Modeling cometabolism of hexavalent chromium by iron reducing bacteria in tertiary substrate system

In this study, a bacterial strain Serratia sp. was employed for the reduction of synthetically prepared different concentration of Cr(VI) solution (10, 25, 40, 50 and 100 mg/L). Cometabolism study have been carried out in the binary substrate system as well as in the tertiary substrate system. The results revealed that when glucose was added as a co-substrate, at low Cr(VI) concentration, complete reduction was achieved followed by increased biomass growth, but when Cr(VI) concentration was increased to 100 mg/L, the reduction decline to 93%. But in presence of high carbon iron filings (HCIF) as co-substrate even at higher Cr(VI) concentration i.e. 100 mg/L, 100% reduction was achieved and the cell growth continued till 124 h. The study was illustrated via Monod growth kinetic model for tertiary substrate system and the kinetic parameters revealed that the HCIF and glucose combination showed least inhibition to hexavalent chromium reduction by Serratia sp.

www.nature.com/scientificreports/ metals presence in wastewater. These problems create difficulty for the non-indigenous microorganisms to effectively treat the wastewater. Cometabolic degradation has been found to be eco-friendly and economically feasible in the process of treating recalcitrant compounds 19 , and coking wastewater (CWW) treatment 20 . Many studies stated that when phenol was used as the cometabolic substrate, the degradation efficiency of p-nitrophenol 21 , carbazole 22 and 4-chlorophenol 23 increased. In cometabolism, metabolic substrates such as glucose, methyl alcohol are sufficient source of carbon and energy for microbes to grow. These substrates induces enzymes synthesis that accelerates metabolism of growth and non-growth substrates, producing NADH as electron donor in metabolic pathway and subsequently these metabolites take part in degradation of various compounds 24 . Little attention has been given to cometabolic reduction of hexavalent chromium.
Earlier in our previous work, Serratia sp. was able to reduce different range of hexavalent chromium. But the inhibition effect of hexavalent chromium was higher for Serratia sp. As a result, the growth of the bacterial species was hindered after few hr 25 . To reduce the inhibitory effect of Cr(VI) on the bacteria, this study demonstrates the use of glucose and high carbon iron filings (HCIF) as cometabolic substrates for microbial reduction of Cr(VI).

Materials and methods
Chemicals. The chemicals, reagents and microbial media were of pure grade. Cr(VI) standard solution of 1000 mg L −1 have been prepared by mixing K 2 CrO 7 in Milli Q water. For measuring hexavalent chromium concentration, Diphenyl carbazide (DPC) solution (0.25%) was prepared by dissolving it in 95%. Microbes were grown in broth media of Lureia Bertani (Hi-Media India). Glucose (Rankem, purity 99.0%) were used as cometabolic substrates.
Experimental process. Bacterial strain was isolated from iron ore mines as discussed in the work done previously by 25 . Commercially available cast iron rod was chipped on a lathe machine and was brought into iron filings in a dough-sized ball mill. HCIF thus obtained were washed with N 2 sparged 1 N HCl for 4-5 times so that the organic material adsorbed on HCIF surface during chipping process may be removed as well as the reactivity of HCIF may be enhanced. Afterwards, HCIF was washed again with N 2 sparged milli Q water for 10-12 times to remove excess HCl. Later, moisture content of HCIF was removed by washing with 95% acetone and HCIF were dried in N 2 atmosphere in a vacuum desiccator and then used for the experimental purpose. Cometabolism studies were conducted for enhancing the reduction capacity of the HCIF-bacteria system. Batch experiments were conducted with two different combination (i) Binary system containing bacteria and glucose (1 g/250 mL) and (ii) Tertiary system with bacteria, HCIF (1 g/250 mL) and glucose (1 g/250 mL) having different Cr(VI) concentration (10,25,40,50, and 100 mg/L) in an incubator shaker at 35 °C and 120 rpm. Samples were taken at every 12 h and analysis of Cr(VI), biomass, and glucose concentration was measured by Uv-Vis spectrophotometer at 540, 600 and 510 nm, respectively. Samples were taken at regular time and concentration of Cr(VI), bacterial growth, and glucose was measured by Uv-Vis spectrophotometer at wavelength of 540, 600 and 510 nm respectively.
