Mycobacterium Vanbaalenii PYR-1 Research Articles

Erratum for Kweon et al., "Substrate Specificity and Structural Characteristics of the Novel Rieske Nonheme Iron Aromatic Ring-Hydroxylating Oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1".

Volume 1, issue 2, e00135-10, 2010, [https://doi:10.1128/mBio.00135-10][1]. The files for Fig. S1 and S2 were incorrect (they were reversed). The files have been corrected online. [1]: /lookup/doi/10.1128/mBio.00135-10

Bioremediation of PAH-contaminated shooting range soil using integrated approaches

Serious contamination of polycyclic aromatic hydrocarbons (PAHs) occurs at outdoor shooting ranges due to the accumulation of clay target fragments containing coal tar or petroleum pitch. These contaminated sites are characterized with high-molecular-weight PAHs that are low in bioavailability and recalcitrant to bioremediation. We evaluated the effectiveness of different remediation strategies, used individually or in combinations, to decontaminate PAHs in a shooting range soil. The treatments included vegetation with bermudagrass [Cynodon dactylon (L.) Pers] or switchgrass [Panicum virgatum]), bioaugmentation of Mycobacterium vanbaalenii PYR-1, and addition of surfactants (Brij-35, rhamnolipid biosurfactant, or Brij-35/sodium dodecyl sulfate mixture). The initial total PAH concentration in the shooting range soil was 373 mg/kg and consisted of primarily high-molecular-weight PAHs (84%). Planting of bermudagrass and switchgrass resulted in 36% and 27% ∑16PAH reduction compared to the non-vegetated control, respectively. Bermudagrass enhanced soil dehydrogenase activity and both vegetation treatments also increased polyphenol oxidase activity. Bioaugmentation of M. vanbaalenii PYR-1 had a significant effect only on the dissipation of high-molecular-weight PAHs, leading to a 15% decrease (∑10PAH) compared to the control. In the non-vegetated soil, Brij-35/sodium dodecyl sulfate mixture increased PAH degradation compared to the no surfactant control. The increased PAH biodegradation in the vegetated and bioaugmented treatments improved lettuce [Lactuca sativa] seed germination, suggesting reduced toxicity in the treated soils. Phytoremediation using bermudagrass or switchgrass with bioaugmentation of M. vanbaalenii PYR-1 was an effective in situ remediation option for shooting range soils with heavy PAH contamination.

A newly defined dioxygenase system from Mycobacterium vanbaalenii PYR-1 endowed with an enhanced activity of dihydroxylation of high-molecular-weight polyaromatic hydrocarbons

NidA3B3 is a terminal dioxygenase whose favorable substrates are high-molecular-weight polyaromatic hydrocarbons (PAHs) from Mycobacterium vanbaalenii PYR-1, a powerful PAHs degradation strain. NidA3B3 was reported to incorporate a dioxygen into the benzene ring of PAHs when equipped with an exogenous electron transport chain components PhdCD from Nocardioides sp. strain KP7 by biotransformation, but this enzyme system was not particularly efficient. In this study, strain PYR-1 was confirmed to utilize four different PAHs at different growth rates. When PhtAcAd, an endogenous electron transport chain ofa phthalate dioxygenase system, was substituted for PhdCD to couple with NidA3B3, the specific activity to convert phenanthrene by strain BL21(DE3) [pNidA3B3-PhAcAd] was 0.15±0.03 U/mg, but the specificactivity of strain BL21(DE3) [pNidA3B3-PhdCD] was only 0.025±0.006 U/mg. In addition, FNidA3, encoded by a newly defined ORF, has a prolonged 19-amino acid sequence at the N-terminus compared with NidA3. FNidA3B3 increased the activity by 50% approximately than NidA3B3 when using PhtAcAd. Components of the electron transport chain PhtAc and PhtAd were purified and characterized. The Km, kcat, kcat/Km values of the PhtAd were 123±26.9 µM, 503±49.9 min−1, 4.1 µM−1 ×min−1, respectively. And the; Km, kcat, kcat/Km values of the ferredoxin PhtAc were 52.5±9.7 µM, 3.8±0.19 min−1 and 0.07 µM−1×min−1 respectively. Basing on the phylogenetic analysis, NidA3/FNidA3 were far from its isoenzyme NidA from the same strain. Combining their primary differences of transcriptional pattern in vivo, it indicated that the functionally similar Rieske dioxygenases NidA3B3/FNidA3B3 and NidAB might originate from different ancestors.

