On Operational Taxonomic Unit Research Articles

Insight into the microbial community of denitrification process using different solid carbon sources: Not only bacteria

There is a lack of understanding about the bacterial, fungal and archaeal communities’ composition of solid-phase denitrification (SPD) systems. We investigated four SPD systems with different carbon sources by analyzing microbial gene sequences based on operational taxonomic unit (OTU) and amplicon sequence variant (ASV). The results showed that the corncob-polyvinyl alcohol sodium alginate-polycaprolactone (CPSP, 0.86±0.04 mg NO3−-N/(g·day)) and corncob (0.85±0.06 mg NO3−-N/(g·day)) had better denitrification efficiency than polycaprolactone (PCL, 0.29±0.11 mg NO3−-N/(g·day)) and polyvinyl alcohol-sodium alginate (PVA-SA, 0.24±0.07 mg NO3−-N/(g·day)). The bacterial, fungal and archaeal microbial composition was significantly different among carbon source types such as Proteobacteria in PCL (OTU: 83.72%, ASV: 82.49%) and Rozellomycota in PVA-SA (OTU: 71.99%, ASV: 81.30%). ASV methods can read more microbial units than that of OTU and exhibit higher alpha diversity and classify some species that had not been identified by OTU such as Nanoarchaeota phylum, unclassified_ f_ Xanthobacteraceae genus, etc., indicating ASV may be more conducive to understand SPD microbial communities. The co-occurring network showed some correlation between the bacteria fungi and archaea species, indicating different species may collaborate in SPD systems. Similar KEGG function prediction results were obtained in two bioinformatic methods generally and some fungi and archaea functions should not be ignored in SPD systems. These results may be beneficial for understanding microbial communities in SPD systems.

Mian: interactive web-based microbiome data table visualization and machine learning platform.

Mian is a web application to interactively visualize, run statistical tools and train machine learning models on operational taxonomic unit (OTU) or amplicon sequence variant (ASV) datasets to identify key taxonomic groups, diversity trends or taxonomic composition shifts in the context of provided categorical or numerical sample metadata. Tools, including Fisher's exact test, Boruta feature selection, alpha and beta diversity, and random forest and deep neural network classifiers, facilitate open-ended data exploration and hypothesis generation on microbial datasets. Mian is freely available at: miandata.org. Mian is an open-source platform licensed under the MIT license with source code available at github.com/tbj128/mian. Supplementary data are available at Bioinformatics online.

In Vitro Incubations Do Not Reflect In Vivo Differences Based on Ranking of Low and High Methane Emitters in Dairy Cows.

Simple SummaryThere is a great interest among the scientific community in breeding animals that emit less methane (CH4) as a strategy to mitigate the environmental impact of ruminants. The present study ranked individual dairy cows as low and high CH4 emitters fed the same diet and evaluated whether the original in vivo ranking was maintained in the in vitro conditions using two contrasting substrates each without or with an antimethanogenic compound. Results do not support a strong effect of rumen microbiome on observed CH4 production in vivo, and thus, ranking was not successfully replicated in in vitro conditions. Instead, it appears that animal-related factors such as digesta passage rate are more important drivers of enteric CH4 production.This study evaluated if ranking dairy cows as low and high CH4 emitters using the GreenFeed system (GF) can be replicated in in vitro conditions using an automated gas system and its possible implications in terms of fermentation balance. Seven pairs of low and high emitters fed the same diet were selected on the basis of residual CH4 production, and rumen fluid taken from each pair incubated separately in the in vitro gas production system. In total, seven in vitro incubations were performed with inoculums taken from low and high CH4 emitting cows incubated in two substrates differing in forage-to-concentrate proportion, each without or with the addition of cashew nutshell liquid (CNSL) as an inhibitor of CH4 production. Except for the aimed differences in CH4 production, no statistical differences were detected among groups of low and high emitters either in in vivo animal performance or rumen fermentation profile prior to the in vitro incubations. The effect of in vivo ranking was poorly replicated in in vitro conditions after 48 h of anaerobic fermentation. Instead, the effects of diet and CNSL were more consistent. The inclusion of 50% barley in the diet (SB) increased both asymptotic gas production by 17.3% and predicted in vivo CH4 by 26.2%, when compared to 100% grass silage (S) substrate, respectively. The SB diet produced on average more propionate (+28 mmol/mol) and consequently less acetate compared to the S diet. Irrespective of CH4 emitter group, CNSL decreased predicted in vivo CH4 (26.7 vs. 11.1 mL/ g of dry matter; DM) and stoichiometric CH4 (CH4VFA; 304 vs. 235 moles/mol VFA), with these being also reflected in decreased total gas production per unit of volatile fatty acids (VFA). Microbial structure was assessed on rumen fluid sampled prior to in vitro incubation, by sequencing of the V4 region of 16S rRNA gene. Principal coordinate analysis (PCoA) on operational taxonomic unit (OTU) did not show any differences between groups. Some differences appeared of relative abundance between groups in some specific OTUs mainly related to Prevotella. Genus Methanobrevibacter represented 93.7 ± 3.33% of the archaeal sequences. There were no clear differences between groups in relative abundance of Methanobrevibacter.

