Advances in Microbiology Research Category: Microbiology Type: Review Article
Gammaproteobacteria and Firmicutes are Resistant to Long-Term Chromium Exposure in Soil
- Dorothea K Thompson1*, Gene S Wickham2
- 1 Department Of Pharmaceutical Sciences, Campbell University, Buies Creek, North Carolina, United States
- 2 Department Of Biological Sciences, Purdue University, West Lafayette, Indiana, United States
*Corresponding Author:Dorothea K Thompson
Department Of Pharmaceutical Sciences, Campbell University, Buies Creek, North Carolina, United States
Received Date: Dec 01, 2017 Accepted Date: Jan 06, 2018 Published Date: Jan 22, 2018
Bacterial phylogenetics; Chromium stress; Small-subunit ribosomal RNA gene clone technology; Soil microbial communities
The U.S. Department of Energy (DOE) is faced with the complex challenge of managing, remediating, and monitoring hazardous mixed wastes present in the subsurface environments of numerous DOE facility sites. Chromium is a risk-driving contaminant found at a number of DOE waste sites. Microbial catalysis of metal reduction constitutes a promising and potentially cost-effective strategy for the in situ remediation of metal-contaminated subsurface environments [16,17]. However, the biotransformation of toxic metal contaminants in natural environments is an inherently complex process that depends on the structure and dynamics of the indigenous microbial community, the types and concentration levels of the contaminants present, and the specific geochemical conditions characterizing the site .
Here we describe the molecular phylogenetic comparison of microbial communities from two contrasting vadose zone soils obtained from the DOE Hanford site in Washington State. The primary objective of this investigation was to characterize the impact of long-term, high-level Cr contamination on soil bacterial abundance, diversity, and community structure in soils under relevant field conditions. Toward that end, Small-Subunit (SSU) rRNA gene cloning technology was used to establish a culture-independent census of the indigenous microbial community. To our knowledge this is the first report that describes the in situmicrobial community structure in two soil samples comparable in texture, pH, and amounts of multiple metal pollution, but contrasting with respect to total Cr concentrations. The results obtained from this study provide valuable insight into bacterial community shifts in response to Cr-induced selective pressure.
MATERIALS AND METHODS
Sampling site and soil collection
Toxic metal content of soil samples
DNA extraction and SSU rRNA gene library construction
Sequencing of SSU rRNA gene clones
Chimera detection and phylogenetic analysis
RESULTS AND DISCUSSION
Toxic metal characteristics of sampling sites
As shown in table 1, the soils extracted from the two sampling sites differed primarily in the amount of Cr. The total chromium concentrations in the control and contaminated soils were determined to be 10.5 ± 2.3 mg kg-1 and 769.9 ± 74.7 mg kg-1, respectively, which constituted a 70-fold difference. Both soil types also contained measurable amounts of Ni, Pb, Fe and Co, but the total concentrations of these toxic metals varied no more than 2.2-fold between the control and contaminated soils (Table 1). The total levels of Fe were particularly high in the 100D samples, attaining levels of 33,988 ± 1089.8 mg kg-1 in the control samples and 25,404 ± 265.2 mg kg-1 in the contaminated samples (Table 1). However, the variance in total Fe concentrations between the control and contaminated soil samples was only approximately 1.3-fold. Contaminated soils, therefore, differed primarily from control samples in terms of total chromium amounts, while the levels of Ni, Pb, Fe and Co were comparable between the two samples.
Community diversity and species richness
Comparison of bacterial phylotype distributions
Members of the Proteobacteria (45%) and Firmicutes (30%) dominated the highly Cr-contaminated bacterial community (Figure 2). The phylogeny of the bacterial community reconstructed from the control samples, however, exhibited a more even distribution of clones across the most abundant phyla present: Proteobacteria (20%), Bacteroidetes (25%), and Actinobacteria (20%) (Figure 2). In 100D soils contaminated with high Cr levels, Proteobacteria were more abundant compared to the control, and the candidate OD1 division contained only DFC phylotypes (Figure 1B). However, the DFU (control) sample had a substantially higher percentage of Actinobacteria, Bacteroidetes, Thermomicrobiae, and Verrucomicrobia than the DFC (contaminated) sample. The bacterial community structure shifted to decreased relative abundances of Thermomicrobiae, Actinobacteria, Acidobacteria, and Bacteroidetes in the presence of elevated chromium levels. By contrast, a high chromium concentration appeared to selectively favor Proteobacteria and Firmicutes. A similar dominance shift from Actinobacteria and Acidobacteria to Proteobacteria was observed for microbial communities in soils contaminated with chromium and arsenic . Interestingly, 25% of the clones from the control soil were closely related to Arthrobacter spp., which are known for their high chromate resistance, while no close relatives of Arthrobacter spp. were identified in the contaminated sample (Figure 1B).
