Transparent soil microcosms for live-cell imaging and non-destructive stable isotope probing of soil microorganisms

Microscale processes are critically important to soil ecology and biogeochemistry yet are difficult to study due to soil\u27s opacity and complexity. To advance the study of soil processes, we constructed transparent soil microcosms that enable the visualization of microbes via fluorescence microscopy and the non-destructive measurement of microbial activity and carbon uptake in situ via Raman microspectroscopy. We assessed the polymer Nafion and the crystal cryolite as optically transparent soil substrates. We demonstrated that both substrates enable the growth, maintenance, and visualization of microbial cells in three dimensions over time, and are compatible with stable isotope probing using Raman. We applied this system to ascertain that after a dry-down/rewetting cycle, bacteria on and near dead fungal hyphae were more metabolically active than those far from hyphae. These data underscore the impact fungi have facilitating bacterial survival in fluctuating conditions and how these microcosms can yield insights into microscale microbial activities

Critical point analysis and biocide treatment in a microbiologically contaminated water purification system of a power plant

Many industries use ultrapure water (UPW) for their operation. In spite of purification efforts, microbial contaminations are the major cause of manufacturing problems in these systems. In our experiments, the water purification system of a power plant was investigated: the microbiologically critical points of the ultrapure water purification system were located by checking the number and diversity of bacteria to determine the optimum operational unit for chemical intervention: the most contaminated site was the mixed-bed ion-exchange resin containing unit. Biocides were tested against bacteria previously isolated from the same system; effect of biocides was checked also in laboratory model systems, and based on the results, a biocide treatment was carried out in the mixed-bed ion-exchange resin columns of the working power plant. Kathon WT was the most effective from the studied chemicals, being effective already in low concentration against most studied microorganisms. In case of the handling of the mixed-bed ion-exchange resin, 8-h treatment with 25ppm biocide concentration was effective. Following the treatment, the quality of the produced UPW met the standards (specific electric conductivity was<1.0x10(-3)mu Scm(-1) at 25 degrees C) and water production capacity increased; moreover, the run-down time of the mixed-bed ion-exchange resin significantly grew

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

Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and pyrosequencing for the characterization of the dentine caries-associated microbiome

Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas, and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture

Anaerobic sulfur oxidation underlies adaptation of a chemosynthetic symbiont to oxic-anoxic interfaces

Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. However, nothing is known about how these variations affect their physiology. Here, by applying omics, Raman microspectroscopy, and stable isotope labeling, we investigated the effect of oxygen on “Candidatus Thiosymbion oneisti.” Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of (13)C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation were upregulated under oxic conditions, together with genes involved in organic carbon assimilation, polyhydroxyalkanoate (PHA) biosynthesis, nitrogen fixation, and urea utilization. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand oxidative stress, and the symbiont appeared to proliferate less. Based on its physiological response to oxygen, we propose that “Ca. T. oneisti” may exploit anaerobic sulfur oxidation coupled to denitrification to proliferate in anoxic sand. However, the ectosymbiont would still profit from the oxygen available in superficial sand, as the energy-efficient aerobic respiration would facilitate carbon and nitrogen assimilation. IMPORTANCE Chemoautotrophic endosymbionts are famous for exploiting sulfur oxidization to feed marine organisms with fixed carbon. However, the physiology of thiotrophic bacteria thriving on the surface of animals (ectosymbionts) is less understood. One longstanding hypothesis posits that attachment to animals that migrate between reduced and oxic environments would boost sulfur oxidation, as the ectosymbionts would alternatively access sulfide and oxygen, the most favorable electron acceptor. Here, we investigated the effect of oxygen on the physiology of “Candidatus Thiosymbion oneisti,” a gammaproteobacterium which lives attached to marine nematodes inhabiting shallow-water sand. Surprisingly, sulfur oxidation genes were upregulated under anoxic relative to oxic conditions. Furthermore, under anoxia, the ectosymbiont appeared to be less stressed and to proliferate more. We propose that animal-mediated access to oxygen, rather than enhancing sulfur oxidation, would facilitate assimilation of carbon and nitrogen by the ectosymbiont

Analysis of 16S rRNA Amplicon Sequencing Options on the Roche/454 Next-Generation Titanium Sequencing Platform

BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies

Ammonia oxidation: Ecology, physiology, biochemistry and why they must all come together

Ammonia oxidation is a fundamental core process in the global biogeochemical nitrogen cycle. Oxidation of ammonia (NH3) to nitrite (NO2 −) is the first and rate-limiting step in nitrification and is carried out by distinct groups of microorganisms. Ammonia oxidation is essential for nutrient turnover in most terrestrial, aquatic and engineered ecosystems and plays a major role, both directly and indirectly, in greenhouse gas production and environmental damage. Although ammonia oxidation has been studied for over a century, this research field has been galvanised in the past decade by the surprising discoveries of novel ammonia oxidising microorganisms. This review reflects on the ammonia oxidation research to date and discusses the major gaps remaining in our knowledge of the biology of ammonia oxidation