2-hydroxymuconic Semialdehyde Research Articles

Synchronous removal of gaseous toluene and benzene and degradation process shifts in microbial fuel cell-biotrickling filter system

Illustrating the biodegradation processes of multi-component volatile organic compounds (VOCs) will expedite the implication of biotechnology in purifying industrial exhaust. Here, performance shifts of microbial fuel cell and biotrickling filter combined system (MFC-BTF) are investigated for removing single and dual components of toluene and benzene. Synchronous removal of toluene (95 %) and benzene (97 %) are achieved by MFC-BTF accompanied with the output current of 0.41 mA. Elevated content of extracellular polymeric substance facilitates the mass transfer of benzene with the presence of toluene. Strains of Bacteroidota, Proteobacteria and Chloroflexi contribute to the removal of dual components VOCs. Empty bed reaction time and the VOCs concentration are the important factors influencing their dissolution in the system. The biodegradation of toluene and benzene proceeds with 2-hydroxymuconic semialdehyde and o-hydroxybenzoic acid as the main intermediates. These results provide a comprehensive understanding of multi-component VOCs removal by MFC-BTF and guide the system design, optimization, and scale-up.

Effect of mixing intensity on biodegradation of phenol in a moving bed biofilm reactor: Process optimization and external mass transfer study

In this work, an effort has been made to design the process variables and to analyse the impact of mixing intensity on mass transfer diffusion in a moving bed biofilm reactor (MBBR). A lab-scale MBBR, filled with Bacillus cereus GS2 IIT (BHU) immobilized-polyethylene biocarriers, was employed to optimize the process variables, including mixing intensity (60–140 rpm), phenol concentration (50–200 mg/L), and hydraulic retention time (HRT) (4–24 h) using response surface methodology. The optimum phenol removal of 87.64 % was found at 100 rpm of mixing intensity, 200 mg/L of phenol concentration, and 24 h of HRT. The higher mixing intensity improved the substrate diffusion between the liquid phase and the surface of the biofilm. The external mass transfer coefficients were found in the range of 1.431 × 10-5-1.845 × 10-5 m/s. Moreover, the detection of catechol and 2-hydroxymuconic semialdehyde revealed that the Bacillus sp. followed the meta-cleavage pathway during the biodegradation of phenol.

Cupriavidus metallidurans CH34 Possesses Aromatic Catabolic Versatility and Degrades Benzene in the Presence of Mercury and Cadmium.

Heavy metal co-contamination in crude oil-polluted environments may inhibit microbial bioremediation of hydrocarbons. The model heavy metal-resistant bacterium Cupriavidus metallidurans CH34 possesses cadmium and mercury resistance, as well as genes related to the catabolism of hazardous BTEX aromatic hydrocarbons. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation. Three chromosome-encoded bacterial multicomponent monooxygenases (BMMs) are involved in benzene catabolic pathways. Growth assessment, intermediates identification, and gene expression analysis indicate the functionality of the benzene catabolic pathway. Strain CH34 degraded benzene via phenol and 2-hydroxymuconic semialdehyde. Transcriptional analyses revealed a transition from the expression of catechol 2,3-dioxygenase (tomB) in the early exponential phase to catechol 1,2-dioxygenase (catA1 and catA2) in the late exponential phase. The minimum inhibitory concentration to Hg (II) and Cd (II) was significantly lower in the presence of benzene, demonstrating the effect of co-contamination on bacterial growth. Notably, this study showed that C. metallidurans CH34 degraded benzene in the presence of Hg (II) or Cd (II).

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-l-phenylalanine (l-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.

Preparation of 2-hydroxymuconic semialdehyde from catechol by combining enzymatic catalysis with bisulfite nucleophilic addition

2-hydroxymuconic semialdehyde (2-HMS) is a versatile intermediate widely used in organic synthesis. However, it is difficult to manufacture 2-HMS by common chemical methods since it has quite a few different functional groups, making it highly reactive and unstable. For the first time, the combination of an enzymatic catalysis with bisulfite addition was used to effectively prepare 2-HMS. Another benefit of the newly developed method is that the aldehyde-bisulfite adduct is much more stable and easier to store for long term. Intracellular enzymes from Pseudomonas stutzeri N2 could quickly and specifically catalyze the conversion of catechol into 2-HMS. The formed 2-HMS was converted to more stable aldehyde-bisulfite adduct via the bisulfite nucleophilic addition. 2-HMS-bisulfite adduct can be readily purified through a simple recrystallization. In the presence of sodium hydroxide, the adduct can be readily converted back to the desired 2-HMS product when needed. The conversion was optimized through the Box-Behnken design. The incubation of the bisulfite adduct with 3 M sodium hydroxide at 50 ℃ for 2 min gave 2-HMS in about 84% yield. The newly established method is considered to environment-friendly and can be easily scaled up for large-scale production.

