Introduction

Cresol and other phenolic compounds enter the environment through wastewater discharged from a variety of industries involved in leather processing, phenol–formaldehyde resin manufacture, oil refinery, pharmaceuticals, coal conversion, paper production, etc., (Aleksieva et al. 2002; Arutchelvan et al. 2005; Saravanan et al. 2008). Of the three isomers of cresols, m-cresol is used quite extensively as a textile-scouring agent (Tsai et al. 2006). Discharge of wastewater containing m-cresol into receiving water bodies leads to several health problems owing to its toxic properties; hence, necessitating its removal from aqueous effluents before final disposal into the environment. Microbial degradation of m-cresol has been shown to be a useful strategy to effectively eliminate these compounds from wastewaters (Yan et al. 2006; Bai et al. 2007). Biodegradation of cresols by pure microbial species, under both aerobic and anaerobic conditions are known (Hao et al. 2002; Kumar et al. 2005); however, a mixed culture of microorganisms is well suited for complete degradation of the compound, and to maintain a high efficiency of degradation in the process. Being highly recalcitrant to biodegradation, m-cresol does not provide necessary energy to sustain the growth of microbial cultures in maintaining high process efficiency. Although an alternative simple carbon source may serve the purpose, kinetics of m-cresol degradation may be affected, and therefore, such type of degradation systems have to be properly dealt with for its better applicability. In this study, an indigenous mixed microbial culture, which failed to grow in media containing m-cresol as the sole source of carbon, was investigated of its m-cresol biodegradation potential in the presence of glucose as an alternative carbon source. The indigenous culture was isolated from petroleum-contaminated soil of a gasoline filling station.

Materials and methods

Chemicals and reagents

m-Cresol and 3,5-dinitrosalicylic acid used in this study were of analytical grade, and obtained from S.D. Fine Chemicals, India. Reagent-grade inorganic salts and other chemicals used in the preparation of microbial growth media were purchased from Himedia Laboratories, India.

Microorganism and culture conditions

Soil, contaminated with petroleum hydrocarbons and collected from a gasoline filling station located in North Guwahati, India, was used in isolation of an indigenous mixed consortium of microbes. The microbial isolation procedure as reported by Nuhoglu and Yalcin (2005) was adopted in this study. Briefly, 10 g (wet weight basis) of the soil was taken in a 250-ml Erlenmeyer flask containing 2 g l−1 glucose in 100 ml of Mineral Salt Medium (MSM) having the following composition (g l−1): K2HPO4 (0.8), KH2PO4 (0.21), (NH4)2SO4 (1), MgSO4 (0.5) and CaCl2 (0.01), pH 7.0. Following incubation of the flasks for 12 h at 30 ± 1°C in a rotating orbital incubator shaker (WIS-S10, DAIHAN Scientific Co., Ltd., Korea) set at 150 rpm, the contents were centrifuged at 5,000 rpm for 15 min to separate the soil and suspended particles; the resulting supernatant containing the microbial biomass was subsequently used in further experiments. Acclimatization of the culture to grow and degrade m-cresol was then performed over a period of more than 1 month with MSM, glucose, at 250 mg l−1 initial concentration, and m-cresol as the main carbon source, up to a maximum initial concentration of 1,000 mg l−1. Addition of glucose during the acclimatization stage was necessary as the culture initially failed to grow in MSM supplemented with only m-cresol as the sole carbon source. m-Cresol degradation profile followed during acclimatization of the culture is presented in Fig. 1, which shows the capability of the culture to degrade m-cresol completely, even at its high concentrations.

Fig. 1
figure 1

m-Cresol degradation profile followed during the acclimatization period

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Biodegradation experiments

All batch biodegradation experiments using the acclimatized indigenous mixed microbial culture were conducted in 250-ml Erlenmeyer flasks with 100 ml of MSM supplemented m-cresol and glucose. Initial m-cresol concentration in the media was varied from 50 to 400 mg l−1 and that of glucose was added, at either 250 or 500 mg l−1. The flasks were agitated at 150 rpm in an orbital incubator shaker maintained at 30°C, and each experimental condition was duplicated. Samples were drawn at regular intervals of time for a period required to complete m-cresol degradation and glucose utilization by the culture, which typically ranged from 10 to 26 h in case of m-cresol degradation and for glucose, it ranged from 4 to 12 h. The samples were then divided into two portions. While one portion was used in the determination of biomass, the other portion was centrifuged at 10,000×g for 10 min using Sigma 4K15 centrifuge, UK, and the resulting supernatant was analyzed for glucose and m-cresol concentrations. The results reported are average of values obtained from the duplicate experiments. Control experiments, in a microorganism-free MSM with m-cresol and glucose, were also carried out in order to evaluate the possible degree of m-cresol removal with volatilization, and it was seen that the m-cresol concentration remained unchanged.