Humic Acid Can Enhance the Mineralization of Phenanthrene Sorbed on Biochars.

Biodegradation of hydrophobic organic contaminants by bacteria has been widely studied, but how dissolved organic matter (DOM) may affect their removal if accumulated on biochars is poorly understood. To address this knowledge gap, microbial mineralization of phenanthrene (PHE) spiked on various biochars by Mycobacterium vanbaalenii PYR-1 in the presence of humic acid (HA, a model DOM) at two concentrations was investigated. Our findings showed that HA greatly increased the rate and extent of PHE mineralization. This could be attributed to enhanced PHE desorption by HA, which facilitated access to it by bacteria in the aqueous phase. Furthermore, the high HA affinity for PHE facilitated PHE flow toward the bacterial cells with HA acting as a carrier in the aqueous phase. The mineralization enhancement of PHE by HA within 480 h was negatively influenced by the aromatic carbon contents and micropore volumes in biochars. This shows the importance of the physicochemical properties of biochars in altering the HA effect. Results of this study provide novel information on how to achieve complete removal of PHE accumulated on biochars with a strong sorption affinity for it, using a microbial technique and natural DOM.

Preparation of dihydroxy polycyclic aromatic hydrocarbons and activities of two dioxygenases in the phenanthrene degradative pathway

Dihydroxy phenanthrene, fluoranthene, and pyrene derivatives are intermediates in the bacterial catabolism of the corresponding parent polycyclic aromatic hydrocarbon (PAH). Ring-opening of the dihydroxy species followed by a series of enzyme-catalyzed reactions generates metabolites that funnel into the Krebs Cycle with the eventual production of carbon dioxide and water. One complication in delineating these pathways and harnessing them for useful purposes is that the initial enzymatic processing produces multiple dihydroxy PAHs with multiple ring opening possibilities and products. As part of a systematic effort to address this issue, eight dihydroxy species were synthesized and characterized as the dimethoxy or diacetate derivatives. Several dihydroxy compounds were examined with two dioxygenases in the phenanthrene degradative pathway in Mycobacterium vanbaalenii PYR-1. One, 3,4-dihydroxyphenanthrene, was processed by PhdF with a kcat/Km of 6.0 × 106 M−1s−1, a value that is consistent with the annotated function of PhdF in the pathway. PhdI processed 1-hydroxy-2-naphthoate with a kcat/Km of 3.1 × 105 M−1s−1, which is also consistent with the proposed role in the pathway. The observations provide the first biochemical evidence for these two reactions in M. vanbaalenii PYR-1 and, to the best of our knowledge, the first biochemical evidence for the reaction of PhdF with 3,4-dihydroxyphenanthrene. Although PhdF is upregulated in the presence of pyrene, it did not process two dihydroxypyrenes. Methodology was developed for product analysis of the extradiol dioxygenases.

Soil bacterial community dynamics following surfactant addition and bioaugmentation in pyrene-contaminated soils

Because of their toxic properties, polycyclic aromatic hydrocarbons (PAHs) are designated as priority pollutants. The low solubility and strong sorption of PAHs in soil often limits bioremediation. To increase PAH bioavailability and enhance microbial degradation, surfactants are often added to contaminated soils. However, the effects of surfactants on the PAH degradation capacities of soil microbes are generally neglected. In this study, 16S rRNA gene high-throughput sequencing was used to evaluate changes in the soil microbial community after the application of rhamnolipid biosurfactant or Brij-35 surfactant and Mycobacterium vanbaalenii PYR-1 bioaugmentation over a 50-d mineralization study in two soils contaminated with pyrene at 10 mg kg−1. The introduction of pyrene in both soils resulted in an increase in Firmicutes and a decrease in microbial richness and Shannon diversity index. Amendment of rhamnolipid at 1,400 μg g−1 to the native clay soil resulted in a decrease in Bacillus from 48% to 2%, which was accompanied with an increase in Mycoplana that accounted for 67% of the total genera relative abundance. Phylogenetic investigation of communities by reconstruction of unobserved states was used to predict the activity of functional genes involved in the PAH degradation KEGG pathway and determined that M. vanbaalenii PYR-1 bioaugmentation resulted in an increased number of functional genes utilized in PAH biodegradation. Results of this study provide a better understanding of the soil microbial dynamics in response to surfactant amendments in addition to bioaugmentation of a PAH-degrading microbe. This knowledge contributes to successful and efficient surfactant-enhanced bioremediation of PAH-contaminated soils.

