Simultaneous degradation profiles of substituted phenols
The biodegradation patterns of the phenolic substrates (4-BP, 4-NP, and 4-CP) in different experimental runs of 22 full factorial design were recorded. Fig. 1 shows their biodegradation patterns at low concentration ranges (Fig. 1 (a), run 1 and 5) and high concentration ranges (Fig. 1 (b), run 4 and 8). It is observed that the microbial culture of A. chlorophenolicus A6 took a longer duration to completely degrade 4-BP and 4-CP as compared to 4-NP in all cases. A prominent lag phase was observed during 4-BP and 4-CP degradation. Further, the lag phase for 4-BP and 4-CP degradation was increased with an increase in initial 4-NP concentration. A similar pattern of lag phase for 4-NP degradation was observed with an increase in 4-CP concentration in the culture media. 4-NP biodegradation was slow down with an increase in the concentration of 4-CP in the culture medium. For instance, in a single substrate system, the culture took 8 h to degrade 4-NP at an initial concentration of 100 mgl-1 (Sahoo et al. 2011b) compared with a minimum culture period of 18 h in a mixed substrate system. The possible reason for slower biodegradation rates in a mixed substrate system might be due to the enhanced toxicity effect exerted by the phenolic compounds on the microbial cells. The phenolic compounds exert their toxicity by uncoupling oxidative phosphorylation (Xie et al. 2018). The formation of dimers between two different substituted phenolics compounds can further aggravate the uncoupling activity (Panigrahyet al. 2018; Escher et al. 2001). Besides, the formation of different intermediates in a mixed substrate degradation system may inhibit enzymes vital for degradation, or its active binding site (Sahoo et al. 2014b). The degradation patterns of individual substrates in the mixed substrate system also differed considerably from those obtained in their respective single substrate systems. Irrespective of the concentration levels of these three substrates, 4-NP was preferentially biodegraded over 4-CP and 4-BP. Similar preferential degradation of aromatic hydrocarbons and phenolics pollutants (100 mgl-1) over free cyanide (>2.5 mgl-1) has been reported by Sharma and Philip (2014). Between 4-BP and 4-CP, 4-BP degradation was found quicker than the other; however, the difference was less significant. Unell et al. (2008) reported that although the same enzyme system is responsible for biodegradation of 4-BP, 4-CP, and 4-NP by A. chlorophenolicus A6 one compound is preferentially biodegraded over the other. For example, in the present case, 4-NP was degraded preferentially over others (Fig. 1). A possible reason could be the fact that enzyme in the biodegradation pathway has a higher affinity towards 4- NP than those to 4-BP and 4-CP, otherwise, there could be transport-level interactions influencing the biodegradation of the phenolics pollutants. This might be due to the differences in pKa values of these substituted phenols. For example, the pKa value of 4-NP is 7.1, while, it is 9.3 for 4-CP and 9.17 for 4-BP. Thus, at pH 7.2–7.5 of the A. chlorophenolicus A6 culture media, almost 65% of the 4-NP might have dissociated to phenolate ion which usually enters into the microbial cells. On the other hand, it is less than 2% in the case of 4-BP and 4-CP. Therefore, due to the different pKa values, 4-NP degraded preferentially over 4-CP and 4-BP. Further, concurrent biodegradation of 4-BP and 4-CP mixture was possible due to their almost alike pKa values of 9.17and 9.3, respectively. Also, this observation was correlated well with the results achieved under these individual substituted phenolic degradation systems. For instance, the values of half-saturation constants (Ks) for 4-CP and 4-BP were nearly the same i.e 30.83 and 30.77 mgl-1, respectively; whereas, the values for 4-NP was lower (20.15 mgl-1) (Sahoo et al. 2011a; Sahoo et al. 2011b; Sahoo et al. 2014a). Many researchers in literature have reported similar observations on the preferential biodegradation of one aromatic pollutant over another in mixed substrate systems, even though the same degradation pathway was followed for these compounds by different microorganisms. For example, the