Most cited article - PubMed ID 22728721
Plant-microorganism interactions in bioremediation of polychlorinated biphenyl-contaminated soil
Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS), which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well-established bacterial identification and classification methods based on nearly complete 16S rRNA gene sequence analyses: a phylotype-based approach, using a closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4-10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS) threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision and recall values of 0.70 and 0.73, respectively. When matched to species-level phylotype assignment, an optimal CS threshold of 0.92 was calculated, with associated precision and recall values of 0.83 and 0.64, respectively. Overall, our research indicates that a similarity-based MALDI-TOF MS approach can be routinely used for efficient dereplication of isolates for downstream analyses, with minimal loss of unique organisms. In addition, MALDI-TOF MS analysis has further improvement potential unlike 16S rRNA gene analysis, whose methodological limits have reached a plateau.
Plant-microbe interactions are of particular importance in polluted soils. This study sought to determine how selected plants (horseradish, black nightshade and tobacco) and NPK mineral fertilization shape the structure of soil microbial communities in legacy contaminated soil and the resultant impact of treatment on the soil microbial community functional potential. To explore these objectives, we combined shotgun metagenomics and 16S rRNA gene amplicon high throughput sequencing with data analysis approaches developed for RNA-seq. We observed that the presence of any of the selected plants rather than fertilization shaped the microbial community structure, and the microbial populations of the root zone of each plant significantly differed from one another and/or from the bulk soil, whereas the effect of the fertilizer proved to be insignificant. When we compared microbial diversity in root zones versus bulk soil, we observed an increase in the relative abundance of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria or Bacteroidetes, taxa which are commonly considered copiotrophic. Our results thus align with the theory that fast-growing, copiotrophic, microorganisms which are adapted to ephemeral carbon inputs are enriched in the vegetated soil. Microbial functional potential indicated that some genetic determinants associated with signal transduction mechanisms, defense mechanisms or amino acid transport and metabolism differed significantly among treatments. Genetic determinants of these categories tend to be overrepresented in copiotrophic organisms. The results of our study further elucidate plant-microbe relationships in a contaminated environment with possible implications for the phyto/rhizoremediation of contaminated areas.
- Keywords
- contaminated soil, fertilization, functional potential, microbial community structure, plants,
- Publication type
- Journal Article MeSH
Despite decades of research there is limited understanding of how vegetation impacts the ability of microbial communities to process organic contaminants in soil. Using a combination of traditional and molecular assays, we examined how phytoremediation with willow and/or fertilization affected the microbial community present and active in the transformation of diesel contaminants. In a pot study, willow had a significant role in structuring the total bacterial community and resulted in significant decreases in diesel range organics (DRO). However, stable isotope probing (SIP) indicated that fertilizer drove the differences seen in community structure and function. Finally, analysis of the total variance in both pot and SIP experiments indicated an interactive effect between willow and fertilizer on the bacterial communities. This study clearly demonstrates that a willow native to Alaska accelerates DRO degradation, and together with fertilizer, increases aromatic degradation by shifting microbial community structure and the identity of active naphthalene degraders.
- Keywords
- Salix alaxensis, bioremediation, diesel range organics, fertilizer, microbial community structure, naphthalene degradation, phytoremediation, stable isotope probing,
- Publication type
- Journal Article MeSH
Aerobic mineralization of PCBs, which are toxic and persistent organic pollutants, involves the upper (biphenyl, BP) and lower (benzoate, BZ) degradation pathways. The activity of different members of the soil microbial community in performing one or both pathways, and their synergistic interactions during PCB biodegradation, are not well understood. This study investigates BP and BZ biodegradation and subsequent carbon flow through the microbial community in PCB-contaminated soil. DNA stable isotope probing (SIP) was used to identify the bacterial guilds involved in utilizing (13)C-biphenyl (unchlorinated analogue of PCBs) and/or (13)C-benzoate (product/intermediate of BP degradation and analogue of chlorobenzoates). By performing SIP with two substrates in parallel, we reveal microbes performing the upper (BP) and/or lower (BZ) degradation pathways, and heterotrophic bacteria involved indirectly in processing carbon derived from these substrates (i.e. through crossfeeding). Substrate mineralization rates and shifts in relative abundance of labeled taxa suggest that BP and BZ biotransformations were performed by microorganisms with different growth strategies: BZ-associated bacteria were fast growing, potentially copiotrophic organisms, while microbes that transform BP were oligotrophic, slower growing, organisms. Our findings provide novel insight into the functional interactions of soil bacteria active in processing biphenyl and related aromatic compounds in soil, revealing how carbon flows through a bacterial community.
