A közösségi anyagcsere vizsgálata anaerob deklorináció során = Investigations on the community metabolism in anaerobe dechlorination

A rövidszénláncú alifás klórozott szénhidrogének gyakori talajvízszennyezők hazánkban. A perklóretén, triklóretén biológiai bontása hatékony folyamat, mégis nagy sebességkülönbségek jellemzők különböző szennyezett területeken. A reduktív deklorinációban a klórozott vegyületek elektronakceptorok, elektrondonor a hidrogén, vagy kis molekulatömegű szerves vegyületek. A folyamat elindulásához a deklorináló baktériumoknak felül kell kerekedniük a kompetítor mikrobákon, amelyek szintén hidrogént és szerves vegyületeket hasznosítanak. Szennyezett területekről származó mikrobaközösségek szerkezetét, tagjainak szerepét laboratóriumi körülmények között polifázikus módszerekkel vizsgáltuk, új molekuláris technikákat vezettünk be a mikrobák közötti anyagcsere-kapcsolatok megértésére. Megállapítottuk, hogy sikeres deklorináló közösségben jellemző a Dehalococcoides ethenogenes dominanciája; a mikrobiális diverzitás csökkenése. A lebontás folyamatát fermentáló szervezetek jelenléte lassítja, akárcsak a kometabolikus partnerek gátlása. Hatékony bontás során biofilm kialakulása jellmező és elengedhetetlen kellő mennyiségű hozzáférhető szerves anyag jelenléte. Négy új módszert (MDA, SNuPE, SEM és FISH) optimáltunk a deklorináló mikroba közösség vizsgálatára. Megállapítottuk, hogy a valós aktivitással jobban korreláló RNS alapú vizsgálatok szükségesek a deklorinációban résztvevő mikrobiális kapcsolatok feltárásához. | Chlorinated short chain aliphatic hydrocarbons are common groundwater pollutants in Hungary. Biological decomposition of perchloroethene and trichloroethene is effective process, though speed of decomposition extremely differ in different sites. At reductive dechlorination chlorinated hydrocarbons serve as electron acceptors, H2 or small organic compounds act as donors. At startup of degradation dechlorinating microbes have to outcompete compeptitor microbes utilising similarly H2 and organic compounds. Microbial communities originating from different polluted sites and the role of their members in the community dechlorinating metabolism were investigated under controlled laboratory microcosm experiments using polyphasic approach, and by introducing new techniques. It could be determined that effective dechlorinating communities are characterised by the dominance of Dehalococcoides ethenogenes, and a simplification of the original diversity. The presence of fermenting microbes retards the speed of degradation; the inhibition of co-metabolic partners acts similarly. Effective degradation is characterised by Dehalococcoides spp. biofilm formation, and presence of adequte available organic compounds is indispensable. Four novel methods (MDA, SNuPE, SEM and FISH) were optimised for the investigation of dechlorinating communities. RNA based investigations better correlate real activities thus their use is indispensable in the investigation of microbial metabolic interactions

A polyphasic study on the species diversity of the sediment microbiota of Lake Hévíz

Lake Hévíz is the largest natural thermal lake of Europe, harboring special bacterial communities. The aim of the present study was to gain information about the distribution and species diversity of the sediment microbiota, with special focus on Actinobacteria, by using cultivation-based and -independent molecular methods. Samples from two depths were taken in two different locations in October 2007. 245 strains were isolated, grouped to 85 OTUs by ARDRA, and identified by 16S rDNA sequencing. Most of the strains showed highest sequence similarity with Bacillus and related genera. Strains belonging to the phylum Actinobacteria were identified as members of Arthrobacter, Brachybacterium, Brevibacterium, Curtobacterium, Friedmanniella, Gordonia, Kocuria, Microbacterium, Micrococcus, Micromonospora, Mycobacterium, Rhodococcus, Streptomyces and Williamsia . Two clone libraries were constructed from H3M and H4M samples, providing 288 and 192 clones which were grouped to 150 and 125 OTUs, respectively, by ARDRA. The two most abundant group of the H4M library were OP8-related. The phylum Proteobacteria was represented mostly by δ -Proteobacteria, other relevant groups were Cyanobacteria, Bacteroidetes, Acidobacteria and β -Proteobacteria. The H3M library was dominated by Cyanobacteria, Verrucomicrobia, β -Proteobacteria, γ -Proteobacteria and δ -Proteobacteria. Chloroflexi, Bacteroidetes, Spirochetes and Firmicutes were scarce. Results from the clone libraries were compared to the length-heterogeneity-PCR fingerprints of the communities

Spatial and temporal changes of bacterial communities inhabiting the well waters of Harkány spa

