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.     

Richness and Diversity of Bacterioplankton Species along an Estuarine Gradient in Moreton Bay, Australia

Bacterioplankton community diversity was investigated in the subtropical Brisbane River-Moreton Bay estuary, Australia (27°25′S, 153°5′E). Bacterial communities were studied using automated rRNA intergenic spacer analysis (ARISA), which amplifies 16S-23S ribosomal DNA internally transcribed spacer regions from mixed-community DNA and detects the separated products on a fragment analyzer. Samples were collected from eight sites throughout the estuary and east to the East Australian Current (Coral Sea). Bacterioplankton communities had the highest operational taxonomic unit (OTU) richness, as measured by ARISA at eastern bay stations (S [total richness] = 84 to 85 OTU) and the lowest richness in the Coral Sea (S = 39 to 59 OTU). Richness correlated positively with bacterial abundance; however, there were no strong correlations between diversity and salinity, NO₃⁻ and PO₄³⁻ concentrations, or chlorophyll a concentration. Bacterioplankton communities at the riverine stations were different from communities in the bay or Coral Sea. The main differences in OTU richness between stations were in taxa that each represented 0.1% (the detection limit) to 0.5% of the total amplified DNA, i.e., the “tail” of the distribution. We found that some bacterioplankton taxa are specific to distinct environments while others have a ubiquitous distribution from river to sea. Bacterioplankton richness and diversity patterns in the estuary are potentially a consequence of greater niche availability, mixing of local and adjacent environment communities, or intermediate disturbance. Furthermore, these results contrast with previous reports of spatially homogeneous bacterioplankton communities in other coastal waters.     

Adaptation of the Cecal Bacterial Microbiome of Growing Pigs in Response to Resistant Starch Type 4

Resistant starch (RS) exacerbates health benefits on the host via modulation of the gut bacterial community. By far, these effects have been less well explored for RS of type 4. This study aimed at gaining a community-wide insight into the impact of enzymatically modified starch (EMS) on the cecal microbiota and hindgut fermentation in growing pigs. Castrated male pigs (n = 12/diet; 29-kg body weight) were fed diets with either 70% EMS or control starch for 10 days. The bacterial profile of each cecal sample was determined by sequencing of the V345 region of the 16S rRNA gene using the Illumina MiSeq platform. EMS diet reduced short-chain fatty acid concentrations in cecum and proximal colon compared to the control diet. Linear discriminant analyses and K means clustering indicated diet-specific cecal community profiles, whereby diversity and species richness were not different among diets. Pigs showed host-specific variation in their most abundant phyla, Firmicutes (55%), Proteobacteria (35%), and Bacteroidetes (10%). The EMS diet decreased abundance of Ruminococcus, Parasutterella, Bilophila, Enterococcus, and Lactobacillus operational taxonomic units (OTU), whereas Meniscus and Actinobacillus OTU were increased compared to those with the control diet (P < 0.05). Quantitative PCR confirmed results for host effect on Enterobacteriaceae and diet effect on members of the Lactobacillus group. The presence of less cecal short-chain fatty acids and the imputed metabolic functions of the cecal microbiome suggested that EMS was less degradable for cecal bacteria than the control starch. The present EMS effects on the bacterial community profiles were different than the previously reported RS effects and can be linked to the chemical structure of EMS.     

Geographical Distribution and Diversity of Bacteria Associated with Natural Populations of Drosophila melanogaster

Drosophila melanogaster is one of the most widely used model systems in biology. However, little is known about its associated bacterial community. As a first step towards understanding these communities, we compared bacterial 16S rRNA gene sequence libraries recovered from 11 natural populations of adult D. melanogaster. Bacteria from these sequence libraries were grouped into 74 distinct taxa, spanning the phyla Proteobacteria, Bacteroidetes, and Firmicutes, which were unevenly spread across host populations. Summed across populations, the distribution of abundance of genera was closely fit by a power law. We observed differences among host population locations both in bacterial community richness and in composition. Despite this significant spatial variation, no relationship was observed between species richness and a variety of abiotic factors, such as temperature and latitude. Overall, bacterial communities associated with adult D. melanogaster hosts are diverse and differ across host populations.     

