Inhibition of dehydrogenase activity in petroleum refinery wastewater bacteria by phenolic compounds

The toxicity of phenol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol on Pseudomonas, Bacillus and Escherichia species isolated from petroleum refinery wastewater was assessed via inhibition of dehydrogenase enzyme activity. At low concentrations, 2-nitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol stimulated dehydrogenase activity and at sufficient concentrations, phenolic compounds inhibited dehydrogenase activities. Generally, phenol is less toxic than substituted phenols. Estimations of the degree of inhibition/stimulation of dehydrogenase activities showed significant dose-dependent responses that are describable by logistic functions. The toxicity thresholds varied significantly (P < 0.05) among the bacterial strains and phenolic compounds. The median inhibitory concentrations (IC50s) ranged from 4.118 ± 0.097 mg.L for 4-nitrophenol against Pseudomonas sp. DAF1 to 1407.997 ± 7.091 mg.L for phenol against Bacillus sp. DISK1. This study suggested that the organisms have moderate sensitivity to phenols and have the potential to be used as indicators for assessment of chemical toxicity. They could also be used as catalysts for degradation of phenols in effluents.


INTRODUCTION
Phenolic compounds from myriads of petrochemical industries are among the pollutants most ubiquitously distributed in industrial effluents.Due to its wide distribution and injurious effects on humans, phenols are considered important environmental pollutants and their removal is of obvious interest.One of the most efficient approaches to phenol removal is biodegradation.
Wide ranges of microorganisms including bacteria, fungi and algae have been reported to degrade phenolic compounds.However, due to their toxicity, microbial degradation of phenolic compounds is usually inhibited at high concentrations (Goudar et al., 2000;Choi and Gu, 2001;Goudar and Delvin, 2001;Acuña-Argüelles et al., 2003;Oboirien et al., 2005;Okpokwasili and Nweke, 2006).In order to evaluate pollution risk of phenolic compounds in wastewater, it is important to assess their toxicity.Rapid and sensitive bioassays have been developed for assessment of toxicity of phenolic compounds.The estimation of respiratory activity is one of the most usually used laboratory screening tests (King, 1984;King and Dutka, 1986;King and Painter, 1986;Cenci et al., 1987;Strotmann et al., 1993;Dalzell et al., 2002;Okolo et al., 2007).In this assay, rates of oxygen uptake and reduction of redox indicators are followed polarographically and spectrophotometrically respectively.In the later approach, activities of dehydrogenase enzymes are determined via reduction of redox indicators to coloured forms whose intensity is measured in a spectrophotometer.
In this study, we assessed the toxicity of eight phenolic compounds to phenol-degrading bacterial strains isolated from petroleum refinery wastewater via reduction of 2,3,5triphenyltetrazolium chloride (TTC) to red-coloured triphenyl formazan (TPF).

Wastewater and bacterial strains
Pure cultures of bacteria were isolated from wastewater of Port Harcourt crude oil refinery.The untreated wastewater samples include the process wastewater derived from the refining process (PWW) and the raw wastewater (RWW) which is a combination of PWW and sewage that is channeled to the dissolved air floatation unit (DAF) for physical removal of oil droplet and then to the rotary biodisk (DISK) for biological treatment.The treated wastewater samples include treated wastewater, which is refinery effluent that has undergone both chemical and biological treatment to eliminate or reduce contents, and the observation pond wastewater (OPWW).Water samples were collected in sterile bottles, stored in a cooler and taken to the laboratory for microbiological analyses.The samples were analyzed within 6 h of collection to avoid deterioration of sample.The phenol-degrading bacteria were isolated on mineral salts agar supplemented with phenol as the only source of carbon and energy (Hill and Robinson, 1975).The phenol-degrading bacteria growing on the mineral salts-phenol agar were purified on nutrient agar (Lab M) and stored in nutrient agar slants at 4 o C. The isolates were characterized biochemically using standard microbiological methods.Identification to generic level followed the scheme of Holt et al. (1994).The phenoldegrading bacterial strains, Pseudomonas sp.DAF1 and Pseudomonas sp.RWW2 were isolated from the dissolved air floatation unit and the raw wastewater respectively.Bacillus sp.DISK1 and Escherichia sp.DISK2 were isolated from the rotary biodisk wastewater.The bacterial strains represent the preponderant morphotypes in their respective sources.