Analytical method

For estimating biomass concentration, absorbance at 600-nm wavelength (OD600) of the samples was measured using a diode array spectrophotometer (Spekol 1200, Analytik Jena, Germany), and the optical density was converted to biomass concentration using a dry weight calibration curve. m-Cresol content in the biomass-free samples was determined quantitatively using Perkin Elmer high performance liquid chromatograph (HPLC) with a UV–visible detector and C18 column. Dimensions of the column were 220 × 4.6 mm with 5-μm-sized particles in the packing. An 80:20 mixture of acetronitrile and water was used as the eluent with a constant flow rate of 1 ml min−1 in the column. At these HPLC settings, m-cresol was detected at 275-nm wavelength and at a retention time of 3.25 min. Glucose in the samples was determined by the standard dinitrosalicylic acid assay (DNS) method (Miller 1959).

Results and discussion

Biomass growth profile of the indigenous mixed culture

Figure 2 shows biomass growth profile (OD600) of the culture with respect to time at each initial m-cresol concentration and at 250 and 500 mg l−1 of glucose. The glucose initial concentrations used were chosen in such a way as to enable the culture growth in presence of m-cresol, and to examine its effect on m-cresol degradation. m-Cresol initial concentrations (50–400 mg l−1) used in the study were based exclusively on the culture capability to degrade the compound. This was based on the previous observation that the culture did not exhibit significantly faster degradation when m-cresol initial concentration was raised above 400 mg l−1 in MSM supplemented either with 250 or 500 mg l−1 initial concentration of glucose. As seen from Fig. 2, the biomass concentration of the culture was generally high at 500 mg l−1 initial concentration of glucose, and increased with initial m-cresol concentration in the media. It should be noted here that in absence of m-cresol, glucose at any of its two concentrations, resulted in low biomass of the culture (data not shown). Therefore, it could be inferred that the mixed culture was able to utilize m-cresol much efficiently along with glucose for its growth. Further, due to the presence of glucose the culture did not show any lag phase in its growth even at higher m-cresol concentrations. Similar enhancement in biomass of Pseudomonas putida strain due to presence of glucose as a co-substrate during 4-chlorophenol degradation was observed by Tarighian et al. (2003).

Fig. 2
figure 2

Time profile of the biomass growth (OD600) at different initial concentrations of m-cresol and glucose; symbols used: solid line 250 mg l−1 glucose, dashed line 500 mg l−1 glucose, open triangle 50 mg l−1 m-cresol, open square 100 mg l−1 m-cresol, filled triangle 200 mg l−1 m-cresol, filled square 400 mg l−1 m-cresol

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m-Cresol degradation and glucose utilization kinetics

Figures 3 and 4 present time profile of m-cresol degradation and glucose utilization by the culture, respectively. m-Cresol degradation, at its various initial concentrations, followed almost a similar trend irrespective of the glucose concentrations used. The culture took a maximum time of ∼24 h for degrading m-cresol at 400 mg l−1 initial concentration, which was the highest used in the study. In literature, degradation time of phenol by the white rot fungi, P. chrysosporium was shown to be decreased in the presence of glucose at concentrations above 20 g l−1 (Toure et al. 1997). In the present study, the glucose concentration was low, either at 250 or 500 mg l−1, and therefore, possibly no adverse effect on m-cresol degradation could be observed. The time period for complete utilization of glucose was slightly higher at 500 mg l−1 compared to that at 250 mg l−1 of its initial concentration. However, glucose utilization at all the combinations was quick and normally preceded m-cresol degradation, which clearly reveals that glucose, as compared with m-cresol, is an easily assimilable source of carbon (Tarighian et al. 2003). At glucose—500 mg l−1 and m-cresol—400 mg l−1, the glucose utilization was complete within ∼12 h itself, m-cresol degradation took place between ∼12 and ∼26 h of time instants of the culture (Figs. 3, 4). The trend was the same for other combinations of glucose and m-cresol in MSM, as well.

