Биодеградация 2,4-дихлорфенола штаммами Pseudomonas sp. PKZNSA и Klebsiella pneumoniae KpKZNSA

2,4-Dichlorophenol Degradation by Indigenous Pseudomonas sp. PKZNSA and Klebsiella pneumoniae KpKZNSA: Kinetics, Enzyme Activity, and Catabolic Gene Detection

B. Setlhare(https://orcid.org/0000-0009-5704-1234), A. Kumar(https://orcid.org/0000-0005-9502-4344), O. A. Aregbesola(https://orcid.org/0000-0004-5503-5678), M. P. Mokoena(https://orcid.org/0000-0005-5503-5698), and A. O. Olaniran(https://orcid.org/0000-0004-5000-5123)*

Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban, 4000 Republic of South Africa

*e-mail: olanirana@ukzn.ac.za

Abstract—In this study, two newly isolated 2,4-dichlorophenol(2,4-DCP)-degrading strains, Pseudomonas sp. KZNSA (PKZNSA) and Klebsiella pneumoniae KZNSA (KpKZNSA), were enriched from an activated sludge sample with a known history of contamination with chlorinated organic compounds collected from a wastewater treatment plant located in Durban, South Africa. The strains could use 2,4-DCP as sole carbon and energy source. PKZNSA and KpKZNSA degraded 64 and 49% of 2,4-DCP, with the degradation rate constant of 0.14 and 0.03 mg L–1 day–1, respectively. Both PKZNSA and KpKZNSA were found to harbor the catabolic genes that encode the enzymes involved in 2,4-DCP degradation via the ortho-pathway which is further confirmed by the specific enzyme activity assays. The strains did not possess genes that encode the enzyme maleylacetate reductase, which is involved in funneling the last intermediate (maleylacetate) in the pathway into the Krebs cycle. Findings from this study will be helpful in the exploitation of these microorganisms and/or their enzymes in developing bioremediation strategies for the chlorophenol-polluted environment.

Keywords: 2,4-dichlorophenol, Pseudomonas sp., Klebsiella pneumoniae, phenol hydroxylase, catechol 1,2-dioxygenase

INTRODUCTION

2,4-Dichlorophenol (2,4-DCP), a derivative of phenol, is used as a precursor in the production of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its derivatives [1] and in the synthesis of higher chlorophenols such as pentachlorophenol [2]. 2,4-DCP is also used as a wood preservative, insecticide, and plant growth regulator [1]. This compound is released during photodegradation of triclosan (antibacterial and antifungal agent) along with the dioxin 2,8-dichlorodibenzo-p-dioxin [3]. 2,4-DCP is also released into the environment as a metabolite of the degradation of chemicals applied in agricultural and food sectors [4]. <…> Due to its potential mutagenicity, carcinogenicity, immunogenicity, fertility, and cytotoxic properties, 2,4-DCP has been classified as a priority pollutant by the Agency for Toxic Substances and Disease Registry, WHO (https://www.iarc.who.int/wp-content/uploads/2018/07/pr236_E.pdf, accessed on February 19, 2021). <…>

Bacterial degradation of 2,4-DCP proceeds via ortho-cleavage pathway [10, 18] or meta-cleavage pathway [10]. In the ortho-pathway, 2,4-DCP is hydroxylated to 3,5-dichlorocatechol by 2,4-DCP hydroxylase [19] and subsequently cleaved to 2,4-dichloro-cis,cis-muconate by catechol 1,2-dioxygenase type II [18, 19]. 2,4-dichloro-cis,cis-muconate is isomerized to 4-carboxymethylbut-2-en-4-olide (dienelactone) by dichloromuconate cycloisomerase. Dienelactone is further hydrolyzed to 2-chloromaleylacetate by dienelactone hydrolases [19]. In the last stage of the degradation, 2-chloromaleylacetate is dechlorinated to maleylacetate and then to 3-oxoadipic acid by maleylacetate reductase [20]. In the meta-cleavage pathway, 2,4-DCP is first oxidized to 3,5-dichlorocatechol and is subsequently degraded to 2-hydroxy-3,5-dichloro-6-oxohexa-2,4-dienoic acid, as illustrated in Cupriavidus necator strain JMP222 [10]. <…>

MATERIALS AND METHODS

Sample collection, enrichment, and isolation of bacterial isolates.

An activated sludge sample with a known history of contamination with chlorinated organic compounds was collected from a wastewater treatment plant located in Durban, South Africa. Samples collected in 500 mL bottle and immediately were stored at 4°C until used for enrichment setup. The mineral salt medium (MSM) used for the culture enrichment was consist of (mg/L): KH2PO4, 800; Na2HPO4, 800; MgSO4·7H2O, 200; (NH4)2SO4, 500. pH was adjusted to 7.5. One mL of trace elements which comprising (mg/L): FeSO4·7H2O, 5.0; ZnSO4·7H2O, 4.0; MnSO4·4H2O, 0.2; NiCl·6H2O, 0.1; H3BO3, 0.15; CoCl2·6H2O, 0.5; ZnCl2, 0.25; EDTA, 2.5 was added using syringe filter (0.2 µm pore) into MSM. Ten percent of the sludge sample was inoculated into MSM supplemented with 40 mg/L of 2,4-DCP in a 250 mL Erlenmeyer flask and incubated at 30°C with shaking at 150 rpm for a week [11]. <…> Pure cultures were obtained by streaking individual morphologically different colonies on nutrient agar plates. The pure colonies were stored at –70°C as 20 vol % glycerol stocks.

