboratory. Benzene was repeatedly degraded in several microcosms accompanied by production of sulfide, leading to stable benzene-degrading enrichment cultures. In control microcosms without filling material, benzene was initially degraded, but the benzene-degrading capacity could not be sustained. The results indicate that long-term physiologically active benzene-degrading microorganisms were attached to surfaces of the solids. The biodiversity and attachment behavior of microorganisms in the in situ microcosms was assessed by confocal laser scanning microscopy and single-strand conformation polymorphism (SSCP) analysis, followed by sequencing of dominant SSCP bands. The microbial community was composed of several different Bacteria, representing members of Clostridia, Bacteroidales, all subgroups of the Proteobacteria, Verrucomicrobia, Nitrospira, Chloroflexi and Chlorobi. Only a few archaeal sequences could be retrieved from the communities. The majority of phylotypes were affiliated to bacterial groups with a possible functional relationship to the bacterial sulfur cycle, thus indicating that the microbial community in the investigated aquifer zone depends mainly on inorganic sulfur compounds as electron donors or acceptors, a finding that corresponds to the geochemical data.

Analysis of 16S rRNA gene clone libraries from acidic mining lake water and sediment, and from an enclosure to which organic carbon was added to stimulate microbial alkalinization processes of sulfate and iron reduction revealed the presence of diverse sequences affiliated with the Acidobacteria subdivision 1. A novel oligonucleotide probe, ACIDO228, was designed that covered most sequences of Acidobacteria subdivision 1. The hybridization conditions were optimized using the type strain Acidobacterium capsulatum. The depth distribution and seasonal dynamics of Acidobacteria in the lake and the enclosure were assessed by whole cell hybridization. Sequence analyses and in situ quantification indicated that Acidobacteria accounted for a substantial part of bacterioplankton communities in both compartments. During the summer stratification distinct maxima of acidobacterial abundance were detected in the hypolimnion (up to 13% of total cell numbers), whereas during spring and autumn circulations no clear depth-dependent differences were visible. These data suggest that Acidobacteria thrive best in the hypolimnion, which is characterized by lower temperatures and higher availability of organic substrates. The application of probe ACIDO228 provided quantitative information on the seasonal and depth distribution of Acidobacteria in a lake environment and in particular in a rather extreme habitat, an acidic mining lake.

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BackgroundMost bacteria present in nature are not culturable in pure culture by means of classic cultivation methods (Pace NR, 1997, Science 276:734–740; Amann RI et al., 1995, Microbiol Rev 59:143–169.). However, it was recently shown that most aerobic heterotrophic bacteria could grow only on artificial media when other micro-organisms are present (Kaeberlein T et al., 2002, Science 296:1127–1129). Because the sulphate reducer Desulfobacula toluolica DSM 7467 and a bacterium (strain MV1) identified as Cellulosimicrobium sp. were not culturable unaccompanied, flow cytometry was used to highlight the strains' relation within the consortium.MethodsDNA patterns were used to provide strain-specific information about population proliferation dynamics. Cells were grown anaerobically and fed with toluene under sulphate-reducing conditions.ResultsOxidation of toluene occurred only in association with sulphate reduction and growth of D. toluolica. A characteristic chromosomal pattern, with at least six subpopulations of D. toluolica, appeared during the stationary phase, and asymmetric cell division was detected. The accompanying strain MV1 grew repeatedly to a high percentage of the culture only in certain growth phases of D. toluolica independently of the feeding substrate toluene.ConclusionsA commensalistic relation between the two strains is suggested. The repeated rapid and frequent changes of the quantities within the community subsets are indicative of very flexible adaptations to changing environmental conditions, reflecting the need for modulated cell states and the ability to use every available source of carbon and energy for survival

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Microbial communities from three Argentinean saline soils were extracted and tested for their ability to degrade diesel fuel in liquid culture at salinities between 0% and 25%. In each case, the degradation process was continuously monitored by measuring oxygen consumption. Two communities (CR1 and CR2) showed nearly equal degrees of degradation across a salinity range of 0%–10% (the former degrading about 63% of the diesel fuel and the latter about 70% after 53 and 80 d, respectively). Furthermore, the degree of degradation was not significantly lower in the presence of 17.5% salt (58% and 65% degraded, respectively). A third community (El Zorro) showed a maximum turnover at 5% salt (79% diesel fuel degraded) and significant degradation (66%) at a salinity of 10%. However, the degree of degradation by this community clearly dropped at 0% and 15% salt. None of the communities were able to degrade diesel fuel in the presence of 25% salt, but the living cell counts showed that components of the microbial population survived the long-term exposure. The surviving portion is obviously sufficient to allow substantial restoration of the original community, as verified by the BIOLOG method. Isolates of the CR1 community were identified as members of the genera Cellulomonas, Bacillus, Dietzia, and Halomonas. In light of our investigations, the bioremediation of contaminated saline soils should be quite possible if the salinity of the soil water is lower than 15% or if it is reduced below this limit by the addition of water

Primers specific for the genes tfdD, tfdE and tfdF, derived from conserved amino acid sequence motifs of the corresponding homologous enzymes, and primers specific for the genes tfdA and tfdB as well as tfdC taken from the literature were applied in PCR reactions using the genomic DNA of Delftia acidovorans P4a as the template. PCR products were obtained with all primer pairs that were similar in size to those found with the genomic DNA of strains harbouring plasmid pJP4 as the carrier of tfd genes. The nucleotide sequences and the corresponding amino acid sequences of the PCR products obtained with Strain P4a were compared with the sequence databases. According to BLAST analyses, the partial sequences of tfdA and tfdB exhibited a 94–99% degree of identity with the homologous sequences of the 2,4-D-degrading strains Achromobacter xylosoxidans subsp. denitrificans EST4002 (pEST4011), Burkholderia sp. RASC, Variovorax paradoxus TV1 (pTV1) and Burkholderia cepacia 2a (pIJB1), whereas the partial sequences of the tfdC, tfdD, tfdE and tfdF genes revealed a 96–100% degree of identity with the homologous sequences of the chlorobenzene-utilizing strains Ralstonia eutropha NH9 (pENH91), Pseudomonas chlororaphis RW71 and Pseudomonas sp. P51 (pP51).

The broad host range plasmid pJP4, which carries genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoic acid, was used in conjugation experiments with mixed cultures enriched from water and sediment samples from an alkaline pond in the area of Szegedi Fehértó, a soda lake in south Hungary. pJP4-encoded mercury resistance was used as a selection marker. One of the transconjugants, the alkaliphilic, moderately halophilic strain EF43, stably maintained the plasmid and was able to degrade 2,4-D and 3-chlorobenzoate under alkaline conditions in the presence of an additional carbon source such as pyruvate, benzoate, or alpha-ketoglutarate, indicating that the degradative genes of pJP4 were expressed in this strain. However, it was unable to grow on these chloroaromatic substrates when the substrate was the sole source of carbon and energy. Chemostat cultivation experiments revealed that the 2,4-D degradation rate during growth on benzoate or pyruvate was limited by the low activity of chlorocatechol-degrading enzymes, particularly chloromuconate cycloisomerase. Strain EF43 was identified as Halomonas sp. on the basis of 16S rRNA sequencing and additional taxonomic studies. 16S rRNA sequence analysis revealed that strain EF43 is closely related to typical soda lake isolates belonging to the genus Halomonas.

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains d

The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA-binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary-phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary-phase-inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprint-ing experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.