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.     

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.     

Identification of anthracene degraders in leachate-contaminated aquifer using stable isotope probing

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in landfill leachate-contaminated aquifer. It is necessary to identify the microorganisms truly responsible for PAH degradation if bioremediation can be applied as an effective technology. DNA-based stable isotope probing (SIP) in combination with terminal restriction fragment length polymorphism (TRFLP) was used to identify the active anthracene degraders in the contaminated aquifer sediment. One kind of degrader was classified as Variovorax species within class ??-proteobacteria, but another belonged to unclassified bacteria. These findings also suggest novel microorganisms involved in PAH-degrading processes.     

Biotreatability of polycyclic aromatic hydrocarbons in brackish sediments: Preliminary studies of an integrated monitoring

An integrated monitoring, of chemical, microbiological and ecotoxicological parameters, was performed for a biotreatability study of polycyclic aromatic hydrocarbons (PAHs)—contaminated brackish sediments. Three slurry reactors were prepared, consisting of (a) a slurry with sediment and seawater called TQ slurry, to evaluate the intrinsic bioremediation potential, (b) a slurry with the addition of a selected microbial consotrium called BIO slurry, to evaluate the bioaugmentation effect, (c) a slurry with the addition of Soya lecithin called LEC slurry, to evaluate the effect of the addition of a natural surfactant. Biodegradation results showed that both BIO and LEC slurries enhanced PAHs removal, increasing the biodegradation rate for 5- and 6-ring PAHs. Furthermore, ecotoxicological response (Microtox® assay on whole sediment, aqueous extract and organic extract) demonstrated a detoxification of the PAHs initial mixture only for BIO slurry. The findings that aerobic PAHs degradation can be stimulated via inoculation with adapted sediment bacteria suggest that a bioaugmentation process may be a useful strategy for ex-situ treatment.     

植物法生物修复PAHs和矿物油污染土壤的调控研究

选择苜蓿草为供试植物,以污染物含量水平、专性细菌和真菌及有机肥为调控因子,进行了植物法生物修复多环芳烃(PAHs)和矿物油污染土壤的调控研究。结果表明,PAHs和矿物油的降解率与有机肥含量呈正相关,增加有机肥5%,可提高矿物油降解率17.6%～25.6%,PAHs降解率9%.在植物存在条件下,土壤微生物降解功能增强。多环芳烃总量的平均降解率比无植物对照土壤提高2.0%～4.7%.投加特性降解真菌可不同程度地提高土壤PAHs总量和矿物油的降解率。真菌对萤蒽、芘和苯(a)蒽/(艹屈)的降解有明显促进作用。而细菌能明显提高苊稀/芴、蒽和苯(a)萤蒽/苯(k)萤蒽的降解率。     

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.     

Tenax TA extraction to understand the rate-limiting factors in methyl-β-cyclodextrin-enhanced bioremediation of PAH-contaminated soil

The effectiveness of many bioremediation systems for PAH-contaminated soil may be constrained by low contaminant bioaccessibility due to limited aqueous solubility or large sorption capacity. Information on the extent to which PAHs can be readily biodegraded is of vital importance in the decision whether or not to remediate a contaminated soil. In the present study the rate-limiting factors in methyl-β-cyclodextrin (MCD)-enhanced bioremediation of PAH-contaminated soil were evaluated. MCD amendment at 10 % (w/w) combined with inoculation with the PAH-degrading bacterium Paracoccus sp. strain HPD-2 produced maximum removal of total PAHs of up to 35 %. The desorption of PAHs from contaminated soil was determined before and after 32 weeks of bioremediation. 10 % (w/w) MCD amendment (M2) increased the Tenax extraction of total PAHs from 12 to 30 % and promoted degradation by up to 26 % compared to 6 % in the control. However, the percentage of Tenax extraction for total PAHs was much larger than that of degradation. Thus, in the control and M2 treatment it is likely that during the initial phase the bioaccessibility of PAHs is high and biodegradation rates may be limited by microbial processes. On the other hand, when the soil was inoculated with the PAH-degrading bacterium (CKB and MB2), the slowly and very slowly desorbing fractions (F _sl and F _vl ) became larger and the rate constants of slow and very slow desorption (k _sl and k _vl ) became extremely small after bioremediation, suggesting that desorption is likely rate limiting during the second, slow phase of biotransformation. These results have practical implications for site risk assessment and cleanup strategies.     

