Biodegradation of phenol and its derivatives by engineered bacteria: current knowledge and perspectives

Biodegradation of phenolic compounds is a promising alternative to physical and chemical methods used to remove these toxic pollutants from the environment. The ability of various microorganisms to metabolize phenol and its derivatives (alkylphenols, nitrophenols and halogenated derivatives) has therefore been intensively studied. Knowledge of the enzymes catalyzing the individual reactions, the genes encoding these enzymes and the regulatory mechanisms involved in the expression of the respective genes in bacteria serves as a basis for the development of more efficient degraders of phenols via genetic engineering methods. Engineered bacteria which efficiently degrade phenolic compounds were constructed in laboratories using various approaches such as cloning the catabolic genes in multicopy plasmids, the introduction of heterologous genes or broadening the substrate range of key enzymes by mutagenesis. Efforts to apply the engineered strains in in situ bioremediation are problematic, since engineered strains often do not compete successfully with indigenous microorganisms. New efficient degraders of phenolic compounds may be obtained by complex approaches at the organism level, such as genome shuffling or adaptive evolution. The application of these engineered bacteria for bioremediation will require even more complex analysis of both the biological characteristics of the degraders and the physico-chemical conditions at the polluted sites.     

Microbial degradation of recalcitrant compounds and synthetic aromatic polymers

The evolution of microbial catabolic enzymes cannot keep pace with the rapid introduction of novel compounds into the environment. These new synthetic compounds that are slowly biodegradable or non-biodegradable are known as recalcitrant compounds, and range from simple halogenated hydrocarbons to complex polymers. Recalcitrant compounds can be made biodegradable by developing microorganisms capable of degrading the compound and by treating the compound to make it more conducive to mirobial attack. Many factors contribute to recalcitrance. The organism may lack the necessary genetic information. The organism can acquire this information by plasmid transfer or de novo enzyme synthesis. Plasmids have been characterized that degrade or transform antibiotics, pesticides, and hydrocarbons. By the use of chemostat techniques or chemical mutagens, organisms have been shown to synthesize de novo enzymes. The compound may be too large to enter the cell, or a transport system may not exist to transport it across the membrane. The compound may be insoluble, either as a solid or a liquid, and the microorganism may lack the proper nutrients. Recalcitrant compounds can be oxygenated prior to degradation, in the presence of a readily assimilable carbon source. In the absence of the assimilable carbon source, the recalcitrant compound is not degraded, or only very slowly. Examples of such co-oxidative metabolism are alkane and lignin degradation. Polymers, particularly synthetic ones, are prime examples of difficult-to-degrade compounds. The initial rate of polymer degradation follows a Freundlich or modified Langmuir isotherm rather than Michaelis-Menten kinetics. Microorganisms can irreversibly bind to solid surfaces by various methods. Soil microorganisms have been found to degrade styrene monomers and dimers. Polystyrene has been shown to be biodegradable by ¹⁴CO₂ evolution but at a very slow rate. In car tyres, styrene as a copolymer of butadiene is co-metabolized in the presence of other assimilable carbon sources.     

The Desulfitobacterium genus

Desulfitobacterium spp. are strictly anaerobic bacteria that were first isolated from environments contaminated by halogenated organic compounds. They are very versatile microorganisms that can use a wide variety of electron acceptors, such as nitrate, sulfite, metals, humic acids, and man-made or naturally occurring halogenated organic compounds. Most of the Desulfitobacterium strains can dehalogenate halogenated organic compounds by mechanisms of reductive dehalogenation, although the substrate spectrum of halogenated organic compounds varies substantially from one strain to another, even with strains belonging to the same species. A number of reductive dehalogenases and their corresponding gene loci have been isolated from these strains. Some of these loci are flanked by transposition sequences, suggesting that they can be transmitted by horizontal transfer via a catabolic transposon. Desulfitobacterium spp. can use H2 as electron donor below the threshold concentration that would allow sulfate reduction and methanogenesis. Furthermore, there is some evidence that syntrophic relationships occur between Desulfitobacterium spp. and sulfate-reducing bacteria, from which the Desulfitobacterium cells acquire their electrons by interspecies hydrogen transfer, and it is believed that this relationship also occurs in a methanogenic consortium. Because of their versatility, desulfitobacteria can be excellent candidates for the development of anaerobic bioremediation processes. The release of the complete genome of Desulfitobacterium hafniense strain Y51 and information from the partial genome sequence of D. hafniense strain DCB-2 will certainly help in predicting how desulfitobacteria interact with their environments and other microorganisms, and the mechanisms of actions related to reductive dehalogenation.     