Fig. 3
figure 3

m-Cresol degradation profile at different initial concentrations of m-cresol and glucose; symbols used: solid line 250 mg l−1 glucose, dashed line 500 mg l−1 glucose, open square 50 mg l−1 m-cresol, open triangle 100 mg l−1 m-cresol, open circle 200 mg l−1 m-cresol, filled circle 400 mg l−1 m-cresol

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Fig. 4
figure 4

Glucose utilization profile at different initial concentrations of m-cresol and glucose; symbols used: solid line 250 mg l−1 glucose, dashed line 500 mg l−1 glucose, filled square 50 mg l−1 m-cresol, filled triangle 100 mg l−1 m-cresol, filled circle 200 mg l−1 m-cresol, open circle 400 mg l−1 m-cresol

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Biomass yield and specific growth rate of the culture

In order to quantify the role played by the two substrates, glucose and m-cresol, on the growth of the culture, the following two kinetic parameters, viz., biomass yield and specific growth rate (Kenneth et al. 2000), were determined:

$$ Y_{X/S} = \frac{{X_{\text{M}} - X_0 }} {{S_0 - S_{\text{M}} }} $$
(1)
$$ \upmu = \frac{1} {X}\frac{{{\text{d}}X}} {{{\text{d}}t}}. $$
(2)

In the above expression for biomass yield (Y X/S ), X M and X 0 are the maximum and initial dry cell concentrations, and S M and S 0 are the corresponding total substrate concentrations with respect to X M and X 0, respectively. The specific growth rate (μ) of the culture was calculated using the experimental data by applying Eq. (2), in which X is the biomass concentration at time t.

Calculated total biomass yield values are presented in Table 1; where, it could be seen that high values of yield coefficients were obtained in the study. However, irrespective of the two glucose concentrations used, the biomass yield value decreased with an increase in m-cresol concentration above 100 mg l−1. Abuhamed et al. (2004) observed a maximum yield value of 0.75 due to benzene at 700 mg l−1 initial concentration on the growth of a P. putida strain. This value is slightly less compared to the highest obtained in our present study—probably due to the fact that both the substrates (m-cresol and glucose) were fully utilized by the mixed culture for its growth, as observed previously in the biomass growth profile of the culture. In a study by Tarighian et al. (2003) using glucose as the co-substrate (at 250 mg l−1 initial concentration), the biomass yield value of a P. putida strain fell from 0.52 to 0.25 when 4-chlorophenol concentration was raised from 30 to 240 mg l−1. The authors attributed this decrease in biomass yield value to inhibition on the growth of the P. putida strain by a high initial concentration of 4-chlorophenol in the media. On similar lines, it may be inferred in this study that above 100 mg l−1 initial concentration, m-cresol inhibited the culture biomass yield.

Table 1 Calculated total biomass yield coefficient (g g− 1) values obtained in the study
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A clear interpretation on substrate inhibition on the growth of the indigenous mixed culture by m-cresol at high concentrations could be obtained from the observed experimental μ values, which are plotted in Fig. 5. The figure shows clearly that above 100 mg l−1, m-cresol inhibited the culture specific growth rate. This inhibitory effect seems to be more pronounced for 250 mg l−1 than for 500 mg l−1 glucose initial concentrations in the media (Tarighian et al. 2003).

