Effect of Exposure Time,Contamination Level,and Type of PAH Compound on Biodegradation Capacity of Mangrove Sediment

Mangrove sediment had high natural attenuation potential with more than 50% of total PAHs being removed within 15 days. The efficiency in degrading PAHs varied with the declining order of phenanthrene (Phe), fluoranthene (Fla), and pyrene (Pyr). The Most Probable Number (MPN) of PAH-degrading bacteria in the PAH-contaminated slurries was 2 to 4 orders of magnitude higher than that in the non-contaminated mangrove slurries. The biodegradation ability of the indigenous microbial community in mangrove sediment slurry was significantly increased after exposure to polycyclic aromatic hydrocarbons. Such enhancement effect was dependent on the level and time of exposure, as well as the types of PAH compounds. The lowest contamination level of 3 mg kg^?1 was effective in promoting the degradation of Phe and Fla after seven days, but the enhancement effect for Pyr degradation was only found in the slurries exposed to contamination levels of 9 mg kg^?1 for 30 days, suggesting a threshold concentration of PAHs to stimulate growth and activity of pyrene-degrading bacteria. The contamination level higher than the threshold concentration did not lead to more degradation. The present study provides insights into the natural attenuation of PAH-contaminated mangrove sediments.     

Bacterial Diversity of a Consortium Degrading High-Molecular-Weight Polycyclic Aromatic Hydrocarbons in a Two-Liquid Phase Biosystem

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. Although several PAH-degrading bacterial species have been isolated, it is not expected that a single isolate would exhibit the ability to degrade completely all PAHs. A consortium composed of different microorganisms can better achieve this. Two-liquid phase (TLP) culture systems have been developed to increase the bioavailability of poorly soluble substrates for uptake and biodegradation by microorganisms. By combining a silicone oil–water TLP system with a microbial consortium capable of degrading HMW PAHs, we previously developed a highly efficient PAH-degrading system. In this report, we characterized the bacterial diversity of the consortium with a combination of culture-dependent and culture-independent methods. Polymerase chain reaction (PCR) of part of the 16S ribosomal RNA gene (rDNA) sequences combined with denaturing gradient gel electrophoresis was used to monitor the bacterial population changes during PAH degradation of the consortium when pyrene, chrysene, and benzo[a]pyrene were provided together or separately in the TLP cultures. No substantial changes in bacterial profiles occurred during biodegradation of pyrene and chrysene in these cultures. However, the addition of the low-molecular-weight PAHs phenanthrene or naphthalene in the system favored one bacterial species related to Sphingobium yanoikuyae. Eleven bacterial strains were isolated from the consortium but, interestingly, only one—IAFILS9 affiliated to Novosphingobium pentaromativorans—was capable of growing on pyrene and chrysene as sole source of carbon. A 16S rDNA library was derived from the consortium to identify noncultured bacteria. Among 86 clones screened, 20 were affiliated to different bacterial species–genera. Only three strains were represented in the screened clones. Eighty-five percent of clones and strains were affiliated to Alphaproteobacteria and Betaproteobacteria; among them, several were affiliated to bacterial species known for their PAH degradation activities such as those belonging to the Sphingomonadaceae. Finally, three genes involved in the degradation of aromatic molecules were detected in the consortium and two in IAFILS9. This study provides information on the bacterial composition of a HWM PAH-degrading consortium and its dynamics in a TLP biosystem during PAH degradation.     