Rate kinetics model. The pseudo first and second order was applied to define the kinetic degradation of microbe's kinetics of hexavalent chromium which is given below: where, C is the concentration at any time, initial concentration is denoted as C O , k is the pseudo first-order rate constant (day −1 ), and reaction time is denoted by t.
Kinetics of microbial growth in system containing single substrate. Growth kinetics in single system comprises of bacteria and glucose and chromium exposure. In a batch system, the specific growth rate of a cell, µ (h −1 ) is defined as where, µ is calculated at the exponential phase of the growth curve. X is the concentration of cell in either cell number at time 't' based on the viable counts (CFU/mL) or in either g/L (dry basis). The specific growth rate of cell on single substrate is expressed in terms of µ in Eq. (2), which is a function of concentration of resource. The Haldane Andrew model was applied here in the single substrate system as it has its wide use in representing the growth kinetics of single substrate: where, µ max is the maximum specific growth rate (day −1 ), substrate concentration is defined by the term 'S' and K s is the substrate affinity constant (mg/L).
Growth kinetics in tertiary substrate system. To stimulate Co-metabolism, for the cell growth on mixtures of substrate, various models have been proposed. In this study, Monod model used in binary substrate system 19 was modified to tertiary substrate system for the analysis of growth kinetic parameters. The equation for the specific growth rate on tertiary inhibitory substrates was as follows:

Results and discussion
Reduction of Cr(VI) in batch reactors in the presence of cometabolic substrate (HCIF and glucose). The strain isolated was identified as Serratia sp. 25 . It was used in our previous study for reduction of different concentration of Cr(VI) in two different ways, i.e. one by the strain alone and the other by co-assistance of HCIF with the strain. This study was performed to demonstrate the cometabolic effect of substrate on reduction of hexavalent chromium. Cometabolism study was conducted to evaluate the cometabolic activity of Serratia sp. in two different sets i.e. when glucose was added (1 g/250 mL) to batch reactors containing Cr(VI) and bacteria (Set 1) and when glucose was added to batch reactors containing Cr(VI), bacteria and HCIF (Set 2) for the reduction of hexavalent chromium by Serratia sp. The biomass concentration, Cr(VI) concentration and glucose concentration was measured in the two sets. Figure 1A,B, shows the biomass abundance at different concentration of Cr(VI) (10, 25, 40, 50, and 100 mg/L) when glucose was added as cometabolic substrate in both the sets. The results of Set 1 indicated that when glucose was added to batch reactors containing only bacteria and Cr(VI), the bacterial growth was highest for 10 mg/L Cr(VI) solution, the growth increased till 50 h and after which it started declining. The lesser the concentration of Cr(VI), the higher was the bacterial growth. For 25 mg/L Cr(VI) concentration, the growth was highest at 24 h and then it declined and lasted till 60 h. In case of 50 mg/L of Cr(VI) concentration, the biomass growth was very low in comparison to other concentrations. However, slight increase in growth was observed at 24 h. In case of 100 mg/L of Cr(VI) concentration, the bacteria attained its log phase at 50 h and after that the stationary phase was observed. In this case the lag phase increased with increase in initial Cr(VI) concentration which may be due to the inhibitory effect of Cr(VI) on the growth of microorganism. At high concentrations, the inhibitory effect increased due to the fact that a fixed amount of inoculum was used for all different concentration of Cr(VI). In the second experimental set (Set 2), it was found out that at all the different concentration of Cr(VI) (10, 25, 40, 50 and 100 mg/L) the log phase started after 50 h and the cell doubling continues till 85 h. After this the decline in biomass concentration was observed.
