Bacterial degradation of chlorophenols and their derivatives

Chlorophenols (CPs) and their derivatives are persistent environmental pollutants which are used in the manufacture of dyes, drugs, pesticides and other industrial products. CPs, which include monochlorophenols, polychlorophenols, chloronitrophenols, chloroaminophenols and chloromethylphenols, are highly toxic to living beings due to their carcinogenic, mutagenic and cytotoxic properties. Several physico-chemical and biological methods have been used for removal of CPs from the environment. Bacterial degradation has been considered a cost-effective and eco-friendly method of removing CPs from the environment. Several bacteria that use CPs as their sole carbon and energy sources have been isolated and characterized. Additionally, the metabolic pathways for degradation of CPs have been studied in bacteria and the genes and enzymes involved in the degradation of various CPs have been identified and characterized. This review describes the biochemical and genetic basis of the degradation of CPs and their derivatives.

CPs have been introduced into the environment via anthropogenic activities [3]. The major sources of contamination are industrial wastes, pesticides, herbicides, and complex chlorinated hydrocarbons [3]. People may be exposed to CPs by eating or drinking substances that contain them or through skin contact [4]. CPs and their derivatives are highly toxic to living beings due to their carcinogenic, mutagenic and cytotoxic properties [5]. The World Health Organization and the International Agency for Research on Cancers have characterized several poly-CPs as potential human carcinogens [5]. Similarly, the United States Environmental Protection Agency has included several CPs in its list of priority pollutants.
Several conventional methods such as adsorption, ion exchange, liquid-liquid extraction, and chemical oxidation and advanced oxidation processes have been used for the removal of CPs from wastewater [3,6]. These methods are expensive and not eco-friendly due to the formation of hazardous compounds as by-products [3]. Conversely, bioremediation is an effective and ecofriendly method of removing CPs from the environment. Biodegradation of CPs has gained attention due to the complete mineralization of CPs by microorganisms in the environment.
Several reviews dealing with the degradation and toxicity of CPs and their derivatives have been published [3,5,7,8]; however, these reviews were focused on the biodegradation/toxicity of MCPs or poly-CPs or both. The present review describes the biochemical and genetic basis of bacterial degradation of CPs and their derivatives including MCPs, poly-CPs, CAPs, CNPs and CMPs. Both aerobic and anaerobic bacterial degradation of CPs are discussed.

Bacterial degradation of CPs
Aerobic degradation of CPs and their derivatives have been extensively investigated in bacteria, and many bacteria with the ability to utilize CPs as their sole carbon and energy sources have been isolated [8]. One of the following mechanisms may be involved in the bacterial degradation of CPs and their derivatives: (i) monooxygenases may catalyze hydroxylation at the ortho-positions of the chlorophenolic rings, which results in the formation of chlorocatechols that may be degraded  further via ortho- [9] or meta-cleavage [10,11] or hydroxylated prior to ring cleavage [12]; (ii) monooxygenases may catalyze the hydroxylation at para-positions of the chlorophenolic rings, resulting in the formation of chlorohydroquinones that may be degraded further via hydroxylation [12] or dehalogenation [13] prior to ring cleavage; (iii) the degradation of CNPs may be initiated via hydroxylation [14], reductive dehalogenation [15] or reduction of the nitro group [16], (iv) The degradation of ACPs may be initiated with the removal of ammonium ions by the enzyme deaminase followed by the ring cleavage [17] or the dehalogenation [18]. In this section, we describe the bacterial degradation pathways for MCPs, poly-CPs, CNPs, CAPs and CMPs.
In addition to the modified-ortho or meta-ring cleavage pathway of 4CC, there is another pathway for degradation of 4CC, which is here designated as the 4CC-Benzenetriol (4CC-BT) pathway. In this pathway, 4CC is first hydroxylated to 1,2,4-benzenetriol (BT) through the release of chloride ion [12]. BT is then further degraded via ring cleavage and the formation of maleylacetate [12]. The 4CC-BT pathway was observed in the degradation of 4CP in A. chlorophenolicus A6 [12].
The 4CP degradation can also occur through the HQ pathway [12,37]. The first step of the HQ pathway is the formation of HQ through the release of chloride ion from 4CP by a 4CP-4-monooxygenase [ Figure 2c]. In the next step, HQ is converted to BT, which is then cleaved into maleylacetate by a BT-dioxygenase [12]. A few bacterial strains degrade 4CP via two pathways. For example, A. chlorophenolicus A6 degrades 4CP via the HQ pathway as well as the 4CC-BT pathway [12].
The degradation of 3CP occurred either via the formation of 3CC or via the formation of 4CC that may be further degraded via the modified ortho-cleavage pathway or the meta-cleavage pathway [7,39].
The first report of the 4C2NP degradation was documented in 1988 with construction of a genetically engineered bacterium, Pseudomonas sp. N31, which utilizes 4C2NP as a sole carbon, nitrogen and energy source [71]. The constructed strain degrades 4C2NP via the formation of 4CC and the release of chloride and nitrite  ions [ Figure 8]. Beunink and Rehm [72] reported 4C2NP degradation via the formation of 4-chloro-2-aminophenol (4C2AP) by a co-culture of Enterobacter cloaceae and Alcaligenes sp. TK-2 [ Figure 8]. A detoxification mechanism for 4C2NP transformation has been proposed for two Bacillus species [73,74]. In this mechanism, detoxification is initiated by the formation of 4C2AP, which acetylates into 4-chloro-2-acetaminophenol (4C2AAP). 4C2AAP is then converted to a non-toxic compound, 5-chloro-2-methylbenzoxazole [ Figure 8].
Another investigation of complete mineralization of 4C2NP was published following the isolation of a 4C2NP-mineralization bacterium, Exiguobacterium sp. PMA [16]. This strain initiates 4C2NP degradation by the formation of 4C2AP via a reduction mechanism, which is further dehalogenated into 2-aminophenol (2AP) through the release of chloride ions [16]. The further degradation of 2AP proceeds via ring cleavage and the removal of ammonium ions [ Figure 8].

