Biosorption and enzymatic reduction of hexavalent chromium by chromium resistant bacteria ameliorate phytotoxicity

Background: Chromium (Cr) (VI) is one of the toxic heavy metals and environmental hazards. Alleviating the levels of contaminants in the environment is imperative, and studying the bioremediation of Cr via reduction or biosorption is an indispensable approach to this cause. In this study, we investigated the efficiency of reduction and biosorption of Cr(VI) by chromate resistant bacteria isolated from tannery wastewater. Results: From screening, 28 Cr resistant bacteria were selected, and only two isolates, SH-1 and SH-2, were found as potential candidates for the reduction of Cr(VI). Post 16s rRNA sequencing, SH-1 isolate was identified to be Klebsiella sp and SH-2 isolate as Lysinibacillus sp. SH-1 could tolerate up to 2000 mg/L of Cr(VI) whereas, SH-2 could tolerate up to 1500 mg/L of Cr(VI). In Luria-Bertani media containing 100 mg/L of Cr(VI), the relative reduction level was 95% (SH-1) and 88.77% (SH-2) but their reduction rate was 63.08% (SH-1) and 49.89% (SH-2) of Cr(VI), respectively in the tannery effluents after 72h period of incubation. In the presence of Cr(VI) at a concentration of 10 mg/L Cr(VI), the cell-free extracts of pre-grown SH-1 and SH-2 showed a reduction of 72.2% and 33%, respectively. This reduction indicates the production or the activity of Cr reducing enzyme being higher in these two isolates than that of control in the presence of Cr(VI). In biosorption study, live and dead biomass of SH-1 biosorbed 51.25 mg and 29.03 mg chromium per gram dry weight, respectively. However, 28.83 mg and 27.65 mg chromium per gram dry weight were biosorbed by live and dead biomass of SH-2, respectively. Both the Langmuir model -for monolayer adsorption- and Freundlich model -for adsorption characteristics for the heterogeneous surface- were suitable for describing biosorption of Cr(VI) by SH-1 live biomass. The chickpea seed germination study confirmed the beneficial environmental effect of Cr(VI) reduction by these two isolates. Conclusion: The bacterial isolates can be exploited for their potential for reduction and biosorption of toxic hexavalent chromium in biological treatment of hexavalent chromium-containing wastes. Abstract Background: Chromium (Cr) (VI) is one of the toxic heavy metals and environmental hazards. Alleviating the levels of contaminants in the environment is imperative, and studying the efficiency of reduction and biosorption of Cr(VI) chromate resistant Results: From screening, 28 Cr resistant bacteria were selected, and only two isolates, SH-1 and SH-2, were found as potential candidates for the reduction of Cr(VI). Post 16s rRNA sequencing, SH-1 isolate was identified to be Klebsiella sp and SH-2 isolate as Lysinibacillus sp.SH-1 could tolerate up to 2000 mg/L of Cr(VI) whereas, SH-2 could tolerate up to 1500 mg/L of Cr(VI). In Luria-Bertani media containing 100 mg/L of Cr(VI), the relative reduction level was 95% (SH-1) and 88.77% (SH-2) but their reduction rate was 63.08% (SH-1) and 49.89% (SH-2) of Cr(VI), respectively in the tannery effluents after 72h period of incubation. In the presence of Cr(VI) at a concentration of 10 mg/L Cr(VI), the cell-free extracts of pre-grown SH-1 and SH-2 showed a reduction of 72.2% and 33%, respectively. This reduction indicates the production or the activity of Cr reducing enzyme being higher in these two isolates than that of control in the presence of Cr(VI). In biosorption study, live and dead biomass of SH-1 biosorbed 51.25 mg and 29.03 mg chromium per gram dry weight, respectively. However, 28.83 mg and 27.65 mg chromium per gram dry weight were biosorbed by live and dead biomass of SH-2, respectively. the Langmuir model -for monolayer adsorption- and Freundlich model -for adsorption characteristics for the heterogeneous surface- for biosorption Cr(VI) by SH-1 live chickpea seed germination study two can be exploited for their potential for reduction and biosorption of toxic hexavalent chromium in biological treatment of hexavalent chromium-containing wastes.

result, we observed that total chromium concentration of 50% (n=7) of the collected effluent water crossed the Bangladesh standard for wastewater disposal.

