Biodegradation of soluble aromatic compounds of jet fuel under anaerobic conditions: laboratory batch experiments

Laboratory batch experiments were performed with contaminated aquifer sediments and four soluble aromatic components of jet fuel to assess their biodegradation under anaerobic conditions. The biodegradation of four aromatic compounds, toluene, o-xylene, 1,2,4-trimethylbenzene (TMB), and naphthalene, separately or together, was investigated under strictly anaerobic conditions in the dark for a period of 160 days. Of the aromatic compounds, toluene and o-xylene were degraded both as a single substrate and in a mixture with the other aromatic compounds, while TMB was not biodegraded as a single substrate, but was biodegraded in the presence of the other aromatic hydrocarbons. Substrate interaction is thus significant in the biodegradation of TMB. Biodegradation of naphthalene was not observed, either as a single substrate or in a mixture of other aromatic hydrocarbons. Although redox conditions were dominated by iron reduction, a clear relation between degradation and sulfate reduction was observed. Methanogenesis took place during the later stages of incubation. However, the large background of Fe(II) masked the increase of Fe(II) concentration due to iron reduction. Thus, although microbial reduction of Fe(III) is an important process, the evidence is not conclusive. Our results have shown that a better understanding of the degradation of complex mixtures of hydrocarbons under anaerobic conditions is important in the application of natural attenuation as a remedial method for soil and groundwater contamination.     

Biodegradation of xenobiotics by anaerobic bacteria

Xenobiotic biodegradation under anaerobic conditions such as in groundwater, sediment, landfill, sludge digesters and bioreactors has gained increasing attention over the last two decades. This review gives a broad overview of our current understanding of and recent advances in anaerobic biodegradation of five selected groups of xenobiotic compounds (petroleum hydrocarbons and fuel additives, nitroaromatic compounds and explosives, chlorinated aliphatic and aromatic compounds, pesticides, and surfactants). Significant advances have been made toward the isolation of bacterial cultures, elucidation of biochemical mechanisms, and laboratory and field scale applications for xenobiotic removal. For certain highly chlorinated hydrocarbons (e.g., tetrachlorethylene), anaerobic processes cannot be easily substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic processes are generally used, anaerobic biodegradation is significant under certain circumstances (e.g., O₂-depleted aquifers, oil spilled in marshes). For persistent compounds including polychlorinated biphenyls, dioxins, and DDT, anaerobic processes are slow for remedial application, but can be a significant long-term avenue for natural attenuation. In some cases, a sequential anaerobic-aerobic strategy is needed for total destruction of xenobiotic compounds. Several points for future research are also presented in this review.     

Degradation of BTEX by anaerobic bacteria: physiology and application

Pollution of the environment with aromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX) is often observed. The cleanup of these toxic compounds has gained much attention in the last decades. In situ bioremediation of aromatic hydrocarbons contaminated soils and groundwater by naturally occurring microorganisms or microorganisms that are introduced is possible. Anaerobic bioremediation is an attractive technology as these compounds are often present in the anoxic zones of the environment. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene and the bacteria involved in anaerobic benzene degradation. Here, we review the existing knowledge on the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria, in particular the physiology and application, including results on the (per)chlorate stimulated degradation of these compounds, which is an interesting new alternative option for bioremediation.     

Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review

Bioremediation of aromatic hydrocarbons in groundwater and sediments is often limited by dissolved oxygen. Many aromatic hydrocarbons degrade very slowly or not at all under anaerobic conditions. Nitrate is a good alternative electron acceptor to oxygen, and denitrifying bacteria are commonly found in the subsurface and in association with contaminated aquifer materials. Providing both nitrate and microaerophilic levels of oxygen may result in oxidation of the stable benzene rings in aromatic contaminants and allow for the intermediates of this oxidation to degrade via denitrification. The effects of using mixed electron acceptors on biodegradation of subsurface contaminants is unclear. Below some critical oxygen threshold, aerobic biodegradation is inhibited, however high levels of oxygen inhibit denitrification. The mechanisms which regulate electron transfer to oxygen and nitrate are complex. This review: 1) describes the factors which may affect the utilization of oxygen and nitrate as dual electron acceptors during biodegradation; 2) summarizes the incidence of dual use of nitrate and oxygen (aerobic denitrification); and 3) presents evidence of the effectiveness of bioremediation under mixed oxygen/nitrate conditions. Received 08 November 1995/ Accepted in revised form 09 June 1996     