Physiological and biochemical responses of rice (Oryza sativa L.) to phenanthrene and pyrene

Phenanthrene (Phe) and pyrene (Pyr) are two typical polycyclic aromatic hydrocarbons (PAHs) found in contaminated soil. This study investigated physiological and biochemical responses of rice (Oryza sativa L.) to PAH stress after they were planted in soils contaminated with Phe and Pyr, in the presence or absence of a PAH-degrading bacteria (Acinetobacteria sp.). A number of parameters including biomass and water, chlorophyll and chlorophyll a/b ratio, electrolyte leakage, activities of superoxide dismutase (SOD) and peroxidase, and soluble carbohydrate and soluble protein contents were monitored. Results show that rice plants have good resistance and tolerance to lower levels of PAHs stress, while adding high levels of PAHs to soils resulted in adverse effects on rice plants such as a reduction in biomass and damage to photosynthetic function. Water content and SOD activities were the most sensitive indicators of PAH stress among the observed parameters. Inoculation with PAH-degrading bacteria promoted growth and photosynthesis of rice.     

Isolation and characterization of pyrene metabolizing microbial consortia from the plant rhizoplane

Most research on the ecology of PAH degrading bacteria in the rhizosphere has focused on individual strains that grow on specific PAHs. Thus, there are fundamental questions as to importance of microbial consortia for PAH degradation in the plant rhizosphere. The study reported here characterized cultivable pyrene degrading rhizoplane microbial communities from two different plant species using a root printing technique on agar plates. Colonies were revealed by formation of clearing zones on medium containing a thin film of pyrene on the surface of a mineral nutrient agar. Prints of the rhizoplane colonies were obtained from roots of Melilotus officinalis (sweet yellow clover) and Andropogon gerardii (big bluestem) plants. Phylogenetic characterizations of selected pyrene degrading colonies were assessed by PCR-DGGE and DNA sequencing. Results showed that different populations of cultivable pyrene degraders were obtained from representative consortia that were examined. Many of the PAH degrading consortia consisted of mixtures of bacterial species that were unable to degrade pyrene by themselves. While this study focused on culturable PAH degraders, the results suggest that pyrene degradation in the rhizosphere commonly involves the activity of bacterial consortia in which various species of bacteria interact to achieve PAH degradation.     

厦门近海海水中多环芳烃降解菌的原位富集与降解菌多样性

摘要：【目的】为了分析厦门近海原位海水中多环芳烃降解菌的多样性。【方法】将涂有菲的聚氯乙烯（PVC）板悬挂在厦门国际邮轮码头的海水中,进行菲降解菌的原位富集。利用变性梯度凝胶电泳（Denaturing gradient gel electrophoresis,DGGE）和16S rRNA基因文库两种方法分析了在PVC板表面富集微生物的菌群结构。之后,在实验室模拟原位条件下,对PVC板表面富集的菲降解菌群进行进一步富集、分离和初步鉴定。【结果】PVC板在海水中浸没6 d后,16S rRNA基因文库分析表明,在涂菲的PVC板表面富集的菌群中解环菌属（Cycloclasticus）对应的克隆子占文库总克隆子的50%;在未涂菲的PVC板表面吸附的菌群中红杆菌科（Rhodobacteraceae）为优势菌,其对应的克隆子占文库总克隆子的47%;而解环菌属的克隆子只占文库总克隆子的2%。DGGE的分析结果也证明解环菌是菲原位富集降解菌群中的优势菌。实验室进一步富集后,从该菌群中分离鉴定出14株细菌,其中一株新鞘氨醇杆菌B14(Novosphingobium sp.B14)具有菲降解能力。但是,解环菌未能获得纯培养。【结论】菲原位富集发现,厦门近海水体中解环菌是多环芳烃的主要降解菌。     

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.     

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas , Halomonas , Marinobacter , Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus , Alteromonas , Thalassospira , Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.     