In this study, changes in the bacterial community composition of the well waters of Harkány Spa were examined. Physical and chemical properties of mixing subsurface cold and thermal karst waters were correlated to shifts in bacterial community structures analyzed by denaturing gradient gel electrophoresis (DGGE) and principal component analysis (PCA). In addition, mineral components of the pellets were investigated by scanning electron microscopy. Samples from the effluent waters of Büdöstapolca I and II, Matty and Thermal VI wells were taken seasonally in 2007 and 2008. The comparison of the results of DGGE and PCA analyses showed that bacterial communities from the Büdöstapolca wells were distinct from those of Matty and Thermal VI, but seasonal changes were not detected. According to the phylogenetic analysis of the excised DGGE bands, presence of chemolithotrophic Proteobacteria (Thiobacillus, Thiothrix, and distant relatives of Sulfurospirillum) were typical in the Büdöstapolca wells, while members of Actinobacteria (Plantibacter, Actinobacterium, Microbacterium) and Firmicutes (Planococcus) were characteristic to the Matty and Thermal VI wells. In the pellets pyrite framboid crystals were observed by electron microscopy, which are minerals known to be biologically induced by dissimilatory iron- and sulfur (sulfate)-reducing bacteria

Single-Nucleotide Primer Extension Assay for Detection and Sequence Typing of “Dehalococcoides” spp.▿ †

A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus “Dehalococcoides”. The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples

The effect of easily degradable substrate feeding on the community structure of laboratory-scale wastewater sludge digesters

The effect of several easily degradable substrates, such as protein, starch and sunflower oil was investigated on the bacterial community of a laboratory-scale biogas model system. Besides measuring gas yield, Denaturing Gradient Gel Electrophoresis (DGGE), Phospholipids Fatty Acid Analysis (PLFA) for Bacteria and T-RFLP analysis of the mcrA gene for Archaea were used. The community of the examined biogas reactors adapted to the new substrates through a robust physiological reaction followed by moderate community abundance shifts. Gas yield data clearly demonstrated the physiological adaptation to substrate shifts. Statistical analysis of DNA and chemotaxonomic biomarkers revealed community abundance changes. Sequences gained from DGGE bands showed the dominance of the phyla Bacteroidetes and the presence of Firmicutes (Clostridia) and Thermotogae. This was supported by the detection of large amounts of branched 15-carbon non-hydroxy fatty acids in PLFA profiles, as common PLFA markers of the Bacteroidetes group. Minor abundance ratios changes were observed in the case of Archaea in accordance with changes of the fed substrates

Comparison of soil microbial communities from two distinct karst areas in Hungary

Karst areas belong to the most exposed terrestrial ecosystems, therefore their study have a priority task in Hungary, as well. The aim of this study was to compare the structure, activity and diversity of soil microbial communities from two distinct Hungarian karst areas (Aggtelek NP and Tapolca-basin). Soil samples were taken three times from 6 distinct sites, from different depths. Soil microbial biomass C (MBC), microbial biomass N (MBN), basal respiration (BRESP) and substrate induced respiration (SIR) were measured. The phylogenetic diversity of bacterial communities was compared by Denaturing Gradient Gel Electrophoresis (DGGE). The highest MBC, MBN, BRESP and SIR values were measured in the rendzina soil from Aggtelek. On the basis of biomass and respiration measurements, microbial communities differentiated mainly according to soil depths whereas DGGE profiles of bacterial communities resulted in groups mainly according to sampling sites

Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions

Stable isotope labeling of microbial taxa of interest and their sorting provide an efficient and direct way to answer the question “who does what?” in complex microbial communities when coupled with fluorescence in situ hybridization or downstream ‘omics’ analyses. We have developed a platform for automated Raman-based sorting in which optical tweezers and microfluidics are used to sort individual cells of interest from microbial communities on the basis of their Raman spectra. This sorting of cells and their downstream DNA analysis, such as by mini-metagenomics or single-cell genomics, or cultivation permits a direct link to be made between the metabolic roles and the genomes of microbial cells within complex microbial communities, as well as targeted isolation of novel microbes with a specific physiology of interest. We describe a protocol from sample preparation through Raman-activated live cell sorting. Subsequent cultivation of sorted cells is described, whereas downstream DNA analysis involves well-established approaches with abundant methods available in the literature. Compared with manual sorting, this technique provides a substantially higher throughput (up to 500 cells per h). Furthermore, the platform has very high sorting accuracy (98.3 ± 1.7%) and is fully automated, thus avoiding user biases that might accompany manual sorting. We anticipate that this protocol will empower in particular environmental and host-associated microbiome research with a versatile tool to elucidate the metabolic contributions of microbial taxa within their complex communities. After a 1-d preparation of cells, sorting takes on the order of 4 h, depending on the number of cells required.ISSN:1750-2799ISSN:1745-2481ISSN:1754-218

Identification of inulin-responsive bacteria in the gut microbiota via multi-modal activity-based sorting

Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.</p