Biodegradation of benzyl butyl phthalate and dibutyl phthalate by Arthrobacter sp. via micellar solubilization in a surfactant-aided system

Endocrine-disrupting chemicals (EDCs) like phthalates, mostly discharged in industrial effluents, cause detrimental effects to different life forms, and hence their removal from constituent wastewater is necessary. This study investigated the kinetics of biomass growth and biodegradation of benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) by Arthrobacter sp. in a surfactant-aided batch system. The effect of different surfactants on aqueous solubility of BBP and DBP was initially examined, which showed that Tween 80 resulted in maximum bioavailability and biodegradation efficiency of the phthalates by the bacterium and without inhibiting the biomass growth. Compared with BBP, DBP was found to be efficiently degraded and supported the bacterial growth within a short period of time over the entire concentration tested in the range 100–1,000 mg L . A maximum biomass concentration of 1.819 g L 1 was obtained at 120 h for a DBP concentration of 600 mg L 1 in the presence of Tween 80, which is 5.66-fold increase in biomass concentration as compared with only DBP as the sole substrate. For evaluating the biokinetic parameters involved in DBP biodegradation, the experimental data on DBP utilization were fitted to various kinetic models as reported in the literature.


).
Hence, different strategies to improve the bioavailability and therefore biodegradation of these compounds have been proposed, which include application of surfactants (Franzetti et al. ; Paria ), decrease in temperature (Thomsen et al. ), solid-dispersion, particle size reduction, nanonization, cosolvency and inclusion complexation (Kumar & Singh ).
Among the afore-mentioned strategies, surfactants are conventionally applied to increase the aqueous solubility of these phthalates for enhancing their bioavailability and biodegradation (Aryal & Liakopoulou-Kyriakides ).
Surfactants distinctively enhance the bioavailability of hydrophobic organic pollutants either by decreasing interfacial tension between aqueous and non-aqueous phases (Cuny et al. ) or by solubilization of hydrophobic organic compounds in the core of the micelles formed by surface-active agents (Sartoros et al. ; Hadibarata & Tachibana ). In the case of poorly water-soluble compounds, it has been reported that bacteria adhere to the liquid-liquid interface for lowering the interfacial tension and causing direct uptake of these pollutants, thereby enhancing their biodegradation rates (Volkering et al.  Moreover, there has been no study carried out to establish the potential of Arthrobacter sp. to degrade EDCs in a surfactant-aided system. Besides, details of kinetics of biodegradation and biomass growth by the organism using phthalates as the carbon source are lacking for establishing its application potential. Hence, this study was aimed at biodegradation of phthalates by Arthrobacter sp. using BBP and DBP as the carbon source. In order to enhance the bioavailability of these compounds, four chemical surfactants, viz. Tween 20, Tween 80, Triton X-100 and sodium dodecyl sulfate (SDS), were assessed as to their capability to solubilize BBP and DBP in aqueous media.
The results of biomass growth and degradation of BBP and DBP by Arthrobacter sp. obtained in the batch experiments conducted at different initial concentrations of the compounds and in the presence of a surfactant were compared with that obtained in experiments without any added surfactant. In order to gain further insight into the biokinetics of DBP biodegradation and biomass growth by Arthrobacter sp. in a surfactant-aided system the experimental data were fitted to different kinetic models reported in the literature and the biokinetic parameters involved were evaluated in the study. To our knowledge, this is the first study to show some new evidence for the biodegradation of both BBP and DBP using Arthrobacter sp. along with a detailed study on the biodegradation of these compounds.

