Polycyclic aromatic hydrocarbon degradation by a Mycobacterium sp. in microcosms containing sediment and water from a pristine ecosystem.

Microcosm studies were conducted to evaluate the survival and performance of a recently discovered polycyclic aromatic hydrocarbon (PAH)-degrading Mycobacterium sp. when this organism was added to sediment and water from a pristine ecosystem. Microcosms inoculated with the Mycobacterium sp. showed enhanced mineralization, singly and as components in a mixture, of 2-methylnaphthalene, phenanthrene, pyrene, and benzo[alpha]pyrene. Studies utilizing pyrene as the sole added PAH showed that the Mycobacterium sp. survived in microcosms for 6 weeks both with and without preexposure to PAH and mineralized multiple doses of pyrene. Pyrene mineralization rates for sterilized microcosms inoculated with the Mycobacterium sp. showed that competition with indigenous microorganisms did not adversely affect survival of or pyrene degradation by the Mycobacterium sp. Pyrene mineralization by the Mycobacterium sp. was not enhanced by inorganic nutrient enrichment and was hindered by organic nutrient enrichment, which appeared to result from overgrowth of indigenous bacteria. This study demonstrates the versatility of the PAH-degrading Mycobacterium sp. and expands its potential applications to include the degradation of two-, three-, four-, and five-ringed PAHs in sediments.     

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.     

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.     

Cross-induction of pyrene and phenanthrene in a Mycobacterium sp. isolated from polycyclic aromatic hydrocarbon contaminated river sediments.

A polycyclic aromatic hydrocarbon (PAH)-degrading culture enriched from contaminated river sediments and a Mycobacterium sp. isolated from the enrichment were tested to investigate the possible synergistic and antagonistic interactions affecting the degradation of pyrene in the presence of low molecular weight PAHs. The Mycobacterium sp. was able to mineralize 63% of the added pyrene when it was present as a sole source of carbon and energy. When the enrichment culture and the isolated bacterium were exposed to phenanthrene, de novo protein synthesis was not required for the rapid mineralization of pyrene, which reached 52% in chloramphenicol-treated cultures and 44% in the absence of the protein inhibitor. In the presence of chloramphenicol, < 1% of the added pyrene was mineralized by the mixed culture after exposure to anthracene and naphthalene. These compounds did not inhibit pyrene utilization when present at the same time as pyrene. Concurrent mineralization of pyrene and phenanthrene after exposure to either compound was observed. Cross-acclimation between ring classes of PAHs may be a potentially important interaction influencing the biodegradation of aromatic compounds in contaminated environments.     

三叶草根系分泌物对多环芳烃微生物降解及加氧酶的影响

为揭示根际效应对多环芳烃降解的影响机制,建立恰当的植物-微生物联合修复模式,本研究向含有微生物及多环芳烃(芘和苯并\[a\]芘)的微宇宙中加入三叶草根系分泌物,分析其对多环芳烃降解的影响,研究降解过程中微生物加氧酶和16S rDNA基因拷贝数的变化,并对具有多环芳烃降解能力的微生物进行鉴定.结果表明： 分枝杆菌M1具有降解多环芳烃的能力;三叶草根系分泌物总有机碳(TOC)浓度为35.5 mg·L^-1时,芘和苯并\[a\]芘降解率明显提高,分枝杆菌加氧酶基因所占比例增加,表明其促进了分枝杆菌对芘和苯并\[a\]芘的降解;在降解过程中,加氧酶基因拷贝数明显增加,而16S rDNA数量增加不明显,表明前者与多环芳烃降解过程有关,而后者和微生物数量有关.三叶草根系分泌物使分枝杆菌加氧酶基因拷贝数明显增加,从而促进了分枝杆菌对多环芳烃的降解.
     

Humic acid effect on pyrene degradation: finding an optimal range for pyrene solubility and mineralization enhancement

The addition of humic acid (HA) to polycyclic aromatic hydrocarbon (PAH) contaminated systems has been shown to enhance, inhibit, or have no effect on the biodegradation of these PAHs. In this study, the surfactant effects of Elliott soil HA (ESHA) at two pH values were tested. At pH 7.0, ESHA did not behave as a surfactant. At pH 11.8, ESHA acted as a surfactant, as displayed by a decrease in surface tension with increasing concentrations of ESHA. The effect of ESHA on pyrene solubility was tested by adding 0 to 800 μg ESHA/g soil to soil-slurries. Enhancement of pyrene apparent solubility demonstrated a dose- and time-related effect. Broader doses from 0 to 10,080 μg ESHA/g soil and three higher doses from 3,360 to 10,080 μg ESHA/g soil were tested for their effects on pyrene mineralization by indigenous soil microorganisms and a novel PAH-degrading Mycobacterium sp. KMS in soil microcosms, respectively. ESHA amendments between 20 and 200 μg ESHA/g soil were found to consistently increase pyrene mineralization by indigenous microorganisms, while the 10,080 μg ESHA/g soil produced inhibition and all other doses presented no effects. Pyrene degradation by M. KMS was significantly inhibited by the addition of the highest dose of ESHA.     

