Phylogenetic characterization of microbial mats and streamers from a Japanese alkaline hot spring with a thermal gradient

Dense microbial mats and streamers of various colors developed in an alkaline-hot spring water at 48-76 degrees C and ~0.077 mm sulfide in Nakabusa, Japan. The microbial community structures with a thermal gradient were compared by denaturing gradient gel electrophoresis (DGGE) analysis of the PCR-amplified 16S ribosomal RNA gene fragments. The sequence analysis revealed that a predominant cyanobacterial DGGE band phylogenetically related to Synechococcus elongatus was detected only from green mats at 48 degrees C. Four DGGE bands were detected commonly from green mats at 48 degrees C, orange mats at 58 degrees C and brown mats at 60 degrees C. The sequence analysis revealed that these were phylogenetically related to Chloroflexaceae group, Rhodothermus group, a candidate division OP10, and an unclassified bacterium. On the other hand, Aquificae-, Thermodesulfobacteria-, Thermus group-, and Crenarchaeota-like sequences were detected as a predominant component of DGGE profiling from the streamers only at temperatures over 66 degrees C, but no phototrophic bacterial bands were detected. Thus, the microbial community structure above 60 degrees C was drastically different from that at the lower temperatures. After the addition of hydrogen into in vitro gray streamers with in situ spring water, sulfide production markedly occurred in the presence of ambient sulfate at 66 degrees C. This result suggests that in situ sulfide is partly produced by Thermodesulfobacteria-like sulfate-reducing bacteria in the streamers.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

Analysis of dissimilatory sulfite reductase and 16S rRNA gene fragments from deep-sea hydrothermal sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5 degrees C and natural vent fluids at 7 degrees C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and gamma- and epsilon-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with "Nanoarchaeota." The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300 degrees C were affiliated with the delta-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4 degrees C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.     

Biotechnological potential of microbial consortia and future perspectives

Design of a microbial consortium is a newly emerging field that enables researchers to extend the frontiers of biotechnology from a pure culture to mixed cultures. A microbial consortium enables microbes to use a broad range of carbon sources. It provides microbes with robustness in response to environmental stress factors. Microbes in a consortium can perform complex functions that are impossible for a single organism. With advancement of technology, it is now possible to understand microbial interaction mechanism and construct consortia. Microbial consortia can be classified in terms of their construction, modes of interaction, and functions. Here we discuss different trends in the study of microbial functions and interactions, including single-cell genomics (SCG), microfluidics, fluorescent imaging, and membrane separation. Community profile studies using polymerase chain-reaction denaturing gradient gel electrophoresis (PCR-DGGE), amplified ribosomal DNA restriction analysis (ARDRA), and terminal restriction fragment-length polymorphism (T-RFLP) are also reviewed. We also provide a few examples of their possible applications in areas of biopolymers, bioenergy, biochemicals, and bioremediation.     

Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high Arctic

The springs at Gypsum Hill and Colour Peak on Axel Heiberg Island in the Canadian Arctic originate from deep salt aquifers and are among the few known examples of cold springs in thick permafrost on Earth. The springs discharge cold anoxic brines (7.5 to 15.8% salts), with a mean oxidoreduction potential of -325 mV, and contain high concentrations of sulfate and sulfide. We surveyed the microbial diversity in the sediments of seven springs by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers. Dendrogram analysis of the DGGE banding patterns divided the springs into two clusters based on their geographic origin. Bacterial 16S rRNA clone sequences from the Gypsum Hill library (spring GH-4) were classified into seven phyla (Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria, Spirochaetes, and Verrucomicrobia); Deltaproteobacteria and Gammaproteobacteria sequences represented half of the clone library. Sequences related to Proteobacteria (82%), Firmicutes (9%), and Bacteroidetes (6%) constituted 97% of the bacterial clone library from Colour Peak (spring CP-1). Most GH-4 archaeal clone sequences (79%) were related to the Crenarchaeota while half of the CP-1 sequences were related to orders Halobacteriales and Methanosarcinales of the Euryarchaeota. Sequences related to the sulfur-oxidizing bacterium Thiomicrospira psychrophila dominated both the GH-4 (19%) and CP-1 (45%) bacterial libraries, and 56 to 76% of the bacterial sequences were from potential sulfur-metabolizing bacteria. These results suggest that the utilization and cycling of sulfur compounds may play a major role in the energy production and maintenance of microbial communities in these unique, cold environments.     