Polycyclic Aromatic Hydrocarbon-Induced Structural Shift of Bacterial Communities in Mangrove Sediment

Mangrove sediment is well known for its susceptibility to anthropogenic pollution, including polycyclic aromatic hydrocarbons (PAHs), but knowledge of the sediment microbial community structure with regards to exposure to PAHs is limited. The study aims to assess the effects of PAHs on the bacterial community of mangrove sediment using both 16s rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and traditional enrichment methods. Both the exposure time and the PAH concentration reduced the microbial diversity, as determined by the DGGE bands. Although PAHs could act as carbon sources for microorganisms, PAHs, at a concentration as low as 20 mg l⁻¹, posed a toxic effect to the microbial community. Sequencing of DGGE bands showed that marine bacteria from the genera of Vibrio, Roseobacter, and Ferrimonas were most abundant after PAH exposure, which suggests that both marine and terrestrial bacteria coexisted in the mangrove sediment, but that the marine microbes were more difficult to isolate using the traditional culture method. DGGE determination further demonstrated that the consistency among triplicates of the enriched consortia was significantly less than that of the sediment slurries. The present study reveals that the mangrove sediment microbial structure is susceptible to PAH contamination, and complex microbial community interactions occur in mangrove sediment.     

大西洋洋中脊深海多环芳烃降解菌群的优势菌分析

摘要：【目的】为了分析大西洋洋中脊深海海水及表层沉积物中多环芳烃(PAHs)降解菌群中的优势菌。【方法】采用富集培养法和平板涂布法从深海样品中分离可培养细菌及PAHs降解菌。通过16S rRNA基因测序完成系统发育分析。采用变性梯度凝胶电泳（DGGE）及DNA测序分析降解菌群中的优势菌。【结果】总共分离到16株细菌,包括一株PAHs降解菌Novosphingobium sp. 4D。系统发育分析发现,可培养细菌中两个最大的类群分别与Alcanivorax dieselolei NO1A（5/16）和Tistrella mobilis TISTR 1108T（5/16）亲缘关系最近。DGGE结果表明,在菌群MC2D中菌株4L（以及4M、4N, Alcanivorax dieselolei NO1A, 99.21％）、4D（Novosphingobium pentaromativorans US6-1T,97.07％）和4B（以及4E、4H、4K,Tistrella mobilis TISTR 1108T,＞99％）是降解菌群中的优势菌。而降解菌群MC3CO中的优势菌是菌株5C（以及5H,Alcanivorax dieselolei NO1A,＞99％）、条带5-8代表的未培养菌株（Novosphingobium aromaticivorans DSM 12444T,99.41%）、5J（Tistrella mobilis TISTR 1108T,99.52％）和5F（以及5G,Thalassospira lucentensis DSM 14000T,＜97％）。【结论】本研究发现在大西洋洋中脊深海海水及表层沉积物中Alcanivorax、Novosphingobium、Thalassospira、Tistrella属的细菌是PAHs降解菌群中的优势菌,其中的主要降解菌是Novosphingobium属的细菌。     

红树林湿地多环芳烃的微生物降解

红树林(mangrove)是海陆交汇带重要的湿地生态系统,也是环境污染物蓄积与转化的热区.多环芳烃(polycyclic aromatic hydrocarbons,PAHs)因其环境蓄积特点在红树林生境中广泛分布,威胁生态系统健康,其降解转化是近年的研究重点.本文聚焦红树林湿地多环芳烃的微生物降解研究现状,从红树林生...     

Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons

The distributions of endophytic bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. grown in soils contaminated with different levels of polycyclic aromatic hydrocarbons (PAHs) were investigated with polymerase chain reaction followed by denaturing gradient gel electrophoresis technology (PCR-DGGE) and cultivation methods. Twelve types of PAHs, at concentrations varying from 0.16 to 180 mg·kg⁻¹, were observed in the roots and shoots of the two plants. The total PAH concentrations in Alopecurus aequalis Sobol obtained from three different PAH-contaminated stations were 184, 197, and 304 mg·kg⁻¹, and the total PAH concentrations in Oxalis corniculata L. were 251, 346, and 600 mg·kg⁻¹, respectively. The PCR-DGGE results showed that the endophytic bacterial communities in the roots and shoots of the two plants were quite different, although most bacteria belonged to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. A total of 68 endophytic bacterial strains were isolated from different tissues of the two plants and classified into three phyla: Firmicutes, Proteobacteria and Bacteroidetes. In both plants, Bacillus spp. and Pseudomonas spp. were the dominant cultivable populations. With an increase in the PAH pollution level, the diversity and distribution of endophytic bacteria in the two plants changed correspondingly, and the number of cultivable endophytic bacterial strains decreased rapidly. Testing of the isolated endophytic bacteria for tolerance to each type of PAH showed that most isolates could grow well on Luria-Bertani media in the presence of different PAHs, and some isolates were able to grow rapidly on a mineral salt medium with a single PAH as the sole carbon and energy source, indicating that these strains may have the potential to degrade PAHs in plants. This research provides the first insight into the characteristics of endophytic bacterial populations under different PAH pollution levels and provides a species resource for the isolation of PAH-degrading endophytic bacteria.     

