Kinetics of Biodegradation of Phenol and p-nitrophenol by Acclimated Activated Sludge

The removal of highly toxic phenolic compounds, such as phenol and p-nitrophenol (PNP) from wastewater through biological approach provides a good cleanup strategy in wastewater treatment. It is recognised as an efficient green treatment with relatively low cost in comparison to conventional physicochemical processes. In this present study, activated sludge was acclimated to phenol and PNP, respectively, using sequencing batch reactor (SBR) system. The growth kinetics of both phenoland PNPacclimated activated sludge were well described by Haldane model. The values of the Haldane kinetic parameters, namely, maximum specific growth rate, μmax, saturation constant, Ks, and inhibition constant, Ki, were found to be 0.4039 and 0.1346 h−1, 5.393 and 110.3 mg l−1, and 550.8 and 63.58 mg l−1, respectively. The smaller ratio of Ks/Ki for phenol degradation compared to that of PNP biodegradation indicates the higher resistance of phenol-acclimated activated sludge to inhibition by its substrate. The pseudo zeroth-order equation was able to describe the phenol degradation kinetics at different initial concentrations. However, the equation was inadequate for the PNP degradation which was attributed to its higher toxicity compared to phenol.


INTRODUCTION
Wastewater is a worldwide concern as it presents a major health and environmental management challenge due to the presence of toxic and hazardous pollutants such as phenol and p-nitrophenol (PNP). 1,2Therefore, it needs to be treated properly prior to being discharged to the environment.In contrast to physicochemical methods, biological approaches provide highly reliable and cost-effective treatment for the complete removal of toxic pollutants. 3Activated sludge process is one of the biological approaches in treating wastewater using mixed culture.Besides its simple operation, it is very efficient in mineralising toxic organic compound. 4 was reported that acclimation process could enhance the ability of activated sludge to achieve complete mineralisation. 5,6Complete removals can be achieved without experiencing inhibition at high concentrations of phenol and PNP at 1050 and 500 mg l −1 , respectively, using acclimated biomass were reported. 5,6In the study by Sam and Ng, phenol biodegradation time was found to decrease from 330-1260 min to 35-330 min for initial phenol concentration of 25-250 mg l −1 after the acclimation of activated sludge to the target compound. 7owledge on biodegradation kinetics of toxic compounds is essential to improve the removal efficiency and process control.The degradation of phenolic compounds has been known to be inhibited by phenolic compounds themselves, especially at high concentrations. 1,7Thus, Haldane model is usually used to describe the growth kinetics of microorganism.Biomass growth can be defined as the increase of quantity of cellular constituents and structure which is accompanied by the increment of size and number of cells.It undergoes several phases, namely lag, exponential, stationary and death phases. 3In view of the wide range of concentrations of phenolic compounds in industrial wastewaters, the aim of this study is to investigate the growth and biodegradation kinetics of phenol and PNP at increasing concentrations in order to comprehend the effect of inhibition on the growth trends of acclimated activated sludges and thus their biodegradation performance.

Culturing and Acclimation of Activated Sludge
The seed of activated sludge was collected from a local municipal sewage treatment plant at Batu Ferringhi, Penang, Malaysia.It was then cultured using two identical laboratory-scale sequencing batch reactors (SBRs), namely, R-P and R-PNP.Each reactor with the total working volume of 5 l was operated with five operational periods of FILL, REACT, SETTLE, DRAW and IDLE with the time ratio of 2: 8: 1: 0.75: 0.25 for a 24 h cycle.The reactors were fed with the base mix of the following compositions (in mg l −1 ): bacto-peptone (188), sucrose (563), KH 2 PO 4 (32), K 2 HPO 4 (180), (NH 4 ) 2 SO 4 (212), NaHCO 3 (500), MgSO 4 (49), FeCl 3 •H 2 O (18.8) and CaCl 2 (40).A volume of 3.5 l of feed solution was added into the reactor and equal volume of treated effluent was withdrawn from the reactor during the FILL and DRAW periods, respectively.The sludge age was maintained at 20 days by wasting 250 ml of mixed liquor at the end of the REACT period.
The activated sludges were then acclimated to the final acclimation concentration of 200 mg l −1 phenol and 250 mg l −1 PNP, respectively, in reactors R-P and R-PNP, without supplementing sucrose and bacto-peptone.Both acclimated sludges were used in the biodegradation studies once the quasi-steady state was attained, indicated by the constant values of mixed liquor suspended solid (MLSS) of 1110 ± 50 and 600 ± 50 mg l −1 for R-P and R-PNP, respectively.