The potential of glycerol in freezing preservation of turbine oil-degrading bacterial consortium and the ability of the revised consortium to degrade petroleum wastes

The turbine oil (TuO)-degrading bacterial consortium Tank-2 (original Tank-2) was preserved as a glycerol stock at −80 °C from 2009 to 2012. Storage methods have been unavailable so far for any TuO-degrading bacterial consortia or isolates. To evaluate the usefulness of glycerol stock, the original Tank-2 consortium frozen in glycerol at −80 °C was thawed and then revived by repeated culture in mineral salts medium (MSM) containing 0.5% (w/w) TuO (revived Tank-2). The revived Tank-2 consortium exhibited a high activity to degrade TuO, which was equivalent to that of original Tank-2. It also degraded car engine oil, used car engine oil, Arabian light and Vityaz crude oils and TuO in wastewater. These results indicated that a glycerol stock at −80 °C was useful for storing Tank-2. PCR-denaturing gradient gel electrophoresis (DGGE) that targeted the V3 regions of 16S rRNA gene sequences showed that the DGGE band profiles of principal bacteria were significantly different between the original and revived Tank-2 consortia and between the revived Tank-2 culture grown in MSM containing TuO and that grown in MSM containing other types of petroleum products. This suggested that bacterial strains inherently residing in Tank-2 could adjust their compositions based on the storage and culture conditions.     

Enhancement of methane production from cassava residues by biological pretreatment using a constructed microbial consortium

In the study, a stable thermophilic microbial consortium with high cellulose-degradation ability was successfully constructed. That several species of microbes coexisted in this consortium was proved by DGGE (denaturing gradient gel electrophoresis) and sequence analysis. The cooperation and symbiosis of these microbes in this consortium enhanced their cellulose-degradation ability. The pretreatment of cassava residues mixing with distillery wastewater prior to anaerobic digestion was investigated by using this microbial consortium as inoculums in batch bioreactors at 55 °C. The experimental results showed that the maximum methane yield (259.46 mL/g-VS) of cassava residues was obtained through 12 h of pretreatment by this microbial consortium, which was 96.63% higher than the control (131.95 mL/g-VS). In addition, it was also found that the maximum methane yield is obtained when the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase) and xylanase activity and soluble COD (sCOD) are produced.     

Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.     

Bacterial composition of microbial mats in hot springs in Northern Patagonia: variations with seasons and temperature

Seasonal shifts in bacterial diversity of microbial mats were analyzed in three hot springs (39?C68 °C) of Patagonia, using culture-independent methods. Three major bacterial groups were detected in all springs: Phyla Cyanobacteria and Bacteroidetes, and Order Thermales. Proteobacteria, Acidobacteria and Green Non-Sulfur Bacteria were also detected in small amounts and only in some samples. Thermophilic filamentous heterocyst-containing Mastigocladus were dominant Cyanobacteria in Porcelana Hot Spring and Geyser, and Calothrix in Cahuelmó, followed by the filamentous non-heterocyst Leptolyngbya and Oscillatoria. Bacteroidetes were detected in a wide temperature range and their relative abundance increased with decreasing temperature in almost all samples. Two Meiothermus populations with different temperature optima were found. Overall, fingerprinting analysis with universal bacterial primers showed high similarities within each hot spring despite differences in temperature. On the other hand, Cahuelmó Hot Spring showed a lower resemblance among samples. Porcelana Hot Spring and Porcelana Geyser were rather similar to each other, possibly due to a common geological substrate given their geographic proximity. This was even more evident with specific cyanobacterial primers. The different geological substrate and the seawater influence in Cahuelmó might have caused the differences in the microbial community structure with the other two hot springs.     