Screening of Cr(VI) reducing bacteria from tannery effluents
We hypothesized that Cr(VI) resistant organism might be found in the tannery effluents as these effluents contain a high amount of chromium (Table S1). We have screened for Cr(VI) resistant as well as reducing bacteria of which the procedure is shown in Figure 1A. At first, all 14 samples were tested for the presence of Cr(VI) resistant bacteria using 100 mg/L Cr(VI) and 28 discrete colonies were found based on morphological observation (Table S3). As shown in Figure 1B, nine isolates were found to be resistant to more than 500 mg/L Cr(VI). We have also screened for Cr(VI) sensitive bacteria from different species that were preserved in the Environmental Microbiology Laboratory to use as a negative control. An environmental isolate of Vibrio cholerae was found most sensitive (100 mg/L Cr(VI), Figure 1B). Two of the isolates (SH-1 and SH-2 can tolerate up to 2000 mg/L and 1500 mg/L, respectively) showed very high resistance to Cr(VI). Representative plates of two isolates from Cr(VI) resistance experiment are shown in Figure 1C. Next, we tested for the Cr(VI) reduction capacity of these isolates and interestingly two isolates that showed the highest resistance also showed the highest rate of reduction (SH-1 showed 97% reduction whereas, SH-2 showed 88% reduction; Figure   1D). After performing ANOVA followed by post-hoc Tukey's Test, all the isolates showed statistically significant differences for the reduction of Cr(VI) as compared to the negative control (Vibrio cholerae). This experiment proceeded with the two isolates (SH-1 and SH-2) that showed a maximum reduction. Besides, we have tested these two isolates for their resistance against different antibiotics and other heavy metals. Isolates SH-1 was resistant only to penicillin and erythromycin whereas sensitive to chloramphenicol, ciprofloxacin, gentamycin, and intermediately resistance to cefotaxime, meropenem, streptomycin (Table S4). Isolates SH-2 was found to be sensitive to all eleven (11) antibiotics except intermediately resistance to amoxicillin and erythromycin (Table S5). Results also revealed the resistance pattern for heavy metals by SH-1 was Cr 6+ > Pb 2+ > Mn 2+ > Ni 2+ , Cu 2+ , Co 2+ > Zn 2+ , Cd 2+ and for SH-2 was Cr 6+ > Pb 2+ > Co 2+ , Cu 2+ , Ni 2+ , Zn 2+ > Cd 2+ ( Figure 1E). The data from primary screening for Cr(VI) resistant bacteria showed that organism with higher resistance has a higher capacity of reduction. This intriguing finding leads us to further study the mechanism of the reduction and possible application for bioremediation.

Identification of the isolates by 16s rRNA sequencing
The analysis of 16S rRNA sequencing identified the two bacterial isolates that were capable of efficiently reducing hexavalent chromium as Klebsiella sp (SH-1) and Lysinibacills sp (SH-2) ( Table 1).
These two bacteria have not been reported in other studies as resistant as well as chromiumreducing. The submitted sequences can be found in the NCBI database, of which accession numbers are MF465571.1 and MF465572.1 ( Table 1).

Effect of chromium under optimum growth conditions
The optimum growth was determined for both isolates. At 37°C, the growth rate of both isolates was greater than that of room temperature, although the growth rate started to decline when the temperature was increased up to 44°C. The bacterial isolates SH-1 and SH-2 can also grow in a wide range of pH (5.5 to 10.0). Interestingly, in the present study, pH 9.0 was found to be optimum for the growth of both isolates. The ability to grow in the presence of Cr(VI) was also determined, in which isolates SH-1 showed similar growth curve as its control, whereas isolates SH-2 showed slightly lower curve pattern than the control. As compared to the negative control (V. cholerae), both of the isolates showed greater growth efficiency in the presence of Cr(VI) (Figure 2A and 2B).