细菌降解多环芳烃上游途径的遗传学研究进展

多环芳烃是一类毒性较大的环境污染物。微生物降解和转化是消除此类污染物的理想方法,已发现多种细菌具有这种功能。主要针对细菌在多环芳烃降解中上游途径的代谢酶及基因簇的组成进行综述,阐述了酶的遗传学特点,并探讨了PAHs代谢基因的进化。这有助于了解PAHs的细菌降解机制,并为有效实施生物修复提供理论依据。     

Polycyclic aromatic hydrocarbons: environmental pollution and bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. Although various physicochemical methods have been used to remove these compounds from our environment, they have many limitations. Xenobiotic-degrading microorganisms have tremendous potential for bioremediation but new modifications are required to make such microorganisms effective and efficient in removing these compounds, which were once thought to be recalcitrant. Metabolic engineering might help to improve the efficiency of degradation of toxic compounds by microorganisms. However, efficiency of naturally occurring microorganisms for field bioremediation could be significantly improved by optimizing certain factors such as bioavailability, adsorption and mass transfer. Chemotaxis could also have an important role in enhancing biodegradation of pollutants. Here, we discuss the problems of PAH pollution and PAH degradation, and relevant bioremediation efforts.     

Microbial biodegradation of polyaromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are widespread in various ecosystems and are pollutants of great concern due to their potential toxicity, mutagenicity and carcinogenicity. Because of their hydrophobic nature, most PAHs bind to particulates in soil and sediments, rendering them less available for biological uptake. Microbial degradation represents the major mechanism responsible for the ecological recovery of PAH-contaminated sites. The goal of this review is to provide an outline of the current knowledge of microbial PAH catabolism. In the past decade, the genetic regulation of the pathway involved in naphthalene degradation by different gram-negative and gram-positive bacteria was studied in great detail. Based on both genomic and proteomic data, a deeper understanding of some high-molecular-weight PAH degradation pathways in bacteria was provided. The ability of nonligninolytic and ligninolytic fungi to transform or metabolize PAH pollutants has received considerable attention, and the biochemical principles underlying the degradation of PAHs were examined. In addition, this review summarizes the information known about the biochemical processes that determine the fate of the individual components of PAH mixtures in polluted ecosystems. A deeper understanding of the microorganism-mediated mechanisms of catalysis of PAHs will facilitate the development of new methods to enhance the bioremediation of PAH-contaminated sites.     

Influence of growth medium on cometabolic degradation of polycyclic aromatic hydrocarbons by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. strain PheB4

The influence of growth medium on cometabolic degradation of polycyclic aromatic hydrocarbons (PAHs) was investigated when Sphingomonas sp. strain PheB4 isolated from surface mangrove sediments was grown in either phenanthrene-containing mineral salts medium (PMSM) or nutrient broth (NB). The NB-grown culture exhibited a more rapid cometabolic degradation of single and mixed non-growth substrate PAHs compared to the PMSM-grown culture. The concentrations of PAH metabolites were also lower in NB-grown culture than in PMSM-grown culture, suggesting that NB-grown culture removed metabolites at a faster rate, particularly, for metabolites produced from cometabolic degradation of a binary mixture of PAHs. Cometabolic pathways of single PAH (anthracene, fluorene, or fluoranthene) in NB-grown culture showed similarity to that in PMSM-grown culture. However, cometabolic pathways of mixed PAHs were more diverse in NB-grown culture than that in PMSM-grown culture. These results indicated that nutrient rich medium was effective in enhancing cometabolic degradation of mixed PAHs concomitant with a rapid removal of metabolites, which could be useful for the bioremediation of mixed PAHs contaminated sites using Sphingomonas sp. strain PheB4.     

Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

Enhancing the intrinsic bioremediation of PAH-contaminated anoxic estuarine sediments with biostimulating agents

Estuarine sediments are frequently polluted with hydrocarbons from fuel spills and industrial wastes. Polycyclic aromatic hydrocarbons (PAHs) are components of these contaminants that tend to accumulate in the sediment due to their low aqueous solubility, low volatility, and high affinity for particulate matter. The toxic, recalcitrant, mutagenic, and carcinogenic nature of these compounds may require aggressive treatment to remediate polluted sites effectively. In petroleum-contaminated sediments near a petrochemical industry in Gwangyang Bay, Korea, in situ PAH concentrations ranged from 10 to 2,900 microg/kg dry sediment. To enhance the biodegradation rate of PAHs under anaerobic conditions, sediment samples were amended with biostimulating agents alone or in combination: nitrogen and phosphorus in the form of slow-release fertilizer (SRF), lactate, yeast extract (YE), and Tween 80. When added to the sediment individually, all tested agents enhanced the degradation of PAHs, including naphthalene, acenaphthene, anthracene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene. Moreover, the combination of SRF, Tween 80, and lactate increased the PAH degradation rate 1.2-8.2 times above that of untreated sediment (0.01-10 microg PAH/kg dry sediment/day). Our results indicated that in situ contaminant PAHs in anoxic sediment, including high molecular weight PAHs, were degraded biologically and that the addition of stimulators increased the biodegradation potential of the intrinsic microbial populations. Our results will contribute to the development of new strategies for in situ treatment of PAH-contaminated anoxic sediments.     

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.     

3株多环芳烃高效降解菌株的分离鉴定及降解特性

筛选分离降解多环芳烃(PAHs)的优势菌种对开展多环芳烃污染生态系统修复具有重要的现实意义。本研究以焦化厂周围受多环芳烃污染的土壤为菌源,经过富集培养驯化和平板分离,获得11株能降解多环芳烃的菌株。通过形态观察、生理生化特征及16S rRNA序列比对对菌株进行鉴定,筛选出3株PAHs高效降解菌,分别命名为DJ-3、DJ-8、DJ-10。经16S rRNA序列分析鉴定,DJ-3为假单胞菌属、DJ-8为克雷伯氏菌属、DJ-10为芽孢杆菌属。对菌株降解能力的研究表明,3株菌(DJ-3、DJ-8、DJ-10)培养7 d后对混合多环芳烃中菲(200 mg·L^-1)、芘(200 mg·L^-1)和萘(160 mg·L^-1)的降解率分别为48.9%～65.9%、38.9%～43.1%和57.6%～64.9%。3株菌对多环芳烃混合样品(1200 mg·L^-1)的降解率分别为49.1%、44.5%、53.9%,远高于其他8株筛选菌,为PAHs高效降解菌株。3种菌株两两之间和三者组合均无拮抗关系。研究结果将为构建高效的多环芳烃降解菌群、提高多环芳烃原位污染土壤的生物修复效果奠定基础。     

多环芳烃降解菌的筛选、鉴定及降解特性

【目的】多环芳烃(PAHs)是一类普遍存在于环境中且具有高毒性的持久性有机污染物,高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。研究拟从供试菌株中筛选多环芳烃高效降解菌,并分析其降解特性,为多环芳烃污染环境的微生物修复提供资源保障和科学依据。【方法】采用平板法从25株供试菌株中筛选出以菲和芘为唯一碳源和能源的高效降解菌,经16S rRNA基因序列进行初步鉴定,通过单因素实验法分析其在液体培养基中的降解特性。【结果】筛选出的3株多环芳烃高效降解菌SL-1、02173和02830经16S rRNA基因序列分析,02173和02830分别与假单胞菌属中的Pseudomonas alcaliphila和Pseudomonas corrugate同源性最近,SL-1为本课题组发表新类群Rhizobium petrolearium的模式菌株;降解实验表明,菌株SL-1 3 d内对单一多环芳烃菲(100 mg/L)和芘(50 mg/L)的降解率分别达到100%和48%,5 d后能够降解74%的芘;而其3 d内对混合PAHs中菲和芘的降解率分别为75.89%和81.98%。菌株02173和02830 3 d内对混合多环芳烃中萘(200 mg/L)、芴(50 mg/L)、菲(100 mg/L)和芘(50 mg/L)的降解率均分别超过97%。【结论】筛选出的3株PAHs降解菌SL-1、02173和02830不仅可以高效降解低分子量PAHs,还对高分子量PAHs具有很好的降解潜力。研究表明,由于共代谢作用低分子量多环芳烃可促进高分子量多环芳烃的降解,而此时低分子量多环芳烃的降解将受到抑制。     