Fig. 5
figure 5

Experimental specific growth rate due to m-cresol and glucose (solid line 250 mg l−1, dashed line 500 mg l−1)

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Sum kinetics model fitting of experimental specific growth rate

Sum kinetics model, proposed by Yoon et al. (1977), was fitted to the experimental data on specific growth rates of the culture to predict its variations due to the co-substrates: glucose and m-cresol. This model was also utilized to evaluate the interaction between glucose and m-cresol on the growth of the culture, and to estimate the relative effects of the two substrates on their individual uptakes (utilization of glucose/degradation of m-cresol). The form of this model is shown in Eq. (3)

$$ \upmu = \frac{{\upmu _{\max ,1} S_{1L} }} {{K_{S,1} + S_{1L} + \frac{{S^2 _{1L} }} {{K_{1i} }} + I_{2,1} S_{2L} }} + \frac{{\upmu _{\max ,2} S_{2L} }} {{K_{S,2} + S_{2L} + \frac{{S^2 _{2L} }} {{K_{2i} }} + I_{1,2} S_{1L} }}. $$
(3)

The interaction parameter I i,j indicates the degree to which substrate i affects the biodegradation of substrate j; a large value of the parameter indicates a strong inhibition on the substrate uptake by the microorganism (Yoon et al. 1977). The other kinetic parameters μmax,j , K s,j , and K ji in Eq. (3) refer to the culture maximum specific growth rate, half-saturation constant and inhibition constant, respectively, due to substrate j in the system. A non-linear regression technique involving constraints for positive integer values of the parameters was employed in solving the model equation using MATLAB 7. Very high determination coefficient (R 2) values of 0.99 and 0.98 were obtained by fitting the model equation to the experimental μ values of the culture, due to different m-cresol concentrations, at 250 and 500 mg l−1 glucose concentrations, respectively. The following interaction parameters (I ij ) values were obtained by solving the model equations for the two cases of glucose concentrations:

$$ \begin{aligned}{} I_{{\text{glucose:}}m{\text{ - cresol}}} & = 10.67{\text{ }}({\text{at }}250\;{\text{mg}}\;{\text{l}}^{ - 1} {\text{ glucose}}){\text{ and }}1.59{\text{ }}({\text{at }}500\;{\text{mg}}\;{\text{l}}^{ - 1} {\text{ glucose}}); \\ I_{m{\text{ - cresol:glucose}}} & = 0.0961{\text{ }}({\text{at }}250\;{\text{mg}}\;{\text{l}}^{ - 1} {\text{ glucose}}){\text{ and }}0.99{\text{ }}({\text{at }}500\;{\text{mg}}\;{\text{l}}^{ - 1} {\text{ glucose}}). \\ \end{aligned} $$

While the interaction parameter I glucose:m-cresol represents the effect of glucose on m-cresol degradation, the other interaction parameter (I m-cresol:glucose) represents the effect of m-cresol on glucose utilization by the culture. From the values of these parameters, it could be said that glucose at 250 mg l−1 exhibits stronger inhibition on m-cresol degradation, compared to its effect at 500 mg l−1. But for m-cresol, its effect on glucose utilization was almost same at both the glucose concentration conditions in the system. In literature, a maximum interaction parameter value of 5.16 has been reported for the effect of toluene on benzene degradation by a P. putida strain (Abuhamed et al. 2004). Considering the fact that glucose being a simple and easily assimilable substrate, a large value of its inhibitory effect on m-cresol degradation by the indigenous mixed culture followed in the present study is not unlikely. Though the kinetic profiles of m-cresol degradation and glucose utilization by the culture did not show a distinct effect of glucose on m-cresol degradation, the interaction parameter values obtained from the model did give a clear understanding on the same.

To further improve the performance of the indigenous culture in degrading higher concentrations of m-cresol, studies employing the microorganism in a suitable bioreactor with necessary controls such as aeration, agitation, temperature and pH may, however, prove more useful, and therefore, have been initiated recently. Moreover, a study pertaining to complete characterization of the indigenous mixed microbial consortium responsible for m-cresol degradation is also currently under way.

Conclusions

An indigenous mixed microbial culture isolated from a soil contaminated with petroleum hydrocarbon and collected from a gasoline filling station was studied of its potential for degrading m-cresol in presence of glucose as an alternative carbon source. Biomass profile of the culture indicated that m-cresol and glucose could be well utilized in the process. Glucose utilization was, however, quick and normally preceded m-cresol degradation. Total biomass yield and specific growth rate of the culture revealed that m-cresol, above 100 mg l−1 initial concentration in the media, inhibited the culture growth. Parameter values for interaction between the two substrates, obtained by fitting a sum kinetic model to the experimental specific growth rates of the culture, showed that glucose had a strong inhibitory effect on m-cresol degradation.