Isolation and Characterization of Novel Hydrocarbon-Degrading Euryhaline Consortia from Crude Oil and Mangrove Sediments

Two novel and versatile bacterial consortia were developed for the biodegradation of hydrocarbons. They were isolated from crude oil from the Cormorant Field in the North Sea (MPD-7) and from sediment associated with mangrove roots (MPD-M). The bacterial consortia were able to degrade both aliphatic and aromatic hydrocarbons in crude oils very effectively in seawater (35 g/L NaCl) and synthetic media containing 0 to 100 g/L NaCl (1.7 M). Salinities over twice that of normal seawater decreased the biodegradation rates. However, even at the highest salinity biodegradation was significant. Ratios of nC17 to pristane and nC18 to phytane were significantly lowered across the range of salinity. The lowest values were at 0 and 20 g/L (0.34 M). Phytane was degraded in preference to pristane. The degradation of these compounds was constant over the salinity range, with evidence of a slight increase for consortium MPD-M with increasing salinity. In general, the consortium isolated from mangrove root sediments was more efficient in metabolizing North Sea crude oil than the consortium isolated from Cormorant crude oil. The 5 strains that comprise MPD-M have been tentatively identified as species of the genera Marinobacter, Bacillus, and Erwinia. This is the first report of hydrocarbon-degrading consortia isolated from crude oil and mangrove sediments that are capable of treating oily wastes over such a wide range of salinity. Received June 30, 1999; accepted May 29, 2000.     

Optimization of high-molecular-weight polycyclic aromatic hydrocarbons' degradation in a two-liquid-phase bioreactor

A microbial consortium degrading the high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) pyrene, chrysene, benzo[a]pyrene and perylene in a two-liquid-phase reactor was studied. The highest PAH-degrading activity was observed with silicone oil as the water-immiscible phase; 2,2,4,4,6,8, 8-heptamethylnonane, paraffin oil, hexadecane and corn oil were much less, or not efficient in improving PAH degradation by the consortium. Addition of surfactants (Triton X-100, Witconol SN70, Brij 35 and rhamnolipids) or Inipol EAP22 did not promote PAH biodegradation. Rhamnolipids had an inhibitory effect. Addition of salicylate, benzoate, 1-hydroxy-2-naphtoic acid or catechol did not increase the PAH-degrading activity of the consortium, but the addition of low-molecular-weight (LMW) PAHs such as naphthalene and phenanthrene did. In these conditions, the degradation rates were 27 mg l-1 d-1 for pyrene, 8.9 mg l-1 d-1 for chrysene, 1.8 mg l-1 d-1 for benzo[a]pyrene and 0.37 mg l-1 d-1 for perylene. Micro-organisms from the interface were slightly more effective in degrading PAHs than those from the aqueous phase.     