Selective enrichment of Pseudomonas spp. defective in catabolism after exposure to halogenated substrates.

Significant selective enrichments of mutants defective in catabolic pathways can be achieved by exposure of pseudomonad cells to halogenated analogs of growth substrates. Between 3 and 95% of viable clones rescued from such enrichments have been defective in specific catabolic pathways. This has been demonstrated for eight different catabolic pathways for aromatic compounds in pseudomonads, in which the genes are located on plasmids or on the chromosome. The plasmid-encoded pathways studied include those for the catabolism of p-cymene (CYM), m- and p-xylenes (TOL), naphthalene (NAH), salicylate (SAL), and 4-methylphthalate (MOP), and the chromosome-encoded pathways include those for p-hydroxybenzoate, monohydric phenols, and p-anisate utilization. The recalcitrance of halogenated compounds may, in part, be explained by these observations, which introduce an as yet not widely recognized factor in assessment of biodegradability of halogenated compounds and their effects on the transformation of the natural substrates.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis.

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Microbial degradation of chlorinated phenols

The chlorinated phenols comprise a large group of toxic, man-made chemicals that are serious environmental pollutants. Microorganisms can degrade many, but not all, of the chlorinated phenols, often using chlorophenol-specific catabolic enzymes. Novel technologies are evolving for using specific microorganisms to clean contaminated soils and waters of chlorophenols.     

Isolation and characterization of indigenous thermophilic bacteria active in natural attenuation of bio-hazardous petrochemical pollutants

Common petrochemical compounds, such as homocyclic polyaromatic hydrocarbons and heterocyclic NOS-polyaromatics (NOS-compounds), were used as the sole carbon and energy source to enrich indigenous bacteria harboring the catabolic ability to degrade these compounds from petroleum-contaminated soils from Kuwait. Chemical analysis of the extracted soil materials revealed residual amounts of oil (<5% w/w), presumably of heavy oil fractions with elevated S-content. Aerobic culturable mesophilic polyaromatic hydrocarbon- and NOS-degraders were abundant in these soils, whereas their moderately thermophilic counterparts constituted only a minor fraction. Glucose stimulated the growth of mesophiles and drastically suppressed the number of thermophiles. 16S rDNA was amplified by PCR from nine of the purified thermophilic strains, using primers specific for eubacteria. Sequencing of 900 bp of the 16S rDNA and database homology search tentatively aligned these isolates to low G+C Gram positive bacteria of the family Bacillaceae. Electron microscopy characterization revealed endospore-forming bacilli varying in size, with well-structured cell walls. Gas chromatography and mass spectrometry (GC/MS) analysis revealed a versatile catabolic ability of the pure and mixed cultures to degrade all tested compounds. The metabolism of the offered substrates does not involve co-metabolism, since all pure cultures consumed the offered substrates completely.     

Isolation and characterization of indigenous thermophilic bacteria active in natural attenuation of bio-hazardous petrochemical pollutants

Common petrochemical compounds, such as homocyclic polyaromatic hydrocarbons and heterocyclic NOS-polyaromatics (NOS-compounds), were used as the sole carbon and energy source to enrich indigenous bacteria harboring the catabolic ability to degrade these compounds from petroleum-contaminated soils from Kuwait. Chemical analysis of the extracted soil materials revealed residual amounts of oil (<5% w/w), presumably of heavy oil fractions with elevated S-content. Aerobic culturable mesophilic polyaromatic hydrocarbon- and NOS-degraders were abundant in these soils, whereas their moderately thermophilic counterparts constituted only a minor fraction. Glucose stimulated the growth of mesophiles and drastically suppressed the number of thermophiles. 16S rDNA was amplified by PCR from nine of the purified thermophilic strains, using primers specific for eubacteria. Sequencing of 900 bp of the 16S rDNA and database homology search tentatively aligned these isolates to low G+C Gram positive bacteria of the family Bacillaceae. Electron microscopy characterization revealed endospore-forming bacilli varying in size, with well-structured cell walls. Gas chromatography and mass spectrometry (GC/MS) analysis revealed a versatile catabolic ability of the pure and mixed cultures to degrade all tested compounds. The metabolism of the offered substrates does not involve co-metabolism, since all pure cultures consumed the offered substrates completely.     