Enhancing denitrifying sulfide removal with functional strains under micro-aerobic condition

The biological denitrifying sulfide reaction (DSR) frequently proceeds in anaerobic environments since excess oxygen inhibits the activity of the denitrifiers. This study isolated a consortium H7, comprising two strains, Penibacillus sp. and Aneurinibacillus aneurinilyticus. It indicated that the DSR performance with the H7 in a mixotrophic medium is significantly enhanced at high sulfide concentrations. The H7 was inhibited by adding >200 mg l⁻¹ of S²⁻ under anaerobic conditions. However, when 280 mg ⁻¹ of S²⁻ was added, H7 could still degrade some of the sulfide, nitrate and acetate under micro-aerobic conditions. Micro-aerobic conditions stimulated the activity of sulfide oxidase and increased the removal rate of highly concentrated sulfide, reducing the inhibition of sulfide on denitrifiers and improving DSR performance.     

Assessment of Microbial Diversity in Saudi Springs by Culture-Dependent and Culture-Independent Methods

Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in two hot springs of the Aljouf region in Saudi Arabia, including Qasr Kaff and Ain Hawas. Physicochemical characteristics of the springs were examined to establish their effect on the biodiversity of thermophilic bacteria and fungi. We employed culture-dependent techniques to study microbial diversity using four different complex media for bacteria and fungi. In addition, the direct count for algal populations from two springs was investigated. We surveyed the microbial diversity in water and sediment samples from both springs by denaturing gradient gel electrophoresis (DGGE) and clone library construction. Bacillariophycaea (18 species) was the most diverse group, followed by Cyanophyceaea. Bacterial isolates closer to the genera Bacillus spp., Geobacillus, Thermoactinomyces, and unidentified actinobacteria were recovered. Fungal isolates were related to Aspergillus, Pezizaceae, Penicillium, Acremonium, Fusarium, Chrysosporium, and Stachybotrys. Using molecular-based techniques, the results were slightly different from those obtained by culture-dependent methods, and more genera were obtained. However, most genera were uncultured microbes, particularly from bacterial communities.     

腾冲热海四种不同颜色菌藻席的DGGE图谱比较

研究热泉水化学成分与菌藻席群落结构的关系。直接提取四种菌藻席(mat)总DNA,PER扩增获得16S rDNA的V8高变区片段,进行DGGE分析。结果：四种菌藻席的细菌组成差异很大,DGGE条带数目最低为20,最高为47;4种菌藻席共有的条带为1。结合文献报道的3个热泉的水化学性质分析,表明水化学成分是热泉生态系统中重要的生态因子,直接影响菌藻席的群落结构和物种组成。     

Microbial consortium-mediated reprogramming of defence network in pea to enhance tolerance against Sclerotinia sclerotiorum

Aims: To evaluate the potentiality of three rhizosphere microorganisms in suppression of Sclerotinia rot in pea in consortia mode and their impact on host defence responses. Methods and Results: Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 from rhizospheric soils were selected based on compatibility, antagonistic and plant growth promotion activities. The microbes were used as consortia to assess their ability to trigger the phenylpropanoid and antioxidant activities and accumulation of proline, total phenol and pathogenesis‐related (PR) proteins in pea under the challenge of the soft‐rot pathogen Sclerotinia sclerotiorum. The triple‐microbe consortium and single‐microbe treatments showed 1·4–2·3 and 1·1–1·7‐fold increment in defence parameters, respectively, when compared to untreated challenged control. Activation of the phenylpropanoid pathway and accumulation of total phenolics were highest at 48 h, whereas accumulation of proline and PR proteins along with activities of the antioxidant enzymes was highest at 72 h. Conclusions: The compatible microbial consortia triggered defence responses in an enhanced level in pea than the microbes alone and provided better protection against Sclerotinia rot. Significance and Impact of the Study: Rhizosphere microbes in consortium can enhance protection in pea against the soft‐rot pathogen through augmented elicitation of host defence responses.     

The development of a DNA microarray-based assay for the characterization of commercially formulated microbial products

Commercially formulated bioproducts containing a complex consortia of bacteria as an active ingredient pose a significant challenge for regulatory agencies and companies seeking to assess the safety and efficacy of these bioproducts. The main challenge stems from how to characterize the bacterial composition of these products, for which there is presently a lack of suitable methods. A prototype DNA microarray composed of oligonucleotide probes for functional genes, virulence factors, and taxonomic genes for a number of bacterial species was developed to examine the utility of microarray technology as a molecular tool for characterizing consortia bioproducts. The genomic DNA from four different products was extracted by two methods and examined with the microarray prototype and by denaturing gradient gel electrophoresis (DGGE). Although the identity of the consortial species remains unknown, the microarray assay provided unique and reproducible hybridization patterns for all four products, and agreed with the fingerprints generated by DGGE. The ability to differentiate between a variety of consortia products demonstrates that DNA microarrays have the potential to be a powerful tool in monitoring complex microbial communities.     