Influence of rhamnolipid biosurfactant and Brij-35 synthetic surfactant on 14C-Pyrene mineralization in soil

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in soil and are considered priority pollutants due to their carcinogenicity. Bioremediation of PAH-contaminated soils is often limited by the low solubility and strong sorption of PAHs in soil. Synthetic surfactants and biosurfactants have been used to enhance the bioavailability of PAHs and to accelerate microbial degradation. However, few studies have compared synthetic and biosurfactants in their efficiency in promoting PAH biodegradation in either native or bioaugmented soils. In this study, we evaluated mineralization of 14C-pyrene in soils with or without the augmentation of Mycobacterium vanbaalenii PYR-1, and characterized the effect of Brij-35 (synthetic) and rhamnolipid biosurfactant at different amendment rates. Treatment of rhamnolipid biosurfactant at 140 or 1400 μg surfactant g-dry soil−1 rates resulted in a significantly longer lag period in 14C-pyrene mineralization in both native and bioaugmented soils. In contrast, amendment of Brij-35 generally increased 14C-pyrene degradation, and the greatest enhancement occurred at 21.6 or 216 μg surfactant g-dry soil−1 rates, which may be attributed to increased bioavailability. Brij-35 and rhamnolipid biosurfactant were found to be non-toxic to M. vanbaalenii PYR-1 at 10X CMC, thus indicating rhamnolipid biosurfactant likely served as a preferential carbon source to the degrading bacteria in place of 14C-pyrene, leading to delayed and inhibited 14C-pyrene degradation. Mineralization of 14C-pyrene by M. vanbaalenii PYR-1 was rapid in the unamended soils, and up to 60% of pyrene was mineralized to 14CO2 after 10 d in the unamended or Brij-35 surfactant-amended soils. Findings of this study suggest that application of surfactants may not always lead to enhanced PAH biodegradation or removal. If the surfactant is preferentially used as an easier carbon substrate than PAHs for soil microorganisms, it may actually inhibit PAH biodegradation. Selection of surfactant types is therefore crucial for the effectiveness of surfactant-aided bioremediation of PAH-contaminated soils.

A comparative intracellular proteomic profiling of Pseudomonas aeruginosa strain ASP-53 grown on pyrene or glucose as sole source of carbon and identification of some key enzymes of pyrene biodegradation pathway