The results from the study indicated that when only glucose acted as co-metabolic substrate, the biomass growth lasted till 60 h but when glucose was added to the HCIF and bacterial set, the bacterial growth continues till 120 h. This may be due to the reason that, HCIF may be acting as a co-metabolic substrate in enhancing the bacterial growth along with the glucose. HCIF too acts as a source of growth to bacteria like glucose, when supplied with low dosing 26 . Experiments were conducted for evaluating the reduction in Cr(VI) concentration with respect to time for the same experimental set as discussed above (Set 1 and Set 2). For Set 1, the results indicated that complete reduction of 10 mg/L and 25 mg/L Cr(VI) concentration was achieved at 40-50 h and 75-76 h, respectively, and similarly for 40 mg/L and 50 mg/L Cr(VI) concentration, the complete reduction was achieved at 80 h. But in case of 100 mg/L Cr(VI) concentration, complete reduction was not achieved till 90 h of experiment ( Fig. 2A). At lower concentration of Cr(VI) (10 and 25 mg/L) the maximum reduction was achieved in less time. The reason may be due to the presence of high concentration gradient of Cr in the solution, makes it more difficult to reduce completely 27 . Although glucose was added as a co-metabolic substrate for enhancing the activity of microbes, complete reduction efficiency was not achieved.
The results from the second experimental set (Set 2) showed that for 10 and 25 mg/L Cr(VI) concentration, the reduction was initiated after 15 h and complete reduction was achieved between 30 and 40 h. In case of 40 and 50 mg/L Cr(VI) concentration, the complete reduction was achieved at 60 h. For 100 mg/L Cr(VI) concentration, www.nature.com/scientificreports/ the reduction was initially slow but after 70 h the reduction was faster and it was completely achieved at 120 h (Fig. 2B). This result was better in comparison to the first experimental set. The results clearly indicated that HCIF was also acting as a co-metabolic substrate in enhancing the reduction efficiency of Cr(VI). The possible mechanism behind improved Cr(VI) reduction is due to coexistence of Serratia sp. with HCIF which helped in reduction of Cr(VI) as well as helped in maintaining the longevity of HCIF. Secondly the strain reduces Fe(III) to Fe(II), resulting in higher dissolved Fe(II) and thus maintaining the structured morphology of HCIF by removing the passivated ferric precipitates on iron surface. It has been confirmed by 28 also that in the presence of NZVI, the Cr(VI) reduction efficiency by strain DIRB HS01could be improved. Similar type of cometabolic study has been performed by 29 in which cometabolic degradation of blended biodiesel by a fungal strain Monilliella wahieum Y12 T was carried out. The results showed that degradation of petroleum diesel (ULSD) was enhanced when the fungal strain was used with biodiesel. Another experiment conducted by 30 demonstrated the effect of hydroxypropyl-β-cyclodextrin on cometabolism of phenol and phenanthrene by Chryseobacterium sp. The results stated that cometabolic activity of phenol and hydroxypropyl-β-cyclodextrin accelerated the degradation of phenol and had great phenanthrene removal rate. Addition of hydroxypropyl-β-cyclodextrin led to increased solubility and phenanthrene toxicity was also reduced thus had improved cometabolic degradation. Glucose concentration was also determined at different Cr(VI) concentration at different time interval. Glucose is highly consumed by microbes for their growth and in enhancement of any kind of bacterial activity. Bacterial growth and glucose consumption are inversely proportional to each other. As bacterial growth tends to increase, the glucose consumption also increases because glucose is needed for growth purpose. The similar trend was observed in this study. For Set 1, with doubling of cell number, the glucose consumption increased and was highest at 24 h when the bacterial growth attended its log phase, as discussed above. The glucose concentration started declining after 24 h and it was negligible at 60 h (Fig. 3A). With decrease in glucose concentration, www.nature.com/scientificreports/ the bacterial growth also declined. The limited amount of glucose available for consumption will automatically destroy the population due to competition for limited amount of food source. In Set 2, the results indicated that glucose concentration declined slowly with respect to time and the complete decline was observed at 120 h (Fig. 3B).This may be due to the presence of HCIF which also served as a source of growth for microbes, so the glucose consumption in this case was less as compared to set 1 where only glucose was present. As there is presence of two co-metabolic substrate, the burden on substrates may get reduced 19 . The rate kinetics model followed pseudo first order in the case when only glucose was present as substrate (Set 1) and in Set 2 when glucose and HCIF was present as substrates, it followed pseudo second order ( Table 1). The rate constant was higher in case of Set 2, when both the substrates were present due to which the Cr(VI) reduced completely. The rate decreased with increase in Cr(VI) concentration in both the Sets 1 and 2. One way ANOVA followed by Duncan's post hoc test results confirmed that the results were statistically different ( Table 2).
