Biodegradation of chlorophenols: a review

Wu Gaofeng , Xu Hong, Jiang Mei
(Beijing University of Chemical Technology, BeiJing,100029; GuangZhou Chemical Technology College , 510730, China)

Abstract The biodegradation of chlorophenols has been reviewed in this paper, including the chlorophenols-degrading microbes, factors for degradabilities and usage of biotechnology, with special emphasis on degradation mechanisms. Dechlorination is the first critical step in the bacteria degradation of many chloroniated pollutants. The mechanism of biodegradation depends on the conditions. Under the aerobic condition, degradation of mono- and dichlorophenols was shown to be initiated by oxygenation into chlorocatechols, and dechlorination occurred only after ring cleavage of the chlorocatechols, while degradation of polychlorinated phenols started with hydrolytic para-hydroxylation, yielding chlorinated para-hydroquinone. Anaerobic biodegradation of chlorophenols occurs by reductive dechlorination, a process by which the chlorines were replaced with hydrogen.
Keywords Chlorophenols, Aerobic degradation, Anaerobic degradation, Biodegradability and Biotechnology.

1. INTRODUCTION
Chlorinated phenols (CPs) are widely used as intermediate products in synthesis, and polychlorinated phenols previously were frequently applied as biocides, fungicides, mainly as wood preservatives. Chlorophenols are also formed as by-products when chlorine is used for bleaching of pulp and for disinfection of drinking water. Therefore, large amounts of chlorinated aromatic compounds, including chlorophenols(CPs), have been discharged into the environment ^[1]. Hundreds of sites in the U.S. have been contaminated with pentachlorophenol(PCP) and many of these sites are on the national priority list for cleaning under the super funded program^[2]. Consideration of the current technology for removal of pollutants, it clearly shows that physical and chemical methods are often uneconomical. Biological treatment may be relatively low-cost, safe and the most promising technologies to this problem and bioremediation has become an accepted technology for restoration of contaminated environments^[3].
    It has been found for long time that microorganisms such as bacteria, fungi, actinomycete, protozoa, and others have performed the function of recycling organic matter from which new plant life can grow. This natural process can be emulated, perhaps even accelerated, and used for detoxification. Microorganisms present in the environment can eventually transform most of the toxic organics, so the subject of biodegradation has been treated critically. Indigenous microbial populations, especially heterotrophic bacteria and fungi, are the chief agents causing biodegradation. Microbes that transform specific compounds can be isolated, cultured, adapted, and enriched under laboratory conditions.
    This paper will review the study and progresses on chlorophenols biodegradation and future research for the development of bioremediation of CPs contaminated environments.

2. THE DEGRADATION MECHANISMS OF CHLORINATED PHENOLS
2.1 Degrading microbes for chlorinated phenols
The focus on the biodegradation of the CPs in recent years has resulted in the isolation of a number of microorganisms that can grow on the compounds as a sole carbon and energy source. Table 1 lists the typical microbes that can degrade the CPs.