Geolocation Inference of Forensic Individual Origin by Soil Metagenomic Analysis.

Objective To preliminarily discuss the feasibility of geolocation inference of forensic individual origin by soil metagenomic analysis. Methods The 33 soil samples from Heilongjiang, Qinghai and Tibet were collected, total bacterial DNA in the samples were extracted, and universal primers were used to amplify the V3 and V4 hypervariable region of bacterial 16S rDNA. The region was sequenced by high-throughput sequencing （HTS） with the MiSeq sequencer. Bioinformatics analysis such as species composition and sample comparison was performed on sequencing data. The richness index and diversity index were calculated based on operational taxonomic unit （OTU） results. Results A total of 2 720 149 sequences were generated by sequencing. Those sequences were clustered into 114 848 OTUs. The Chao1 indexes of soil microorganisms in Heilongjiang, Qinghai, and Tibet were 797.45, 745.11 and 535.98, respectively, and Shannon indexes were 6.46, 6.36 and 6.25, respectively. The number of bacterial species and the community diversity in the soil from high to low were Heilongjiang > Qinghai > Tibet. The composition of soil bacteria in three provinces at various classification levels were obtained, the dominant genuses in Heilongjiang were Chthoniobacteraceae DA101 and an unannotated genus of Thermogemmatisporaceae; the dominant genuses in Qinghai were an unannotated genus of Cytophagaceae and an unannotated genus of Nocardioidaceae; the dominant genuses in Tibet were an unannotated genus of Comamonadaceae and Verrucomicrobiaceae Luteolibacter. The results of principal co-ordinates analysis demonstrated that, according to the weighted UniFrac analysis, the three principle components represented 56.36% of the total variable, and according to the unweighted UniFrac analysis, the three principle components represented 34.81% of the total variable. The samples from the same province could be clustered together, and the species and content of soil microorganisms from different provinces were significantly different. Conclusion Based on the metagenomic analysis method, soil samples from different regions can be effectively distinguished, which has potential application value in geolocation inference of forensic individual origin in the future.

Association of high-risk human papillomavirus infection duration and cervical lesions with vaginal microbiota composition.