Bacterial degradation of CAPs and CMPs
CAPs are amino derivatives of MCPs that are used in the manufacture of dyes. Examples include 4-chloro-2-aminophenol (4C2AP) and 2-chloro-4-aminophenol (2C4AP). Bacterial degradation of 4C2AP was studied in the Gram negative bacterium, Burkholderia sp. RKJ 800, which utilizes 4C2AP as a sole carbon and energy source [17]. The degradation of 4C2AP is initiated by the release of ammonium ion and the formation of 4CC by a deaminase. In the next step, 4CC is cleaved to cis, cis-chloromuconic acid by 4CC-1,2-dioxygenase ( Figure 10a). Conversely, the bacterial degradation of 2C4AP was studied in a Gram positive bacterium, Arthrobacter sp. SPG, which utilized 2C4AP as its sole source of carbon and energy [18]. The first step of 2C4AP degradation involves removal of the ammonium ion by deaminase, which leads to formation of CHQ that is then dehalogenated to HQ by a CHQ-dehalogenase ( Figure 10b). In the next step, HQ is cleaved to γ-hydroxymuconic semialdehyde by HQ-1,2-dioxygenase (EC = 1.13.11.66) [18].
The mineralization of CPs has been found to be coupled with sulfate reduction. Haggblom and Young [88] developed a CPs-mineralizing sulfate reducing consortia from estuarine sediment that was maintained on 2CP, 3CP or 4CP as the only source of carbon and energy for several years [89]. Their experiments utilizing a 4CP-utilizing consortium revealed that mineralization of 4CP into CO 2 was coupled to sulfate reduction, and that 4CP depletion did not occur in the absence of sulfate. In this reaction, sulfate, thiosulfate or sulfite were used as electron acceptors [89]. The coupling of sulfate reduction with mineralization of CPs was also observed in degradation of 2CP or 4CP by sulfate reducing enrichment cultures derived from Hudson River sediment [90].
Under denitrifying conditions, the 2CP degradation was studied in enrichment cultures derived from activated sludge samples [91]. The presence of nitrate was essential as electron acceptors for the mineralization of 2CP into CO 2 [91]. Sanford and Tiedje [92] studied dechlorination and subsequent degradation of MCPs and DCPs in anaerobic microcosms supplemented with 1 mM or 5 mM nitrate. CPs degradation is associated with reduction of Fe 3+ to Fe 2+ . An anaerobic enrichment culture derived from Hudson River sediments mineralized 2CP, 3CP and 4CP with concomitant reduction of Fe 3+ to Fe 2+ [93]. Several factors may affect dechlorination of CPs and reduction of Fe. For example, a low amount of nitrate enhances reductive dechlorination of PCP and Fe(III) reduction, while high concentrations of nitrate inhibit reductive dechlorination and Fe(III) reduction [94].
The reductive dehalogenation of MCPs and DCPs was investigated in the anaerobic sediment samples of estuarine Lake Shinji and Lake Nakaum [95]. Estuarine sediment enrichment cultures of lake Shinji dehalogenated 2CP, 3CP and 2,6-DCP, whereas enrichment cultures of Lake Nakaum dehalogenated 3CP and 2,6-DCP [95]. The dehalogenated product of MCPs was phenol, which was further degraded by the formation of benzoic acid. Itoh et al. [96] identified the bacterial consortia involved in dehalogenation of MCP into phenol and transformation of phenol to benzoic acid using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of the 16S rRNA gene in the enrichment sample of Lake Shinji. The 4CP-dechlorinating culture had two dominant bacteria, in which one belonged to Dehalobacter sp. In the phenol transforming culture, Cryptanaerobacter phenolicass was present.
Li et al. [97] established a simple anaerobic upflow column system (15 cm long, 5 cm inner diameter) for complete PCP-mineralization using a microbial consortium requiring only lactate as an external nutrient. Anaerobic microbes dehalogenated PCP to 3CP and phenol using external lactate as an electron donor [97]. The further degradation of 3CP and phenol proceeded without an external electron donor and the nitrogen required for degradation was supplied by nitrogen-fixation [97]. The potential dechlorinators, Dehalobacter and Desulfitobacterium, and the phenol/3CP fermentative or syntrophic degraders, Cryptanaerobacter and Syntrophus, were found at the bottom of the column, whereas the nitrogen-fixing facultative anaerobe, Rhizobiales, was detected in the top of the upflow column, and other possible nitrogen-fixers were found at both the bottom and top of the upflow column [97].
A 4CP-degradation gene cluster (cph genes) was identified in A. chlorophenolicus [12]. This gene cluster contains 10 open reading frames that show similarity to the genes encoding the enzymes involved in CP degradation. Several open reading frames encode enzymes with similar functions. For example, two genes, cphA-1 and cph-11, encode functional hydroxyquinol-1,2-dioxygenase. A mutant strain constructed by disturbing the gene cphA-1 by site-directed mutagenesis was unable to utilize 4CP as the sole source of carbon energy. Other genes present on this cluster include cphC-I, cphC-II, cphF-I, cphF-II, Cph B, CphX, CphR and CphS. The genes cphC-I and cphC-II encode putative monooxygenase, whereas cphF-1 and cphF-11 encode putative maleylacetate reductase and cphB encodes a NADH:flavin adenine dinucleotide oxidoreductase. The roles of the remaining genes in the cph gene cluster have yet to be determined [112].