Kinetics of microbial growth in single and tertiary substrate system. The experimentally acquired data for the specific growth rate of the strain at its growth in single substrate system consisting of different chromium concentration as substrate 1, different glucose concentration as substrate 2 and different HCIF concentration as substrate 3 were used to fit the stated kinetic models using the nonlinear regression analysis in GraphPad Prism 6 software for evaluating the kinetic parameters. Among other inhibitory growth kinetic models, Haldane Andrews's model gave the best fit for the experimentally acquired data for all substrates (Fig. 4A-C). The R 2 value for only chromium as a substrate was found to be 0.977, µmax value 0.002346 and the inhibition coefficient value to be 134.12. When only glucose was added as a substrate, the µmax and inhibition coefficient were found to be 0.1129 and 1.01 respectively with R 2 0.9916. In case of HCIF, the µmax and the inhibition coefficient were 0.1923   www.nature.com/scientificreports/ and 11.99 respectively and R 2 value was 0.9876. The values of specific growth rate obtained from fitting the values of single substrate system was later applied to the various inhibitory growth models to check for the cometabolic activity in tertiary substrate system. Out of the other inhibitory models used for fitting, Monod model was best fitted for the growth kinetics in tertiary substrate system in order to evaluate the interaction between the three co-metabolic substrate (i.e. HCIF and glucose and chromium) in enhancing Cr(VI) reduction. Previous studies 31 showed that addition of some co-metabolic substrate to the system was effective in dealing the degradation of refractory compounds. Table 2. One way ANOVA analysis. All values are represented as mean ± SD. Different alphabets represents difference in mean according to one way ANOVA followed by Duncan's post hoc test at probability level (p ≤ 0.05). 25 mg/L 0.154667 ± 0.066154a 0.232444 ± 0.021461bc 0.095822 ± 0.026619c 0.042578 ± 0.01024d 0.029522 ± 0.005303c 40 mg/L 0.056667 ± 0.015275b 0.266 ± 0.012b 0.131 ± 0.009849c 0.145333 ± 0.049075c 0.02287 ± 0.001499c 50 mg/L 0.030489 ± 0.004575b 0.059444 ± 0.017519c 0.038778 ± 0.014206c 0.049667 ± 0.027099d 0.026333 ± 0.005774c 100 mg/L 0.019667 ± 0.002309b 0.258778 ± 0.213544b 0.106611 ± 0.131977c 0.076889 ± 0.019357d 0.017444 ± 0.001711c  www.nature.com/scientificreports/ To understand the co-metabolism between glucose, Cr(VI) and HCIF in the tertiary system, Eq. (3) was used for the cell growth kinetic analysis. The relative kinetic parameters obtained from the analysis are presented in Table 3.The model was best fitted with the R 2 value of 0.988 (Fig. 5). The values of K 2 CS P (Inhibition of glucose in the presence of Cr(VI)), K 3 C S h (Inhibition of glucose in the presence of HCIF) and K 4 C S P S h (Inhibition of glucose in the presence of Cr(VI) and HCIF) increased with increasing Cr(VI) concentration. Amongst the three kinetic constants, the value of K 3 C S h is lower as compared to K 4 C S P S h and K 2 C S P . Lower values of K 3 C S h , indicates that the inhibition of glucose consumption is low in presence of HCIF due to the fact that HCIF was also providing food source for the microbial growth other than glucose. The values of K 2 C S P and K 4 C S P S h revealed that when only Cr was present (K 2 C S P ) the inhibition on glucose consumption was higher as compared to when both Cr and HCIF (K 4 C S P S h ) were present. Similarly, amongst the other three kinetic constant values K 2 P S C (Inhibition of Cr(VI) reduction in the presence of glucose), K3P Sh (Inhibition of Cr(VI) reduction in the presence of HCIF) and K 4 P S P S C (Inhibition of Cr(VI) reduction in the presence of glucose and HCIF), K 2 P S C values were higher which indicates that when only glucose was