Isolates showed different Chromium (VI) reduction capacity in different media
This study has found that these isolates have very high chromium (Cr) resistance; hence, we hypothesized that these isolates might have Cr reducing ability. Afterward, we tried to confirm this hypothesis by conducting a time course study of Cr(VI) reduction by these isolates in LB broth containing 100 mg/L of Cr(VI) at the optimum temperature (37°C) and pH (9.0). It has been reported that the optimal Cr(VI) reduction capacity is directly associated with the optimum pH condition (8.0) for the growth of Bacillus isolates [30]. In the present study, we observed that both isolates could reduce Cr(VI) rapidly at 37°C and pH 9. Isolates SH-1 could reduce Cr(VI) approximately 81.12%, 91.66%, and 95% from the medium after 24h, 48h, and 72h, respectively ( Figure 2C). On the other hand, SH-2 was also able to reduce 69.14%, 77.28%, and 88.77% from the medium after 24h, 48h, and 72h, respectively ( Figure 2C). Statistical significance was determined by paired two-tailed student's t-test. SH-1 was found to be more effective for chromate reduction compared to SH-2.
Furthermore, we have found that Cr(VI) reduction also occurred in the controls, which were cell-free ( Figure 2C).
To determine the reduction efficiency of both isolates in surface water, they were inoculated in pond water and tannery effluents containing 100 mg/L of Cr(VI) without the addition of any nutrients. Their reduction efficiency was also compared with the indigenous microorganism present in the water samples during these periods. After 72h of incubation in pond water with initial Cr(VI) concentration of 100 mg/L, SH-1 and SH-2 showed 45% and 38.37% reduction, respectively, whereas the control (with indigenous organisms) showed only 13.52% reduction ( Figure 2D). Therefore after subtracting the reduction percentage (13.52%) by the indigenous organism, the increase in reduction percentage via the addition of our isolates was calculated to be 31.48% and 24.85% (SH-1 and SH-2, respectively).
Using one way ANOVA, we obtained a statistically significant difference (p<0.05) for different concentration reduction of both isolates followed by Tukey's Post Hoc Test. Additionally, in tannery effluents, SH-1 showed 63.08% reduction after 72h of incubation; likewise, SH-2 reduced 49.89%, ( Figure 2E). However, the indigenous organisms present in the effluents exhibited only 36.65% reduction after 72h ( Figure 2E). The reduction by indigenous microorganisms was observed, but the reduction was much lower compared to the isolates. Since SH-1 can itself reduce 26.34% Cr(VI) in the presence of indigenous organisms after 72h of incubation, it indicates that higher Cr(VI) resistance may have helped SH-1 to outgrow the indigenous organisms in the presence of 100 mg/L Cr(VI).
Isolates showed enhanced chromate reductase activity in the presence of Cr(VI) The chromate reductase activity of crude cell extracts was obtained by using proteins collected from cells pre-grown; both in the presence and absence of Cr(VI). The soluble proteins from SH-1 and SH-2, in presence and absence of Cr(VI), could reduce Cr(VI) as indicated in Table 2, whereas protein sample boiled at 100°C and negative control (V. cholerae) failed to show any significant chromate reduction. The reduction caused by protein sample boiled at 100°C was used as blank control and was subtracted from the other reduction data. Protein samples from isolates that were treated with Cr(VI) showed 72.2% (SH-1) and 33% (SH-2) reduction, but protein samples from isolates that were not treated with Cr(VI) showed 52% (SH-1) and 26.6% (SH-2) reduction. Therefore, reduction caused by Cr(VI) treated proteins was higher than the reduction of untreated proteins of the isolates ( Table 2).
As we have used the same amount of protein for every sample, we can deduce that chromate reductase activity increased with Cr(VI) reduction ability ( Table 2).