Degradation of fluoranthene in a soil matrix by indigenous and introduced bacteria

Microbial growth and degradation of fluoranthene in amended soil microcosms by the indigenous microbial population and a PAH degrading mixed culture inoculum were characterised. Percentages of fluoranthene disappearance ranged from 14.4 % in sterilised uninoculated soil microcosms to 52.1 % in unsterilized inoculated microcosms. Inoculated soils had initial microbial counts approximately one order of magnitude higher than the indigenous soil count and exhibited enhanced fluoranthene degradation. Over a nine week incubation period, total viable counts in inoculated non-sterile soil declined to the levels observed for the original indigenous population.     

Mycobacterium diversity and pyrene mineralization in petroleum-contaminated soils

Degradative strains of fast-growing Mycobacterium spp. are commonly isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soils. Little is known, however, about the ecology and diversity of indigenous populations of these fast-growing mycobacteria in contaminated environments. In the present study 16S rRNA genes were PCR amplified using Mycobacterium-specific primers and separated by temperature gradient gel electrophoresis (TGGE), and prominent bands were sequenced to compare the indigenous Mycobacterium community structures in four pairs of soil samples taken from heavily contaminated and less contaminated areas at four different sites. Overall, TGGE profiles obtained from heavily contaminated soils were less diverse than those from less contaminated soils. This decrease in diversity may be due to toxicity, since significantly fewer Mycobacterium phylotypes were detected in soils determined to be toxic by the Microtox assay than in nontoxic soils. Sequencing and phylogenetic analysis of prominent TGGE bands indicated that novel strains dominated the soil Mycobacterium community. Mineralization studies using [(14)C]pyrene added to four petroleum-contaminated soils, with and without the addition of the known pyrene degrader Mycobacterium sp. strain RJGII-135, indicated that inoculation increased the level of degradation in three of the four soils. Mineralization results obtained from a sterilized soil inoculated with strain RJGII-135 suggested that competition with indigenous microorganisms may be a significant factor affecting biodegradation of PAHs. Pyrene-amended soils, with and without inoculation with strain RJGII-135, experienced both increases and decreases in the population sizes of the inoculated strain and indigenous Mycobacterium populations during incubation.     

Degradation of pyrene, benz[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site.

The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [14C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysene-dicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH.     

糙皮侧耳和鞘脂菌NS7对多环芳烃污染土壤的生物强化与协同作用

【背景】真菌和细菌被认为在多环芳烃污染土壤生物修复过程中发挥协同作用,目前在真实土壤体系中开展真菌-细菌协同降解研究较少。【目的】研究真菌和细菌对不同种类多环芳烃降解的差异及对蒽和苯并[a]蒽的生物强化与协同作用。【方法】选用多环芳烃降解真菌和细菌各一株,在液体纯培养体系下分析它们对不同种类多环芳烃降解的差异,在土壤体系中采用放射性同位素示踪技术研究2种微生物对蒽和苯并[a]蒽的生物强化与协同作用。【结果】供试细菌鞘脂菌NS7能够很好地降解低环种类多环芳烃,以蒽作为唯一碳源时可以将其完全降解,在复合污染条件下对菲、蒽、荧蒽、芘等降解效果突出(>90%),对苯并[a]芘降解效果较差(9.76%)。相比而言,供试真菌糙皮侧耳菌对苯并[a]芘具有更好的降解效果(21.18%),对低环多环芳烃降解效果明显不如降解菌NS7。在自然土壤中,蒽和苯并[a]蒽具有明显不同的矿化效率,分别为18.61%和4.28%,在蒽污染土壤中加入鞘脂菌NS7并未显著提高蒽的矿化率(P>0.05),相比而言,苯并[a]蒽污染土壤中加入糙皮侧耳显著提高了污染物矿化效率(2.24倍),表明真菌和细菌在土壤环境...     