rate of benzene biodegradation was faster than o-xylene by fungal species might be because benzene is the preferred substrate in a benzene-xylene mixture (Khoramfar et al. 2020). Fu et al. (2017) have reported the simultaneous biodegradation of 3-NP, 2-NP, and 4-NP by the microbial consortium of Cupriavidusnecator JMP134, Pseudomonas sp. WBC-3 and Alcaligenes sp. NyZ2015, in a sequential batch reactor. Their results showed that almost complete degradation of this phenolic mixture at 0.5 mM each was attained by the microbial consortium in 84 h. Interestingly, 4-NP preferentially degraded over 3-NP and 4-NP by the Pseudomonas sp. WBC-3. Dey and Mukherjee (2013) investigated the mixture of phenol and resorcinol biodegradation under an aerobic batch reactor. They reported complete degradation of phenol and resorcinol at an initial concentration of 400 mgl-1 each within 58 h. Further, the inhibition effect of resorcinol on specific substrate degradation rate is larger than the inhibition effect caused by phenol. Bai et al. (2007) evaluated the performance of Alcaligenes faecalis in the degradation of m-cresol and phenol in a mixed substrate system. The researchers reported that faster biodegradation of m-cresol occurred towards the exhaustion of the phenol in the culture medium. Zou et al. (2018) have investigated the competition for molecular oxygen (O2) and an electron donor (2H) in concurrent degradation of quinoline and phenol using a vertical baffled bioreactor (VBBR). They reported the existence of mutual inhibition between quinoline and phenol, which competed for electron donor (2H) and molecular oxygen (O2) during the simultaneous degradation process. Xiao et al., (2019) studied the degradation of cresol and phenol by Chlorella vulgaris. They observed that low concentrations of initial phenol (60 – 100 mgl-1) improved the rate of p-cresol degradation. In the present study, a similar finding was observed; where the rates of 4-BP and 4-CP degradation were improved with a low concentration of 4-NP and towards its depletion in the culture medium. For instance, 250 mgl-1 of 4-BP or 4-CP takes about 16 h for completely degradation in single substrate systems (Sahoo et al. 2011a: Sahoo et al. 2014a) whereas, in mixed substrate systems, 125 mgl-1 4-BP and 125 mgl-1 4-CP took only 12 h in presence of 50 mgl-1 4-NP.
Biomass Growth of the Culture in the Mixed Substrate System
Biomass profiles obtained at different concentration combinations of these phenolics are presented in Fig. 2 in the form of OD600nm of the culture. It is clear from Fig. 2 that the actinomycetes required more time to grow when a higher concentration of 4-NP (> 100 mgl-1) was present in the medium (together with 4-CP and 4-BP); along with a poor biomass yield. The observation on the biomass profile of the culture in the mixture of pollutants' system differed considerably with the patterns observed in the single substrate system (Sahoo et al. 2011a; Sahoo et al. 2011b; Sahoo et al. 2014a). This phenomenon is obvious owing to a difference in the accessibility of carbon source in the culture medium along with increased toxicity. These observations were in close agreement with that of Bai et al. (2007), who studied similarly mixed substrate effect on the growth of Alcaligenes faecalis. Moreover, a lag phase was observed beyond certain concentrations combination of 100 mgl-1 for 4-BP, 4-CP, each, and 75 mgl-1 of 4-NP, probably due to their combined toxicity effect of the phenolics. On the other hand, as discussed earlier, 4-BP and 4-CP were degraded simultaneously. In the present study, the growth of the actinomycetes did not reveal any plateau phase in the transition point of the different substituted phenols indicating no diauxic growth as depicted in Fig. 2. Unell et al. (2008), employed a mutant strain of A. chlorophenolicus A6 (T99) carrying a transposon in a hydroxyquinol 1,2-dioxygenase gene (T99 mutant) which seriously impaired the microbial growth on 4-CP. They reported that the T99 mutant behaved the same way when 4- BP or 4-NP was used as the only source of carbon. This phenomenon revealed that the same enzyme system was used by the actinomycetes for biodegradation of 4-CP and thus confirmed no diauxic growth.
