Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times ( 2 S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.

Recent Advances in Petroleum Microbiology

Microbial degradation of petroleum hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) include a group of organic priority pollutants of critical environmental and public health concern due to their toxic, genotoxic, mutagenic and/or carcinogenic properties and their ubiquitous occurrence as well as recalcitrance. The increased awareness of their various adverse effects on ecosystem and human health has led to a dramatic increase in research aimed towards removing PAHs from the environment. PAHs may undergo adsorption, volatilization, photolysis, and chemical oxidation, although transformation by microorganisms is the major neutralization process of PAH-contaminated sites in an ecologically accepted manner. Microbial degradation of PAHs depends on various environmental conditions, such as nutrients, number and kind of the microorganisms, nature as well as chemical property of the PAH being degraded. A wide variety of bacterial, fungal and algal species have the potential to degrade/transform PAHs, among which bacteria and fungi mediated degradation has been studied most extensively. In last few decades microbial community analysis, biochemical pathway for PAHs degradation, gene organization, enzyme system, genetic regulation for PAH degradation have been explored in great detail. Although, xenobiotic-degrading microorganisms have incredible potential to restore contaminated environments inexpensively yet effectively, but new advancements are required to make such microbes effective and more powerful in removing those compounds, which were once thought to be recalcitrant. Recent analytical chemistry and genetic engineering tools might help to improve the efficiency of degradation of PAHs by microorganisms, and minimize uncertainties of successful bioremediation. However, appropriate implementation of the potential of naturally occurring microorganisms for field bioremediation could be considerably enhanced by optimizing certain factors such as bioavailability, adsorption and mass transfer of PAHs. The main purpose of this review is to provide an overview of current knowledge of bacteria, halophilic archaea, fungi and algae mediated degradation/transformation of PAHs. In addition, factors affecting PAHs degradation in the environment, recent advancement in genetic, genomic, proteomic and metabolomic techniques are also highlighted with an aim to facilitate the development of a new insight into the bioremediation of PAH in the environment.

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Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1574-6941.2008.00629.x

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

Geobacter species specialize in making electrical contacts with extracellular electron acceptors and other organisms. This permits Geobacter species to fill important niches in a diversity of anaerobic environments. Geobacter species appear to be the primary agents for coupling the oxidation of organic compounds to the reduction of insoluble Fe(III) and Mn(IV) oxides in many soils and sediments, a process of global biogeochemical significance. Some Geobacter species can anaerobically oxidize aromatic hydrocarbons and play an important role in aromatic hydrocarbon removal from contaminated aquifers. The ability of Geobacter species to reductively precipitate uranium and related contaminants has led to the development of bioremediation strategies for contaminated environments. Geobacter species produce higher current densities than any other known organism in microbial fuel cells and are common colonizers of electrodes harvesting electricity from organic wastes and aquatic sediments. Direct interspecies electron exchange between Geobacter species and syntrophic partners appears to be an important process in anaerobic wastewater digesters. Functional and comparative genomic studies have begun to reveal important aspects of Geobacter physiology and regulation, but much remains unexplored. Quantifying key gene transcripts and proteins of subsurface Geobacter communities has proven to be a powerful approach to diagnose the in situ physiological status of Geobacter species during groundwater bioremediation. The growth and activity of Geobacter species in the subsurface and their biogeochemical impact under different environmental conditions can be predicted with a systems biology approach in which genome-scale metabolic models are coupled with appropriate physical/chemical models. The proficiency of Geobacter species in transferring electrons to insoluble minerals, electrodes, and possibly other microorganisms can be attributed to their unique "microbial nanowires," pili that conduct electrons along their length with metallic-like conductivity. Surprisingly, the abundant c-type cytochromes of Geobacter species do not contribute to this long-range electron transport, but cytochromes are important for making the terminal electrical connections with Fe(III) oxides and electrodes and also function as capacitors, storing charge to permit continued respiration when extracellular electron acceptors are temporarily unavailable. The high conductivity of Geobacter pili and biofilms and the ability of biofilms to function as supercapacitors are novel properties that might contribute to the field of bioelectronics. The study of Geobacter species has revealed a remarkable number of microbial physiological properties that had not previously been described in any microorganism. Further investigation of these environmentally relevant and physiologically unique organisms is warranted.