Biodegradation of neutralized sarin.

This research investigated the biotransformation of IMPA, the neutralization product of the nerve agent Sarin, by a microbial consortia. As mandated by the Chemical Weapons Convention signed by 132 countries in 1993, all chemical warfare agents are to be destroyed within ten years of ratification. Technologies must be developed to satisfy this commitment. This paper presents data from a biodegradation kinetics study and background information on the biological transformation of IMPA. Microbial transformation of organophosphate nerve agents and organophosphate pesticide intermediates can be incorporated into a treatment process for the fast and efficient destruction of these similar compounds. Sarin (isopropyl methylphosphonofluoridate), also known as GB, is one of several highly neurotoxic chemical warfare agents that have been developed over the past 50 to 60 years. Four mixed cultures were acclimated to the Sarin hydrolysis product, isopropyl methylphosphonic acid (IMPA). Two of these cultures, APG microorganisms and SX microorganisms, used IMPA as the sole phosphorus source. Extended exposure to IMPA improved the cultures' abilities to degrade IMPA to form methylphosphonic acid (MPA) and inorganic phosphate. The presence of free phosphate in the reactor suppressed the degradation of IMPA. IMPA did not inhibit either cultural consortia within the tested concentration range (0 to 1250 mg/L). The numax was 120.9 mg/L/day for the SX microorganisms and 118.3 mg/L/day for the APG microorganisms. Initial IMPA concentrations of 85 to 90 mg/L were degraded to nondetectable levels within 75 h. These results demonstrate the potential for biodegradation to serve as a complementary treatment process for the destruction of stockpiled Sarin.     

Biodegradation and biotransformation of organofluorine compounds

The carbon–fluorine bond is one of the strongest in nature, and the increasing use of organofluorine compounds in agriculture, human and veterinary medicine, and industry has raised concerns about their fate in the environment. Microorganisms can degrade organofluorine compounds, either via specific enzymatic hydrolysis of the C–F bond, or through transformation by catabolic enzymes with broad substrate specificities. Here our current understanding of organofluorine catabolism in microorganisms is summarised.     

Biodegradation and Rhodococcus--masters of catabolic versatility

The genus Rhodococcus is a very diverse group of bacteria that possesses the ability to degrade a large number of organic compounds, including some of the most difficult compounds with regard to recalcitrance and toxicity. They achieve this through their capacity to acquire a remarkable range of diverse catabolic genes and their robust cellular physiology. Rhodococcus appear to have adopted a strategy of hyper-recombination associated with a large genome. Notably, they harbour large linear plasmids that contribute to their catabolic diversity by acting as 'mass storage' for a large number of catabolic genes. In addition, there is increasing evidence that multiple pathways and gene homologues are present that further increase the catabolic versatility and efficiency of Rhodococcus.     

Microbial reductive dehalogenation.

A wide variety of compounds can be biodegraded via reductive removal of halogen substituents. This process can degrade toxic pollutants, some of which are not known to be biodegraded by any other means. Reductive dehalogenation of aromatic compounds has been found primarily in undefined, syntrophic anaerobic communities. We discuss ecological and physiological principles which appear to be important in these communities and evaluate how widely applicable these principles are. Anaerobic communities that catalyze reductive dehalogenation appear to differ in many respects. A large number of pure cultures which catalyze reductive dehalogenation of aliphatic compounds are known, in contrast to only a few organisms which catalyze reductive dehalogenation of aromatic compounds. Desulfomonile tiedjei DCB-1 is an anaerobe which dehalogenates aromatic compounds and is physiologically and morphologically unusual in a number of respects, including the ability to exploit reductive dehalogenation for energy metabolism. When possible, we use D. tiedjei as a model to understand dehalogenating organisms in the above-mentioned undefined systems. Aerobes use reductive dehalogenation for substrates which are resistant to known mechanisms of oxidative attack. Reductive dehalogenation, especially of aliphatic compounds, has recently been found in cell-free systems. These systems give us an insight into how and why microorganisms catalyze this activity. In some cases transition metal complexes serve as catalysts, whereas in other cases, particularly with aromatic substrates, the catalysts appear to be enzymes.     

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichlorethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms.     

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10.

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichlorethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms.     

Epoxy coenzyme a thioester pathways for degradation of aromatic compounds

Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.     

Bacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities

Bacterial dehalogenases catalyse the cleavage of carbon-halogen bonds, which is a key step in aerobic mineralization pathways of many halogenated compounds that occur as environmental pollutants. There is a broad range of dehalogenases, which can be classified in different protein superfamilies and have fundamentally different catalytic mechanisms. Identical dehalogenases have repeatedly been detected in organisms that were isolated at different geographical locations, indicating that only a restricted number of sequences are used for a certain dehalogenation reaction in organohalogen-utilizing organisms. At the same time, massive random sequencing of environmental DNA, and microbial genome sequencing projects have shown that there is a large diversity of dehalogenase sequences that is not employed by known catabolic pathways. The corresponding proteins may have novel functions and selectivities that could be valuable for biotransformations in the future. Apparently, traditional enrichment and metagenome approaches explore different segments of sequence space. This is also observed with alkane hydroxylases, a category of proteins that can be detected on basis of conserved sequence motifs and for which a large number of sequences has been found in isolated bacterial cultures and genomic databases. It is likely that ongoing genetic adaptation, with the recruitment of silent sequences into functional catabolic routes and evolution of substrate range by mutations in structural genes, will further enhance the catabolic potential of bacteria toward synthetic organohalogens and ultimately contribute to cleansing the environment of these toxic and recalcitrant chemicals.     

细菌吸附及转运芳香族化合物的研究进展

芳香族化合物是一类具有苯环结构的有机物,它们结构稳定,不易分解,并可通过食物链进行生物富集和生物放大,对生态环境及人类健康造成极大危害。细菌具有超强的分解代谢能力,能降解多环芳烃(polycyclic aromatic hydrocarbons, PAHs)等多种难降解芳香族污染物。吸附和转运是细菌进行芳香族化合物细胞内代谢的前提。虽然芳香族化合物的细菌降解已取得较为显著的研究进展,但吸附和转运机理仍不甚清楚。本文讨论了细菌对芳香族化合物的吸附有积极作用的细胞表面疏水性、生物被膜形成和细菌趋化性等影响因素,总结了FadL家族、TonB依赖性受体蛋白、OmpW家族等外膜转运系统和主要协同转运蛋白超家族(major facilitator superfamily, MFS)转运体、ATP结合盒(ATP-binding cassette, ABC)转运蛋白等内膜转运系统对该类化合物跨膜运输作用,并对跨膜转运机制进行了讨论和阐述,旨在为芳香族污染物的防控和治理提供一定理论参考。     

Microbial degradation of organophosphorus compounds

Synthetic organophosphorus compounds are used as pesticides, plasticizers, air fuel ingredients and chemical warfare agents. Organophosphorus compounds are the most widely used insecticides, accounting for an estimated 34% of world-wide insecticide sales. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field or accidental release may lead occasionally to contamination of surface and ground water. Several reports suggest that a wide range of water and terrestrial ecosystems may be contaminated with organophosphorus compounds. These compounds possess high mammalian toxicity and it is therefore essential to remove them from the environments. In addition, about 200,000 metric tons of nerve (chemical warfare) agents have to be destroyed world-wide under Chemical Weapons Convention (1993). Bioremediation can offer an efficient and cheap option for decontamination of polluted ecosystems and destruction of nerve agents. The first micro-organism that could degrade organophosphorus compounds was isolated in 1973 and identified as Flavobacterium sp. Since then several bacterial and a few fungal species have been isolated which can degrade a wide range of organophosphorus compounds in liquid cultures and soil systems. The biochemistry of organophosphorus compound degradation by most of the bacteria seems to be identical, in which a structurally similar enzyme called organophosphate hydrolase or phosphotriesterase catalyzes the first step of the degradation. organophosphate hydrolase encoding gene opd (organophosphate degrading) gene has been isolated from geographically different regions and taxonomically different species. This gene has been sequenced, cloned in different organisms, and altered for better activity and stability. Recently, genes with similar function but different sequences have also been isolated and characterized. Engineered microorganisms have been tested for their ability to degrade different organophosphorus pollutants, including nerve agents. In this article, we review and propose pathways for degradation of some organophosphorus compounds by microorganisms. Isolation, characterization, utilization and manipulation of the major detoxifying enzymes and the molecular basis of degradation are discussed. The major achievements and technological advancements towards bioremediation of organophosphorus compounds, limitations of available technologies and future challenge are also discussed.