Biodegradation of central intermediate compounds produced from biodegradation of aromatic compounds

In this study I consider the incomplete biodegradation of aromatic compounds during the wastewater cycle between aerobic or anaerobic zones in biological nutrient removal processes, including aerobic biodegradation of compounds (such as cyclohex-1-ene-1-carboxyl-CoA) produced during the incomplete anaerobic biodegradation of aromatic compounds, and anaerobic biodegradation of compounds (such as catechol, protocatechuate, and gentisic acid) produced during the incomplete aerobic biodegradation of aromatic compounds. Anaerobic degradation of the aerobic central intermediates that result from the incomplete aerobic degradation of aromatic compounds usually leads to benzoyl-CoA. On the other hand, aerobic degradation of the anaerobic central intermediates that result from the incomplete anaerobic degradation of aromatic compounds usually leads to protocatechuate.     

Anaerobic biodegradation of natural gas condensate can be stimulated by the addition of gasoline

Biodegradation of a broad range of linear and branched alkanes, parent and alkyl alicyclic hydrocarbons, and benzene and alkyl-substituted benzenes was observed when sediment and groundwater samples collected from a gas condensate-contaminated aquifer were incubated under methanogenic and especially under sulfate-reducing conditions, even though no exogenous nitrogen or phosphorus was added. This finding expands the range of hydrocarbon molecules known to undergo anaerobic decay and confirms that natural attenuation is an important process at this site. The addition of 1 μl of gasoline to the samples (∼10 ppm) had minimal impact on the biodegradation of saturated compounds, but substantially increased the diversity and extent of aromatic compounds undergoing transformation. We attribute this to the promotion or induction of biodegradation pathways in the indigenous microflora following the addition of the gasoline components. The promoting compounds are not precisely known, but may have been present in the initial condensate and reduced in concentration by various mechanisms (dissolution, biodegradation, etc.) such that their concentration in the aquifer fell below necessary levels. A variety of aromatic hydrocarbons would appear to be likely candidates.     

Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches

Biodegradation can achieve complete and cost-effective elimination of aromatic pollutants through harnessing diverse microbial metabolic processes. Aromatics biodegradation plays an important role in environmental cleanup and has been extensively studied since the inception of biodegradation. These studies, however, are diverse and scattered; there is an imperative need to consolidate, summarize, and review the current status of aromatics biodegradation. The first part of this review briefly discusses the catabolic mechanisms and describes the current status of aromatics biodegradation. Emphasis is placed on monocyclic, polycyclic, and chlorinated aromatic hydrocarbons because they are the most prevalent aromatic contaminants in the environment. Among monocyclic aromatic hydrocarbons, benzene, toluene, ethylbenzene, and xylene; phenylacetic acid; and structurally related aromatic compounds are highlighted. In addition, biofilms and their applications in biodegradation of aromatic compounds are briefly discussed. In recent years, various biomolecular approaches have been applied to design and understand microorganisms for enhanced biodegradation. In the second part of this review, biomolecular approaches, their applications in aromatics biodegradation, and associated biosafety issues are discussed. Particular attention is given to the applications of metabolic engineering, protein engineering, and “omics” technologies in aromatics biodegradation.     