Genetics of reductive dehalogenation
Reductive dehalogenation of CPs and poly-CPs is generally carried out by chlorophenol reductive dehalogenases (CprA) encoded by the cprA gene, which have been well-studied in Desulfitobacterium hafniense PCP-1, D. dehalognase IW/IU-DC1, and D. chlororespirans [98]. The cprA genes are associated with cpr gene clusters that also encode several accessory proteins (e.g., CprAanchor protein [98,125], chaperones, regulators [126]). The cpr gene clusters composed of eight genes (cprT, cprK, cprZ, cprE, cprB, cprA, cprC, and cprD) have been identified in the genome of Desulfitobacterium dehalogenans IW/IU-DC1 and Desulfitobacterium hafniense DCB-2 [126,127]. CprK, a member of the CRP-FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family regulators, control transcription of the cpr genes [128]. The mechanism responsible for regulation of transcription of cpr genes has been investigated [128]. An effector domain of CprK interacts with a chlorinated aromatic compound with high affinity which induces its binding to an upstream target DNA sequence known as the "dehalobox to activate the transcriptions of the cpr genes [128].
A non-CprA reductive dehalogenase known as CrdA from D. hafniense strain PCP-1 cultures amended with 2,4,6 TCP has been isolated and characterized. CrdA catalyzes ortho-dehalogenation of PCP and 2,4,6-TCP [134]. The gene (crdA) encoding CrdA has been cloned and sequenced from strain PCP-1 and also detected in several other strains of Desulfitobacterium [134]. Gauthier et al. [129] monitored the expression of the crd gene in Desulfitobacterium strains and transcripts of crdA were detected in D. hafniense strains PCP-1, DCB-2 and TCE-1.

Conclusions
The bacterial degradation of MCPs and poly-CPs has been extensively studied and several pathways have been proposed for degradation of MCPs and poly-CPs. The bacterial degradation of CPs and poly-CPs proceeded via formation of the corresponding CCs or the corresponding (chloro)HQs. The genes involved in the degradation of MCPs and poly-CPs have also been identified and characterized from CPs-degrading bacteria.
CAPs and CMPs are highly toxic compounds, and few studies have been conducted to investigate the biodegradation of these compounds. More CAPs and CMPsdegrading bacteria must be isolated to investigate the genetic and biochemical mechanism by which these compounds are degraded.
Anaerobic degradation of CPs has also been studied, and it has been established that MCPs and poly-CPs are initially dehalogenated to phenol, which is further transformed to benzoic acid and then mineralized to CO 2 under anaerobic conditions. However, further study is needed to elucidate the genetic and enzymatic basis of this mechanism. Furthermore, anaerobic degradation of other CPs such as CNPs, CAPs and CMPs should also be studied.