Growth and degradation studies.

Growth and degradation assays for the 2,4-DCP-degrading isolates were set up in an Erlenmeyer flask. The isolates were grown to their late log phase in nutrient broth (NB) composed of (g/L): beef extract, 1.0; yeast extract, 2.0; peptone, 5.0 and NaCl, 5.0 at 30°C with shaking at 150 rpm, harvested by centrifugation at 8000 × g for 10 min, washed twice with MSM, resuspended in the same medium and standardized to OD600 of 1.0 based on McFarland turbidity standards. About 10 vol % of the standardized inoculum was added separately into 90 mL of MSM supplemented with 40 mg/L of 2,4-DCP and the flasks were incubated for 22 days at 30°C at 150 rpm. Cupriavidus necartor JMP134 was used as a positive control while MSM plus 2,4-DCP without the inoculum was used as a negative control to test for abiotic loss. Unless otherwise stated, all growth and degradation assays in this study were conducted in triplicates. The growth of the cultures in the flasks was monitored spectrophotometrically at 600 nm. To measure the 2,4-DCP concentration in the flask, 1.5 mL aliquots were withdrawn from each flask at 48 h interval and centrifuged at 20000 × g for 10 min. The supernatants were transferred into a quartz cuvette (Hellma Analytics, Germany) and the OD200-350 was measured using a UV–Vis spectrophotometer at scanning mode. 2,4-DCP having absorption maximum at 284 nm used to perform calibration curve. 2,4-DCP concentrations in sample at each sampling period were extrapolated from the linear regression equation. <…>

nd centrifuged at 20000 × g for 10 min. The supernatants were transferred into a quartz cuvette (Hellma Analytics, Germany) and the OD200-350 was measured using a UV–Vis spectrophotometer at scanning mode. 2,4-DCP having absorption maximum at 284 nm used to perform calibration curve. 2,4-DCP concentrations in sample at each sampling period were extrapolated from the linear regression equation. <…>

Determination of chloride released during degradation.

Chloride release during 2,4-DCP degradation was quantified spectrophotometrically as described previously [11] with some modifications. Two hundred µL 250 mM Fe(NH4)(SO4)2·12H2O in 6 M nitric acid was added to the supernatant, followed by the addition of 400 µL of saturated mercuric thiocyanate in ethanol. The development of a red color is an indication of the presence of free halide which was measured at OD460. NaCl standard curve was plotted, and the concentration of the chloride released during degradation studies was calculated by extrapolation from the linear regression equation.

Identification and phylogenetic analysis of the bacterial isolates.

Two isolates showing highest degradation for 2,4-DCP were identified by PCR amplification, sequencing, and analysis of 16S rRNA gene sequences. Genomic DNA was isolated and purified (Genomic DNA Purification Kit, Thermo Fisher Scientific, USA), and used as a template. The primers used were: 63F-5′-CAGGCCTAACACATGCAAGTC-3′ and 1387R-5′-GGGCGGTGTGTACAAGGC-3′ [21]. <…>

Preparation of crude extract to determine enzyme activities.

The crude extract was prepared as described previously [11] with some modifications. The modified MSM (pH 7.0) contained (g/L): K2HPO4, 2.75; KH2PO4, 0.1; NH4Cl, 0.2; MgSO4·7H2O, 0.01; CaCl2·2H2O, 1.0; NH4Cl, 0.5, yeast extract, 1.0, and 1 mL of filter-sterilized micronutrients. The micronutrients contained (mg/L): FeSO4·7H2O, 5.0; ZnSO4·7H2O, 4.0; MnSO4·4H2O, 0.2; NiCl·6H2O, 0.1; H3BO3, 0.15; CoCl2·6H2O, 0.5; ZnCl2, 0.25, and EDTA, 2.0. The inoculum was prepared as described above and 10 vol % inoculated in 1 L MSM supplemented either with 600 mg/L phenol or 40 mg/L 2,4-DCP in separate flasks. The flask was left for 36 h at 30oC at 200 rpm. The cells were harvested at the late exponential phase of growth by centrifugation at 12096 × g for 15 min at 4°C. The cells were washed twice with 50 mM Na-phosphate buffer, pH 7.5 containing 1 mM EDTA and 1 mM β-mercaptoethanol to halt the protease activity. The cell pellet was collected and re-suspended in 100 mL of the same buffer. Cell-free extracts were prepared by lysing the pellet by sonication with 400 ultrasonicator (OMNI International, USA) using 8 cycles each with a pulse of the 30 s on/off for 4 min. The cell extract was centrifuged at 20442 × g for 30 min at 4°C. The clear supernatant was kept on ice to prevent inactivation of the enzymes and used as a crude extract for enzyme assays or stored at –20°C.

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