Experimental and Genomic Evaluation of the Oestrogen Degrading Bacterium Rhodococcus equi ATCC13557

Rhodococcus equi ATCC13557 was selected as a model organism to study oestrogen degradation based on its previous ability to degrade 17α-ethinylestradiol (EE2). Biodegradation experiments revealed that R. equi ATCC13557 was unable to metabolise EE2. However, it was able to metabolise E2 with the major metabolite being E1 with no further degradation of E1. However, the conversion of E2 into E1 was incomplete, with 11.2 and 50.6% of E2 degraded in mixed (E1-E2-EE2) and E2-only conditions, respectively. Therefore, the metabolic pathway of E2 degradation by R. equi ATCC13557 may have two possible pathways. The genome of R. equi ATCC13557 was sequenced, assembled, and mapped for the first time. The genome analysis allowed the identification of genes possibly responsible for the observed biodegradation characteristics of R. equi ATCC13557. Several genes within R. equi ATCC13557 are similar, but not identical in sequence, to those identified within the genomes of other oestrogen degrading bacteria, including Pseudomonas putida strain SJTE-1 and Sphingomonas strain KC8. Homologous gene sequences coding for enzymes potentially involved in oestrogen degradation, most commonly a cytochrome P450 monooxygenase (oecB), extradiol dioxygenase (oecC), and 17β-hydroxysteroid dehydrogenase (oecA), were identified within the genome of R. equi ATCC13557. These searches also revealed a gene cluster potentially coding for enzymes involved in steroid/oestrogen degradation; 3-carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxy-phenyl)propionate hydroxylase, cytochrome P450 monooxygenase, and 3-oxosteroid 1-dehydrogenase. Further, the searches revealed steroid hormone metabolism gene clusters from the 9, 10-seco pathway, therefore R. equi ATCC13557 also has the potential to metabolise other steroid hormones such as cholesterol.

Extraction and purification of 2-hydroxymuconic semialdehyde accumulated in phenol degradation by Pseudomonas stutzeri N2

2-Hydroxymuconic semialdehyde (2-HMS) is a useful intermediate in organic synthesis, but there are few reports for its preparation. 2-HMS accumulates in phenol-containing wastewater treated with Pseudomonas stutzeri N2. This work has studied the extraction of 2-HMS so that the phenolic wastes of high concentration may be used to produce 2-HMS. When 4.25 mmol/L phenol was degraded by Pseudomonas stutzeri N2, the accumulated 2-HMS can reach 3.79 ± 0.08 mmol/L within 30 h. A saturated solution of NaHSO3 was added into the 2-HMS culture, and white rod-like precipitates formed gradually. Through SEM, XRD and FT-IR, the precipitates were identified as monoclinic hexagonal crystals of α-hydroxy sulfonate sodium with high purity. In the presence of NaOH, α-hydroxy sulfonate sodium could be reduced to the desired 2-HMS product. The optimized conditions were characterized to be 3 mol/L NaOH solution, 5 min and 50 °C. The conversion rate of α-hydroxy sulfonate sodium was up to 87.3 ± 0.2%, and 0.42 ± 0.02 mmol/L 2-HMS were obtained.

Effect of lignite activated coke packing on power generation and phenol degradation in microbial fuel cell treating high strength phenolic wastewater