Dehydrogenase assay
Dehydrogenase activity (DHA) was determined using 2,3,5-triphenyltetrazolium chloride (TTC) as the artificial electron acceptor, which is reduced to red-coloured triphenyl formazan (TPF).The assay was done in 3-ml volume of nutrient broth-glucose-TTC medium supplemented with varying concentrations of phenolic compounds in separate screw-capped test tubes.Portions (0.3 ml) of washed bacterial suspensions (A 420 = 0.5) were inoculated into triplicate glass tubes containing 2.5 ml of phthalate-buffered (pH 7.0) nutrient broth glucose medium amended with each phenolic compound.Thereafter, 0.2 ml of 0.4% (w/v) TTC in deionized distilled water was added to each tube to obtain final concentrations of 20 -200 mg.L -1 (substituted phenols) and 200 -2000 mg.L -1 (phenol).The final concentrations of nutrient broth and glucose in the medium were 2 mg/ml each.The controls consisted of the isolates and the media without phenolic compound.The reaction mixtures were incubated under static conditions at room temperature (28 ± 2°C) for 24 h.The TPF produced was extracted in 4 ml of amyl alcohol and determined spectrophotometrically.The dehydrogenase activities as percent of control were computed.

Data analysis
Data were expressed as the mean and standard deviations.The effect of phenolic compounds on dehydrogenase activity was calculated relative to the control as shown in equation 1.To estimate the toxicity thresholds (IC 20 , IC 50 and IC 80 ), the data generated from equation 1 were fitted into logistic dose-response model (equation 2).For the responses with stimulation of dehydrogenase activity, data were fitted into asymmetric logistic dose-response model (equation 3).Curve fitting was done by iterative minimization of least squares using Levenberg-Marquardt algorithm of Table Curve 2D.All regression was done using the mean data and standard deviation.The toxicity thresholds for each bacterium and phenolic compound was compared pairwise using student's t-test with the levels of significance set at P < 0.05.
[1] [2] where C A is the absorbance of triphenyl formazan produced in uninhibited control (without phenolic compound), T A the absorbance of triphenyl formazan produced in inhibited test (with different concentrations of phenolic compound), x is the concentration of phenolic compound, a the uninhibited value of enzyme activity (100 %), b is IC 50 and c is dimensionless toxicity parameter.
[3] where a, b, c and d are model parameters.
Generally, phenol was less toxic to the organisms than the substituted phenols.In Pseudomonas sp.DAF1, Pseudomonas sp.RWW2 and Escherichia sp.DISK2, phenol inhibited dehydrogenase activity with successive increase in the concentration of phenol.Total inhibition of dehydrogenase activity occurred at 1200 and 1400 mg.L -1 in Escherichia sp.DISK2 and Pseudomonas sp.DAF1 respectively.At concentrations ranging from 200 to 900 mg.L -1 , phenol stimulated dehydrogenase activity in Bacillus sp.DISK1 and thereafter inhibited it until total inhibition at 2000 mg.L -1 .Stimulation of dehydrogenase activity in some bacteria by phenolic compounds could indicate the use of the phenol as a growth substrate.Pseudomonas, Bacillus and Escherichia species have been reported to degrade phenol and other substituted phenols (Dapaah and Hill, 1992;Hollender et al., 1994;Monserrate and Hăggblom, 19997;Jain et al., 1994).Similar stimulation of dehydrogenase activity in a soil Acinetobacter species by 4-nitrophenol and 2,4-dinitrophenol was reported by Okolo et al. (2007).The progressive inhibition of dehydrogenase activity with increasing concentration of phenols is in line with the well documented inhibitory nature of phenols at high concentrations for organisms which can use phenols as growth substrates (Acuña-Argüelles et al., 2003;Ruiz-Ordaz et al., 1998).The substituted phenols inhibited dehydrogenase activity more than phenol.This greater toxicity of substituted phenols have been reported.For instance, Cenci et al. (1987) reported that chlorophenols inhibited dehydrogenase activity in bacteria more than phenol.In a similar dehydrogenase activity assay using Pseudomonas putida, nitrophenols and chlorophenols was reported to be more toxic than phenol (Gül and Öztürk, 1998).In a bioluminescence assay, phenol was reported to be less toxic than 4-bromophenol, chlorophenols and nitrophenols (Ren and Frymier, 2002).