Revealing potential functions of VBNC bacteria in polycyclic aromatic hydrocarbons biodegradation

The bioremediation of polycyclic aromatic hydrocarbon (PAH)‐contaminated sites is not running smoothly, because of the lower activity of PAH‐degrading bacteria in actual bioremediation applications. The phenomenon of “viable but nonculturable” (VBNC) state may be a main limiting factor for their poor biodegradation capabilities of PAHs. Due to their abilities of entering into the VBNC state, most of bacterial populations with PAH‐degradation potential remain unculturable. Resuscitation of VBNC bacteria will enhance the degradation capability of indigenous bacteria which will eventually obtain their better capabilities in environmental bioremediation. Although evidences have been presented indicating that resuscitation of VBNC bacteria in polychlorinated biphenyl (PCB)‐contaminated environments not only significantly enhanced PCB degradation, but also obtained novel highly efficient PCB‐degrading bacteria, scanty information is available on the VBNC bacteria in PAH‐contaminated sites. VBNC bacteria, as a vast majority of potential microbial resource could be the repository of novel highly efficient PAH‐biodegraders. Therefore, studies need to be done on resuscitation of VBNC bacteria to overcome key bottlenecks in bioremediation of PAH‐contaminated sites. This mini‐review provides a new insight into the potential functions of VBNC bacteria in PAHs biodegradation.

Significance and Impact of the Study

As the vast majority microbial resource, viable but nonculturable (VBNC) bacteria, which showed their potential functions in polycyclic aromatic hydrocarbons (PAHs) biodegradation, can be of great significance in environmental bioremediation. It is therefore important to resuscitate VBNC bacteria for their better capabilities. Meanwhile, preventing the indigenous functional community from entering into the VBNC state will also maintain the high activity of PAH‐degrading bacteria in actual bioremediation applications. Undoubtedly, much more work needs to be done to reveal indigenous micro‐organisms in the VBNC state from the perspective of environmental functions.     

细菌降解低分子量多环芳烃的研究进展

具有"三致"效应的多环芳烃污染造成了巨大的环境危害,威胁人类健康和生存。目前能够降解低分子量多环芳烃的细菌已有广泛的研究。细菌通过多层次的调控分析和适应性进化提高它们的降解能力。本文基于国内外文献调研,简要总结了生物修复在低分子量多环芳烃降解领域的研究进展。拟通过多层次的调控分析和适应性进化来产生多种分解代谢途径,为生物降解能力强化的未来降解技术提供支撑。     

红树林厌氧环境对多环芳烃类有毒物的降解预测

红树林是连接陆地和海洋的重要生态系统,由于潮汐活动,氧化还原条件表现出明显的昼夜间的交替,这一生态体系中不但有大量的动植物种类,同时还有数量极高的不同种类的细菌,包括好氧和厌氧类型,厌养的硫酸(盐)还原菌已证实在降解多环芳烃有机物方面有其独特的生化优势,但从红树林中分离出的此类纯细菌还很少,在降解方面,已初步确定萘的厌氧降解途径异于好氧细菌,厌氧降解时的一系列代谢中间产物也有明显的专一性,羰基化反应是开始的一个重要步骤,而后的每步生化反应还有待进一步验证。从现有的结果可以看出,红树林中厌养的硫酸还原菌应在降解多环芳烃有机物中起到非常重要的作用。