Microbial fuel cell (MFC) is commonly used for easily biodegradable substances, yet its application for high strength phenolic wastewater treatment remains a challenge. This study incorporates lignite activated coke (LAC) into MFC to alleviate the inhibition of phenol and facilitate power generation of MFC. The power generation and phenol degradation in both LAC-MFC and control MFC were investigated at phenol concentrations of 500 mg/L, 700 mg/L and 1000 mg/L. The results showed that the maximum power density in LAC-MFC was maintained in the range of 252.3–278.7 mW/m2 when the phenol concentration increased from 500 mg/L to 1000 mg/L, whereas it was decreased from 246.5 mW/m2 to 167.5 mW/m2 in control-MFC. This corresponds to a high phenol removal of 89.4% to 94.5% in LAC-MFC but a significant decrease from 80.7% to 50.8% in control MFC. The inhibition coefficient (Ki) calculated from Haldane inhibition model decreased by 24.3% in LAC-MFC compare with control MFC, suggesting that the stress of high concentrations of phenol on MFC was largely relieved. In addition, the phenol degradation intermediate products of 2-hydroxymuconic semialdehyde, 4-hydroxybenzoic acid, 4-hydroxy-3-methylbenzoic acid were apparently lower in LAC-MFC than control MFC, indicating that the biodegradation of phenol was enhanced in LAC-MFC. High-throughput sequencing analysis revealed that Pseudomonas, Desulfuromonas, and Hydrogenophaga might be associated with phenol degradation and power generation in LAC-MFC. Because of its effectiveness in alleviating the toxic effects of phenol, LAC packing is a promising MFC technology for high strength phenolic wastewater treatment.

Evaluating metabolic potential of Thauera sp. M9 for the transformation of 4-chloroaniline (4-CA)

Abstract Thauera sp. M9, was isolated after enrichment of microbial diversity in nitrogen free mineral salt medium (NFMSM) supplemented with 300 mg/l of 4-chloroaniline (4-CA). The isolate M9 was able to achieve complete degradation of 300 mg/l 4-CA supplement to NFMSM, after 30 h incubation. In addition to 4-CA, isolate M9 was able to achieve complete degradation of 300 mg/l 3-chloroaniline (3-CA), 21% degradation of 300 mg/l 2-chloroaniline (2-CA), and complete transformation of 50 mg/l of 3, 4-dichloroaniline (3, 4-DCA), was observed only in the presence of 0.02% (w/v) yeast extract (YE) supplement to NFMSM. The presence of catechol 2, 3-dioxygenase (C23O) activity in cell free extract and detection of 4-chlorocatechol (4-CC); 2-hydroxy muconic semialdehyde (2-HMS) in biologically transformed samples by mass spectroscopy (MS) analysis indicated to meta-cleavage of the aromatic ring. A plug flow reactor (PFR) was developed using polyurethane foam (PUF) and refractory brick pieces (RBP) as immobilization matrix. The treatment of simulated effluent supplemented with 300 mg/l 4-CA through PFR, resulted in 88% transformation of 4-CA and 70% lowering of chemical oxygen demand (COD). These observations suggest that Thauera sp. M9 could be a potential inoculum for developing bioremediation protocols for waste rich in chloroanilines (CAs).

Co-metabolism for enhanced phenol degradation and bioelectricity generation in microbial fuel cell

Co-metabolism is one of the effective approaches to increase the removal of refractory pollutants in microbial fuel cells (MFCs), but studies on the links between the co-substrates and biodegradation remain limited. In this study, four external carbon resources were used as co-substrates for phenol removal and power generation in MFC. The result demonstrated that acetate was the most efficient co-substrate with an initial phenol degradation of 78.8% and the voltage output of 389.0 mV. Polarization curves and cyclic voltammogram analysis indicated that acetate significantly increased the activity of extracellular electron transfer (EET) enzyme of the anodic microorganism, such as cytochrome c OmcA. GC-MS and LC-MS results suggested that phenol was biodegraded via catechol, 2-hydroxymuconic semialdehyde, and pyruvic acid, and these intermediates were reduced apparently in acetate feeding MFC. The microbial community analysis by high-throughput sequencing showed that Acidovorax, Geobacter, and Thauera were predominant species when using acetate as co-substrate. It can be concluded that the efficient removal of phenol was contributed to the positive interactions between electrochemically active bacteria and phenolic degradation bacteria. This study might provide new insight into the positive role of the co-substrate during the treatment of phenolic wastewater by MFC.

Biokinetic aspects for biocatalytic remediation of xenobiotics polluted seawater.