Pseudomonas aeruginosa strain ASP-53, isolated from a petroleum oil-contaminated soil sample, was found to be an efficient degrader of pyrene. PCR amplification of selected hydrocarbon catabolic genes (alkB gene, which encodes for monooxygenase, and the C12O, C23O, and PAH-RHDα genes encoding for the dioxygenase enzyme) from the genomic DNA of P. aeruginosa strain ASP-53 suggested its hydrocarbon degradation potential. The GC–MS analysis demonstrated 30.1% pyrene degradation by P. aeruginosa strain ASP-53 after 144h of incubation at pH6.5, 37°C. Expressions of 115 and 196 intracellular proteins were unambiguously identified and quantitated by shotgun proteomics analysis when the isolate was grown in medium containing pyrene and glucose, respectively. The pyrene-induced uniquely expressed and up-regulated proteins in P. aeruginosa strain ASP-53 in addition to substrate (pyrene) metabolism are also likely to be associated with different cellular functions for example-related to protein folding (molecular chaperone), stress response, metabolism of carbohydrate, proteins and amino acids, and fatty acids; transport of metabolites, energy generation such as ATP synthesis, electron transport and nitrate assimilation, and other oxidation-reduction reactions. Proteomic analyses identified some important enzymes involved in pyrene degradation by P. aeruginosa ASP-53 which shows that this bacterium follows the salicylate pathway of pyrene degradation. SignificanceThis study is the first report on proteomic analysis of pyrene biodegradation pathway by Pseudomonas aeruginosa, isolated from a petroleum-oil contaminated soil sample. The pathway displays partial similarity with deduced pyrene degradation mechanisms of Mycobacterium vanbaalenii PYR-1. The GC–MS analysis as well as PCR amplification of hydrocarbon catabolic genes substantiated the potency of the bacterium under study to effectively degrade high molecular weight, toxic PAH such as pyrene for its filed scale bioremediation experiments. The proteomics approach (LC-MS/MS analysis) identified the differentially regulated intracellular proteins of the isolate P. aeruginosa ASP-53 when grown in pyrene medium. This study identified some important pyrene biodegradation enzymes in Pseudomonas aeruginosa ASP-53 and highlights that the bacterium follows salicylate pathway for pyrene degradation.

Microbial Transformation of Multiwalled Carbon Nanotubes by Mycobacterium vanbaalenii PYR-1.

Carbonaceous nanomaterials are widely used in industry and consumer products, but concerns have been raised regarding their release into the environment and subsequent impacts on ecosystems and human health. Although many efforts have been devoted to understanding the environmental fate of carbonaceous nanomaterials, information about their microbial transformation is still rare. In this study, we found that within 1 month a polycyclic aromatic hydrocarbon-degrading bacterium, Mycobacterium vanbaalenii PYR-1, was able to degrade both pristine and carboxyl-functionalized multiwalled carbon nanotubes (p-MWCNT and c-MWCNT), as demonstrated by consistent results from high resolution transmission electron microscopy, Raman spectroscopy, and confocal Raman microspectroscopy. Statistical analysis of Raman spectra identified a significant increase in the density of disordered or amorphous carbon in p-MWCNT and c-MWCNT after biodegradation. Microbial respiration further suggested potential mineralization of MWCNTs within about 1 month. All of our analyses consistently showed higher degradation or mineralization of c-MWCNT compared to p-MWCNT. These results highlight the potential of using bacteria in engineered systems to remove residual carbonaceous nanomaterials and reduce risk of human exposure and environmental impact. Meanwhile, our finding suggests possible transformation of carbonaceous nanomaterials by polycyclic aromatic hydrocarbon-degrading bacteria in the natural environment, which should be accounted for in predicting the environmental fate of these emerging contaminants and in nanotechnology risk regulation.

Bioavailability of phenanthrene and nitrobenzene sorbed on carbonaceous materials

To better understand the mechanisms regarding effects of the interactions between organic contaminants (ORCs) and carbonaceous materials (CMs) on bioavailability of ORCs, 22 mg/kg of phenanthrene and nitrobenzene were spiked onto various CMs, and their microbial mineralization by the bacterium Mycobacterium vanbaalenii PYR-1 was investigated. The results showed that as sorption of phenanthrene and nitrobenzene to CMs increased 4.5 and 42.3 times, their freely dissolved concentrations (Cfree) decreased by 80.4% and 97.0%, respectively. Correspondingly, the maximum mineralization rate was reduced by 99.6% for phenanthrene and 98.2% for nitrobenzene. Functionalization of multiwalled carbon nanotubes (CNTs) with hydrophilic polar moieties greatly weakened sorption of phenanthrene and nitrobenzene. Correspondingly, the reduction in mineralization of them preloaded on CNTs was more modest. However, bioavailability of CM-sorbed phenanthrene and nitrobenzene was not solely controlled by Cfree. Model calculations of mineralization kinetics data and electron microscopy observations of CMs evidenced that bacteria access to sorbed phenanthrene and nitrobenzene through cell attachment to the particle surfaces, but this process was CM dependent. This work highlights the factors and underlying mechanisms governing microbial availability of ORCs sorbed on CMs, which is significant for risk assessment of ORCs and possible application of CMs for ORC pollution control through sequestration.