微生物降解石油烃的功能基因研究进展

微生物对石油烃的降解在自然衰减去除土壤和地下水石油烃污染的过程中发挥了重要作用。微生物通过其产生的一系列酶来利用和降解这类有机污染物,其中,编码关键降解酶的基因称为功能基因。功能基因可作为生物标志物用于分析环境中石油烃降解基因的多样性。因此,研究石油降解功能基因是分析土著微生物群落多样性、评价自然衰减潜力与构建基因工程菌的重要基础。本文主要介绍了烷烃和芳香烃在有氧和无氧条件下的微生物降解途径,重点总结了烷烃和芳香烃降解的主要功能基因及其作用,包括参与羟化作用的单加氧酶和双加氧酶基因、延胡索酸加成反应的琥珀酸合酶基因以及中心中间产物的降解酶基因等。     

Biodegradation of Aliphatic and Aromatic Hydrocarbons by Nocardia sp. H17-1

We investigated the biodegradation of hydrocarbon components by Nocardia sp. H17-1 and the catabolic genes involved in the degradation pathways of both aliphatic and aromatic hydrocarbons. After 6 days of incubation, the aliphatic and aromatic fractions separated from Arabian light oil were degraded 99.0 ± 0.1% and 23.8 ± 0.8%, respectively. Detection of the catabolic genes involved in the hydrocarbon degradation indicated that H17-1 possessed the alkB genes for n-alkane biodegradation and catA gene for catechol 1,2-dioxygenase. However, H17-1 had neither the C23O gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity. The investigation of the genes involved in the biodegradation of hydrocarbons supported the low degradation activity of H17-1 on the aromatic fractions.     

Stable isotope probing identifies anthracene degraders under methanogenic conditions

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in groundwater. The remediation of PAH-contaminated groundwater often involves anaerobic biodegradation. The knowledge about the microorganisms responsible for PAH degradation in anaerobic subsurface environment is still lacking. DNA-based stable isotope probing (SIP) was applied to discover the microorganisms responsible for anaerobic anthracene degradation within microcosms inoculated with aquifer sediment from landfill leachate-contaminated site. Three phylotypes were identified as the degraders, all falling within the phylum Proteobacteria. Two anthracene degraders were classified within the genera Methylibium and Legionella, while another one was an unclassified Rhizobiales species. They all were first linked to PAH degradation. These findings also provide an illustration of the utility of SIP to discover the roles of uncultured microorganisms in PAH-degrading processes.     

多环芳烃厌氧生物降解研究进展

多环芳烃（PAHs）是环境中广泛分布的一类持久性有机污染物,对生态环境和公众健康具有极大危害。微生物降解是环境中去除多环芳烃污染的有效途径,近年来PAHs厌氧生物降解研究逐渐取代好氧降解成为人们关注的重点。本文从PAHs厌氧生物降解的研究背景出发,从不同厌氧还原反应体系、厌氧降解微生物、PAHs厌氧生物转化途径等方面阐述了PAHs厌氧生物降解的研究概况,归纳了对PAHs厌氧生物降解有积极作用的影响因素,提出了PAHs厌氧降解研究目前存在的问题,并对该领域未来研究方向作了简述和展望。希望为多环芳烃厌氧生物降解与环境修复研究与实践提供参考。     

Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotype

The anaerobic biodegradation of benzene, a common constituent of petroleum and one of the least reactive aromatic hydrocarbons, is insufficiently understood with respect to the involved microorganisms and their metabolism. To study these aspects, sulfate-reducing bacteria were enriched with benzene as sole organic substrate using marine sediment as inoculum. Repeated subcultivation yielded a sediment-free enrichment culture constituted of mostly oval-shaped cells and showing benzene-dependent sulfate reduction and growth under strictly anoxic conditions. Amplification and sequencing of 16S rRNA genes from progressively diluted culture samples revealed an abundant phylotype; this was closely related to a clade of Deltaproteobacteria that includes sulfate-reducing bacteria able to degrade naphthalene or other aromatic hydrocarbons. Cell hybridization with two specifically designed 16S rRNA-targeted fluorescent oligonucleotide probes showed that the retrieved phylotype accounted for more than 85% of the cells detectable via DAPI staining (general cell staining) in the enrichment culture. The result suggests that the detected dominant phylotype is the 'candidate species' responsible for the anaerobic degradation of benzene. Quantitative growth experiments revealed complete oxidation of benzene with stoichiometric coupling to the reduction of sulfate to sulfide. Suspensions of benzene-grown cells did not show metabolic activity towards phenol or toluene. This observation suggests that benzene degradation by the enriched sulfate-reducing bacteria does not proceed via anaerobic hydroxylation (mediated through dehydrogenation) to free phenol or methylation to toluene, respectively, which are formerly proposed alternative mechanisms for benzene activation.     