The dose-response patterns of the organisms are describable by logistic functions with high coefficient of regression (R 2 > 0.9).The toxicity threshold concentrations are shown in Table 1.The median inhibitory concentrations (IC 50 s) ranged from 4.118 ± 0.097 mg.L -1 for 4-nitrophenol against Pseudomonas sp.DAF1 to 1407.997 ± 7.091 mg.L -1 for phenol against Bacillus sp.DISK1.The IC 50 of phenol varied significantly (P < 0.05) with that of other phenolic compounds.Also, the statistical analysis indicated that the toxicity thresholds varied significantly among the phenolic compounds and bacteria.Variable toxicity thresholds estimated from inhibition of dehydrogenase activity have been reported.These are shown in Table 2.The IC 50 of phenol against Escherichia coli reported by Cenci et al. (1987) is comparable with those reported in this study.However, the toxicity thresholds in this study were lower than the values reported by Gül andÖztürk, 1998. Abbondanzi et al. (2003) reported lower IC 50 of 210 mg.L -1 phenol for Pseudomonas fluorescens.Based on 5-day oxygen consumption during biodegradation of peptone by mixed bacterial culture, Tišler and Zagorc-Končan (1995) reported phenol IC 50 of 487 mg.L -1 .The artificial electron acceptor, 2,3,5-triphenyltetrazolium chloride (TTC) has been widely used as a measure of microbial growth (Tengerdy et al., 1967;Ghaly and Ben-Hassan, 1993).Abbondanzi et al. (2003) has also suggested good correlation of TTC-dehydrogenase activity with microbial growth.Thus, the toxicity thresholds obtained from growth inhibition data were compared with that obtained in this study.In this regard, the growth inhibition IC 50 s reported for phenol by Dutka and Kwan (1981) are comparable with the values reported in this study (Table 2).Similarly, the toxicity thresholds obtained for substituted phenols through inhibition of respiration and growth were in some cases comparable with our TTC-dehydrogenase thresholds (see Tables 1 and 2).Pseudomonas and Escherichia species in this study seem to be of moderate sensitivity.Bacillus sp.DISK1 with IC 50 of 1407.997 ± 7.091 mg.L -1 could be considered a resistant strain.Dehydrogenase activity in this bacterium was stimulated by phenol concentrations up to 800 mg.L -1 , and respiration still occur in the presence of 1800 mg.L -1 phenol.Thus, the Bacillus species have potential to be used in biotreatment of phenolic wastewater.Staphylococcus, Corynebacterium, Bacillus and Proteus species have been found to resist 10 mM (941.08 mg.L -1 ) phenol (Ajaz et al., 2004).

CONCLUSION
The results of the in vitro toxicity assays indicate that increasing concentrations of phenolic compound are potentially toxic to phenol-degrading bacteria in petroleum refinery wastewater.However, the organisms tolerated low concentrations of phenols and stimulated dehydrogenase activities.Thus, they could be used as catalysts for degradation of phenolic compounds in effluents.Nevertheless, in order to achieve biological oxidation and mineralization of phenolic compounds in the wastewater, the concentrations of phenols must be finely adjusted to reduce toxicity in wastewater treatment plants.

Table 1 .
Toxicity threshold concentrations of phenolic compounds for inhibition of dehydrogenase activity in the bacterial strains.

Table 2 .
Some reported toxicity threshold concentrations for phenolompcompounds.