BackgroundCervical cancer is reportedly caused by the synergistic effects of persistent high-risk human papillomavirus (HPV) infection. Cervical microbiota represent a unique and dynamically changing microecological system that is directly exposed to the vagina. The relationship between HPV and the composition of the cervical microbiome has long been a primary focus of research.MethodsTo determine the specific differential florae throughout the process of cervical cancer development, in the present study, 16S rRNA sequencing was combined with KEGG pathway enrichment analysis to analyse five groups of cervical scraping samples with increasing durations of HPV infection and cervical intraepithelial neoplasia pathological classification.ResultsThe findings revealed that decreasing levels of probiotics, including Shuttleworthia, Prevotella, Lactobacillus, and Sneathia, and increasing levels of pathogenic bacteria, including Dispar, Streptococcus, and Faecalibacterium prausnitzii, could be the direct result of early HPV infection. Other pathogenic bacteria, such as Bifidobacteriaceae, might represent key factors in cancer progression. Additionally, KEGG pathway enrichment analysis indicated that HPV infection directly inhibits multiple pathways, including those of sporulation, porphyrin and chlorophyll metabolism, arginine and proline metabolism, isoquinoline alkaloid biosynthesis, and ansamycin biosynthesis, which may lead to the development of early symptoms of cervical cancer. Biomarkers were predicted based on operational taxonomic unit (OTU) abundance data, and OTU851726 and OTU715913 were undoubtedly the best potential indicators of cervical cancer.ConclusionsThe findings of the present study could assist with the development of a guideline for screening new clinical drugs for cervical cancer.

Comparative analysis of free-living and particle-associated bacterial communities of Lake Baikal during the ice-covered period

Free-living (FLC) and particle-associated (PAC) bacterial communities contribute differentially to the cycling of matter in the environment. In this work we have for the first time studied the taxonomic composition and diversity of FLC and PAC in the oligotrophic freshwater Lake Baikal during the under-ice plankton peak. The communities on the bottom ice surface and in the water column under the ice were studied. Analysis by light microscopy showed that the bottom surface of the ice was dominated by green algae Chlorella spp. A complex of microalgae developed in the water column: green algae Monoraphidium griffithii, Monoraphidium contortum, Chlorella spp., chrysophyte Dinobryon cylindricum, haptophyte Chrysochromulina parva, and diatom Synedra acus. Algal abundance on the ice sub-surface was several orders of magnitude higher than that in the water column. According to pyrosequencing data of the bacterial 16S rRNA V3-V4 region, diversity of FLC (as measured by Shannon, Simpson, Chao1 and ACE indices) was higher than that of PAC. Proteobacteria (Burkholderiaceae) dominated the FLC associated with ice; FLC of the water column under ice were dominated by Actinobacteria (Sporichthyaceae and Ilumatobacteraceae) and Verrucomicrobia (Methylacidiphilaceae). PAC associated with ice were dominated by Bacteroidetes (Flavobacteriaceae), while water column-associated PAC were dominated by Verrucomicrobia (FukuN18_freshwater_group, Methylacidiphilaceae) and cyanobacteria (Cyanobiaceae). The difference between free-living and particle-associated, as well as between water column and ice sub-surface bacterial communities, were explored on operational taxonomic unit (OTU) and phylotype levels.

Involvement and mechanism of fecal microbiota transplantation on endotoxic acute lung injury in rats