Table 1 Degrading microbes for chlorinated phenols

    As indicated above, Pseudomonas spp. can dechlorinate various CPs in suspension cultures. In fact, Pseudomonas spp. also can degrade many other aromatic compounds, such as other chlorinated aromatic compounds, nitrified aromatic compounds, aminophenols, and polycyclic aromatic hydrocarbon^[4,5]. Desulfomonile tiedjei, a strictly anaerobic Gram-negative sulfate-reducing bacterium, is the best-described dechlorinating anaerobic bacterium to date[6]. The basidiomycete Phanerochaete chrysosporium that commonly is known as a white rot fungus, can mineralize a variety of hazardous organic chemicals including chlorinated phenols.
2.2 The degradation mechanisms of CPs
Dechlorination is the first critical step in the bacteria degradation of many chlorinated pollutants. Krooneman J. et al. believed the presence or absence of molecular oxygen plays a crucial role in determining the fate and biodegradation mechanisms of aromatic compounds^[7]. In general, under aerobic conditions, the CPs are transformed via oxidative dechlorination, while in anaerobic ambience via reductive dechlorination.
2.2.1 Oxidative dechlorination
For the aerobic biodegradation of CPs, two different mechanisms have so far been described. Degradation of mono- and dichlorophenols was shown to be initiated by oxygenation into chlorocatechols, and dechlorination occurred only after ring cleavage of the chlorocatechols. A different mechanism has been described for the degradation of polychlorinated phenols. In the presence of pentachlorophenol-metabolizing Ralstonia chlorophenolicus and in a Flavobacterium sp., degradation of PCP started by hydrolytic para–hydroxylation, yielding chlorinated para-hydroquinone. This hydroquinone pathway was also described for 2,4,5-trichlorophenoxyacetate-degrading Pseudomonas cepacia.. It may be more interesting that Li Deng-Yu et. al. have found the existence of different catabolic pathways for phenolic compounds in Azotobacter sp. strain GP1: the catechol pathway for phenol degrading and the hydroquinone pathway for chlorinated phenol degradation^[8].
    It is deserved to be mentioned that breakdown processes of chlorocatechols occured in the catechol pathway. In general, after the formation of catechols, methyl-substituted aromatic compounds are degraded via the meta ring cleavage pathway by catechol 2,3-dioxygenases, whereas chloroaromatic compounds are mineralized via the ortho-cleavage pathway by 1,2-dioxygenases ^[9]. Figure 1 illustrates the degradation pathway for the mechanism of chlorocathol via ortho ring. However, Juliane Hollender et al. proved the pure culture in Comamonas testosteroni JH5, which could completely mineralize a mixture consisting of 4-CP and 4-methylphenol (4-MP), could mineralize 4-CP via meta ring fission. The metabolic pathways are displayed in Figure 2 . In result, it is possible to transform the chlorinated and methyl-substituted aromatic compounds simultaneously.

Fig.1 Degradation pathway for chlorocathol via ortho ring fission

Fig.2 Illustration of degradation pathway for 4-CP via meta ring fission

2.2.2 Reductive dechlorination
Anaerobic biodegradation of CPs occurs by reductive dechlorination. In this process, chlorines are replaced with hydrogen, while degrading microorganisms use chlorinated chlorophenols as terminal electron acceptors in an anaerobic respiration. Therefore reductive dechlorination are partially or completely inhibited by the presence of other electron acceptors such as sulfate, nitrate, O₂ and CO₂. Sulfate may be the important electron acceptor influencing dehalogenation and anaerobic degradation of chlorinated aromatic compounds ^[10].
    Anaerobic dechlorination is very important for biodegradation of CPs, especially for polychlorinated phenols[11]. Most of the polychlorinated phenols that are resistant to aerobic microbial metabolism can be biodegraded under anaerobic conditions. And the less chlorinated metabolic products from reductive dechlorination of polychlorinated phenols are generally less toxic and degraded more easily than the parent compound. The reductive dechlorination process under the anaerobic condition is of environmental importance also because anoxic condition in soils, as well as bottom layers of aquatic sediments and freshwater and marine ecosystems are often prevailing ^[11].
    Studies on the distribution and fate of PCP in Japanese rice paddy fields provide some of the earliest information concerning anaerobic CPs biotransformation[2]. Since then, anaerobic enrichment cultures, which are active on various chlorinated phenols, have been obtained by a number of researchers^[12,13]. Reductive dechlorination of CPs has been observed for unacclimated and acclimated anaerobic sewage sludge, sediments, and the microbial communities, such as methanogenic consortia and sulfate-reducing bacteria^[14].
    Due to its toxicity to a wide variety of organisms and wide distribution in the environment, the effects and fate of pentachlorophenol (PCP) in nature have been well investigated[2,10]. At the same time, degradation efficiency and anaerobic degradation mechanisms of PCP have been studied with isolates such as Flavobacterium sp., Arthrobactor sp. and Rohodococous chlorophenolius, all of which had PCP as their sole source of carbon. Meanwhile, the degradation efficiency and mechanism was also be studied at microbial community such as methanogenic consortia and sulfate-reducing bacteria[15]. Biotransformation pathways of PCP vary with the characteristics of microbial consortium. In general, chlorines in positions ortho to the hydroxy group were removed more readily than those in the meta or para position. David K. et al. reported that acclimated microbial consortia had yielded biotransformation pathways different from those of unacclimated. They observed that the methanogenic consortium having been exposing to PCP for 10 days dechlorinated PCP principally at the ortho position. However, acclimation of the organisms to PCP over period of 6 months produced a microbial consortium with the ability to remove chlorines from the ortho, meta and para positions of PCP. The alternative processes are shown in Figure 3^[2].