Biodegradation Kinetic Study
Biodegradations of phenol and PNP were carried out by using phenol-and PNPacclimated activated sludges, respectively, in a batch mode.The degradation studies were investigated by varying the initial concentrations of phenol (0 to 1000 mg l −1 ) or PNP (0 to 500 mg l −1 ) in the presence of 400 mg l −1 of acclimated activated sludges and nutrients with the same composition as base mix.The residual concentration of phenol was determined using 4-aminoantipyrine method and measured spectrometrically at 506 nm while the PNP concentration was determined at 401 nm. 8,9The biomass growth in terms of mixed liquor vapour suspended solid (MLVSS) was determined using optical density measurement at 600 nm.The complete mineralisation of phenolic compounds was ascertained by chemical oxygen demand (COD) concentrations using closed reflux and titrimetric method. 8e specific biomass growth data was fitted into Haldane model using MATLAB software R2017a.The Haldane equation is given as below: where S o is the initial substrate concentration (mg l −1 ), µ max is the maximum specific growth rate (h −1 ), K s is the half-saturation constant (mg l −1 ) and K i is the substrate inhibition constant (mg l −1 ).The value of specific growth rate, µ, was determined using a graph of biomass versus time.The kinetic data of phenolic compound degradation was fitted into pseudo zeroth-order equation shown below: where S o is the initial substrate concentration (mg l −1 ), S is substrate concentration at time, t and k is the pseudo zeroth-order rate constant.The ability of Haldane model in describing the growth kinetics in the presence of inhibitory effects was also reported in other studies. 1,10In this study, it was observed that specific growth rates, µ, increased with the increase of phenol and PNP concentrations up to 100 and 50 mg l −1 , respectively.The µ values decreased subsequently with the increase in substrate concentrations.This revealed the inhibition behaviour of phenolic compounds on acclimated activated sludges at higher concentrations. 7,11e values of kinetic parameters obtained from the fitting of Haldane model are: μ max = 0.4039 and 0.1346 h −1 , K s = 5.393 and 110.3 mg l −1 , K i = 550.8and 63.58 mg l −1 with the R 2 = 0.9677 and 0.9764 for phenol and PNP degradations, respectively.Based on the K i values obtained, the inhibition effect occurred at phenol and PNP concentrations higher than 550.8 and 63.58 mg l −1 , respectively.Furthermore, it was reported that the degree of inhibition can be illustrated by the K s /K i ratio, in which the resistance of acclimated activated sludge towards the inhibitory effects was indicated by a small K s /K i ratio. 7,12In this study, the K s /K i ratio for phenol degradation (0.0098) was found to be much smaller compared to that of PNP degradation (1.735).Thus, phenol-acclimated activated sludge was able to grow within the phenol concentration range studied without any observable inhibition.

RESULTS AND DISCUSSION
The kinetics of biodegradation of phenol and PNP fitted well to the pseudo-zerothorder equation with R 2 values > 0.9.The value of rate constant, k, was determined from the slope of the plot of different phenol or PNP initial concentration against time and the values are presented in Table 1.For the cases of phenol biodegradation, the highest value of k was obtained at the initial phenol concentration of 50 mg l −1 .This result is in agreement with the study of growth kinetics in which the μ max for phenol-acclimated activated sludge was obtained at this concentration (Figure 1(a)).In general, above 50 mg l −1 a gradual decrease of k value was observed with increasing phenol concentration.The decreasing trend is again in agreement with the Haldane fitting.For PNP biodegradation, the increasing trend of k values from 50 to 250 mg l −1 contradicts the growth kinetics results obtained using Haldane model (Figure 1(b)).Higher toxicity of PNP (EC 50 =64 mg l −1 ) compared to phenol (EC 50 =270 mg l −1 ) resulted in significant inhibitory effect at PNP concentration as low as 63.58 mg l −1 as predicted based on the Haldane model. 13Thus, the use of pseudo-zeroth order equation in describing the biodegradation kinetics of PNP is not sastifactory as it does not take into account the effect of inhibition at high PNP concentration.

CONCLUSION
Specific biomass growth was well descibed by Haldane model with the kinetic parameters, μ max = 0.4039 and 0.1346 h −1 , K s = 5.393 and 110.3 mg l −1 and K s = 550.8and 63.58 mg −1 for both phenol and PNP degradations, respectively.Low degree of inhibition for phenol biodegradation in the concentration range studied was reflected by the smaller K s /K i ratio compared to that of PNP biodegradation.Therefore, phenol-acclimated activated sludge was able to grow at phenol concentration up to 1000 mg l −1 without experiencing inhibition.In addition, pseudo zeroth-order kinetics was used to describe the biodegradation kinetics of phenol biodegradation.However, this equation does not incorporate the inhibition effect and thus it is inadequate to explain the cases of PNP degradation at high concentrations which is attributed to its high toxicity.

Figure 1 :
Figure 1: Experimental and Haldane predicted specific growth rates at different initial (a) phenol and (b) PNP concentrations.Complete mineralisations of phenol and PNP were achieved as indicated by the COD concentrations below 20 mg l −1 .The experimental data of specific growth rate was plotted against various phenol (Figure 1(a)) and PNP concentrations (Figure 1(b)).The data was observed to be well-fitted into the Haldane model.The ability of Haldane model in describing the growth kinetics in the presence of inhibitory effects was also reported in other studies.1,10In this study, it was observed that specific growth rates, µ, increased with the increase of phenol and PNP concentrations up to 100 and 50 mg l −1 , respectively.The µ values decreased subsequently with the increase in substrate concentrations.This revealed the inhibition behaviour of phenolic compounds on acclimated activated sludges at higher concentrations.7,11

Table 1 :
Mean values of the pseudo zeroth-order rate constants for phenol and PNP biodegradations.