Objective To observe the effects of fecal microbiota transplantation (FMT) on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rats and to investigate the possible mechanism in order to provide new thoughts for the treatment of acute lung injury. Methods Forty-five adult healthy male SPF level SD rats were randomly divided into three groups of normal saline (NS), LPS and FMT with 15 in each group. ALI was induced by intraperitoneal injection of rats with 5 mg/kg of LPS. The FMT group was given 10 ml/kg of fecal microbiota solution intervention (twice a day for two consecutive days) after ALI induction. Five rats in each group were randomly selected and sacrificed at 24 h, 48 h and 72 h after intervention. Pathological changes in lung tissues were examined with hematoxylin and eosin (HE) staining and pathological scores were assessed. Right lung samples were weighed to measure wet/dry (W/D) ratios. Abdominal aorta blood samples were collected for PaO2 analysis. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin 6 (IL-6) in bronchoalveolar lavage fluid (BALF) were detected by enzyme linked immunosorbent assay (ELISA). Expression of transforming growth factor-β (TGF-β) and intracellular signal transduction protein Smads (Smad3 and Smad7) at mRNA level was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Expression of ERK1/2 and p-ERK1/2 at protein level was detected by Western blot (WB). Rat fecal samples were collected to extract DNA and the PCR products of V3 and V4 regions were sequenced by Illumina MiSeq. Bioinformatics analysis on microbiota was conducted with Illumina MiSeq based on operational taxonomic unit (OTU) clustering. Results Compared with the NS group, the LPS group showed significantly widened alveolar septa, massive inflammatory cell infiltration and some alveolar collapse in lung tissues. Compared with the LPS group, the FMT group showed significantly alleviated inflammatory cell infiltration, reduced swelling in pulmonary interstitial tissues, and less damage to pulmonary structure. Compared with the NS group, the W/D lung ratio, PaO2 concentration and TNF-α, IL-1 and IL-6 levels in BALF in the LPS group significantly increased at 24 h after intervention and then gradually decreased, but still higher than those in the NS group (P<0.05). The W/D lung ratio, PaO2 concentration and TNF-α, IL-1 and IL-6 levels in BALF of the FMT group were significantly lower than those of the LPS group (P<0.05). Compared with the NS group, the expression of TGF-β1 and Smad3 at mRNA level in lung tissues increased at all time points in the LPS group, while the expression of Smad7 at mRNA level decreased (P<0.05). The expression of TGF-β1 and Smad3 at mRNA level in the FMT group were significantly lower than that in the LPS group (P<0.05), while the expression of Smad7 at mRNA level was higher (P<0.05). Compared with the NS group, p-ERK expression significantly increased in the LPS group with the highest expression at 24 h and gradually decreased at 48 h and 72 h (P<0.05). The expression of p-ERK in the FMT group was significantly lower than that in the LPS group (P<0.05). Results of the intestinal microbiota sequencing revealed 1 362 OTU in the NS group, 1 443 OTU in the LPS group and 1 510 OTU in the FMT group. There were 336 OTU shared by them accounting for 26.4%. The constitution of intestinal microbiota in the LPS group was significantly different with that of normal rats and characterized by high diversity with increased Firmicutes/Bacteroidetes ratio and decreased gene abundance of intestinal microbiota. After the intervention of fecal bacteria transplantation, the constitution of intestinal microbiota in the FMT group was similar to that of the NS group, showing decreased Firmicutes/Bacteroidetes ratio and increased gene abundance of the bacterial community (P<0.05). Conclusions FMT might inhibit immune inflammation, reduce the production and secretion of inflammatory markers in the body, alleviate alveolar epithelial damage and abnormal repair through regulating intestinal microbiota and TGF-β1/Smads/ERK pathway to improve the LPS-induced endotoxic ALI in rats. Key words: Acute lung injury; Fecal microbiota transplantation; High-throughput Sequencing; Lipopolysaccharide

Implications of Soil Pollution with Diesel Oil and BP Petroleum with ACTIVE Technology for Soil Health.

Grass Elymus elongatus has a potential in phytoremediation and was used in this study in a potted experiment, which was performed to determine the effect of polluting soil (Eutric Cambisol) with diesel oil (DO) and unleaded petroleum (P) on the diversity of soil microorganisms, activity of soil enzymes, physicochemical properties of soil, and on the resistance of Elymus elongatus to DO and P, which altogether allowed evaluating soil health. Both petroleum products were administered in doses of 0 and 7 cm3 kg−1 soil d.m. Vegetation of Elymus elongatus spanned for 105 days. Grasses were harvested three times, i.e., on day 45, 75, and 105 of the experiment. The study results demonstrated a stronger toxic effect of DO than of P on the growth and development of Elymus elongatus. Diesel oil caused greater changes in soil microbiome compared to unleaded petroleum. This hypothesis was additionally confirmed by Shannon and Simpson indices computed based on operational taxonomic unit (OTU) abundance, whose values were the lowest in the DO-polluted soil. Soil pollution with DO reduced the counts of all bacterial taxa and stimulated the activity of soil enzymes, whereas soil pollution with P diminished the diversity of bacteria only at the phylum, class, order, and family levels, but significantly suppressed the enzymatic activity. More polycyclic aromatic hydrocarbons (PAHs) were degraded in the soil polluted with P compared to DO, which may be attributed to the stimulating effect of Elymus elongatus on this process, as it grew better in the soil polluted with P than in that polluted with DO.