A Doubling of Microphytobenthos Biomass Coincides with a Tenfold Increase in Denitrifier and Total Bacterial Abundances in Intertidal Sediments of a Temperate Estuary

Surface sediments are important systems for the removal of anthropogenically derived inorganic nitrogen in estuaries. They are often characterized by the presence of a microphytobenthos (MPB) biofilm, which can impact bacterial communities in underlying sediments for example by secretion of extracellular polymeric substances (EPS) and competition for nutrients (including nitrogen). Pyrosequencing and qPCR was performed on two intertidal surface sediments of the Westerschelde estuary characterized by a two-fold difference in MPB biomass but no difference in MPB composition. Doubling of MPB biomass was accompanied by a disproportionately (ten-fold) increase in total bacterial abundances while, unexpectedly, no difference in general community structure was observed, despite significantly lower bacterial richness and distinct community membership, mostly for non-abundant taxa. Denitrifier abundances corresponded likewise while community structure, both for nirS and nirK denitrifiers, remained unchanged, suggesting that competition with diatoms for nitrate is negligible at concentrations in the investigated sediments (appr. 1 mg/l NO₃^-). This study indicates that MPB biomass increase has a general, significantly positive effect on total bacterial and denitrifier abundances, with stimulation or inhibition of specific bacterial groups that however do not result in a re-structured community.     

Influence of glacial melt and Atlantic water on bacterioplankton community of Kongsfjorden,an Arctic fjord

The Kongsfjorden, an Arctic fjord is experiencing warming due to increased input of Atlantic water masses. High-throughput sequencing was performed to examine bacterial diversity from the outer and inner zone of the fjord in summer and fall of 2012. A total of 11,999 operational taxonomic units (OTUs) were assigned into 19 known phyla and 5 genera incertae sedis. Significant variation (p = 0.001, n = 4) was observed between the bacterial community structure of outer and inner fjord while variation between summer and fall was minimum. Proteobacteria was the most abundant phylum (55.9-61.0%) in summer and fall. The most dominant alphaproteobacterial member of this phylum (OTU 263 Pelagibacteriaceae) contributed maximum to the observed dissimilarity between the outer and inner fjord community. Characterised by relatively fresher and warmer water, glacial meltwater input could be a major source of predominance of OTU 6968 Flavobacteriaceae, OTU 5552 Psychrobacter, OTU 7148 Sphingomonadales and OTU 5011 Loktanella in the inner fjord in summer. Thus, the significant variation in the bacterioplankton community composition of outer and inner fjord indicates strong and localized influence of glacial melt water in shaping the community structure.     

Understanding the Linkage between Elevation and the Activated-Sludge Bacterial Community along a 3,600-Meter Elevation Gradient in China

To understand the relationship between elevation and bacterial communities in wastewater treatment plants (WWTPs), bacterial communities in 21 municipal WWTPs across China, located 9 to 3,660 m above sea level (masl), were investigated by 454 pyrosequencing. A threshold for the association of elevation with bacterial community richness and evenness was observed at approximately 1,200 masl. At lower elevations, both richness and evenness were not significantly associated with elevation. At higher elevations, significant declines with increased elevations were observed for community richness and evenness. The declining evenness trend at the phylum level was reflected by distinct trends in relative abundance for individual bacterial phyla. Betaproteobacteria, Bacteroidetes, and Firmicutes displayed significant increases, while most other phyla showed declines. Spearman correlation analysis indicated that the community richness and evenness at high elevations were more correlated with elevation than with any other single environmental variable. Redundancy analysis indicated that the contribution of elevation to community composition variances increased from 3% at lower elevations to 11% at higher elevations whereas the community composition variance at higher elevations remained much more explained by operational variables (39.2%) than by elevation. The influent total phosphorus concentration, food/microorganism ratio, and treatment process were the three shared dominant contributors to the community composition variance across the whole elevation gradient, followed by effluent ammonia nitrogen and temperature at higher elevations.     

Changes in bacterial and archaeal communities during the concentration of brine at the graduation towers in Ciechocinek spa (Poland)

