16S rDNA pyrosequencing analysis of bacterial community in heavy metals polluted soils

Soil contamination with heavy metals is a widespread problem, especially prominent on grounds lying in the vicinity of mines, smelters, and other industrial facilities. Many such areas are located in Southern Poland; they are polluted mainly with Pb, Zn, Cd, or Cu, and locally also with Cr. As for now, little is known about most bacterial species thriving in such soils and even less about a core bacterial community—a set of taxa common to polluted soils. Therefore, we wanted to answer the question if such a set could be found in samples differing physicochemically and phytosociologically. To answer the question, we analyzed bacterial communities in three soil samples contaminated with Pb and Zn and two contaminated with Cr and lower levels of Pb and Zn. The communities were assessed with 16S rRNA gene fragments pyrosequencing. It was found that the samples differed significantly and Zn decreased both diversity and species richness at species and family levels, while plant species richness did not correlate with bacterial diversity. In spite of the differences between the samples, they shared many operational taxonomic units (OTUs) and it was possible to delineate the core microbiome of our sample set. The core set of OTUs comprised members of such taxa as Sphingomonas, Candidatus Solibacter, or Flexibacter showing that particular genera might be shared among sites ~40 km distant

Characterization of the core microbiota of the drainage and surrounding soil of a brazilian copper mine

The core microbiota of a neutral mine drainage and the surrounding high heavy metal content soil at a Brazilian copper mine were characterized by 16S rDNA pyrosequencing. The core microbiota of the drainage was dominated by the generalist genus Meiothermus. The soil samples contained a more heterogeneous bacterial community, with the presence of both generalist and specialist bacteria. Both environments supported mainly heterotrophic bacteria, including organisms resistant to heavy metals, although many of the bacterial groups identified remain poorly characterized. The results contribute to the understanding of bacterial communities in soils impacted by neutral mine drainage, for which information is scarce, and demonstrate that heavy metals can play an important role in shaping the microbial communities in mine environments384484489CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP550240/2010-12012/05429-

Changes in the bacterial community of soil from a neutral mine drainage channel

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments95CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP550240/2010-12012/05429-

Characteristics of Sediment Bacterial Community in Response to Environmental Impacts in a Sewage Polluted River

The Jiaolai River is the main source of industrial and irrigation water for its catchment of 3900 km(2). Anthropogenic activities have caused heavy pollution of this river, but their impacts on biota have never been evaluated. In this study, molecular techniques were applied to investigate the impacts of environmental pollution on the river. Quantitative PCR revealed that total bacterial abundance ranged from 2.90x10(7) to 2.12x10(8) copies/g, with no significant differences among sampling sites or seasons. Bacterial abundance and pore water ammonium concentration were negatively correlated. Cluster analysis revealed that bacterial communities were mainly distributed into groups corresponding to nitrate concentration. Two clone libraries were constructed to compare the bacterial composition of samples with high (J308) and moderate (J304) nitrate impact. Sample J308 was characterized by more members in Clostridia and disappearance of Betaproteobacteria members, which are the primary contributors to nitrogen biogeochemical cycling. Bacterial communities in the sediment were clearly differentiated by environmental nitrogen pollution, suggesting that nitrogen eutrophication was the main environmental problem influencing the Jiaolai River

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.Paikallinen lyijykontaminaatio esim. ampumarata-alueilla on saanut suhteellisen vähän huomiota verrattuna laaja-alaiseen teollisuusperäiseen kontaminaatioon. Ampumarata-alueille päätynyt lyijy muodostaa riskin eliöille, pohjavesille ja myös ihmisten terveydelle. Metsittyneet ampumaradat eivät kuitenkaan usein erotu ulkonäöltään läheisistä terveistä ympäristöistä. Tässä väitöskirjassa tutkittiin Hälvälän ampumarata-aluetta Etelä-Suomessa. Saastuneen maaperän oli aiemmin osoitettu vaikuttavan mm karikkeenhajotukseen. Maan akuutista toksisuudesta saatiin kuitenkin ristiriitaista tietoa, esim. änkyrimatoihin vaikutus oli selvä mutta bakteeritestit osoittivat, että lyijyn biosaatavuus on vähäistä. Lyijyn vesiliukoisuus oli myös pientä. Lyijyresistenttien bakteerien osuus yhteisössä oli kasvanut. Väitöskirjatyössä käytettiin useita erilaisia DNA-tekniikoita tutkittaessa maaperän bakteereiden ja sienten, erityisesti ektomykorritsasienten yhteisöijä. Bakteerien ja sienten diversiteetti kontaminoituneessa maaperässä oli suuri eikä bakteerien osalta poikennut puhtaasta maasta. Sienten diversiteetti sen sijaan oli pienentynyt kontaminoituneessa maaperässä. Tämä näkyi erityisesti kantasienissä (Basidiomycota). Oordinaatioanalyysillä osoitettiin, että ektomykorritsasienten (EcM) yhteisörakenne oli muuttunut. Thelephora- suku selkeästi yleistyi kontaminoituneessa maassa, kun Pseudotomentella, Suillus ja Tylospora -sukujen frekvenssit vähenivät. Väitöskirjan yhtenä osatyönä kehitettiin tietokoneohjelma suurten DNA-aineistojen käsittelyyn ja analysoimiseen

Effect of Silene vulgaris and Heavy Metal Pollution on Soil; Microbial Diversity in Long-Term Contaminated Soil