Fig.3 Illustration of the reductive dechlorination pathway for PCP and its metabolites by a PCP-acclimated methanogenic consortium

    Most of reductive dehalogenation studies have used with mixed cultures, and only a few stable enrichments or pure cultures of dechlorinating anaerobes exist. Why reductive dechlorination of CPs is liable to take place in microbial consortium? Ferguson et. al. believed that the anaerobic transformation of chlorinated aromatic compounds depends on a close association between different bacteria for the following understandings: Firstly, strict anaerobic bacterium needs an anaerobic or reductive circumstance which can be supplied by other bacteria; Secondly, the electron donors needed by anaerobic bacteria may be the final products of other anaerobic bacteria metabolism; Finally, the toxic products of biodegradation of CPs can be eliminated by other bacteria.
2.3 Influence factors on biodegradabilities of CPs
The use of microbes for degradation of the CPs is limited by many factors ^[10]. Some of them are as follows: (1) Chemical structure of CPs ; (2) The microbes; (3)  Environmental factors.
    The less chlorinated phenols usually show less toxic, and aerobic biodegradation is more easily. It has been reported that MCPs and DCPs could be degraded and mineralized to CO₂ and H₂O, release of chloride by pure culture. Under aerobic conditions, but it is hard for polychlorophenols.
    The degradabilities of microorganisms to chlorophenols were highly specific. Haggblom M.M. et. al. used to observe the degradation of the three monochlorophenols (MCP) under methanogenic cultures. 4-MCP was degraded the fastest, 3-MCP somewhat slower and 2-MCP the slowest. However, the reverse biodegradation rates of isomers MCPs were observed under sulfate-reducing conditions[11,14]. Generally, concentration of bacterium cells can affect degradation rate of CPs. Sometimes it can change degradation pathway of substrate and final products. Li,Deng-Yu et al. once reported resting cells of Azotobacter sp. strain GP1, an isolate which uses 2,4,6-trichlorophenol (TCP) as carbon source for growth, degraded 2,4,6-TCP only and transform it to 2,6-DCHQ at low cell density, However it could degrade completely or partially MCPs and DCPs into CO₂ and H₂O at high cell densities^[8]. Such an expansion of the spectrum for CPs degradation via increasing cell concentration was earlier identified by Chu and Kirsch with a PCP degrader^[8].
    Furthermore, environmental factors such as substrate concentration, medium pH, temperature and mineral salt components are important for biodegradation of CPs^[10].