Inoculating Bacteria into Polycyclic Aromatic Hydrocarbon-Contaminated Oil Sands Soil by Means of Electrokinetics

Electrokinetics (EK) is commonly used in bioremediation studies to aid mobilization of amendments in low permeable soils. This study evaluated the possibility of using EK as a delivery mechanism to emplace organic contaminant degrading bacteria, Sphingomonas paucimobilis EPA505 and Mycobacterium vanbaalenii PYR-1, into low permeability soils without disturbing the soils. Bacterial cultures were grown to exponential phase with a polycyclic aromatic hydrocarbon (PAH) carbon source, and then transferred to EK reservoirs. Direct current was applied to induce EK movement, and soil DNA evaluated for the inoculated bacteria. Electroosmotic transport efficacy for a silty and an oil sands soil was compared. Results show that EK can be used to inoculate S. paucimobilis EPA505 into soils under electroosmotic flow, but the hydrophobic nature of soils especially enabled electroosmosis. For the first time, we demonstrate that in situ inoculation of non-native bacterial species using EK is possible. The results of this study demonstrate the potential for future applications of this technique for in situ bioaugmentation and remediation of PAH-contaminated soils.

Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a Stenotrophomonas sp: Characterization of nid Genes and Effect of Surfactants on Degradation.

A polyaromatic hydrocarbon degrading bacterium was isolated from a petroleum contaminated site and designated as Stenotrophomonas sp. strain IITR87. It was found to utilize pyrene, phenanthrene and benzo(a)pyrene as sole carbon source, but not anthracene, chrysene and fluoranthene. Gas chromatography and mass spectroscopy analysis resulted in identification of pyrene metabolites namely monohydroxypyrene, 4-oxa-pyrene-5-one, dimethoxypyrene and monohydroxyphenanthrene. Southern hybridization using naphthalene dioxygenase gene (nidA) as probe against the DNA of strain IITR87 revealed the presence of nidA gene. PCR analysis suggests dispersed occurrence of nid genes in the genome instead of a cluster as reported in a PAH-degrading Mycobacterium vanbaalenii PYR-1. The nid genes in strain IITR87, dioxygenase large subunit (nidA), naphthalene dioxygenase small subunit (nidB) and aldehyde dehydrogenase gene (nidD) showed more than 97% identity to the reported nid genes from Mycobacterium vanbaalenii PYR-1. Most significantly, the biodegradation of PAHs was enhanced 25-60% in the presence of surfactants rhamnolipid and Triton X-100 due to increased solubilization and bioavailability. These results could be useful for the improved biodegradation of high-molecular-weight PAHs in contaminated habitats.

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil.

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C12 and C13 being degraded at the highest rate, although alkanes up to C28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications.

Polymorphism of the bphA genes in bacteria destructing biphenyl/chlorinated biphenils

Polychlorinated biphenyls (PCBs) are persistent organic pollutants. Biphenyl 2,3-dioxygenase (BDO) is a key enzyme that determines the range of PCBs oxidized by a bacterial strain. BDO subunit α (BphA1) plays an essential role in substrate recognition and binding. The genes for dioxygenases that hydroxylate aromatic rings were screened and analyzed phylogenetically. Genes found in biphenyl-oxidizing Rhodococcus erythropolis strains G12a, B7b, and B106a proved to be similar to the published nucleotide sequences of the Rhodococcus sp. HA99 and R04 and Novosphingobium aromaticivorans F199 bphA1 genes, which code for the α-subunits that do not belong to the biphenyl/toluene dioxygenase (B/TDO) family. PCB-destructing R. ruber P25 was found to possess a unique bphA1 gene, which clusters together with the phenylpropionate dioxygenase (PPDO) α-subunits of Mycobacterium vanbaalenii PYR-1 and Frankia sp. EuI1c. The deduced amino acid sequences of the genes were analyzed. The amino acids of the BDO active site in R. wratislaviensis P1, P12, P13, and P20 (bphA1 genes of the B/TDO family) were identical to those of the active PCB degrader R. jostii RHA1. The Rhodococcus strains in question were shown to be active toward both orthoand parachlorinated ring of 2,4'-dichlorobiphenyl. The α-subunit amino acids responsible for the substrate specificity of the enzyme in Pseudomonas sp. S9, S13, S210, S211, and S212 (B/TDO family) were the same as in P. pseudoalcaligenes KF707. The Pseudomonas strains were active toward the para-chlorinated ring of 2,4'-dichlorobiphenyl. The results of screening bacterial strains for bphA1 can be used to identify the biotechnologically promising PCB destructors.

