Biodegradation of pentachlorophenol by Bacillus tropicus isolated from activated sludge of a wastewater treatment plant in Durban, South Africa.

Abstract

Pentachlorophenol (PCP) is a persistent organic compound that bio-accumulates in the environment due to its recalcitrant nature and has been listed as a priority pollutant due to its toxicological properties. The recent incidences of xenobiotic at different sites and provinces in South Africa, other African Countries and Europe is worrisome and required a proactive measure. Biodegradation has been projected as one of the best ways to ameliorate recalcitrant impacted sites. This study thus aims to isolate and characterize PCP-degrading microorganism from the environment; degrade PCP and other compounds with the isolate in batch culture; optimize biotransformation processes for effective and efficient transformation; determine biodegradation kinetic parameters; profile metabolites produced; detect and amplify PCPdegrading genes from the selected isolate; map the degradation pathway; clone and overexpress the catabolic genes heterologous; purified and characterized the protein both biologically and structurally and sequence the whole-genome of the isolate with the view to determine the evolution and arrangements of PCP-catabolic genes in the genome of the isolate as well as exploring other potentials of the isolates. A PCP-degrading strain was isolated and characterized using a PCR amplification and analysis of the 16S rRNA. Biodegradation process parameters were optimized using response surface methodology. Degradation kinetics were determined via substrate inhibition models, while PCP transformation was evaluated by spectrophotometric and GC-MS analysis. Catabolic genes were detected and amplified via PCR. Genes were cloned via heat-shock technique using chemically competent cells. Proteins purification, digestion and sequencing were done using affinity chromatography, tryptic digestion, and Liquid Chromatography–Mass Spectrometry (LC-MS) techniques respectively. Pacific Biosciences RS II sequencer with the Single Molecule, Real-Time (SMRT) Link was used to sequence the whole genome of the isolate. Coting’s were assembled and analysed using the HGAP4-de-novo assembly application. Genes were annotated on the Rapid Annotation using Subsystem Technology tool kit (RASTtk) and ab initio prediction (PROKKA) using the prokaryotic genome annotation pipelines. Metabolic model pathways of the bacterium was reconstructed using the RAST SEED Viewer. Primarily, the isolate was identified as Bacillus cereus strain AOA-CPS1 (BcAOA) based on the 16S rDNA sequence analysis. However, a quality control test by NCBI for the submitted whole genome sequence of the strain, using an average nucleotide identity (which compares the submitted genome sequence against the whole genomes of the type strains that are already in GenBank) resulted in the renaming of BcAOA as Bacillus tropicus strain AOA-CPS1 (BtAOA). BcAOA was renamed as BtAOA (based on the whole genome data submitted at NCBI under accession number CP049019). The bacterium degraded 74% of PCP (within 9 days at initial PCP concentration of 350 mg l-1 in a batch culture) and other chlorophenolic compounds in co-metabolism. The degradation followed first and zero-order kinetics at low and high PCP concentration, respectively with biokinetic constants: maximum degradation rate (0.0996 mg l-1 h-1); substrate inhibition constant (723.75 mg l-1) and a halfsaturation constant (171.198 mg l-1) and R2 (0.98). The genes (pcpABCDE, cytochrome P450) encoding the enzymes involved in the biodegradation of PCP were amplified from the genomic DNA of BcAOA. Further, depending upon the genes amplified and identified metabolites using GC-MS, there are two different PCP transformation pathways were proposed in this study. At optimized conditions, BtAOA transformed 98.2% of 500 mg L-1 of PCP in 6 days which represent a significant 59.2% increase in PCP transformation compared to the unoptimized conditions. The kinetic parameters for PCP transformation at optimized conditions were: 1.064 ± 0.114 mg l-1 h-1 (maximum biodegradation rate); 229 ± 19.5 mg l-1 (half-saturation constant); 535 mg l-1 (inhibition constant); and R2 = 0.96. Each of the catabolic genes shared >99% sequences homologies with the corresponding genes in the genomes of their ancestors, however, their biological functions remain putative to date. The optimum temperature and pH of CpsB were 30oC and 7.0. CpsB showed functional stability between pH 6.0-7.5 and temperature 25oC- 30oC. CpsB activity was stimulated by Fe2+, Ca2+, EDTA (0.5-1.5 mM) and Dithiothreitol (0.5- 1.0 mM) but inhibited by sodium azide and sodium dodecyl sulphate (>0.5 mM). CpsB enzyme substrate reaction kinetics studies showed allosteric nature of the enzyme and followed presteady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465 μmol.s-1, 140 