present (K 2 P S C ) inhibition on Cr reduction was higher followed by inhibition on Cr reduction when only HCIF was present (K 3 P S h ). But when both substrates (glucose and HCIF) were present (K 4 P S P S C ), the inhibition on Cr reduction was lowest. Inhibition of HCIF consumption due to glucose (K 2 h S C ) is lowest as compared to inhibition of HCIF consumption in presence of both Cr and glucose (K 4 h S P S C ) and when only Cr was present (K 3 h S P ). When HCIF and glucose were present, microbes were utilizing both glucose and HCIF for their growth. The higher values of K 2 P and K 3 P suggested high degree of inhibition of glucose and HCIF on Cr(VI) reduction and diauxic growth of the strain. Similarly lower values of K 2 C and K 3 C indicated the negligible impact of Cr(VI) imposed to glucose and HCIF consumption. It has been studied by 32 that for the growth of cell on mixed substrates, there are categorization of the interaction between the growth substrates which is defined as competitive, noncompetitive and uncompetitive inhibition of growth. On the basis of these observations, the results showed that glucose and Cr(VI) were not structurally similar, so uncompetitive inhibition was the best interpretation to the interaction between glucose and Cr(VI). The interaction constants K 2 h and K 3 h (inhibition of iron due to glucose and Cr(VI) respectively) were one to threefold larger than the corresponding K 2 C and K 3 C. Thus it can be inferred that another possible mixed pattern of interaction exists involving both competitive and uncompetitive inhibition. The values of kinetic constants K 4 C, K 4 P and K 4 h indicated that K 4 P and K 4 h values are lower as compared to the value of K 4 C. This may be due to the reason that the inhibition factor was more in case when there were presence of Cr and HCIF which poses competitive inhibition. The results confirmed that both glucose and HCIF as a co-metabolic substrate had a great role in Cr(VI) reduction, but when HCIF was added as another substrate, it increased the reduction efficiency of the system. The strain utilized glucose as well as  www.nature.com/scientificreports/ HCIF for maintaining their activity and for their growth. It has been reported by 33,34 that two substrates (glucose and biphenyl) had great role in bioremediation of PBDEs. However, a large number of reports 35 indicated that optimum dose of co-substrates is essential to use because excessive dose of any of the co-substrates hampers the degradation efficiency and was attributed for the inhibition between the substrates. Excess dosage of substrates would cause self-inhibition on growth of the cell, thus decrease reduction efficiency. Therefore, it is extremely important to choose appropriate co-substrate and optimum dosage of substrate.

Conclusion
The efficiency of Cr(VI) reduction by Serratia sp. was improved by cometabolism activity. Meanwhile, it is known that presence of diversity of carbon sources impose effects on the interactive activity of metabolic pathways and substrates. Glucose had great role as anti-competitive inhibitor in Cr(VI) reduction. However, on the other side, another substrate HCIF had both competitive and uncompetitive inhibition on Cr(VI) reduction by enhancing the strain ability to help in complete reduction. Both glucose and HCIF as a co-metabolic substrate had a great role in Cr(VI) reduction, but when HCIF was added as another substrate, it increased the reduction efficiency of the system and combination of HCIF and glucose showed least inhibition to hexavalent chromium reduction by Serratia sp. The outcomes of the current work enlightens a noteworthy approach to carry out treatment of other compounds as well and a great finding for enhancing long term performance of ZVI PRBs in combination with Serratia Sp. for remediation of Cr(VI) contaminated sites.