In this study, we analysed the impact of heavy metals and plant rhizodeposition on the structure of indigenous microbial communities in rhizosphere and bulk soil that had been exposed to heavy metals for more than 150 years. Samples of the rhizosphere of Silene vulgaris and non-rhizosphere soils 250 and 450 m from the source of emission that had different metal concentrations were collected for analyses. The results showed that soils were collected 250 m from the smelter had a higher number of Cd-resistant CFU compared with the samples that were collected from 450 m, but no significant differences were observed in the number of total and oligotrophic CFU or the equivalent cell numbers between rhizosphere and non-rhizosphere soils that were taken 250 and 450 m from the emitter. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of the denaturing gradient gel electrophoresis (DGGE) profiles, as well as a cluster analysis that was generated on the phospholipid fatty acid (PLFA) profiles, showed that the bacterial community structure of rhizosphere soils depended more on the plant than on the distance and metal concentrations. The sequencing of the 16S rDNA fragments that were excised from the DGGE gel revealed representatives of the phyla Bacteroidetes, Acidobacteria, Gemmatimonadetes, Actinobacteria and Betaproteobacteria in the analysed soil with a predominance of the first three groups. The obtained results demonstrated that the presence of S. vulgaris did not affect the number of CFUs, except for those of Cd-resistant bacteria. However, the presence of S. vulgaris altered the soil bacterial community structure, regardless of the sampling site, which supported the thesis that plants have a higher impact on soil microbial community than metal contamination.Peer reviewe

Plant growth-promoting rhizobacteria enhance the growth and Cd uptake of Sedum plumbizincicola in a Cd-contaminated soil

This study aimed to isolate plant growth-promoting rhizobacteria (PGPR) that exhibit heavy metal resistance to examine their influence on Cd uptake and soil microbial community structure during phytoremediation. Heavy metal-tolerant PGPR were isolated from the roots of possible hyperaccumulators using plates with 1-aminocyclopropane-1-carboxylate (ACC) as sole nitrogen source. Minimal inhibitory concentrations (MICs) of each isolate were determined by the plate dilution method. The impacts of isolated PGPR on the growth and Cd accumulation of Sedium plumbizincicola were conducted in a pot experiment. In addition, the effect of PGPR inoculation on the microbial community during phytoextraction by S. plumbizincicola was studied by 454 pyrosequencing. A total of nine Cd-resistant strains were isolated from the roots of Cd accumulators, and their plant growth-promoting activities were characterized. Isolates were able to produce indole-3-acetic acid (IAA) (28-133 mg L-1) and solubilize phosphate (65-148 mg L-1). In a pot experiment, the inoculation of isolates NSX2 and LCR1 significantly enhanced the growth of and uptake of Cd by the Cd hyperaccumulator S. plumbizincicola. 454 pyrosequencing revealed that the inoculation of the PGPR lead to a decrease in microbial community diversity in the rhizopshere during phytoextraction. Specifically, indigenous heavy metal-tolerant PGPR such as Actinospica, Bradyrhizobium, Rhizobium, Mesorhizobium, and Mycobacterium were selectively enriched in the treatments in which PGPR were added. It is suggested that a unique constitution of microbial communities in inoculated treatments plays a key role in enhancing Cd phytoremediation. Inoculation of strains Rhodococcus erythropolis NSX2 and Cedecea davisae LCR1 could promote S. plumbizincicola growth and enhance the remediation efficiency. The introduced PGPR could also affect the indigenous microbial community structure and the diversity in Cd-contaminated soil during phytoremediation.This study aimed to isolate plant growth-promoting rhizobacteria (PGPR) that exhibit heavy metal resistance to examine their influence on Cd uptake and soil microbial community structure during phytoremediation

Molecular tools to characterise plant-associated bacterial communities in soils naturally enriched or contaminated with trace metals

The thesis focuses on the (1) characterisation and comparison of the bacterial communities associated with Ni-hyperaccumulating and metal-excluding plants growing in ultramafic soils naturally enriched in TE and (2) analysis of bacterial communities in soils contaminated with TE and treated with phytoremediation strategies. The work planned aimed to obtain information about bacteria potentially involved in the mechanisms of TE (hyper)accumulation and/or exclusion and to study the relationships between the bacterial communities and the changes induced in soil properties by phytomanagement

Microbial ecological response of the intestinal flora of Peromyscus maniculatus and P. leucopus to heavy metal contamination

Heavy metal contamination negatively affects natural systems including plants, birds, fish and bacteria by reducing biodiversity at contaminated sites. At the Tri-State Mining District, efforts have been made to remediate sites to mitigate the detrimental effects that contamination has caused on human health. While the remediation effort has returned the site to within federal safety standards, it is unclear if this effort is sufficient to restore floral and faunal communities. Intrinsic to ecosystem and organism health is the biodiversity and composition of microbial communities. We have taken advantage of recent advances in sequencing technology and surveyed the bacterial community of remediated and reference soils as well as the intestinal microbial community of two ubiquitous rodent species to provide insight on the impacts of residual heavy metal contamination on the ecosystem. Rodents found on the remediated site had reduced body mass, smaller body size and lower body fat than animals on reference sites. Using bar-coded, massively parallel sequencing, we found that bacterial communities in both the soil and Peromyscus spp. gastrointestinal tracts had no difference in diversity between reference and remediated sites but assemblages differed in response to contamination. These results suggest that niche voids left by microbial taxa that were unable to deal with the remnant levels of heavy metals on remediated sites were replaced by taxa that could persist in this environment. Whether this replacement provided similar ecosystem services as ancestral bacterial communities is unknown.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79374/1/MEC_4485_sm_Supporting.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/79374/2/j.1365-294X.2009.04485.x.pd