Unicellular Eukaryotic Community Response to Temperature and Salinity Variation in Mesocosm Experiments.

Climate change has profound impacts on marine biodiversity and biodiversity changes in turn might affect the community sensitivity to impacts of abiotic changes. We used mesocosm experiments and Next Generation Sequencing to study the response of the natural Baltic and Mediterranean unicellular eukaryotic plankton communities (control and +6°C heat shock) to subsequent salinity changes (-5 psu, +5 psu). The impact on Operational Taxonomic Unit (OTU) richness, taxonomic and functional composition and rRNA:rDNA ratios were examined. Our results showed that heat shock leads to lower OTU richness (21% fewer OTUs in the Baltic and 14% fewer in the Mediterranean) and a shift in composition toward pico- and nanophytoplankton and heterotrophic related OTUs. Heat shock also leads to increased rRNA:rDNA ratios for pico- and micrograzers. Less than 18% of shared OTUs were found among the different salinities indicating the crucial role of salinity in shaping communities. The response of rRNA:rDNA ratios varied highly after salinity changes. In both experiments the diversity decrease brought about by heat shock influenced the sensitivity to salinity changes. The heat shock either decreased or increased the sensitivity of the remaining community, depending on whether it removed the more salinity-sensitive or the salinity-tolerant taxa.

Characterization of the Stool Microbiome in Hispanic Preschool Children by Weight Status and Time.

Variations in gut microbiota composition and diversity have been associated with childhood adiposity, although most studies describing this have been cross-sectional in nature. Our objective was to evaluate associations between body weight and the gut microbiota over time in obese preschool-age children. Obese, preschool-age, Hispanic children provided stool samples at baseline and following a 6-month behavioral intervention. Normal-weight (NW) children also provided stool samples. Stool microbial community composition was characterized using 16S rRNA gene sequencing. Estimates of within-sample diversity were calculated on operational taxonomic unit (OTU) count data, and the Firmicutes:Bacteroidetes (F:B) ratio was determined on per-sample basis. Estimates of between-sample diversity were generated using the weighted Unifrac metric, differential abundances were evaluated using Wilcoxon rank-sum tests, and associations of microbiome features with clinical data were quantified using Spearman rank correlations. For the 30 obese children sampled preintervention and postintervention, a decrease in body mass index (BMI) z-score from 2.55 to 2.34 (p = 0.004, paired t-test) was observed. Bacteroides massiliensis was significantly enriched in obese children, while B. plebius was significantly enriched in NW controls. We identified significant correlations between multiple Bacteroides-like OTUs and BMI z-score, but neither F:B ratios nor OTU-level abundances were altered in conjunction with weight change in the obese children. Rather, highly individualized OTU-level responses were observed. Although differences exist between the gut microbiota of obese and NW children, we detected highly individualized responses of the gut microbiota of obese children over time and following weight loss.

Methane Production in Dairy Cows Correlates with Rumen Methanogenic and Bacterial Community Structure.

Methane (CH4) is produced as an end product from feed fermentation in the rumen. Yield of CH4 varies between individuals despite identical feeding conditions. To get a better understanding of factors behind the individual variation, 73 dairy cows given the same feed but differing in CH4 emissions were investigated with focus on fiber digestion, fermentation end products and bacterial and archaeal composition. In total 21 cows (12 Holstein, 9 Swedish Red) identified as persistent low, medium or high CH4 emitters over a 3 month period were furthermore chosen for analysis of microbial community structure in rumen fluid. This was assessed by sequencing the V4 region of 16S rRNA gene and by quantitative qPCR of targeted Methanobrevibacter groups. The results showed a positive correlation between low CH4 emitters and higher abundance of Methanobrevibacter ruminantium clade. Principal coordinate analysis (PCoA) on operational taxonomic unit (OTU) level of bacteria showed two distinct clusters (P < 0.01) that were related to CH4 production. One cluster was associated with low CH4 production (referred to as cluster L) whereas the other cluster was associated with high CH4 production (cluster H) and the medium emitters occurred in both clusters. The differences between clusters were primarily linked to differential abundances of certain OTUs belonging to Prevotella. Moreover, several OTUs belonging to the family Succinivibrionaceae were dominant in samples belonging to cluster L. Fermentation pattern of volatile fatty acids showed that proportion of propionate was higher in cluster L, while proportion of butyrate was higher in cluster H. No difference was found in milk production or organic matter digestibility between cows. Cows in cluster L had lower CH4/kg energy corrected milk (ECM) compared to cows in cluster H, 8.3 compared to 9.7 g CH4/kg ECM, showing that low CH4 cows utilized the feed more efficient for milk production which might indicate a more efficient microbial population or host genetic differences that is reflected in bacterial and archaeal (or methanogens) populations.

