Bacteria-mediated PAH degradation in soil and sediment

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the natural environment and easily accumulate in soil and sediment due to their low solubility and high hydrophobicity, rendering them less available for biological degradation. However, microbial degradation is a promising mechanism which is responsible for the ecological recovery of PAH-contaminated soil and sediment for removing these recalcitrant compounds compared with chemical degradation of PAHs. The goal of this review is to provide an outline of the current knowledge of biodegradation of PAHs in related aspects. Over 102 publications related to PAH biodegradation in soil and sediment are compiled, discussed, and analyzed. This review aims to discuss PAH degradation under various redox potential conditions, the factors affecting the biodegradation rates, degrading bacteria, the relevant genes in molecular monitoring methods, and some recent-year bioremediation field studies. The comprehensive understanding of the bioremediation kinetics and molecular means will be helpful for optimizing and monitoring the process, and overcoming its limitations in practical projects.     

Effects of humic substances and soya lecithin on the aerobic bioremediation of a soil historically contaminated by polycyclic aromatic hydrocarbons (PAHs)

The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) strongly reduces their bioavailability in aged contaminated soils, thus limiting their bioremediation. The biodegradation of PAHs in soils can be enhanced by employing surface-active agents. However, chemical surfactants are often recalcitrant and exert toxic effects in the amended soils. The effects of two biogenic materials as pollutant-mobilizing agents on the aerobic bioremediation of an aged-contaminated soil were investigated here. A soil historically contaminated by about 13 g kg(-1) of a large variety of PAHs, was amended with soya lecithin (SL) or humic substances (HS) at 1.5% w/w and incubated in aerobic solid-phase and slurry-phase reactors for 150 days. A slow and only partial biodegradation of low-molecular weight PAHs, along with a moderate depletion of the initial soil ecotoxicity, was observed in the control reactors. The overall removal of PAHs in the presence of SL or HS was faster and more extensive and accompanied by a larger soil detoxification, especially under slurry-phase conditions. The SL and HS could be metabolized by soil aerobic microorganisms and enhanced the occurrence of both soil PAHs and indigenous aerobic PAH-degrading bacteria in the reactor water phase. These results indicate that SL and HS are biodegradable and efficiently enhance PAH bioavailability in soil. These natural surfactants significantly intensified the aerobic bioremediation of a historically PAH-contaminated soil under treatment conditions similar to those commonly employed in large-scale soil bioremediation.     

Bioremediation of polyaromatic hydrocarbon contaminated soils by native microflora and bioaugmentation with Sphingobium chlorophenolicum strain C3R: A feasibility study in solid- and slurry-phase microcosms

The aim of the research was to verify if a Sphingobium chlorophenolicum strain C3R was effective in the degradation of phenanthrene (Ph) in agricultural soil co-contaminated by metals and mixtures of PAHs. The presence of PAHs in mixtures produced interactive effects which could either increase or decrease the utilization rate of Ph by C3R and by the native bacterial microflora. Bioaugmentation significantly improved the biodegradation rate of Ph in the presence of both cadmium and arsenic and PAH mixtures. The augmented C3R strain persisted in inoculated microcosms as monitored by the DGGE analysis and outcompeted some indigenous bacteria. The potential role of the soil bacteria in PAH degradation could be envisaged. The results indicate the applicability of S. chlorophenolicum C3R toward in situ bioremediation of sites contaminated with phenanthrene alone or co-contaminated with low molecular weight PAHs and with cadmium and arsenate.     

微生物降解多环芳烃(PAHs)的研究进展

从多环芳烃(PAHs)的降解菌株的筛选、降解机制以及PAHs污染的生物修复等方面介绍了微生物降解PAHs的最新研究进展。     

土壤中高环多环芳烃微生物降解的研究进展

微生物修复是去除土壤中多环芳烃(PAHs)的主要措施。本文以微生物修复PAHs污染土壤的理论基础及其难点为主线,全面综述了土壤中高环PAHs的微生物降解机理。近年来,富集分离得到的以高环PAHs为唯一碳源和能源的优势降解菌逐渐增多,其中,主要是代谢降解四环PAHs的单株降解菌,一些降解菌还能以共代谢方式利用五环PAHs。高环PAHs污染土壤修复的一个难点是其低生物可利用性,微生物通过释放生物表面活性剂、形成生物膜以及分泌胞外多糖提高高环PAHs的生物可利用性,从而加速其降解。真菌和细菌联合作用能增强污染土壤实地修复的效果。因此,通过微生物修复技术来去除土壤中PAHs具有环境友好性、经济适用性以及可持续应用性。     