- MeSH
- Hydrocarbons, Aromatic metabolism MeSH
- Bacteria genetics metabolism MeSH
- Benzoates metabolism MeSH
- Biphenyl Compounds metabolism MeSH
- Biodegradation, Environmental MeSH
- Soil Pollutants metabolism MeSH
- Hazardous Waste MeSH
- Polychlorinated Biphenyls metabolism MeSH
- Soil chemistry MeSH
- Soil Microbiology * MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Environmental Pollution * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Names of Substances
- Hydrocarbons, Aromatic MeSH
- Benzoates MeSH
- Biphenyl Compounds MeSH
- biphenyl MeSH Browser
- Soil Pollutants MeSH
- Hazardous Waste MeSH
- Polychlorinated Biphenyls MeSH
- Soil MeSH
- RNA, Ribosomal, 16S MeSH
Functional gene ecological analyses using amplicon sequencing can be challenging as translated sequences are often burdened with shifted reading frames. The aim of this work was to evaluate several bioinformatics tools designed to correct errors which arise during sequencing in an effort to reduce the number of frameshifts (FS). Genes encoding for alpha subunits of biphenyl (bphA) and benzoate (benA) dioxygenases were used as model sequences. FrameBot, a FS correction tool, was able to reduce the number of detected FS to zero. However, up to 44% of sequences were discarded by FrameBot as non-specific targets. Therefore, we proposed a de novo mode of FrameBot for FS correction, which works on a similar basis as common chimera identifying platforms and is not dependent on reference sequences. By nature of FrameBot de novo design, it is crucial to provide it with data as error free as possible. We tested the ability of several publicly available correction tools to decrease the number of errors in the data sets. The combination of maximum expected error filtering and single linkage pre-clustering proved to be the most efficient read processing approach. Applying FrameBot de novo on the processed data enabled analysis of BphA sequences with minimal losses of potentially functional sequences not homologous to those previously known. This experiment also demonstrated the extensive diversity of dioxygenases in soil. A script which performs FrameBot de novo is presented in the supplementary material to the study or available at https://github.com/strejcem/FBdenovo. The tool was also implemented into FunGene Pipeline available at http://fungene.cme.msu.edu/FunGenePipeline/.
- Keywords
- FrameBot, Frameshift, amplicon sequencing, benzoate dioxygenase, biphenyl dioxygenase, functional genes,
- Publication type
- Journal Article MeSH
Given that the degradation of aromatic pollutants in anaerobic environments such as sediment is generally very slow, aeration could be an efficient bioremediation option. Using stable isotope probing (SIP) coupled with pyrosequencing analysis of 16S rRNA genes, we identified naphthalene-utilizing populations in aerated polyaromatic hydrocarbon (PAH)-polluted sediment. The results showed that naphthalene was metabolized at both 10 and 20°C following oxygen delivery, with increased degradation at 20°C as compared to 10°C-a temperature more similar to that found in situ. Naphthalene-derived (13)C was primarily assimilated by pseudomonads. Additionally, Stenotrophomonas, Acidovorax, Comamonas, and other minor taxa were determined to incorporate (13)C throughout the measured time course. The majority of SIP-detected bacteria were also isolated in pure cultures, which facilitated more reliable identification of naphthalene-utilizing populations as well as proper differentiation between primary consumers and cross-feeders. The pseudomonads acquiring the majority of carbon were identified as Pseudomonas veronii and Pseudomonas gessardii. Stenotrophomonads and Acidovorax defluvii, however, were identified as cross-feeders unable to directly utilize naphthalene as a growth substrate. PAH degradation assays with the isolated bacteria revealed that all pseudomonads as well as Comamonas testosteroni degraded acenaphthene, fluorene, and phenanthrene in addition to naphthalene. Furthermore, P. veronii and C. testosteroni were capable of transforming anthracene, fluoranthene, and pyrene. Screening of isolates for naphthalene dioxygenase genes using a set of in-house designed primers for Gram-negative bacteria revealed the presence of such genes in pseudomonads and C. testosteroni. Overall, our results indicated an apparent dominance of pseudomonads in the sequestration of carbon from naphthalene and potential degradation of other PAHs upon aeration of the sediment at both 20 and 10°C.
- Keywords
- Comamonas testosteroni, Pseudomonas gessardii, Pseudomonas veronii, biodegradation, dioxygenase, naphthalene, polyaromatic hydrocarbons, stable isotope probing,
- Publication type
- Journal Article MeSH