Survival and naphthalene-degrading activity of Rhodococcus sp. strain 1BN in soil microcosms

AIMS: The survival and activity of Rhodococcus sp. strain 1BN, inoculated into naphthalene-contaminated sandy-loam soil microcosms, were studied using classical and molecular methods. METHODS AND RESULTS: The naphthalene-degrading activity of 1BN in microcosms was examined through viable counts, CO2 production and naphthalene consumption, while its survival after inoculation was monitored by detecting the contemporary presence of alkane and naphthalene degradative genes and by analysing the 16S rDNA specific restriction profile. The inoculation of 1BN did not significantly enhance naphthalene degradation in the naphthalene-contaminated native soil, where 1BN maintained its catabolic activity also when in the presence of indigenous microflora. Instead the rate of naphthalene degradation by the inoculated 1BN was greater in sterile naphthalene-contaminated soil. The level of 1BN was only slightly higher after inoculation regardless of whether indigenous naphthalene-degrading bacteria were present or not and 1BN remained viable even when the substrate was depleted. CONCLUSIONS: This study documents the colonization and growth of 1BN in a non-sterile, naphthalene-added, sandy-loam soil having an active indigenous naphthalene-degrading population. SIGNIFICANCE AND IMPACT OF THE STUDY: An active and well-established naphthalene-degrading bacterial population in the native soil did not hamper the survival of the introduced 1BN that, through its activity, enhanced the mineralization rate of naphthalene.     

Bioremediation of a polyaromatic hydrocarbon contaminated soil by native soil microbiota and bioaugmentation with isolated microbial consortia

Biodegradation of a mixture of PAHs was assessed in forest soil microcosms performed either without or with bioaugmentation using individual fungi and bacterial and a fungal consortia. Respiratory activity, metabolic intermediates and extent of PAH degradation were determined. In all microcosms the low molecular weight PAH’s naphthalene, phenanthrene and anthracene, showed a rapid initial rate of removal. However, bioaugmentation did not significantly affect the biodegradation efficiency for these compounds. Significantly slower degradation rates were demonstrated for the high molecular weight PAH’s pyrene, benz[a]anthracene and benz[a]pyrene. Bioaugmentation did not improve the rate or extent of PAH degradation, except in the case of Aspergillus sp. Respiratory activity was determined by CO₂ evolution and correlated roughly with the rate and timing of PAH removal. This indicated that the PAHs were being used as an energy source. The native microbiota responded rapidly to the addition of the PAHs and demonstrated the ability to degrade all of the PAHs added to the soil, indicating their ability to remediate PAH-contaminated soils.     

Characterization of cultures enriched from acidic polycyclic aromatic hydrocarbon-contaminated soil for growth on pyrene at low pH

Two polycyclic aromatic hydrocarbon (PAH)-contaminated soils of pH 2 were successfully used as inoculum to enrich cultures growing on phenanthrene and pyrene at different pHs, including pH 3. Selected pyrene-utilizing cultures obtained at pH 3, pH 5, and pH 7 were further characterized. All showed rapid [14C]pyrene mineralization at pH 3 and pH 5 and grew on pyrene at pH values ranging from 2 to 6. Eubacterial and mycobacterial 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and sequencing indicated that the cultures were dominated by a single bacterium closely related to Mycobacterium montefiorense, belonging to the slow-growing Mycobacterium sp. In contrast, a culture enriched on pyrene at pH 7 from a slightly alkaline soil sampled at the same site was dominated by Pseudomonas putida and a fast-growing Mycobacterium sp. The M. montefiorense-related species dominating the pyrene-utilizing cultures enriched from the acidic soils was also the dominant Mycobacterium species in the acidic soils. Our data indicate that a slow-growing Mycobacterium species is involved in PAH degradation in that culture and show that bacteria able to degrade high-molecular-weight PAHs at low pH are present in acidic PAH-contaminated soil.     

Study of biochemical pathways and enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS

Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthroic acid, were identified during pyrene degradation. Pyrene-4,5-dione, which accumulates as an end product in some gram-negative bacterial cultures, was further utilized and degraded by Mycobacterium sp. strain KMS. Enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS were studied, using 2-D gel electrophoresis. The first protein in the catabolic pathway, aromatic-ring-hydroxylating dioxygenase, which oxidizes pyrene to cis-4,5-pyrene-dihydrodiol, was induced with the addition of pyrene and pyrene-4,5-dione to the cultures. The subcomponents of dioxygenase, including the alpha and beta subunits, 4Fe-4S ferredoxin, and the Rieske (2Fe-2S) region, were all induced. Other proteins responsible for further pyrene degradation, such as dihydrodiol dehydrogenase, oxidoreductase, and epoxide hydrolase, were also found to be significantly induced by the presence of pyrene and pyrene-4,5-dione. Several nonpathway-related proteins, including sterol-binding protein and cytochrome P450, were induced. A pyrene degradation pathway for Mycobacterium sp. strain KMS was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of pyrene degradation.     

Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.     

Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10, 201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO(2) by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [(14)C]benzo[a]pyrene was recovered as (14)CO(2) in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.     

Enhanced Biodegradation of Anthracene in Acidic Soil by Inoculated <Emphasis Type="Italic">Burkholderia</Emphasis> sp. VUN10013

The ability of Burkholderia sp. VUN10013 to degrade anthracene in microcosms of two acidic Thai soils was studied. The addition of Burkholderia sp. VUN10013 (initial concentration of 10(5) cells g(-1) dry soil) to autoclaved soil collected from the Plew District, Chanthaburi Province, Thailand, supplemented with anthracene (50 mg kg(-1) dry soil) resulted in complete degradation of the added anthracene within 20 days. In contrast, under the same test conditions but using autoclaved soil collected from the Kitchagude District, Chanthaburi Province, Thailand, only approximately 46.3% of the added anthracene was degraded after 60 days of incubation. In nonautoclaved soils, without adding the VUN10013 inocula, 22.8 and 19.1% of the anthracene in Plew and Kitchagude soils, respectively, were degraded by indigenous bacteria after 60 days. In nonautoclaved soil inoculated with Burkholderia sp. VUN10013, the rate and extent of anthracene degradation were considerably better than those seen in autoclaved soils or in uninoculated nonautoclaved soils in that only 8.2 and 9.1% of anthracene remained in nonautoclaved Plew and Kitchagude soils, respectively, after 10 days of incubation. The results showed that the indigenous microorganisms in the pristine acidic soils have limited ability to degrade anthracene. Inoculation with the anthracene-degrading Burkholderia sp. VUN10013 significantly enhanced anthracene degradation in such acidic soils. The indigenous microorganisms greatly assisted the VUN10013 inoculum in anthracene degradation, especially in the more acidic Kitchagude soil.     

Products from the incomplete metabolism of pyrene by polycyclic aromatic hydrocarbon-degrading bacteria

Pyrene is a regulated pollutant at sites contaminated with polycyclic aromatic hydrocarbons (PAH). It is mineralized by some bacteria but is also transformed to nonmineral products by a variety of other PAH-degrading bacteria. We examined the formation of such products by four bacterial strains and identified and further characterized the most apparently significant of these metabolites. Pseudomonas stutzeri strain P16 and Bacillus cereus strain P21 transformed pyrene primarily to cis-4,5-dihydro-4,5-dihydroxypyrene (PYRdHD), the first intermediate in the known pathway for aerobic bacterial mineralization of pyrene. Sphingomonas yanoikuyae strain R1 transformed pyrene to PYRdHD and pyrene-4,5-dione (PYRQ). Both strain R1 and Pseudomonas saccharophila strain P15 transform PYRdHD to PYRQ nearly stoichiometrically, suggesting that PYRQ is formed by oxidation of PYRdHD to 4,5-dihydroxypyrene and subsequent autoxidation of this metabolite. A pyrene-mineralizing organism, Mycobacterium strain PYR-1, also transforms PYRdHD to PYRQ at high initial concentrations of PYRdHD. However, strain PYR-1 is able to use both PYRdHD and PYRQ as growth substrates. PYRdHD strongly inhibited phenanthrene degradation by strains P15 and R1 but had only a minor effect on strains P16 and P21. At their aqueous saturation concentrations, both PYRdHD and PYRQ severely inhibited benzo[a]pyrene mineralization by strains P15 and R1. Collectively, these findings suggest that products derived from pyrene transformation have the potential to accumulate in PAH-contaminated systems and that such products can significantly influence the removal of other PAH. However, these products may be susceptible to subsequent degradation by organisms able to metabolize pyrene more extensively if such organisms are present in the system.     

多环芳烃降解菌筛选及其降解特性

通过选择性富集培养,从辽河油田稠油污染土壤4号土样中,获得了能以高浓度菲(2000mg·L^-1)为唯一碳源和能源快速生长的优势菌系和优良菌株ZL5．16S rDNA核苷酸序列分析表明,ZL5菌株归类于鞘氨醇单胞菌属,分得的菌系和菌株有较强的降解菲能力,120h混合菌系降解了投加菲的95．28％,菌株降解了69．24％,但它们对芘的降解能力均较低,外加碳源葡萄糖可提高菌系和菌株的菲、芘降解能力,加量多。提高幅度大,但超过一定量。降解速率开始下降,表现出抑制效应。所以,应用时需控制适宜的浓度。     

Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.