大西洋洋中脊深海多环芳烃降解菌群的优势菌分析

摘要：【目的】为了分析大西洋洋中脊深海海水及表层沉积物中多环芳烃(PAHs)降解菌群中的优势菌。【方法】采用富集培养法和平板涂布法从深海样品中分离可培养细菌及PAHs降解菌。通过16S rRNA基因测序完成系统发育分析。采用变性梯度凝胶电泳（DGGE）及DNA测序分析降解菌群中的优势菌。【结果】总共分离到16株细菌,包括一株PAHs降解菌Novosphingobium sp. 4D。系统发育分析发现,可培养细菌中两个最大的类群分别与Alcanivorax dieselolei NO1A（5/16）和Tistrella mobilis TISTR 1108T（5/16）亲缘关系最近。DGGE结果表明,在菌群MC2D中菌株4L（以及4M、4N, Alcanivorax dieselolei NO1A, 99.21％）、4D（Novosphingobium pentaromativorans US6-1T,97.07％）和4B（以及4E、4H、4K,Tistrella mobilis TISTR 1108T,＞99％）是降解菌群中的优势菌。而降解菌群MC3CO中的优势菌是菌株5C（以及5H,Alcanivorax dieselolei NO1A,＞99％）、条带5-8代表的未培养菌株（Novosphingobium aromaticivorans DSM 12444T,99.41%）、5J（Tistrella mobilis TISTR 1108T,99.52％）和5F（以及5G,Thalassospira lucentensis DSM 14000T,＜97％）。【结论】本研究发现在大西洋洋中脊深海海水及表层沉积物中Alcanivorax、Novosphingobium、Thalassospira、Tistrella属的细菌是PAHs降解菌群中的优势菌,其中的主要降解菌是Novosphingobium属的细菌。     

Anaerobic degradation of p-xylene in sediment-free sulfate-reducing enrichment culture

Anaerobic degradation of p-xylene was studied with sulfate-reducing enrichment culture. The enrichment culture was established with sediment-free sulfate-reducing consortium on crude oil. The crude oil-degrading consortium prepared with marine sediment revealed that toluene, and xylenes among the fraction of alkylbenzene in the crude oil were consumed during the incubation. The PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene for the p-xylene degrading sulfate-reducing enrichment culture showed the presence of the single dominant DGGE band pXy-K-13 coupled with p-xylene consumption and sulfide production. Sequence analysis of the DGGE band revealed a close relationship between DGGE band pXy-K-13 and the previously described marine sulfate-reducing strain oXyS1 (similarity value, 99%), which grow anaerobically with o-xylene. These results suggest that microorganism corresponding to pXy-K-13 is an important sulfate-reducing bacterium to degrade p-xylene in the enrichment culture.     

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis,RNA-DNA membrane hybridization,and DNA microarray technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.     

Changes in the composition of an acid mine drainage microbial community upon successive transfers in medium containing low-grade copper sulfide

A consortium of microorganisms from acid mine drainage samples was cultured in modified 9 K medium containing low-grade copper sulfide. The culture was maintained for sixty days and then transferred to fresh medium. This process was repeated three more times and a final consortium exhibiting a copper extraction rate of 89.3% was obtained. RFLP and microarrays analysis of 16S rRNA sequences retrieved from the consortia showed that Acidithiobacilluscaldus, Leptospirillumferriphilum, Sulfobacillus sp., Acidiphilium sp., and Sulfolobus spp. were represented in higher numbers in the consortia obtained in the copper-containing medium than in the original consortium. In contrast, a decrease in Acidithiobacillus ferrooxidans, Alicyclobacillus sp., Pseudomonas sp., and Sulfobacillus thermosulfidooxidans was observed. The abundance of genes related to sulfur metabolism from At. caldus and Sulfolobus spp., iron oxidation from Leptospirillum sp. and metal resistance from most of the detected microorganisms increased as the consortium was successively transferred into fresh medium.