Statistical Analysis of a mixture of substituted phenol biodegradation
Experimental results obtained on the growth of the microorganism and phenolics degradation were used for calculating the pollutant degradation rates of these substrates in the mixture. Fig. 3 shows the degradation rates of these substituted phenols at different concentration combinations (run order number) as per the 22-level full factorial design of experiment (Table 2). The figure reveals that the degradation rates of 4-BP and 4-CP were higher than that of 4-NP except at their lower (experimental run number-1) concentration combination (75+75+50 mgl-1 of 4-BP, 4-CP, and 4-NP, respectively). In general, rates of 4-BP biodegradation were higher than that of 4-CP followed by 4-NP. Moreover, higher biodegradation rates of 4-CP and 4-BP were obtained in the higher concentration range of 4-NP which may be due to high initial biomass produced during the 4-BP and 4-CP degradation process. Further, these values were very low when the concentration ranges of the substrates were high. For instance, degradation of 4-NP at lower concentration range (75+75+50 mgl-1of 4-BP, 4-CP, and 4-NP, respectively) was found to be 0.139h-1; on the contrary, the value was 0.043h-1 at higher concentration range (125+125+100 mgl-1 of 4-BP, 4-CP, and 4-NP, respectively). This finding confirmed the fact that the growth of the culture, as well as its phenolics degradation rates, was inhibited at higher concentration ranges of these pollutants.
Statistical analysis of biodegradation of phenolics mixture using ANOVA and Student's t-test
Based on the above-obtained results were the statistical analyzed in terms of ANOVA and Student’s ‘t’ test. This statistical analysis was performed primarily to interpret the roles of individual variables played on phenolic biodegradation. Tables 2 (a), (b), and (c) present the results of ANOVA of 4-CP, 4-BP, and 4-NP degradation rates in the study. From this statistical analysis of the result, it can be seen that both the main (individual) and two-way interaction terms for these phenolics pollutants were very much significant on the biodegradation activity at greater than 97.3% confidence level (P<0.027). On the other hand, except 4-NP degradation rate, the ANOVA results presented in Table 2 (a) and (b) revealed that the three-way interaction term was insignificant (P>0.05).The ANOVA table for each pollutants degradation rate also represents an error term, which revealed that the experimental error in this study was quite insignificant. Further, the higher values of the determination coefficient (R2>99) indicate that the polynomial model is highly accurate in predicting phenolics biodegradation. Further, while the Pareto charts illustrated in Fig. 4 (a), (b), and (c) revealed a significant negative (inhibitory) individual effect of 4-CP on 4-NP as well as on its degradation performance. Fig. 4 (c) demonstrates that the negative main effect of 4-NP on its degradation was comparatively lesser than that of 4-CP. Further, the interaction effect between 4-NP and 4-CPon 4-NP biodegradation activity and that between 4-CP and 4-BP on 4-BP degradation was considerably negative than that of between 4-BP and 4-CP on 4-CP degradation (Fig. 4 a, b, c). To analyze the main and interaction effects that exist among these substituted phenols student's t-test was executed. Table 3 represents the calculated coefficients of individual and interaction terms along with the associated t and P values. Generally, a larger t value with a smaller P-value of a variable designates a higher significance of the respective model term. The coefficient of ‘t’ value for 4-NP degradation reveals strong inhibition mainly due to 4-CP (P = 0.001) followed by 4-BP (P= 0.015). Hence, it was concluded that 4-NP degradation performance was considerably inhibited compared to 4-BP due to the presence of 4-CP. On the other hand, the interaction between the other two factors viz. 4-CP and 4-BP (X1 X2) were found insignificant (P = 0.613) on 4-NP degradation but was negatively significant on their degradation performance (P =.0.004 and 0.017). This analysis demonstrated that 4-NP was significantly inhibited by 4-CP concentration in the mixture whereas the vice-versa was not correct (P = 0.757). Other interaction effects except three-way interaction effects were found to be significant at a 95% confidence level. Similar statistical analysis and interpretation have been reported in the literature for biodegradation of different pollutants (Mohanty and Jena 2018; Farag et al. 2018; Khanpour-Alikelayeh et al. 2020; Nam et al. 2017; Khatoon and Rai 2020).