μmol, 0.099 s-1 and 7.07 x 10-4 μmol-1.s-1, respectively, for the substrate PCP and 0.259 μmol.s-1, 224 μmol, 0.055 s-1 and 2.47 x 10-4 μmol-1.s-1, respectively, for co-substrate NADH. The Hill plots and M-W-C model reveal CpsB allosteric nature and belong to K-System. CpsB shared 100% sequence homology with aromatic amino acid hydroxylase and is classified as aromatic amino acid hydroxylase superfamily with multiple putative conserved domains and metal ion binding sites confirming its allosteric nature. Bacillus tropicus AOA-CPS1 Cytochrome P450 monooxygenase (P450CPS1) exhibited optimum activity at 40oC and pH 7.5. The P450CPS1 was stable between 25oC-30oC retaining 100% of its residual activity after 240 min of incubation. The activity of P450CPS1 was stimulated by Mn2+, Fe2+, and Fe3+ typical of an oxidoreductase but inhibited by 2.0 mM piperonyl butoxide and sodium dodecyl sulphate. The reaction kinetics studies showed allosteric nature of P450CPS1 showing apparent vmax, Km, kcat and kcat/Km values of 0.069 μmol.s-1, 200 μmol, 0.011 s-1 and 5.42 × 10-5 μmol-1.s-1, respectively, for the substrate PCP and 0.385 μmol.s-1, 56.46 μmol, 0.06 s-1 and 1.77 × 10-3 μmol- 1.s-1, respectively, for co-substrate NADH. CpsD showed optimal activity at pH 7.5 and temperature between 30oC–40oC. The enzyme was stable between pH 7.0 – 7.5 and temperature between 30oC and 35oC. CpsD activity was enhanced by Fe2+ ion and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 μmol s-1, 94 μmol, 0.029 s-1 and 3.13x10-4s-1μmol-1, respectively, for substrate tetrachloro-1,4-benzoquinone. CpsD belongs to the pterin-4α-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF-1 (DCoH) superfamily, with specific conserved protein domains of pterin-4α dehydratase (PCD), validated Pterin-4α-carbinolamine dehydratase (DCoH), and coenzyme transport and metabolism proteins. CpsA showed optimum activity at 30oC and pH 9.0. CpsA was stable between 20oC-40oC, and also retained about 90% of its activity at 60oC. The enzyme retained about 90% activity between pH 9.0 and 11.5 and 60% activity at pH 13.0. CpsA was found to be Fe2+ dependent as about 90% increased activity was observed in the presence of FeSO4. CpsA showed apparent Vmax, Km, Kcat and Kcat/Km of 27.77±0.9 μM s-1, 0.990±0.03 mM, 4.20±0.04 s-1 and 4.24±0.03 s-1 mM-1, respectively at pH 9.0. CpsA 3D structure revealed a conserved 2-His-1-carboxylate facial triad motif (His 9, His 244 and Thr 11), with Fe3+ at the centre. The whole genome of the isolate comprises one chromosome and one plasmid. The metabolic reconstruction for Bacillus tropicus strain AOACPS1 showed that the organism has been exposed to various chlorophenolic compounds including 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane, 1,2-dichloroethane, 1,4- dichlorobenzene, 2,4-dichlorobenzene, atrazine, and other xenobiotics previously and it has recruited enzymes for their degradation. PCP degradation by the isolate was independent of substrate concentration but highly dependent on the maximum specific growth and degradation rates. The low-affinity coefficient and high inhibition constant obtained in this study showed that the bacterium has a high affinity and tolerance to PCP, which could be explored for bulk remediation of PCP. The combination of the recombinant plasmid’s vector harbouring the PCP catabolic genes can be used for direct bioremediation of PCP in a bioreactor optimized for the growth of the hosts for overexpression of the proteins. Alternatively, concoction of the enzymes can be produced and immobilised for direct enzymatic bioremediation of PCP and other related recalcitrant xenobiotics. The study proposed that CpsD catalysed the reduction of tetrachlorobenzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine-hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in Bacillus tropicus AOA-CPS1 PheOHS using a probable Yin-Yang mechanism. CpsD may play a catalytic and regulatory role in Bacillus cereus PheOHS and PCP degradation pathway. Findings from this study provide new insights into the biological role of CpsA in PCP degradation and suggest alternate possible mechanism of ring-cleavage by dioxygenases. The study also provides the first experimental evidence of the involvement of a putative cytochrome p450 from Bacillus tropicus group in PCP transformation. Sequence mining and comparative analysis of the metabolic reconstruction of BtAOA with the closest strain and other closely related strains suggests that the operon encoding the first two enzymes in the PCP pathway were acquired from a pre-existing pterin-carbinolamine dehydratase subsystem. The next two enzymes were recruited (via horizontal gene transfer) from the pool of hypothetical proteins with no previous specific function while the last enzyme was recruited from pre-existing enzymes from the tricarboxylic acid cycle or serine-glyoxalase cycle via horizontal gene transfer events.