This research was conducted to investigate the biocatalytic remediation of xenobiotics polluted seawater using two biocatalysts; whole bacterial cells of facultative aerobic halotolerant Corynebacterium variabilis Sh42 and its extracted crude enzymes. One-Factor-at-A-Time technique and statistical analysis were applied to study the effect of initial substrate concentrations, pH, temperature, and initial biocatalyst concentrations on the batch biocatalytic degradation of three xenobiotic pollutants (2-hydroxybiphenyl (2-HBP), catechol and benzoic acid) in artificial seawater (salinity 3·1%). HPLC and gas-chromatography mass spectroscopy analyses were utilized to illustrate the quantitative removal of the studied aromatic xenobiotic pollutants and their catabolic pathway. The results revealed that the microbial and enzymatic cultures followed substrate inhibition kinetics. Yano and Koga's equation showed the best fit for the biokinetic degradation rates of 2-HBP and benzoic acid, whereas Haldane biokinetic model adequately expressed the specific biodegradation rate of catechol. The biokinetic results indicated the good efficiency and tolerance of crude enzyme for biocatalytic degradation of extremely high concentrations of aromatic pollutants than whole C. variabilis Sh42 cells. The monitored by-products indicated that the catabolic degradation pathway followed an oxidation mechanism via a site-specific monooxygenase enzyme. Benzoic acid and catechol were identified as major intermediates in the biodegradation pathway of 2-HBP, which were then biodegraded through meta-cleavage to 2-hydroxymuconic semialdehyde. With time elapsed, the semialdehyde product was further biodegraded to acetaldehyde and pyruvic acid, which would be further metabolized via the bacterial TCA cycle. The batch enzymatic bioreactors performed superior-specific biocatalytic degradation rates for all the studied xenobiotic pollutants. The enzymatic system of C. variabilis Sh42 is tolerable for toxic xenobiotics and different physicochemical environmental parameters. Thus, it can be recommended as an effective biocatalyst for biocatalytic remediation of xenobiotics polluted seawater.

Mutation of Arthrospira platensis by gamma irradiation to promote phenol tolerance and CO2 fixation for coal-chemical flue gas reduction

In order to promote phenol tolerance and the carbon fixation rate of cells during CO2 sequestration in coal chemical flue gas, γ-ray irradiation from cobalt-60 (60Co) was used to mutate Arthrospira platensis to generate efficient strains. A phenol-tolerant strain Mutant 11k was obtained with 11,000 Gy of gamma irradiation. The phenol degradation efficiency of the mutant was 34.7 % higher than that of wild strain, attributing to the increased activity of phenol hydroxylase that catalyzed phenol to generate catechol. The catechol 1,2-dioxygenase activity in mutant cells was enhanced by 39.5 % while catechol 2,3-dioxygenase was weakened by 23.8 %, revealing that the β-ketoadipate pathway plays a more important role than 2-hydroxymuconic semialdehyde pathway in phenol degradation. The activity of superoxide dismutase and catalase increased by 6.1 % and 39.8 %, respectively, reducing peroxide damage and maintaining rapid cell growth. The contents of chlorophyll-a and maximum quantum yield of photosystem II in mutant cells were higher than those in wild strain. The biomass yield and carbon fixation rate of mutant were 20.8 % and 22.4 % higher, respectively, than those of the wild strain.

Metabolic profile analysis and kinetics of p-cresol biodegradation by an indigenous Pseudomonas citronellolis NS1 isolated from coke oven wastewater

A p-cresol degrading bacterial strain was isolated from coke oven wastewater of an iron and steel plant industry. The isolated strain was identified as Pseudomonas citronellolis NS1 by 16S rRNA gene sequencing method. The isolated Pseudomonas species could degrade 1200 mg l−1 of p-cresol completely in 40 h. Further, at 1200 mg l−1 of initial p-cresol more than 95.4% TOC and 98.5% toxicity removal were also achieved by the microbial culture. The metabolic pathway of the Pseudomonas species was investigated using HPLC, UV visible spectra, FT-IR and GC–MS analysis. The major metabolic intermediate products identified in the bacterial culture were 4-methyl catechol, protocatechuic acid, 3-oxadipic acid, 4- carboxy muconolactone, 2-hydroxy muconicsemialdehyde and maleic acid. Based on the identified metabolic intermediate products, a tentative new meta-cleavage 3-oxoadipate pathway has been proposed. The biodegradation of p-cresol by the culture followed first order kinetic. The biokinetic parameters associated with biodegradation of p-cresol have been estimated as follow; μ = 0.353 h−1, Ksi = 464 mg l−1, Ks = 99.5 mg l−1. It was observed that, when the initial concentrations of p-cresol exceeded more than 1000 mg l−1, about 70% of the total substrate carbon was utilized towards microbial maintenance energy.

Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas chlororaphis Strain UFB2.

Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaver–Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.

Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 via ipso-Hydroxylation of 3-(2,4-Dihydroxyphenyl)-propionic Acid.

A gene cluster, denoted as hcdABC, required for the degradation of 3-(2,4-dihydroxyphenyl)-propionic acid has been cloned from 7-hydroxycoumarin-degrading Pseudomonas mandelii 7HK4 (DSM 107615), and sequenced. Bioinformatic analysis shows that the operon hcdABC encodes a flavin-binding hydroxylase (HcdA), an extradiol dioxygenase (HcdB), and a putative hydroxymuconic semialdehyde hydrolase (HcdC). The analysis of the recombinant HcdA activity in vitro confirms that this enzyme belongs to the group of ipso-hydroxylases. The activity of the proteins HcdB and HcdC has been analyzed by using recombinant Escherichia coli cells. Identification of intermediate metabolites allowed us to confirm the predicted enzyme functions and to reconstruct the catabolic pathway of 3-(2,4-dihydroxyphenyl)-propionic acid. HcdA catalyzes the conversion of 3-(2,4-dihydroxyphenyl)-propionic acid to 3-(2,3,5-trihydroxyphenyl)-propionic acid through an ipso-hydroxylation followed by an internal (1,2-C,C)-shift of the alkyl moiety. Then, in the presence of HcdB, a subsequent oxidative meta-cleavage of the aromatic ring occurs, resulting in the corresponding linear product (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid. Here, we describe a Pseudomonas mandelii strain 7HK4 capable of degrading 7-hydroxycoumarin via 3-(2,4-dihydroxyphenyl)-propionic acid pathway.

Strategy of Pseudomonas pseudoalcaligenes C70 for effective degradation of phenol and salicylate.

Phenol- and naphthalene-degrading indigenous Pseudomonas pseudoalcaligenes strain C70 has great potential for the bioremediation of polluted areas. It harbours two chromosomally located catechol meta pathways, one of which is structurally and phylogenetically very similar to the Pseudomonas sp. CF600 dmp operon and the other to the P. stutzeri AN10 nah lower operon. The key enzymes of the catechol meta pathway, catechol 2,3-dioxygenase (C23O) from strain C70, PheB and NahH, have an amino acid identity of 85%. The metabolic and regulatory phenotypes of the wild-type and the mutant strain C70ΔpheB lacking pheB were evaluated. qRT-PCR data showed that in C70, the expression of pheB- and nahH-encoded C23O was induced by phenol and salicylate, respectively. We demonstrate that strain C70 is more effective in the degradation of phenol and salicylate, especially at higher substrate concentrations, when these compounds are present as a mixture; i.e., when both pathways are expressed. Moreover, NahH is able to substitute for the deleted PheB in phenol degradation when salicylate is also present in the growth medium. The appearance of a yellow intermediate 2-hydroxymuconic semialdehyde was followed by the accumulation of catechol in salicylate-containing growth medium, and lower expression levels and specific activities of the C23O of the sal operon were detected. However, the excretion of the toxic intermediate catechol to the growth medium was avoided when the growth medium was supplemented with phenol, seemingly due to the contribution of the second meta pathway encoded by the phe genes.

PYRENE BIODEGRADATION POTENTIALS OF AN ACTINOMYCETE, MICROBACTERIUM ESTERAROMATICUM ISOLATED FROM TROPICAL HYDROCARBON-CONTAMINATED SOIL

A novel pyrene-degrading actinomycete, phylogenetically identified as Microbacterium esteraromaticum strain SL9 was isolated from a polluted hydrocarbon-contaminated soil in Lagos, Nigeria. Growth of the isolate on pyrene was assayed using total viable counts, pyrene degradation was monitored using gas chromatography (GC-FID) while UV-Vis spectrophotometry was used to detect metabolites of pyrene degradation. The isolate tolerated salt concentration of up to 6%, grew luxuriantly on crude oil and exhibited weak utilization of fluorene, acenaphthene and engine oil. It resisted cefotaxime, ciprofloxacin and amoxicilin, but was susceptible to meropenem, linezolid and vancomycin. It also resisted elevated concentrations of heavy metals such as 1-5 mM lead and nickel. On pyrene, the isolate exhibited growth rate and doubling time of 0.023 h-1 and 1.25 h, respectively. It degraded 55.16 (27.58 mg L-1) and 89.28% (44.64 mg L-1) of pyrene (50 mg L-1) within 12 and 21 days respectively, while the rate of pyrene utilization was 0.09 mg L-1h-1. Catechol dioxygenase assay using UV-Vis spectrophotometry revealed the detection of meta cleavage compound, 2-hydroxymuconic semialdehyde in the crude cell lysate. The results of this study showed the catabolic versatility of Microbacterium species on hydrocarbon substrates and their potential as seeds for bioremediation of environments co-contaminated with polycyclic aromatic hydrocarbons and heavy metals.