微生物VBNC状态形成及复苏机制

99%以上的微生物因处于活的但非可培养(viable but non-culturable,VBNC)状态而无法分离培养。复苏促进因子(resuscitation-promoting factors,Rpfs)是培养获取VBNC菌的最重要突破。结合课题组近十余年从环境功能视角利用Rpf复苏培养VBNC菌的研究,本文在阐述微生物VBNC状态的形成及复苏进展的基础上,从VBNC菌形成及复苏过程出发,探究"探索因子"与群体感应的内在关系。并总结了课题组利用Rpf所复苏培养的具有潜在环境功能的VBNC菌种。本论文将为揭示微生物VBNC状态的形成及复苏机制提供新的思路,并为认识和重新评价Rpf法复苏培养VBNC菌在污染环境微生物修复中的作用提供理论依据。     

Culture‐dependent and culture‐independent characterization of potentially functional biphenyl‐degrading bacterial community in response to extracellular organic matter from Micrococcus luteus

Biphenyl (BP)‐degrading bacteria were identified to degrade various polychlorinated BP (PCB) congers in long‐term PCB‐contaminated sites. Exploring BP‐degrading capability of potentially useful bacteria was performed for enhancing PCB bioremediation. In the present study, the bacterial composition of the PCB‐contaminated sediment sample was first investigated. Then extracellular organic matter (EOM) from Micrococcus luteus was used to enhance BP biodegradation. The effect of the EOM on the composition of bacterial community was investigated by combining with culture‐dependent and culture‐independent methods. The obtained results indicate that Proteobacteria and Actinobacteria were predominant community in the PCB‐contaminated sediment. EOM from M. luteus could stimulate the activity of some potentially difficult‐to‐culture BP degraders, which contribute to significant enhancement of BP biodegradation. The potentially difficult‐to‐culture bacteria in response to EOM addition were mainly Rhodococcus and Pseudomonas belonging to Gammaproteobacteria and Actinobacteria respectively. This study provides new insights into exploration of functional difficult‐to‐culture bacteria with EOM addition and points out broader BP/PCB degrading, which could be employed for enhancing PCB‐bioremediation processes.     

Diversity and PAH growth abilities of bacterial strains isolated from a contaminated soil in Slovakia

Different abandoned industrial areas contaminated by polycyclic aromatic hydrocarbons (PAHs) are present in Slovakia. These environmental burdens are very dangerous to the health of human and environment. The bioremediation, based on the use of hydrocarbons degrading microorganisms, is a promising strategy to sanitize these polluted sites. The aim of this investigation was to assess the bacterial diversity of a PAHs-contaminated soil and to select the potential hydrocarbonoclastic bacteria which can be used for different bioremediation approaches. The bacterial strains were isolated on minimal medium agar supplemented with a mixture of PAHs. Seventy-three isolated strains were grouped by ribosomal interspacer analysis in 15 different clusters and representatives of each cluster were identified by 16S rRNA sequencing. The PAHs degradation abilities of all bacterial isolates were estimated by the 2,6-dichlorophenol indophenol assay and by their growth on minimal broth amended with a mixture of PAHs. Different kinds of strains, members of the genus Pseudomonas, Enterobacter, Bacillus, Arthrobacter, Acinetobacter and Sphingomonas, were isolated from the contaminated soil. Four isolates (Pseudomonas putida, Arthrobacter oxydans, Sphingomonas sp. and S. paucimobilis) showed promising PAHs-degrading abilities and therefore their possible employing in bioremediation strategies.     

氧化胁迫诱导苍白杆菌JP1形成VBNC状态及其特征

石油降解菌在各种有害环境因素作用下会进入活的非可培养(viable but non-culturable, VBNC)状态，从而影响其生长及石油降解率。为了研究有害环境因素对石油降解菌生长及石油降解率的影响，采用分光光度法、荧光染色-激光共聚焦显微镜观测H₂O₂胁迫下苍白杆菌(Ochrobactrum sp.)JP1细胞的生长及VBNC状态形成情况。结果表明，不同浓度H₂O₂对其生长有一定抑制作用，当培养液中H₂O₂浓度为75.0 mmol/L时，可有效抑制苍白杆菌JP1生长，处理12 h后苍白杆菌JP1进入VBNC状态。VBNC状态的苍白杆菌JP1细胞缩小变成球体，周质间隙增大；在适宜条件下，VBNC状态苍白杆菌JP1能够复苏为可培养状态，添加丙酮酸钠能够促进VBNC状态细菌细胞的复苏。复苏后的苍白杆菌RJP1具有良好的环境适应性和石油降解能力，为石油污染生物修复的菌种筛选及应用提供了新的策略。     