Improving Rice Ability to Degrade Different Polycyclic Aromatic Hydrocarbons Through Multigene Transformation of a Hybrid Dioxygenase System

The widespread presence and potential harm conferred by polycyclic aromatic hydrocarbons (PAHs) to various organisms have raised increased research interest on these pollutants. Plants lack of efficient PAH degradation genes so that they had to suffer from PAH stress. To improve the ability of plants to tolerate various PAHs, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as nahAa and nahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were ectopically expressed in rice. The hybrid multicomponent naphthalene dioxygenase can assemble correctly and break down PAHs powerfully. Transgenic rice plants exhibited enhanced tolerance toward two- to four-ring PAHs. PAH tolerance in transgenic plants was associated with high chlorophyll levels and photosynthesis efficiency, normal development, and minimal ion leakage under PAH stress. The naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Our results indicate that grafting the naphthalene dioxygenase system into plants is useful to degrade PAHs efficiently in vivo and improve the plant tolerance to the pollutants. This study had introduced a suitable strategy to accelerate the bioremediation process of PAH-contaminated soil under waterlogged conditions and improve the food safety in contaminated area.

Metabolic engineering of Arabidopsis for remediation of different polycyclic aromatic hydrocarbons using a hybrid bacterial dioxygenase complex

The widespread presence of polycyclic aromatic hydrocarbons (PAHs) and their potential harm to various organisms has generated interest in efficiently eliminating these compounds from the environment. Phytoremediation is an efficient technology for cleaning up pollutants. However, unlike microorganisms, plants lack the catabolic pathway for complete degradation of these dangerous groups of compounds. One way to enhance the potential of plants for remediation of these compounds is by transferring genes involved in xenobiotic degradation from microbes to plants. In this paper, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as NahAa and NahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were transferred and ectopically expressed in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing the heterozygous naphthalene dioxygenase system exhibited enhanced tolerance toward 2–4 rings PAHs. Transgenic plants assimilated PAHs from the culture media faster and accumulated less in vivo than wild-type plants. Furthermore, examination of metabolic intermediates by gas chromatography–mass spectrometry revealed that the naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Taken together, our findings suggest that grafting the naphthalene dioxygenase complex into plants is a possible strategy to breed PAH-tolerant plants to efficiently degrade PAHs in the environment.

Pleiotropic and epistatic behavior of a ring-hydroxylating oxygenase system in the polycyclic aromatic hydrocarbon metabolic network from Mycobacterium vanbaalenii PYR-1.

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.

Mid-infrared spectroscopic assessment of nanotoxicity in Gram-negative vs. Gram-positive bacteria

Nanoparticles appear to induce toxic effects through a variety of mechanisms including generation of reactive oxygen species (ROS), physical contact with the cell membrane and indirect catalysis due to remnants from manufacture. The development and subsequent increasing usage of nanomaterials has highlighted a growing need to characterize and assess the toxicity of nanoparticles, particularly those that may have detrimental health effects such as carbon-based nanomaterials (CBNs). Due to interactions of nanoparticles with some reagents, many traditional toxicity tests are unsuitable for use with CBNs. Infrared (IR) spectroscopy is a non-destructive, high throughput technique, which is unhindered by such problems. We explored the application of IR spectroscopy to investigate the effects of CBNs on Gram-negative (Pseudomonas fluorescens) and Gram-positive (Mycobacterium vanbaalenii PYR-1) bacteria. Two types of IR spectroscopy were compared: attenuated total reflection Fourier-transform infrared (ATR-FTIR) and synchrotron radiation-based FTIR (SR-FTIR) spectroscopy. This showed that Gram-positive and Gram-negative bacteria exhibit differing alterations when exposed to CBNs. Gram-positive bacteria appear more resistant to these agents and this may be due to the protection afforded by their more sturdy cell wall. Markers of exposure also vary according to Gram status; Amide II was consistently altered in Gram-negative bacteria and carbohydrate altered in Gram-positive bacteria. ATR-FTIR and SR-FTIR spectroscopy could both be applied to extract biochemical alterations induced by each CBN that were consistent across the two bacterial species; these may represent potential biomarkers of nanoparticle-induced alterations. Vibrational spectroscopy approaches may provide a novel means of fingerprinting the effects of CBNs in target cells.