Initial characterization of new bacteria degrading high-molecular weight polycyclic aromatic hydrocarbons isolated from a 2-year enrichment in a two-liquid-phase culture system

AIMS: To characterize some polycyclic aromatic hydrocarbons (PAH)-degrading microorganisms isolated from an enriched consortium degrading high molecular weight (HMW) PAHs in a two-liquid-phase (TLP) soil slurry bioreactor, and to determine the effect of low molecular weight (LMW) PAH on their growth and HMW PAH-degrading activity. METHODS AND RESULTS: Several microorganisms were isolated from a HMW-PAH (pyrene, chrysene, benzo[a]pyrene and perylene) degrading consortium enriched in TLP cultures using silicone oil as the organic phase. From 16S rRNA analysis, four isolates were identified as Mycobacterium gilvum B1 (99% identity),Bacillus pumilus B44 (99% identity), Microbacterium esteraromaticum B21 (98% identity), and to the genus Porphyrobacter B51 (96% identity). The two latter isolates have not previously been associated with PAH degradation. Isolate B51 grew strongly in the interfacial fraction in the presence of naphthalene vapours and phenanthrene compared with cultures without LMW PAHs. Benzo[a]pyrene was degraded in cultures containing a HMW PAH mixture but pyrene had no effect on its degradation. The growth of isolates B1 and B21 was improved in the aqueous phase than in the interfacial fraction for cultures with naphthalene vapours. Pyrene was required for benzo[a]pyrene degradation by isolate B1. For isolate B21, pyrene and chrysene were degraded only in cultures without naphthalene vapours. CONCLUSION: Consortium enriched in a TLP culture is composed of microorganisms with different abilities to grow at the interface or in the aqueous phase according to the culture conditions and the PAH that are present. Naphthalene vapours increased the growth of the microorganisms in TLP cultures but did not stimulate the HMW PAH degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: New HMW PAH-degrading microorganisms and a better understanding of the mechanisms involved in HMW PAH degradation in TLP cultures.     

Effect of bioaugmentation and supplementary carbon sources on degradation of polycyclic aromatic hydrocarbons by a soil-derived culture

The degradation of polycyclic aromatic hydrocarbons (PAHs) by an undefined culture obtained from a PAH-polluted soil and the same culture bioaugmented with three PAH-degrading strains was studied in carbon-limited chemostat cultures. The PAHs were degraded efficiently by the soil culture and bioaugmentation did not significantly improve the PAH degrading performance. The presence of PAHs did, however, influence the bacterial composition of the bioaugmented and non-bioaugmented soil cultures, resulting in the increase in cell concentration of sphingomonad strains. the initial enhancement of the degradation of the PAHs by biostimulation gradually disappeared and only the presence of salicylate in the additional carbon sources had a lasting slightly stimulating effect on the degradation of phenanthrene. The results suggest that bioaugmentation and biostimulation have limited potential to enhance PAH bioremediation by culture already proficient in the degradation of such contaminants.     

Improved Enrichment and Isolation of Polycyclic Aromatic Hydrocarbons (PAH)-Degrading Microorganisms in Soil Using Anthracene as a Model PAH

Lack of attention to soil and microbial characteristics that influence PAHs degradation has been a leading cause of failures in isolation of efficient PAH degraders and bioaugumentation processes with microbial consortia. This study compared the classic method of isolation of PAHs-degraders with a modified method employing a pre-enrichment respirometric analysis. The modified enrichment of PAH degrading microorganisms using in vitro microcosm resulted to reduced enrichment period and more efficient PAH-degrading microbial consortia. Results indicate that natural soils with strong heterotrophic microbial activity determined through pre-enrichment analysis, are better suited for the isolation of efficient PAH degrading microorganisms with significant reduction of the enrichment period.     

Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria isolated from PAH-contaminated soils were analyzed genotypically and phenotypically for their capacity for metabolism of naphthalene and other PAH substrates. The methods used for the analyses were DNA hybridization using NAH7-derived gene probes, PAH spray plate assays, ¹⁴C-PAH mineralization assays, and dioxygenase activity assays. The results of the analyses showed a dominant number of PAH-degrading bacteria with a NAH7-like genotype. The results support the continued use of the nahA probe for contaminated soils to monitor the genetic potential of indigenous microorganisms to degrade PAHs. However, the finding of non-it nahA-hybridizing PAH-degrading bacteria show the limitation of NAH7-derived gene probes. Fifteen percent (13/89) of PAH-degrading bacteria isolated were not detected with the nahA gene probe. Four isolates (designated A5PH1, A8AN3, B1PH2, and B10AN1) did not hybridize with any of the NAH7-derived gene probes ( nahA, nahG, nahH, and nahR) used in this study. Considering the numerous unculturable microorganisms in nature and their potential genotypes, NAH7-derived gene probes may underestimate the microbial potential to catabolize PAHs. This necessitates development of new gene probes for enumeration and isolation of PAH-degrading bacteria to better understand the in situ microbial potential to degrade PAHs.     

A pyrene-degrading consortium from deep-sea sediment of the West Pacific and its key member Cycloclasticus sp. P1

A pyrene-degrading bacterial consortium was obtained from deep-sea sediments of the Pacific Ocean. The consortium degraded many kinds of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, pyrene, acenaphthene, fluorene, anthracene, fluoranthene, 2-methylnaphthalene and 2,6-dimethylnaphthalene, but it did not grow with chrysene and benzo[alpha]pyrene. With methods of plate cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 72 bacteria belonging to 22 genera were detected from this consortium. Among the detected bacteria, the following genera frequently occurred: Flavobacterium, Cycloclasticus, Novosphingobium, Halomonas, Achromobacter, Roseovarius and Alcanivorax. The first two genera showed the strongest bands in denaturing gradient gel electrophoresis (DGGE) profiles and appeared in all PAH treatments. By now, only one isolate designated P1 was confirmed to be a pyrene degrader. It was identified to be Cycloclasticus spirillensus (100%). Although P1 can degrade pyrene independently, other bacteria, such as Novosphingobium sp. (Band 14), Halomonas sp. (Band 16) and an unidentified bacterium (Band 35), were involved in pyrene degradation in some way; they persist in the consortium in the test of dilution to extinction if only the consortium was motivated with pyrene. However, the secondary most important member Flavobacterium sp. evaded from the community at high dilutions. As a key member of the consortium, P1 distinguished itself by both cell morphology and carbon source range among the isolates of this genus. Based on intermediate analyses of pyrene degradation, P1 was supposed to take an upper pathway different from that previously reported. Together with the results of obtained genes from P1 homology with those responsible for naphthalene degradation, its degradation to pyrene is supposed to adopt another set of genes unique to presently detected. Summarily, an efficient pyrene-degrading consortium was obtained from the Pacific Ocean sediment, in which Cycloclasticus bacterium played a key role. This is the first report to exploit the diversity of pyrene-degrading bacteria in oceanic environments.     

Microbial proteases in peptide synthesis: approaches and applications

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in the environment is often limited due to unfavorable nutrient conditions for the bacteria that use these PAHs as sole source of carbon and energy. Mycobacterium and Sphingomonas are 2 PAH-degrading specialists commonly present in PAH-polluted soil, but not much is known about their specific nutrient requirements. By adding different inorganic supplements of nitrogen (N) and phosphorus (P), affecting the overall carbon/nitrogen/phosphorus ratio of soil in soil slurry degradation tests, we investigated the impact of soil inorganic N and P nutrient conditions on PAH degradation by PAH-degrading Sphingomonas and Mycobacterium strains. The general theoretically calculated C/N/P ratio of 100/10/1 (expressed in moles) allowed rapid PAH metabolization by Sphingomonas and Mycobacterium strains without limitation. In addition, PAH-degradation rate and extent was not affected when ca. ten times lower concentrations of N and P were provided, indicating that Sphingomonas and Mycobacterium strains are capable of metabolizing PAHs under low nutrient conditions. Nor does PAH-degradation seem to be affected by excesses of N and P creating an imbalanced C/N/P ratio. However, supplements of N and P salts increased the salinity of soil slurry solutions and seriously limited or even completely blocked biodegradation.