Further analysis of the distribution of DFU and DFC clones across classes within the phylum Proteobacteria revealed some striking features. Within the Proteobacteria, bacterial community overlaps were observed for the Alpha (α) and Beta (β) classes (Figure 3). Although 16S rRNA gene phylotypes assigned to Alphaproteobacteria were evenly distributed across the control and contaminated bacterial communities, 71% of the DFU clones derived from the control soil were closely related to Betaproteobacteria compared to 2% of the DFC clones, suggesting that these bacteria as a group are much less resistant to high chromium concentrations. The bacterial community structure appeared to shift from a dominance of Betaproteobacteria to a dominance of Gammaproteobacteria, presumably in response to chromium-induced selective pressure. Unexpectedly, there was a complete absence of Gammaproteobacteria in the control soil sample, where as Gammaproteobacteria comprised a total representation of 30% in the clone library derived from the contaminated soil sample (Figure 3). Representatives of the Epsilon (ε) class of Proteobacteria were not recovered from either the control or contaminated soils, where as a single DFU clone affiliated with the Delta (δ) class of Proteobacteria was recovered from the control soil (Figure 3).
Prevalence of Pseudomonas spp. in Cr-contaminated soil
The DFC clones comprising Phylotype II were most closely related to Pseudomonas mendocina and Pseudomonas pseudoalcaligenes, with pair wise similarities of greater than 99.5%. In previous work using soil artificially contaminated with 1000 mg Cr(VI) kg-1, resistant bacteria isolates were identified as members of . These isolates were not only resistant to 40 mM Cr(VI) in pure culture, but showed high chromate reduction activity that was growth-phase dependent . For other strains of P. mendocina, studies demonstrated that biotransformation of hexavalent chromium to its trivalent form was plasmid-mediated  and catalyzed by a periplasmic chromate reductase . Additionally, an isolate belonging to P. pseudoalcaligenes based on 16S rDNA sequence similarity exhibited resistance to multiple toxic metals, including chromium .
The findings from this research are noteworthy because a number of Pseudomonas spp. have the capacity to mediate the complete bio reduction of Cr(VI) to Cr(III), including Pseudomonas putida [40-42], Pseudomonas sp. G1DM21 , Pseudomonas ambigua , Pseudomonas gessardii strain LZ-E , and Pseudomonas aeruginosa PCN-2 . Collectively, the genus Pseudomonas is remarkable for its high degree of metabolic versatility, including hydrocarbon degradation and resistance to various toxic metals . Presumably, the dominance of the genus Pseudomonas with in the soil bacterial communities subjected to high chromium stress in this study is due to their diverse functional capacity to resist and reduce high Cr (VI) concentrations. The high Cr(VI) resistance of Pseudomonas spp., together with their Cr(VI) reduction capability, indicate the potential utility of this genus as a bio augmentation organism for remediation of complex contaminated soils, such as those present at U.S. Department of Energy sites. The development of effective remediation strategies will require knowledge of the effects of long-term chromium exposure on resident microbial communities.
In conclusion, this study demonstrated a substantial shift in community dominance from Actinobacteria and Bacteroidetes in control soils to Firmicutes and Proteobacteria in chromium-contaminated soils. Further analysis showed that Gammaproteobacteria dominated contaminated soils, while Betaproteobacteria dominated the control sample. These shifts in phylum level and proteobacterial class were dependent on differences in the chromium content of the soil samples, suggesting that Gammaproteobacteria exhibit some of the highest level of resistance to toxic metals at metal-contaminated sites.
POTENTIAL CONFLICTS OF INTEREST:
Authors report no conflicts of interest relevant to this research.
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Citation:Thompson DK, Wickham GS (2018) Gammaproteobacteria and Firmicutes are Resistant to Long-Term Chromium Exposure in Soil. Adv Microb Res 2: 002.
Copyright: © 2018 Dorothea K Thompson, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.