Brett R. Baldwin

Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

ABSTRACT Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 × 102 copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.

Soil Microbial Community Responses to Additions of Organic Carbon Substrates and Heavy Metals (Pb and Cr)

ABSTRACT Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the Actinomycetales were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear.

Enumeration of aromatic oxygenase genes to evaluate biodegradation during multi-phase extraction at a gasoline-contaminated site

Multi-phase extraction (MPE) is commonly used at petroleum-contaminated sites to volatilize and recover hydrocarbons from the vadose and saturated zones in contaminant source areas. Although primarily a physical treatment technology, the induced subsurface air flow can potentially increase oxygen supply and promote aerobic biodegradation of benzene, toluene, ethylbenzene, and xylenes (BTEX), the contaminants of concern at gasoline-contaminated sites. In this study, real-time PCR enumeration of aromatic oxygenase genes and PCR-DGGE profiles were used to elucidate the impact of MPE operation on the aquifer microbial community structure and function at a gasoline-contaminated site. Prior to system activation, ring-hydroxylating toluene monooxygenase (RMO) and naphthalene dioxygenase (NAH) gene copies were on the order of 10(6) to 10(10)copies L(-1) in groundwater samples obtained from BTEX-impacted wells. Aromatic oxygenase genes were not detected in groundwater samples obtained during continuous MPE indicating decreased populations of BTEX-utilizing bacteria. During periods of pulsed MPE, total aromatic oxygenase gene copies were not significantly different than prior to system activation, however, shifts in aromatic catabolic genotypes were noted. The consistent detection of RMO, NAH, and phenol hydroxylase (PHE), which catabolizes further oxidation of hydroxylated BTEX metabolites indicated the potential for aerobic biodegradation of dissolved BTEX during pulsed MPE.

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

ABSTRACT Bacterial multicomponent monooxygenase gene targets in Pseudonocardia dioxanivorans CB1190 were evaluated for their use as biomarkers to identify the potential for 1,4-dioxane biodegradation in pure cultures and environmental samples. Our studies using laboratory pure cultures and industrial activated sludge samples suggest that the presence of genes associated with dioxane monooxygenase, propane monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase are promising indicators of 1,4-dioxane biotransformation; however, gene abundance was insufficient to predict actual biodegradation. A time course gene expression analysis of dioxane and propane monooxygenases in Pseudonocardia dioxanivorans CB1190 and mixed communities in wastewater samples revealed important associations with the rates of 1,4-dioxane removal. In addition, transcripts of alcohol dehydrogenase and aldehyde dehydrogenase genes were upregulated during biodegradation, although only the aldehyde dehydrogenase was significantly correlated with 1,4-dioxane concentrations. Expression of the propane monooxygenase demonstrated a time-dependent relationship with 1,4-dioxane biodegradation in P. dioxanivorans CB1190, with increased expression occurring after over 50% of the 1,4-dioxane had been removed. While the fraction of P. dioxanivorans CB1190-like bacteria among the total bacterial population significantly increased with decrease in 1,4-dioxane concentrations in wastewater treatment samples undergoing active biodegradation, the abundance and expression of monooxygenase-based biomarkers were better predictors of 1,4-dioxane degradation than taxonomic 16S rRNA genes. This study illustrates that specific bacterial monooxygenase and dehydrogenase gene targets together can serve as effective biomarkers for 1,4-dioxane biodegradation in the environment.

Multilevel Samplers as Microcosms to Assess Microbial Response to Biostimulation

Passive multilevel samplers (MLS) containing a solid matrix for microbial colonization were used as in situ microcosms in conjunction with a push-pull biostimulation experiment designed to promote biological U(VI) and Tc(VII) reduction. MLS were deployed at 24 elevations in the injection well and two downgradient wells to investigate the spatial variability in microbial community composition and growth prior to and following biostimulation. The microbial community was characterized by real-time quantitative polymerase chain reaction (Q-PCR) quantification of bacteria, NO(3)(-)-reducing bacteria (nirS and nirK), delta-proteobacteria, Geobacter sp., and methanogens (mcrA). Pretest cell densities were low overall but varied substantially with significantly greater bacterial populations detected at circumneutral pH (t-test, alpha= 0.05), suggesting carbon substrate and low pH limitations of microbial activity. Although pretest cell densities were low, denitrifying bacteria were dominant members of the microbial community. Biostimulation with an ethanol-amended ground water resulted in concurrent NO(3)(-) and Tc(VII) reduction, followed by U(VI) reduction. Q-PCR analysis of MLS revealed significant (1 to 2 orders of magnitude, Mann-Whitney U-test, alpha= 0.05) increases in cell densities of bacteria, denitrifiers, delta-proteobacteria, Geobacter sp., and methanogens in response to biostimulation. Traditionally, characterization of sediment samples has been used to investigate the microbial community response to biostimulation; however, collection of sediment samples is expensive and not conducive to deep aquifers or temporal studies. The results presented demonstrate that push-pull tests with passive MLS provide an inexpensive approach to determine the effect of biostimulation on contaminant concentrations, geochemical conditions, and the microbial community composition and function.