Influence of single-walled carbon nanotubes on microbial availability of phenanthrene in sediment

Increasing production and use of single-walled carbon nanotubes (SWCNT) will inevitably lead to release of these nanoparticles to aquatic ecosystems. Similar to black carbon (BC) particles, SWCNT have a high affinity for hydrophobic organic contaminants (HOCs) and therefore the presence of SWCNT in sediment may lead to altered bioavailability of HOCs. We compared SWCNT with biochar and charcoal on their effect on the microbial degradability of 0.05 mg kg(-1) (14)C-phenanthrene (PHE) by Mycobacterium vanbaalenii PYR-1 in two sediments with different organic carbon (OC) contents. When the amendment rate of SWCNT or BC was 1 mg g(-1), PHE mineralization was inhibited much more significantly by SWCNT than by either biochar or charcoal. After 360 h of incubation, the mineralized fraction of PHE in the presence of SWCNT was 59.5% of the non-amended control in the sediment with low OC content, and only 42.4% in the other sediment with a higher OC content. Analysis of the freely dissolved concentration (C (free)) using disposable polydimethylsiloxane (PDMS) fibers showed that SWCNT decreased C (free) by 85-95%, apparently due to preferential sorption of PHE to SWCNT particles that had a much larger specific surface area and pore volume than biochar or charcoal. However, pre-interaction of SWCNT with dissolved organic matter (peptone, tannic acid, and humic acid) led to attachment of polar functional groups and reduced surface area on SWCNT, resulting in decreased PHE sorption and an alleviated effect on PHE biodegradation in the order of peptone > tannic acid > humic acid.

Biodegradation of pyrene in sand, silt and clay fractions of sediment

Microbial degradation is the dominant pathway for natural attenuation of PAHs in environmental compartments such as sediments, which in turn depends on the bioavailability of PAHs. The bioavailability of PAHs has seldom been studied at the sediment particle size scale. We evaluated biodegradation of pyrene by Mycobacterium vanbaalenii PYR-1 as a function of sediment particle sizes, and investigated the relationship between the rate of degradation on sand, silt and clay particles with their individual desorption kinetics measured with the Tenax extraction method. Regression analysis showed that the total organic carbon (TOC), black carbon (BC), and specific surface area (SSA) of the specific particle size fractions, instead of the particle size scale itself, were closely related (P < 0.01) with the mineralization rate. While the fraction in the rapid desorption pool (F rapid) ranged from 0.11 to 0.38 for the whole sediments and different size groups, the fractions mineralized after 336-h incubation (0.52 to 0.72) greatly surpassed the F rapid values, suggesting utilization of pyrene in the slow desorption pool (F slow). A biodegradation model was modified by imbedding a two-phase desorption relationship describing sequential Tenax extractions. Model analysis showed that pyrene sorbed on silt and clay aggregates was directly utilized by the degrading bacteria. The enhanced bioavailability may be attributed to the higher chemical concentration, higher TOC or larger SSA in the silt and clay fractions, which appeared to overcome the reduced bioavailability of pyrene due to sorption, making pyrene on the silt and clay particles readily available to degrading microbes. This conjecture merits further investigation.Electronic supplementary materialThe online version of this article (doi:10.1007/s10532-010-9399-z) contains supplementary material, which is available to authorized users.