Biomass yield on biodegradation of mixture of substituted phenols
The calculated biomass yield values for different concentration ranges in the mixed substrate system are presented in Table 4. Though 4-NP was biodegraded before 4-BP and 4-CP, the biokinetic data revealed that 4-NP had a large negative effect on the microbial cells than the other two pollutants. Similarly, the actinomycetes degraded 4-NP faster; however, the biomass yield was very poor. This might be due to the presence of a nitro group on the phenolic ring impart greater toxicity to the microorganism compared with the other two phenolic pollutants (Tian et al. 2020). This phenomenon revealed that the microbial cells are in a more harsh condition when cultured in 4-NP than that of 4-BP and 4-CP. 4-NP possibly a more intoxicating mitochondrial uncoupling agent compared to 4-BP and 4-CP and hence affects the microbial cells more negatively. However, irrespective of the different combinations of initial 4-BP,4-CP, and 4-NP concentrations, the biomass yield was increased with an increase in the initial 4-BP and 4-CP concentration at a fixed 4-NP concentration in the mixture except in the case of experimental run number-8. The lower biomass yield due to 4-BP, 4-CP, and 4-NP at 125, 125, and 100 mgl-1 may be attributed due to high initial concentration combination exerted elevated toxicity level on the microbial cells as a consequence of which most of the carbon sources is diverted to maintenance energy rather than biomass growth (Sahoo et al. 2011b). Panigrahy et al. (2020b) reported that the biomass yield coefficient increases from 0.57 -0.73 g dry cell mass g-1 of cresol with a change in the initial concentration of cresol from 100–1200 mgl-1 using an indigenous Pseudomonas citronellolis NS1. In another study, Dionisi and Etteh (2019) investigated a mixture of paracetamol and phenol biodegradation by a mixed microbial culture. They reported a higher biomass yield of 0.51 gg-1 using paracetamol than that of phenol (0.20 gg-1 COD). Similar biomass yield coefficient ranged from 0.293 to 0.64 gg-1 of phenol has been reported in the literature using Alcaligenes strain TW1 and Bacillus brevis (Essam et al. 2010). In this study, the highest yield value of 0.2152 gg-1 was obtained at a higher concentration combination of 4-CP and 4-BP with a low level of 4-NP (125+125+50 mgl-1of 4-BP, 4-CP, and 4-NP, respectively). On the other hand, a very low yield value of 0.1673 was achieved at the same range of 4-BPand 4-CP but a higher 4-NP concentration of 100 mgl-1. This value indicates that the concentration of 4-BP and 4-CP particularly influenced the biomass yield in the experiments. The lower biomass yield achieved in this study may be attributed to the elevated toxicity effect of substituted phenol on microbial cells, as the aromatic ring of the phenolic compounds with its shared resonance electrons offers higher stability and resistance to the microbial enzymatic attack. Halogen substituents on the aromatic ring further improved the stabilization, as the halogens promote electron-withdrawing effect and create a steric hindrance to enzymes (Uberoi and Bhattacharya 1997; Panigrahy et al. 2018). Similar higher microbial toxicity of substituted phenols has been reported in the literature. For instance, Aktaş (2012) investigated a mixture of 2-CP, 2-NP, and phenol biodegradation using activated sludge. The researcher observed the substrate to biomass ratio for 2-CP, 2-NP (1.5 and 5.5 mg CODeq/mg MLSS) is higher than that of phenol (8.5 mg CODeq/mg MLSS), which indicating higher microbial toxicity of substituted phenol compared to phenol. Therefore, the necessity of higher maintenance energy to overcome the toxic effect of these substituted phenols cannot be ignored.