This study evaluates the changes in bacterial and archaeal community structure during the gradual evaporation of water from the brine (extracted from subsurface Jurassic deposits) in the system of graduation towers located in Ciechocinek spa, Poland. The communities were assessed with 16S rRNA gene sequencing (MiSeq, Illumina) and microscopic methods. The microbial cell density determined by direct cell count was at the order of magnitude of 10⁷ cells/mL. It was found that increasing salt concentration was positively correlated with both the cell counts, and species-level diversity of bacterial and archaeal communities. The archaeal community was mostly constituted by members of the phylum Euryarchaeota, class Halobacteria and was dominated by Halorubrum-related sequences. The bacterial community was more diverse, with representatives of the phyla Proteobacteria and Bacteroidetes as the most abundant. The proportion of Proteobacteria decreased with increasing salt concentration, while the proportion of Bacteroidetes increased significantly in the more concentrated samples. Representatives of the genera Idiomarina, Psychroflexus, Roseovarius, and Marinobacter appeared to be tolerant to changes of salinity. During the brine concentration, the relative abundances of Sphingobium and Sphingomonas were significantly decreased and the raised contributions of genera Fabibacter and Fodinibius were observed. The high proportion of novel (not identified at 97% similarity level) bacterial reads (up to 42%) in the 16S rRNA gene sequences indicated that potentially new bacterial taxa inhabit this unique environment.

     

Biochar,Bentonite and Zeolite Supplemented Feeding of Layer Chickens Alters Intestinal Microbiota and Reduces Campylobacter Load

A range of feed supplements, including antibiotics, have been commonly used in poultry production to improve health and productivity. Alternative methods are needed to suppress pathogen loads and maintain productivity. As an alternative to antibiotics use, we investigated the ability of biochar, bentonite and zeolite as separate 4% feed additives, to selectively remove pathogens without reducing microbial richness and diversity in the gut. Neither biochar, bentonite nor zeolite made any significant alterations to the overall richness and diversity of intestinal bacterial community. However, reduction of some bacterial species, including some potential pathogens was detected. The microbiota of bentonite fed animals were lacking all members of the order Campylobacterales. Specifically, the following operational taxonomic units (OTUs) were absent: an OTU 100% identical to Campylobacter jejuni; an OTU 99% identical to Helicobacter pullorum; multiple Gallibacterium anatis (>97%) related OTUs; Bacteroides dorei (99%) and Clostridium aldenense (95%) related OTUs. Biochar and zeolite treatments had similar but milder effects compared to bentonite. Zeolite amended feed was also associated with significant reduction in the phylum Proteobacteria. All three additives showed potential for the control of major poultry zoonotic pathogens.     

The Diverse Bacterial Community in Intertidal, Anaerobic Sediments at Sapelo Island, Georgia

The phylogenetic diversity and composition of the bacterial community in anaerobic sediments from Sapelo Island, GA, USA were examined using 16S rRNA gene libraries. The diversity of this community was comparable to that of soil, and 1,186 clones formed 817 OTUs at 99% sequence similarity. Chao1 estimators for the total richness were also high, at 3,290 OTUs at 99% sequence similarity. The program RDPquery was developed to assign clones to taxonomic groups based upon comparisons to the RDP database. While most clones could be assigned to describe phyla, fewer than 30% of the clones could be assigned to a described order. Similarly, nearly 25% of the clones were only distantly related (<90% sequence similarity) to other environmental clones, illustrating the unique composition of this community. One quarter of the clones were related to one or more undescribed orders within the γ-Proteobacteria. Other abundant groups included the δ-Proteobacteria, Bacteroidetes, and Cyanobacteria. While these phyla were abundant in other estuarine sediments, the specific members at Sapelo Island appeared to be different from those previously described in other locations, suggesting that great diversity exists between as well as within estuarine intertidal sediments. In spite of the large differences in pore water chemistry with season and depth, differences in the bacterial community were modest over the temporal and spatial scales examined and generally restricted to only certain taxa. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

耐铜植物茵陈蒿根际细菌群落结构及影响因素

采用Mi Seq高通量测序技术对耐铜植物茵陈蒿根际的细菌16S r DNA基因V3—V4区片段进行了测序,研究了细菌群落结构的变化,并分析了其与土壤环境因子的关系。研究表明,采样点Cu3中细菌群落的多样性、丰富度、均匀度、ACE指数、Chao1指数等均显著低于Cu1和Cu2,但Cu3的覆盖度高于Cu1。排名前10的优势细菌门总相对丰度均在95%以上,其中8个优势细菌门在3个采样点中是相同的,包括Proteobacteria(变形菌门)、Acidobacteria(酸杆菌门)、Bacteroidetes(拟杆菌门)、Gemmatimonadetes(芽单胞菌门)、Actinobacteria(放线菌门)、Verrucomicrobia(疣微菌门)、Planctomycetes(浮霉菌门)和Unclassified(未分类门)等。采样点Cu1中变形菌门、拟杆菌门和芽单胞菌门的相对丰度显著高于其他两个采样点,而酸杆菌门、放线菌门、疣微菌门、绿弯菌门(Chloroflexi)和未分类门则刚好相反,表明细菌对胁迫环境的适应能力有明显差异。主坐标和冗余分析表明,3个采样点的细菌群落结构发生了明显改变。土壤环境因子与细菌群落的变化关系密切,8个因子的特征值共解释了97.5%的总方差。其中,总铜、总磷、p H、有效磷和有机质为显著性因子,可以解释93.9%的群落变化,但影响不同采样点细菌群落的主导因子有所差异。     