Broad substrate specificity of naphthalene-and biphenyl-utilizing bacteria

Abstract Although aromatic compounds are most often present in the environment as components of complex mixtures, biodegradation studies commonly focus on the degradation of individual compounds. The present study was performed to investigate the range of aromatic substrates utilized by biphenyl- and naphthalene-degrading environmental isolates and to ascertain the effects of co-occurring substrates during the degradation of mono-aromatic compounds. Bacterial strains were isolated on the basis of their ability to utilize either biphenyl or naphthalene as a sole source of carbon. Growth and transformation assays were conducted on each isolate to determine the range of substrates degraded. One isolate, Pseudomonas putida BP18, was tested for the ability to biodegrade benzene, toluene, ethylbenzene and xylene isomers (BTEX) individually and as components of mixtures. Overall, the results indicate that organisms capable of growth on multi-ring aromatic compounds may be particularly versatile in terms of aromatic hydrocarbon biodegradation. Furthermore, growth and transformation assays performed with strain BP18 suggest that the biodegradation of BTEX and biphenyl by this strain is linked to a catabolic pathway with overlapping specificities. The broad substrate specificity of these environmental isolates has important implications for bioremediation efforts in the field.

Monitoring gene expression to evaluate oxygen infusion at a gasoline-contaminated site.

Increasingly, molecular biological tools, most notably quantitative polymerase chain reaction (qPCR), are being employed to provide a more comprehensive assessment of bioremediation of petroleum hydrocarbons and fuel oxygenates. While qPCR enumeration of key organisms or catabolic genes can aid in site management decisions, evaluation of site activities conducted to stimulate biodegradation would ideally include a direct measure of gene expression to infer activity. In the current study, reverse-transcriptase (RT) qPCR was used to monitor gene expression to evaluate the effectiveness of an oxygen infusion system to promote biodegradation of BTEX and MTBE. During system operation, dissolved oxygen (DO) levels at the infusion points were greater than 30 mg/L, contaminant concentrations decreased, and transcription of two aromatic oxygenase genes and Methylibium petroleiphilum PM1-like 16S rRNA copies increased by as many as 5 orders of magnitude. Moreover, aromatic oxygenase gene transcription and PM1 16s rRNA increased at downgradient locations despite low DO levels even during system operation. Conversely, target gene expression substantially decreased when the system was deactivated. RT-qPCR results also corresponded to increases in benzene and MTBE attenuation rates. Overall, monitoring gene expression complemented traditional groundwater analyses and conclusively demonstrated that the oxygen infusion system promoted BTEX and MTBE biodegradation.

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes

Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55±0.64 × 108 cells were measured per μmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.

Monitoring Microbial Community Dynamics to Evaluate Bioremediation

Bioremediation can be an effective treatment strategy at contaminated sites when applied appropriately and implemented properly. Thus it is important to have methods that can be used to reliably assess biodegradation potential and ultimately the feasibility and performance of bioremediation as a treatment approach. While an important component of site monitoring, decreasing trends in contaminant concentrations can result from physical processes, do not document that loss is due to biodegradation, and can be difficult to discern in practice. Therefore direct measures of microbes responsible for biodegradation of compounds have been developed. These methods fall into two categories: direct measure of biodegradation functional genes using methods such as quantitative PCR and microarrays and indirect measures in changes in microbial community structure using methods such as PCR-DGGE and T-RFLP when target genes are unknown. This chapter describes the various molecular methods being used and examples where they have been applied for the assessment of environmental samples.

Normalized Quantitative PCR Measurements as Predictors for Ethene Formation at Sites Impacted with Chlorinated Ethenes

Quantitative PCR (qPCR) targeting Dehalococcoides mccartyi ( Dhc) biomarker genes supports effective management at sites impacted with chlorinated ethenes. To establish correlations between Dhc biomarker gene abundances and ethene formation (i.e., detoxification), 859 groundwater samples representing 62 sites undergoing monitored natural attenuation or enhanced remediation were analyzed. Dhc 16S rRNA genes and the vinyl chloride (VC) reductive dehalogenase genes bvcA and vcrA were detected in 88% and 61% of samples, respectively, from wells with ethene. Dhc 16S rRNA, bvcA, vcrA, and tceA (implicated in cometabolic reductive VC dechlorination) gene abundances all positively correlated with ethene formation. Significantly greater ethene concentrations were observed when Dhc 16S rRNA gene and VC RDase gene abundances exceeded 107 and 106 copies L-1, respectively, and when Dhc 16S rRNA- and bvcA + vcrA-to-total bacterial 16S rRNA gene ratios exceeded 0.1%. Dhc 16S rRNA gene-to- vcrA/ bvcA ratios near unity also indicated elevated ethene; however, no increased ethene was observed in 19 wells where vcrA and/or bvcA gene copy numbers exceeded Dhc cell numbers 10- to 10 000-fold. Approximately one-third of samples with detectable ethene lacked bvcA, vcrA, and tceA, suggesting that comprehensive understanding of VC detoxification biomarkers has not been achieved. Although the current biomarker suite is incomplete, the data analysis corroborates the value of the available Dhc DNA biomarkers for prognostic and diagnostic groundwater monitoring at sites impacted with chlorinated ethenes.