3. RECOMMENDATIONS FOR FUTURE WORK
The article has attempted to emphasize the progress on biodegradation of CPs and profitable lines of future work, which may aid in realizing the technology's full potential, are delineated as follows:
    1. Further understanding of the mechanism of chlorophenols transformation and the bacteria responsible for the degradation activity. Knowledge of biotransformation pathways is essential to predict the environmental fate of toxic or recalcitrant compounds, and it is also necessary to assess risks at contaminated sites or design bioremediation strategies. Though the progress on biodegradation mechanism of CPs has been magnitude. There are many questions unanswered. It can be illustrated with a simple example: Ralstonis sp strain RK1 is the first pure culture capable of growing on and mineralizing 2,6-DCP, but both of the ortho positions of 2,6-DCP are occupied by chlorine atoms, so the degradation pathway by PK1 must be different from the pathway hydroxylating in the ortho position and producing chlorocatechol^[1,16].
    2. The microbes of degrading incompatible compounds should be achieved. Biological treatment of wastewater and contaminated soils always involves degradation of some incompatible mixtures, rather than degradation of single compounds^[10]. Methyl- and chlorinated aromatic compounds are known to be incompatible substrates and often exist together. But the observed degradation capability of certain microbe is specific for certain or similar compounds. Therefore, achieving such microbes with the ability to degrade incompatible substrate mixtures is vital. Up to date, there are several ways to get such organisms: the first one is genetic engineering. Rojo, F. et al. once construct a bifunctional bacterium through a patchwork assembly of pathway segments from A.eutrophus JMP134 in recipient Pseudomonas strain B13 and the bacterium could degrade the mixtures of chloro- and methylaromatics. Another way is the enrichment by selection against the MCPs degrading or induction of suitable enzymes in a single strain by exposing to the mixtures of CPs and methylaromatics. The third way is mutagenesis. Recently Hailgler BE et al described that Pseudomonas strain JS150, which was obtained by mutagenesis of a 1,4-dichlorobenzene-degrading organism, degraded simultaneously chloro- and methyllphenols in the presence of phenol.^[17]
    3. Further research should emphasize large-scale pilot studies to evaluate the effectiveness of CPs-degrading microbes^[1]. During last several years, researchers have primarily focused their studies on the isolation and activities of the CPs-degrading bacteria in shaken liquid media, and reported data mainly are limited to laboratory experiments. However, the results observed in the laboratory often differ from those observed in the field or in treatment processes in practice. For instance, bioavailability of organic chemicals in soil can be limited or controlled by physicochemical processes including diffusion, sorption/desorption and dissolution[18]. And the answer, whether the strains isolated in laboratory are actually able to bioremediate CPs contaminated environments efficiently, awaits further research[19]. So, it is necessary to take large-scale pilot studies to evaluate the effectiveness of CPs-degrading microbes.
    Bioremediation of contaminated environments could be achieved by finding ways to stimulate the activities of indigenous microorganisms, also by introducing high efficient laboratory strains. The latter process is known as bioaugmentation. The use of high efficient laboratory strains for biodegradation has been described by Crawford R L et al. They introduced the laboratory strains, Flavobacterium sp., Arthrobater s.p, and Rhodococcus chlorophenolicus to bioremediate PCP contaminated soil, and in each case removal of PCP was accelerated.
    4. Enzymes instead of the microbes should be studied to degrade CPs. Compared to physical and chemical methods, biodegradation is cost-competitive and safe. However, the use of microbes for bioremediation is beset with many rate-limiting factors. Furthermore, severe conditions such as chemical shock, extremes of pH and temperature, toxins, predators and high concentrations of the pollutants or their products may irreversibly damage or metabolically inactivate microbial cells.
  Biotransformation involves a series of enzyme-catalyzed reactions^[20]. Thus, most of these adverse factors can either be eliminated or mitigated if the enzymes are used instead of the microbes. Compared to the microbe, enzyme (especially immobilized enzyme which is bound to solid supports,) is active in a wider pH and temperature range, and is more resistant to proteolytic degradation. Furthermore enzymes may prove more economical treatments because they are biochemically stable and can be used repeatedly to detoxify xenobiotics.

氯酚类化合物的生物降解研究进展
吴高峰 徐鸿 姜梅
(北京化工大学 100029；广州市化工中专 510730)
摘要 本文对近年来在具有降解氯酚类化合物能力的微生物的筛选、氯酚类化合物的好氧和厌氧降解机制、氯酚类化合物可生物降解性主要影响因素以及现代生物技术的开发利用四个方面的研究进行综述.并阐述了氯酚类化合物在不同条件下的降解路径.在好氧条件下,单氯酚和二氯酚在氧化酶的攻击下形成氯代邻二酚,邻二酚开环生成相应的氯代粘康酸或半醛,粘康酸内酯化过程中释放氯离子;高度氯代的化合物则是在氢氧化酶作用下生成氯代醌,并逐步脱去所有的氯原子生成苯酚后才开环的。在厌氧或缺氧条件下,氯酚进行还原脱氯,在得到电子的同时去掉一个氯取代基.
关键词 氯酚类化合物 好氧降解 厌氧降解 生物降解性 生物技术