Geobacter: The Microbe Electric's Physiology, Ecology, and Practical Applications

The adoption of molecular genetic techniques has greatly improved our ability to characterize microbial populations in situ, leading to more accurate assessments of pollutant biodegradation and the impact of remediation technologies. Initially, biases associated with culturing were avoided by using DNA hybridization techniques with probes containing the catabolic gene of interest. Cross-hybridization at low stringency (46) and exclusion of related but not identical genes at high stringency (15, 30, 56) may produce false-positive and false-negative results with environmental samples. PCR amplification with primers based on conserved regions within the gene of interest, however, will permit amplification despite variability in the overall sequence and detect genotypes not detected by hybridization alone. Molecular methods, particularly in quantitative PCR (Q-PCR), continue to advance rapidly. Our laboratory described a competitive Q-PCR technique to enumerate catechol 2,3-dioxygenase genes as a means to evaluate bioremediation of aromatic hydrocarbons at petroleum-contaminated sites (35). Here we present primer sets targeting additional aromatic catabolic genes and the use of real-time PCR for quantification to extend our ability to enumerate genotypes involved in biodegradation of aromatic pollutants.

The two main approaches to Q-PCR are competitive and real-time PCR. In competitive PCR, a comparison of the intensities of the standard and target amplicons allows quantification (42). One limitation of this method is that amplification efficiency of the target and standard are not always equivalent. Furthermore, post-PCR analysis of the products is labor-intensive and not amenable to the large-scale screening of environmental samples. Real-time PCR offers an accurate, sensitive method of quantification without the labor-intensive postamplification analysis and assumptions required by competitive PCR. Detection of product during amplification can be accomplished by several means, including molecular beacons (55), hybridization probes (60), and BODIPY FL-labeled probes and primers (27), but hydrolysis probes and DNA-binding dyes are the most commonly reported methods. The hydrolysis probe (TaqMan) assay takes advantage of the 5′-nuclease ability of DNA polymerase to hydrolyze a labeled probe bound to its target amplicon to produce a signal. The SYBR green assay relies on fluorescence signal produced as the dye binds to double-stranded DNA during the extension step. While the hydrolysis probe provides an additional degree of specificity, identifying an additional highly conserved region for the probe may not always be possible. Furthermore, both the probe and the primers must be from a highly conserved region of the target to avoid biasing quantitation (2). Simpler methods using DNA-staining dyes, such as SYBR green I, have been successful (36, 62), do not require the design of an internal hybridization probe, and thus can be used with any established PCR primers with only minor modifications of the described protocols.

This study was conducted to develop multiplex and real-time PCR procedures to quantify aromatic catabolic genes in environmental samples and eventually improve our understanding of biodegradation in the field. The large subunit of aromatic oxygenase genes was chosen as the indicator gene, because it has been implicated in substrate specificity and is one of the rate-limiting steps in aromatic hydrocarbon biodegradation (14) and its DNA sequence is conserved for oxygenases targeting the same substrate. Alignments were constructed from groups of related oxygenase genes, and each primer set was chosen from a conserved region unique to that group of oxygenases. Thus, a single primer set will detect an entire subfamily of related oxygenase genes rather than a species-specific catabolic gene. In all, PCR primer sets which targeted biphenyl dioxygenase, naphthalene dioxygenase, toluene dioxygenase, toluene/xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. Testing and optimization with genetically well-characterized bacterial strains demonstrated the specificity of each primer set. Multiplex PCR protocols were developed to permit simultaneous detection of aromatic oxygenase genes and facilitate rapid screening of environmental samples. Real-time PCR with SYBR green I was used to determine the gene copy number with a quantification limit of 2 × 102 copies of target per reaction mixture. The primer sets and real-time PCR methods presented will be used to assess natural attenuation, investigate the ecology of contaminated sites, and aid in optimization of bioremediation in the field.

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

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.

Soil microbial community responses to additions of organic carbon substrates and heavy metals (Pb and Cr).

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.

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

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.

Brett R. Baldwin

Papers

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

Soil microbial community responses to additions of organic carbon substrates and heavy metals (Pb and Cr).

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

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

Enumeration of aromatic oxygenase genes to evaluate monitored natural attenuation at gasoline-contaminated sites.