Benzene biodegradation by indigenous mixed microbial culture: Kinetic modeling and process optimization

Benzene is one of hazardous pollutants generated from paint, chemical, and petrochemical industries have a harmful impact on human health and the atmosphere. This study reports benzene biodegradation by indigenous mixed microbial culture in shake flasks over a concentration ranging from 25 to 600 mg/l, and the kinetics involved in the process has been modeled. Experimental data obtained were fitted to both inhibition and noninhibition models to determine the biokinetic constants. Haldane model was best to predict the experimental data. The central composite design was further used for optimization of pH and benzene concentration to enhance the benzene biodegradation. At an optimum pH of 7.05 and benzene concentration of 332.82 mg/l, the maximum estimated specific growth rate and degradation rate were 0.05 1/h and 6.01 mg/l h, respectively. The LC-MS analysis of sample indicate the presence of catechol, cis-1,2-dihydrobenzene-1,2-diol, and 2-hydroxymuconate semialdehyde as intermediates, which justifies the developed pathway of benzene biodegradation. The predominant microorganism in the mixed culture responsible for benzene degradation was later identified to be Enterobacter cloacae SG208. The results provide insight into the process of benzene biodegradation and prove the potential of indigenous mixed culture for treatment of benzene.

Meta-cleavage pathway of phenol degradation by Acinetobacter sp. strain AQ5NOL 1

The characterization of bacterial enzymatic pathways of phenol metabolism is important to better understand phenol biodegradation. Phenol hydroxylase is the first enzyme involved in the oxidative metabolism of phenol, followed by further degradation via either meta- or ortho-pathways. In this study, the first known instance of phenol degradation via the meta-pathway by a member of the genus Acinetobacter (Acinetobacter sp. strain AQ5NOL 1) is reported. Phenol hydroxylase converts phenol to catechol, which is then converted via the meta-pathway to 2-hydroxymuconic semialdehyde by the catechol 2,3-dioxygenase enzyme. Phenol hydroxylase extracted from strain AQ5NOL 1 was fully purified using DEAE-Sepharose®, DEAE-Sephadex®, Q-Sepharose® and Zorbax® Bioseries GF-250 gel filtration and was demonstrated by SDS-PAGE to have a molecular weight of 50 kDa. The phenol hydroxylase was purified to about 210.51 fold. The optimum pH and temperature for enzyme activities are 20 °C and 7–7.5, respectively. The apparent K m and V max values of phenol hydroxylase with phenol as the substrate were 13.4 µM and 2.5 µmol min−1 mg−1, respectively. The enzyme was stable at −20 °C for 36 days.

Biodegradation of 4-chlorophenol in an airlift inner loop bioreactor with mixed consortium: effect of HRT, loading rate and biogenic substrate.

In the present work, removal of 4-chlorophenol (4-CP) by the mixed microbial consortium was evaluated in an airlift inner loop bioreactor. During the study, the effect of various reactor parameters such as hydraulic retention time (HRT), biogenetic substrate concentration, loading rate, and initial substrate concentration on the removal efficiency of 4-CP was investigated. Bioreactor showed a maximum removal rate of 16.59 mg/L/h at the optimum conditions of 24 h HRT, 400 mg/L initial 4-CP, and 0.2 g/L peptone. The optimum HRT found was 24 h after that the washout occured, and the degradation efficiency almost dropped to 50 % at 18 h HRT. Effect of peptone showed that lower concentration of peptone improves 4-CP removal efficiency of the bioreactor. Also, the mixed consortium had utilized 4-CP as a carbon source, as evidenced by the increasing biomass concentration with 4-CP at constant peptone concentration. The presence of 5-chloro 2-hydroxymuconic semialdehyde in the reactor infers that the mixed consortium has followed the meta-cleavage pathway for 4-CP degradation.