Search of Microorganisms that Degrade PAHs under Alkaline Conditions

Bacterial strains were enriched from building rubble contaminated with polycyclic aromatic hydrocarbons (PAHs). These strains were studied as an inoculum in bioremediation processes with contaminated building rubble. The selection criteria for the bacteria were broad profiles in PAH degradation, stable expression of the traits and tolerance to alkaline conditions. Various strains of Micrococcus sp., Dietzia sp., Rhodococcus sp. and Pseudomonas sp. met the selection criteria. In general, degradative activity was limited at higher pH values. Strains of Micrococcus were suitable for practical use as complete degradation of various PAHs was observed at pH values exceeding 10. Strains of Dietzia sp. showed broad PAH degradation profile, but in some cases degradation came to a halt leaving some of the PAHs unutilized. With Dietzia sp. this could be due to inhibitory effects from the accumulation of toxic PAH metabolic products and/or growth‐limiting media conditions.     

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.     

Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia

Aims: To characterize polycyclic aromatic hydrocarbon (PAH)‐degrading bacteria from sediments of the Bizerte lagoon, and to determine their ability to resist other pollutants such as antibiotics and heavy metals. Methods and Results: More than 100 strains were isolated for their ability to use fluoranthene as the sole carbon and energy source. Most of them showed antibiotic and heavy metal resistance; 20 representative strains were selected for further analysis. 16S rRNA coding sequences analysis showed that the majority of the selected bacteria (75%) were affiliated to the Gammaproteobacteria. The selected strains also utilized high molecular weight PAHs containing up to four benzene rings and showed different profiles of PAH substrate usage suggesting different PAH degradation pathways. These results are consistent with the fact that nah‐like genes and idoA‐like genes, involved in PAH degradation, were detected in 6 and 1 strains respectively. Conclusions: The Bizerte lagoon, polluted by many human activities, leads to the co‐selection of strains able to cope with multiple contaminants. Significance and Impact of the Study: Polluted areas are often characterized by the concomitant presence of organic pollutants, heavy metals and antibiotics. This study is one of the first showing bacterial strains adapted to multiple contaminants, a promising potential for the development of bioremediation processes.     

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.     

Nutrient-limited biodegradation of PAH in various soil strata at a creosote contaminated site

The effects of nutrient addition on the in situ biodegradation of polycyclic aromatic hydrocarbons in creosote contaminated soil were studied in soil columns taken from various soil strata at a wood preserving plant in Norway. Three samples were used: one from the topsoil (0–0.5 m), one from an organic rich layer (2–2.5 m) and one from the sandy aquifer (4.5–5 m). The addition of inorganic nitrogen and phosphorous stimulated the degradation of polycyclic aromatic hydrocarbons (PAHs) in the top soil and the aquifer sand. These two soils, which differed strongly in contamination levels, responded similarly to nutrient addition with the corresponding degradation of 4-ring PAHs. The ratio between available nitrogen (N) and phosphorous (P) might explain the degree of degradation observed for the 4-ring PAHs. However, the degree of degradation of 3-ring PAHs did not significantly increase after nutrient addition. An increase in the respiration rate, after nutrient addition, could only be observed in the topsoil. In the aquifer sand, 4-ring PAH degradation was not accompanied by an increase in the respiration rate or the number of heterotrophic micro-organisms. PAH degradation in the organic layer did not respond to nutrient addition. This was probably due to the low availability of the contaminants for micro-organisms, as a result of sorption to the soil organic matter. Our data illustrate the need for a better understanding of the role of nutrients in the degradation of high molecular weight hydrocarbons for the successful application of bioremediation at PAH contaminated sites.     

Exploring the potential environmental functions of viable but non-culturable bacteria

A conventional plate count is the most commonly employed method to estimate the number of living bacteria in environmental samples. In fact, judging the level of viable culture by plate count is limited, because it is often several orders of magnitude less than the number of living bacteria actually present. Most of the bacteria are in “viable but non-culturable” (VBNC) state, whose cells are intact and alive and can resuscitate when surrounding conditions are more favorable. The most exciting recent development in resuscitating VBNC bacteria is a bacterial cytokine, namely, the resuscitation-promoting factor (Rpf), secreted by Micrococcus luteus, which promotes the resuscitation and growth of high G+C Gram-positive organisms, including some species of the genus Mycobacterium. However, most of studies deal with VBNC bacteria only from the point of view of medicine and epidemiology. It is therefore of great significance to research whether these VBNC state bacteria also possess some useful environmental capabilities, such as degradation, flocculation, etc. Further studies are needed to elucidate the possible environmental role of the VBNC bacteria, rather than only considering their role as potential pathogens from the point view of epidemiology and public health. We have studied the resuscitation of these VBNC bacteria in polluted environments by adding culture supernatant containing Rpf from M. luteus, and it was found that, as a huge microbial resource, VBNC bacteria could provide important answers to dealing with existing problems of environmental pollution. This mini-review will provide new insight for considering the potentially environmental functions of VBNC bacteria.     