Enhanced anaerobic bioremediation of groundwater contaminated by fuel hydrocarbons at Seal Beach, California

Enhanced anaerobic biodegradation of groundwater contaminated by fuel hydrocarbons has been evaluated at a field experiment conducted at the Naval Weapons Station, Seal Beach, California. This experiment included the establishment of three different remediation zones in situ: one zone was augmented with sulfate, one was augmented with sulfate and nitrate, and the third was unaugmented. This enables a comparison of hydrocarbon biodegradation under sulfate-reducing, sequential denitrifying/sulfate-reducing, and methanogenic conditions, respectively. In general, the results from the field experiment are: (1) Certain fuel hydrocarbons were removed preferentially over others, but the order of preference is dependent upon the geochemical conditions; and (2) In the zones that were augmented with sulfate and/or nitrate, the added electron acceptors were consumed quickly, indicating that enhancement via electron acceptor injection accelerates the biodegradation process. More specifically, in the sulfate-reducing zone, sulfate was utilized with an apparent first-order rate coefficient of approximately 0.1 day^-1. In the combined denitrifying/sulfate-reducing zone, nitrate was utilized preferentially over sulfate, with an apparent first-order rate coefficient of 0.1–0.6 day^-1. However, the data suggest that slow sulfate utilization does occur in the presence of nitrate, i.e., the two processes are not strictly sequential. With regard to the aromatic BTEX hydrocarbons, toluene was preferentially removed under intrinsic conditions; biodegradation of benzene was slow if it occurred at all; augmentation with sulfate preferentially stimulated biodegradation of o-xylene; and ethylbenzene appeared recalcitrant under sulfate-reducing conditions but readily degradable under denitrifying conditions.     

Biodegradation and bioremediation of hydrocarbons in extreme environments

Many hydrocarbon-contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, high salt concentrations, or high pressure, Hydrocarbon-degrading microorganisms, adapted to grow and thrive in these environments, play an important role in the biological treatment of polluted extreme habitats. The biodegradation (transformation or mineralization) of a wide range of hydrocarbons, including aliphatic, aromatic, halogenated and nitrated compounds, has been shown to occur in various extreme habitats. The biodegradation of many components of petroleum hydrocarbons has been reported in a variety of terrestrial and marine cold ecosystems. Cold-adapted hydrocarbon degraders are also useful for wastewater treatment. The use of thermophiles for biodegradation of hydrocarbons with low water solubility is of interest, as solubility and thus bioavailability, are enhanced at elevated temperatures. Thermophiles, predominantly bacilli, possess a substantial potential for the degradation of environmental pollutants, including all major classes. Indigenous thermophilic hydrocarbon degraders are of special significance for the bioremediation of oil-polluted desert soil. Some studies have investigated composting as a bioremediation process. Hydrocarbon biodegradation in the presence of high salt concentrations is of interest for the bioremediation of oil-polluted salt marshes and industrial wastewaters, contaminated with aromatic hydrocarbons or with chlorinated hydrocarbons. Our knowledge of the biodegradation potential of acidophilic, alkaliphilic, or barophilic microorganisms is limited.     