Bacterial Diversity in Shallow Oligotrophic Marine Benthos and Overlying Waters: Effects of Virus Infection,Containment, and Nutrient Enrichment

Little is known of the factors shaping sediment bacterial communities, despite their high abundance and reports of high diversity. Two factors hypothesized to shape bacterial communities in the water column are nutrient (resource) availability and virus infection. The role these factors play in benthic bacterial diversity was assessed in oligotrophic carbonate–based sediments of Florida Bay (USA). Sediment–water mesocosm enclosures were made from 1-m diameter clear polycarbonate cylinders which were pushed into sediments to 201 cm sediment depth enclosing 80 L of water. Mesocosms were amended each day for 14 d with 10 µM NH ₄ ⁺ and 1 µM PO ₄ ^3– . In a second experiment, viruses from a benthic flocculent layer were concentrated and added back to flocculent layer samples which were collected near the mesocosm enclosures. Photosynthesis by microalgae in virus-amended incubations was monitored by pulse-amplitude modulated (PAM) fluorescence. In both experiments, bacterial diversity was estimated using automated rRNA intergenic spacer analysis (ARISA), a high-resolution fingerprinting approach. Initial sediment bacterial operational taxonomic unit (OTU) richness (236 ± 3) was higher than in the water column (148 ± 9), where an OTU was detectable when its amplified DNA represented >0.09% of the total amplified DNA. Effects on bacterial diversity and operational taxonomic unit (OTU) richness in nutrient-amended mesocosms may have been masked by the effects of containment, which stimulated OTU richness in the water column, but depressed OTU richness and diversity in sediments. Nutrient addition significantly elevated virus abundance and the ratio of viruses to bacteria (p < 0.05 for both) in the sediments, concomitant with elevated bacterial diversity. However, water column bacterial diversity (in unamended controls) was not affected by nutrient amendments, which may be due to rapid nutrient uptake by sediment organisms or adsorption of P to carbonate sediments. Addition of live viruses to benthic flocculent layer samples increased bacterial OTU diversity and richness compared with heat-killed controls; however, cluster analyses showed that the community structure in the virus-amended mesocosms varied greatly between replicates. Despite the strong effects upon eubacterial communities, photosynthesis of co-occurring protists and cyanobacteria was not significantly altered by the presence of virus concentrates. This study supports the hypothesis that nutrient availability plays a key role in shaping sediment bacterial communities, and also that viruses may regulate the abundance of the dominant competitors and allow less dominant organisms to maintain or increase their abundance in a community due to decreased competition for resources.     

Effects of operational taxonomic unit inference methods on soil microeukaryote community analysis using long‐read metabarcoding

Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communities of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing. We tested how three different methods for generation of operational taxonomic units (OTUs) effected estimated richness and identified taxa, and how well large‐scale ecological patterns associated with shifting environmental conditions were recovered in data from the three methods. The field site at Kungsängen Nature Reserve has drawn frequent visitors since Linnaeus''s time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic‐dry part that has a species rich grass‐dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigate how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30%–40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.     

Comparative analysis of biodiversity in the planktonic and biofilm bacterial communities in Lake Baikal

Bacterial communities of the water and the biofilm formed during five years on an artificial substrate in Lake Baikal were studied by the pyrosequencing of 16S rRNA gene fragments; taxonomic diversity of bacterial communities and differences in their structure were revealed. The biofilm community contained mainly representatives of three phyla: Cyanobacteria, Bacteroidetes, and Proteobacteria; the amounts of other groups were within 1%. Bacterial community of the plankton was more heterogeneous; along with the dominant phyla (Bacteroidetes, Actinobacteria, and Proteobacteria) 15% of the members were of the other phyla. The use of pyrosequencing allowed to reveal 35 bacterial phyla in Lake Baikal, some of which were identified for the first time; moreover, minor groups of microorganisms (including only several sequences), which were not earlier determined by other molecular methods were found.     

Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([¹⁴C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and Actinobacteria. Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.     

Bacterial and archaeal populations at two shallow hydrothermal vents off Panarea Island (Eolian Islands, Italy)

The aim of this study was to investigate the microbial community thriving at two shallow hydrothermal vents off Panarea Island (Italy). Physico-chemical characteristics of thermal waters were examined in order to establish the effect of the vents on biodiversity of both Bacteria and Archaea. Water and adjacent sediment samples were collected at different times from two vents, characterised by different depth and temperature, and analysed to evaluate total microbial abundances, sulphur-oxidising and thermophilic aerobic bacteria. Total microbial abundances were on average of the order of 10⁵ cells ml⁻¹, expressed as picoplanktonic size fraction. Picophytoplanktonic cells accounted for 0.77–3.83% of the total picoplanktonic cells. The contribution of bacterial and archaeal taxa to prokaryotic community diversity was investigated by PCR–DGGE fingerprinting method. The number of bands derived from bacterial DNA was highest in the DGGE profiles of water sample from the warmest and deepest site (site 2). In contrast, archaeal richness was highest in the water of the coldest and shallowest site (site 1). Sulphur-oxidising bacteria were detected by both culture-dependent and -independent methods. The primary production at the shallow hydrothermal system of Panarea is supported by a complex microbial community composed by phototrophs and chemolithotrophs.     

Fungal control of early-stage bacterial community development in decomposing wood

The earliest stages of bacterial colonisation of wood have received little attention, particularly with respect to how the colonisation process may be affected by the presence of wood-decay fungi. This study used 16s rRNA gene sequencing to examine the bacterial community in wood that had been incubated in the field for 14 or 84 d, either in wood uncolonised by fungi or pre-colonised by Vuilleminia comedens, Trametes versicolor or Hypholoma fasciculare. All three fungal species significantly delayed bacterial colonisation of the wood. V. comedens and H. fasciculare also reduced bacterial OTU richness and altered bacterial community composition, increasing the relative abundance of Burkholderiales and reducing the proportion of Enterobacteriaceae and Bacteroidetes. Wood that had not been pre-colonised showed seasonal differences between autumn and spring: bacterial richness increased between 14 d and 84 d in the spring, but not in the autumn. Community composition at 84 d in spring was also different to the other time points, with reduced dominance of Gamma-proteobacteria. Archaea were also detected in nearly a third of samples, but with no apparent pattern, and always at low abundances.     

A Limited Microbial Consortium Is Responsible for Extended Bioreduction of Uranium in a Contaminated Aquifer

Subsurface amendments of slow-release substrates (e.g., emulsified vegetable oil [EVO]) are thought to be a pragmatic alternative to using short-lived, labile substrates for sustained uranium bioimmobilization within contaminated groundwater systems. Spatial and temporal dynamics of subsurface microbial communities during EVO amendment are unknown and likely differ significantly from those of populations stimulated by soluble substrates, such as ethanol and acetate. In this study, a one-time EVO injection resulted in decreased groundwater U concentrations that remained below initial levels for approximately 4 months. Pyrosequencing and quantitative PCR of 16S rRNA from monitoring well samples revealed a rapid decline in groundwater bacterial community richness and diversity after EVO injection, concurrent with increased 16S rRNA copy levels, indicating the selection of a narrow group of taxa rather than a broad community stimulation. Members of the Firmicutes family Veillonellaceae dominated after injection and most likely catalyzed the initial oil decomposition. Sulfate-reducing bacteria from the genus Desulforegula, known for long-chain fatty acid oxidation to acetate, also dominated after EVO amendment. Acetate and H₂ production during EVO degradation appeared to stimulate NO₃⁻, Fe(III), U(VI), and SO₄²⁻ reduction by members of the Comamonadaceae, Geobacteriaceae, and Desulfobacterales. Methanogenic archaea flourished late to comprise over 25% of the total microbial community. Bacterial diversity rebounded after 9 months, although community compositions remained distinct from the preamendment conditions. These results demonstrated that a one-time EVO amendment served as an effective electron donor source for in situ U(VI) bioreduction and that subsurface EVO degradation and metal reduction were likely mediated by successive identifiable guilds of organisms.