Microbial Factors Rather Than Bioavailability Limit the Rate and Extent of PAH Biodegradation in Aged Crude Oil Contaminated Model Soils

The rate and extent of polynuclear aromatic hydrocarbons (PAH) biodegradation in a set of aged model soils that had been contaminated with crude oil at the high concentrations (i.e.,>20,000?mg/kg) normally found in the environment were measured in 90-week slurry bioremediation experiments. Soil properties such as organic matter content, mineral type, particle diameter, surface area, and porosity did not significantly influence the PAH biodegradation kinetics among the 10 different model soils. A comparison of aged and freshly spiked soils indicates that aging affects the biodegradation rate and extent only for higher-molecular-weight PAHs, while the effects of aging are insignificant for 4-ring PAHs and total PAHs. In all model soils with the exception of kaolinite clay, the rate of abiotic desorption was faster than the rate of biodegradation during the initial phase of bioremediation treatment, indicating that PAH biodegradation was limited by microbial factors. Similarly, any of the higher-molecular-weight PAHs that were still present after 90 weeks of treatment were released rapidly during abiotic desorption tests, which demonstrates that bioavailability limitations were not responsible for the recalcitrance of these hydrocarbons. Indeed, an analysis of microbial counts indicates that a severe reduction in hydrocarbon degrader populations may be responsible for the observed incomplete PAH biodegradation. Therefore, it can be concluded that the recalcitrance of PAHs during bioremediation is not necessarily due to bioavailability limitations and that these residual contaminants therefore might pose a greater risk to environmental receptors than previously thought.     

Activity and viability of polycyclic aromatic hydrocarbon‐degrading Sphingomonas sp. LB126 in a DC‐electrical field typical for electrobioremediation measures

There has been growing interest in employing electro‐bioremediation, a hybrid technology of bioremediation and electrokinetics for the treatment of contaminated soil. Knowledge however on the effect of weak electrokinetic conditions on the activity and viability of pollutant‐degrading microorganisms is scarce. Here we present data about the influence of direct current (DC) on the membrane integrity, adenosine triphosphate (ATP) pools, physico‐chemical cell surface properties, degradation kinetics and culturability of fluorene‐degrading Sphingomonas sp. LB126. Flow cytometry was applied to quantify the uptake of propidium iodide (PI) and the membrane potential‐related fluorescence intensities (MPRFI) of individual cells within a population. Adenosine tri‐phosphate contents and fluorene biodegradation rates of bulk cultures were determined and expressed on a per cell basis. The cells' surface hydrophobicity and electric charge were assessed by contact angle and zeta potential measurements respectively. Relative to the control, DC‐exposed cells exhibited up to 60% elevated intracellular ATP levels and yet remained unaffected on all other levels of cellular integrity and functionality tested. Our data suggest that direct current (X = 1 V cm^?1; J = 10.2 mA cm^?2) as typically used for electrobioremediation measures has no negative effect on the activity of the polycyclic aromatic hydrocarbon (PAH)‐degrading soil microorganism, thereby filling a serious gap of the current knowledge of the electrobioremediation methodology.     

Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments

Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.     

Biodegrader metabolic expansion during polyaromatic hydrocarbons rhizoremediation

Root-microbe interactions are considered to be the primary process of polyaromatic hydrocarbon (PAH) phytoremediation, since bacterial degradation has been shown to be the dominant pathway for environmental PAH dissipation. However, the precise mechanisms driving PAH rhizostimulation symbiosis remain largely unresolved. In this study, we assessed PAH degrading bacterial abundance in contaminated soils planted with 18 different native Michigan plant species. Phenanthrene metabolism assays suggested that each plant species differentially influenced the relative abundance of PAH biodegraders, though they generally were observed to increase heterotrophic and biodegradative cell numbers relative to unplanted soils. Further study of >1800 phenanthrene degrading isolates indicated that most of the tested plant species stimulated biodegradation of a broader range of PAH compounds relative to the unplanted soil bacterial consortia. These observations suggest that a principal contribution of planted systems for PAH bioremediation may be via expanded metabolic range of the rhizosphere bacterial community.