Composition of the microbial communities of bituminous constructions at natural oil seeps at the bottom of Lake Baikal

Microbial communities of two bituminous constructions at the bottom of Lake Baikal in the region of natural oil seeps at a depth of 900 m have been investigated. Construction 8 contained biodegraded hydrocarbons, and construction 3, through which oil seeped, contained material that experienced biodegradation to a lesser degree. The composition of the microbial communities was studied by means of pyrosequencing of 16S rRNA gene fragments. Most of the bacterial 16S rRNA gene sequences identified in both bituminous constructions were attributed to proteobacteria, along with which Actinobacteria, Acidobacteria, Bacteroidetes, and TM7 were revealed. About 40% of the bacterial sequences in bituminous construction 3 belonged to representatives of uncultured groups within the classes Alphaproteobacteria and Betaproteobacteria and the phylum Bacteroidetes. The 16S rRNA gene sequences of archaea belonged to aceticlastic and hydrogenotrophic methanogens of the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales. The 16S rRNA genes of various groups of bacteria carrying out aerobic biodegradation of aromatic compounds and n-alkanes were found; their compositions differed between the constructions. Neither known groups of denitrifying betaproteobacteria nor known groups of sulfate-reducing deltaproteobacteria capable of carrying out anaerobic degradation of n-alkanes were found, which agrees with the low content of nitrate and sulfate in the water. In the anaerobic zone of bituminous constructions, the processes of biodegradation of hydrocarbons are probably carried out in the absence of alternative electron acceptors by the syntrophic community, including deltaproteobacteria of the genus Syntrophus and methanogenic archaea.     

Influence of adhesion on aerobic biodegradation and bioremediation of liquid hydrocarbons

Biodegradation of poorly water-soluble liquid hydrocarbons is often limited by low availability of the substrate to microbes. Adhesion of microorganisms to an oil–water interface can enhance this availability, whereas detaching cells from the interface can reduce the rate of biodegradation. The capability of microbes to adhere to the interface is not limited to hydrocarbon degraders, nor is it the only mechanism to enable rapid uptake of hydrocarbons, but it represents a common strategy. This review of the literature indicates that microbial adhesion can benefit growth on and biodegradation of very poorly water-soluble hydrocarbons such as n-alkanes and large polycyclic aromatic hydrocarbons dissolved in a non-aqueous phase. Adhesion is particularly important when the hydrocarbons are not emulsified, giving limited interfacial area between the two liquid phases. When mixed communities are involved in biodegradation, the ability of cells to adhere to the interface can enable selective growth and enhance bioremediation with time. The critical challenge in understanding the relationship between growth rate and biodegradation rate for adherent bacteria is to accurately measure and observe the population that resides at the interface of the hydrocarbon phase.     

Biodegradation of polycyclic aromatic hydrocarbons

The intent of this review is to provide an outline of the microbial degradation of polycyclic aromatic hydrocarbons. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons are examined in some detail. The pathways of polycyclic aromatic hydrocarbon catabolism are discussed. Studies are presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.     

Enhanced Biodegradation of Casablanca Crude Oil by A Microbial Consortium in Presence of a Rhamnolipid Produced by Pseudomonas Aeruginosa AT10

The biodegradation of oil products in the environment is often limited by their low water solubility and dissolution rate. Rhamnolipids produced by Pseudomonas aeruginosa AT10 were investigated for their potential to enhance bioavailability and hence the biodegradation of crude oil by a microbial consortium in liquid medium. The characterization of the rhamnolipids produced by strain AT10 showed the effectiveness of emulsification of complex mixtures. The addition of rhamnolipids accelerates the biodegradation of total petroleum hydrocarbons from 32% to 61% at 10 days of incubation. Nevertheless, the enhancement of biosurfactant addition was more noticeable in the case of the group of isoprenoids from the aliphatic fraction and the alkylated polycyclic aromatic hydrocarbons (PHAS) from the aromatic fraction. The biodegradation of some targeted isoprenoids increased from 16% to 70% and for some alkylated PAHs from 9% to 44%.     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.