Chemicals and reagents
Benzyl butyl phthalate (>97% pure) and dibutyl phthalate (>97% pure) used in the study were obtained from TCI Chemicals Pvt. Ltd (Chennai, India). Analytical grade chemicals for preparing the bacterial growth media were obtained from Merck (Mumbai, India). Solvents (HPLC grade), including methanol, acetonitrile, and water were procured from Finar Limited (Ahmedabad, India). The surfactants Tween 20, Tween 80, Triton X-100 and SDS were purchased from either Hi-Media, Merck or Sigma-Aldrich.  Biomass dry weight was expressed as cell dry weight (CDW, mg L À1 ), and the biomass specific growth rate (μ) was calculated as per the following Equation (1):

Aqueous solubility of phthalates with surfactants
where μ is the biomass specific growth rate (h À1 ), X is the biomass concentration (g L À1 ) corresponding to time t (h).
The biokinetics of DBP utilization by Arthrobacter sp.
were analyzed by fitting the experimental data to first order, logarithmic and Logistic kinetic models (Equations (2)-(4)).
First order where S and S 0 are the instantaneous and initial DBP concentrations (g L À1 ), respectively. X 0 is the initial biomass concentration (g L À1 ), K is the first-order rate constant (h À1 ), μ max is the maximum DBP specific utilization rate (h À1 ), and K S is the half-saturation coefficient (mg L À1 ). In addition, the following modified form of the Gompertz model was used to explain DBP utilization by Arthrobacter sp.
where r smax is the maximum rate of DBP utilization (mg L À1 h À1 ), S max is the maximum utilizable DBP concentration (mg L À1 ) and λ is lag time for DBP utilization (h).
The effect of initial DBP concentration on its specific utilization rate (q, h À1 ) by the bacterium was calculated by using the following Equation (6), and the experimental data fitted to different bio-kinetic models reported in the literature (Equations (7)-(13)): Yano and Koga where q is the specific DBP utilization rate (h À1 ), q max is the maximum specific DBP utilization rate (h À1 ), S is the DBP concentration (mg L À1 ), S m is the DBP concentration above which net growth ceases (mg L À1 ), K s is the half-saturation constant (mg L À1 ), K i is the inhibition constant (mg L À1 ), K 1 is a positive constant and n and m are empirical constants.
In addition to the above, the following two bio-kinetic models (Equations (14) and (15)) were further applied to simulate the biomass growth.
where X o is the initial biomass concentration, X is the biomass concentration at time t, X max is the maximum biomass concentration (g L À1 ) and r xmax is the maximum rate of biomass growth (g L À1 h À1 ).   (Figure 2(a) and 2(b)). These results clearly reveal that the biomass growth rate is relatively lower in the absence of surfactant, probably due to poor solubility and therefore very low bioavailability of these compounds in the media for an efficient utilization by the bacterium. All these results clearly reveal that in the presence of surfactant, the bacterium is very capable of utilizing phthalates as the sole carbon source. However, in the presence of Tween 80, Arthrobacter sp. showed a lag period in its growth due to BBP, which was not observed with DBP.

Residual
The lag phase in growth of the bacterium due to BBP can be attributed to its greater recalcitrance compared to DBP.
Hence, in order to further confirm the uptake of BBP and DBP by the bacterium for its growth, their biodegradation in the presence of Tween 80 was analyzed.     bacter sp. is found to be highly efficient in quickly degrading DBP even up to 1,000 mg L À1 . In order to gain further insight into DBP degradation by the bacterium, kinetics of the biodegradation process were further analyzed.

Kinetics of DBP utilization and biomass growth by
Arthrobacter sp.
Experimental and predicted DBP utilization by Arthrobacter sp. obtained using different biokinetic models are shown in Figure 6, and estimated values of biokinetic parameters from these models are presented in Table 2. Among the three different models tested -First order, Logarithmic, and Logistic kinetic models, the Logistic model accurately fitted the experimental data for all the initial concentrations of DBP with a coefficient of determination (R 2 ) value greater than 0.98 ( within 3 days of culture, and kinetic analysis of the results revealed that DBP biodegradation followed first-order kinetics but only for an initial concentration less than 100 mg L À1 .
In order to further understand the growth-associated

The effect of different initial DBP concentration on
Arthrobacter sp. biomass growth was further modelled using modified Gompertz and Logistic models. Figure 8 presents the experimental and the predicted Arthrobacter sp. biomass growth at different initial DBP concentrations; the biokinetic parameters assessed from the two models are presented in Table 4. It can be clearly observed that the modified Gompertz model is better than the Logistics model in explaining the experimental results (R 2 > 0.97).
The predicted maximum specific growth rate of Arthrobacter  Moreover, identification of metabolites formed during