Influence of Chemical- and Natural-Based Lotions on Bacterial Communities in Human Forearm Skin

Purpose of this study was to evaluate the influence of a lotion on the bacterial community in the human forearm skin. The chemical- and natural-based lotions were applied on the left and right inner forearm skins, respectively, of 14 participants, who cleansed forearm skin using sterilized cotton swabs. The germs on cotton swabs were analyzed using libraries of PCR amplicons. The genetic diversity of the bacterial communities detected on the natural-based lotion-applied skin (NLS) was significantly higher than that of the bacterial communities on the chemical-based lotion-applied skin (CLS) in all participants, except two. The diversity was estimated based on operational taxonomic unit (OTU), Chao1, Shannon, and Simpson indices. Bacterial communities obtained from the CLS and NLS were phylogenetically separated into 5 and 3 monophyletic groups, respectively, based on lotion types. The taxonomic distribution of the bacterial communities, which were composed of 198 genera in 14 phyla in the CLS and NLS, respectively, was irregularly and biasedly separated into 2 groups based on the lotion types. Among the 14 phyla, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were found to be relatively dominant, and 15 of the 198 genera, including Methylobacterium, Propionibacterium, Pseudomonas, Staphylococcus, Streptococcus, and Bacillus were relatively dominant (>0.5%). The taxonomic distribution of dominant bacterial communities from CLS and NLS was irregularly and biasedly separated without relation to the lotion types. In conclusion, the chemical- and natural-based lotions were responsible for changing or influencing the genetic diversity, phylogenetic separation, and taxonomic distribution of skin bacterial communities.

Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

Selection for Cu-Tolerant Bacterial Communities with Altered Composition, but Unaltered Richness, via Long-Term Cu Exposure

Toxic metal pollution affects the composition and metal tolerance of soil bacterial communities. However, there is virtually no knowledge concerning the responses of members of specific bacterial taxa (e.g., phyla or classes) to metal toxicity, and contradictory results have been obtained regarding the impact of metals on operational taxonomic unit (OTU) richness. We used tag-coded pyrosequencing of the 16S rRNA gene to elucidate the impacts of copper (Cu) on bacterial community composition and diversity within a well-described Cu gradient (20 to 3,537 μg g(-1)) stemming from industrial contamination with CuSO(4) more than 85 years ago. DNA sequence information was linked to analysis of pollution-induced community tolerance (PICT) to Cu, as determined by the [(3)H]leucine incorporation technique, and to chemical characterization of the soil. PICT was significantly correlated to bioavailable Cu, as determined by the results seen with a Cu-specific bioluminescent biosensor strain, demonstrating a specific community response to Cu. The relative abundances of members of several phyla or candidate phyla, including the Proteobacteria, Bacteroidetes, Verrumicrobia, Chloroflexi, WS3, and Planctomycetes, decreased with increasing bioavailable Cu, while members of the dominant phylum, the Actinobacteria, showed no response and members of the Acidobacteria showed a marked increase in abundance. Interestingly, changes in the relative abundances of classes frequently deviated from the responses of the phyla to which they belong. Despite the apparent Cu impacts on Cu resistance and community structure, bioavailable Cu levels did not show any correlation to bacterial OTU richness (97% similarity level). Our report highlights several bacterial taxa responding to Cu and thereby provides new guidelines for future studies aiming to explore the bacterial domain for members of metal-responding taxa.