SH-1 live biomass showed better biosorption capacity in response to different conditions
The biosorption capacities of the bacterial biomass (SH-1 live, SH-2 live, SH-1 dead, SH-2 dead) for the removal of Cr(VI) were found as a function of initial metal ion concentration, pH and time.
(i) Low pH showed better biosorption. As wastewater containing metal ion have different pH values, that's why biosorption study of Cr(VI) was performed at different pH. Initial pH values were set from 1 to 8 of 100 mg/L Cr(VI) solution. As illustrated in Figure 3A, the capacity of adsorption increased from pH 1.0 to pH 2.0, and then decreased with the increase of pH. We observed a negative correlation of Cr(VI) adsorption individually between the variables (SH-1 live, SH-1 dead, SH-2 live and SH-2 dead) and the increasing pH (r 1 = -0.89, r 2 = -0.94, r 3 = -0.88 and r 4 = -0.96 respectively); which were statistically significant (p <0.01). The maximum rates of biosorption were 33.28 mg/g of live biomass (SH-1 live) at pH 2 and 16.77 mg/g of dead biomass (SH-2 dead) at pH 1 ( Figure 3A).
(ii) Biosorption increased with the increasing initial concentration of Cr(VI). The biosorption process was conducted at optimized pH (pH 2 for live and pH 1 for dead) at different initial concentrations (20 mg/L to 200mg/L). When the initial Cr(VI) concentration was increased from 20 to 200 mg/L, the absorbing capacity was also increased from 8.76 to 51.25 mg/g (SH-1 live), 3.46 to 28.83 mg/g (SH-2 live), 6.21 to 29.03 mg/g (SH-1 dead), 1.8 to 27.65 mg/g (SH-2 dead) biomass ( Figure 3B). We observed a positive correlation of Cr(VI) adsorption individually between the variables (SH-1 live, SH-1 dead, SH-2 live and SH-2 dead) and the increasing Cr(VI) concentration (r 1 = 0.99, r 2 = 0.96, r 3 = 0.95 and r 4 = 0.93 respectively); which were statistically significant (p <0.01). The maximum biosorption capacity was found in the SH-1 live biomass at 200 mg/L.
(iii) Effect of time on biosorption process. Figure 3C illustrates the contact time of Cr(VI) by the live and dead biomass at a concentration of 200 mg/L at the optimized pH mentioned above. Higher adsorption capacity was found in live biomass than the dead biomass ( Fig 3C). Adsorption by the biomass (SH-1 live, SH-1 dead, SH-2 live and SH-2 dead) was increased rapidly throughout the first 60 min and continued to be constant until 90 min. Here, a positive correlation of Cr(VI) adsorption individually between the variables (SH-1 live, SH-1 dead, SH-2 live and SH-2 dead) and the time up to 90 min was found (r 1 = 0.94, r 2 = 0.86, r 3 = 0.91 and r 4 = 0.88 respectively); which were statistically significant (p <0.01). Subsequently, after acquiring the equilibrium, no significant differences of Cr(VI) adsorption with time were observed, though the experiments were performed until 360 min. In the Freundlich model, the value of 1 n is 0.77 and n is 1.30 which reveals that the sorption of Cr(VI) ion unto live SH-1 biomass is favorable and the R 2 value is 0.994 ( Figure 3E and 3F). So the value of K f and n is the indication of easy uptake of Cr(VI) ion with a high adsorptive ability of live biomass.
The isotherm models for SH-2 live biomass is added as a supplementary figure ( Figure S6).
Phytotoxicity assay using chickpea seed germination Toxicity of hexavalent chromium on seed germination along with the growth of roots and shoots was examined in this study. As shown in Figure 4A and 4B growth of shoot and root length have gradually decreased with increasing concentration of Cr(VI). Calculating correlation of Cr(VI) concentration individually with the shoot and root length we observed a negative correlation (r 1 = -0.74 and r 2 = -0.91 respectively), which indicates that Cr(VI) affected the growth of shoots and roots of chickpea seeds. Although Cr(VI) did not affect the rate of seed germination. Above 50 mg/L seedling growth was greatly hampered, and no seedling was observed at 100 mg/L ( Figure 4A and 4B). A similar test was conducted to assess the mitigation of toxicity of bacterial treated and untreated Cr(VI) samples ( Figure 4C). In control, the mean shoot and root length was observed as 14.9 cm and 7.7 cm, respectively ( Figure 4D); while the mean shoot and root length of the seeds with Cr(VI) sample that was treated with SH-1 bacteria were 10.1 cm and 3.2 cm and Cr(VI) sample that were treated with SH-2 bacteria were 6.7 cm and 3.6 cm, respectively. On the other hand, the mean shoot and root lengths were 0.7 cm and 0.8 cm, respectively in case of 50 mg/L Cr(VI). These results indicate that 50 mg/L Cr(VI) treatment significantly reduces the growth of shoot and root (95.3% and 89.6%, respectively). Of note, Cr(VI) sample that was treated with SH-1 bacteria significantly recovered 93% and 75% shoot and root length respectively as compared to the 50mg/L Cr(VI) sample. Similarly, SH-2 treated Cr(VI) sample also significantly recovered 89% and 77% shoot and root length respectively as compared to the 50mg/L Cr(VI) ( Figure 4C and 4D).

Discussion
Prokaryotes can tolerate toxic Cr better than the eukaryotes, and thus, bacteria can survive in a higher concentration of toxic Cr(VI) [31]. Cr(III) sulfate ([Cr(H 2 O) 6 ] 2 (SO 4 ) 3 ) has long been used as a tanning agent and being leached into the effluents [32]. Though Cr(III) sulfate is being used, the transformation of Cr(III) of tanning agent into Cr(VI) is occurred through oxidation due to the repeated exposure of skin to direct sunlight and during other leather tanning processes [33,34]. In this study, we investigated the screening of 28 Cr(VI)-resistant bacteria from tannery effluent, followed by Cr(VI) reduction and absorption by the two selected isolates.
The potentiality of efficient remediation of toxic metals by utilizing living microorganisms is a key factor which may enhance the application of heavy metals bioremediation [35]. In some previous studies, the bacteria which were tested showed a lower reduction of Cr(VI) over a relatively long period of time. For example, it was reported that with 120h of incubation, Alcaligens faecalis reduced 70%, while Pseudochromobactrum saccharolyticum reduced 40% of Cr(VI) [36]. Another study by Sharma et al. revealed that Bacillus sp (Accession number FM334108.1), Bacillus subtilis, and Bacillus sp (Accession number FJ3480004.1) reduced 73.41%, 42.15% and 60% of 100 mg/L of Cr(VI) after 120h of incubation, respectively [37]. After 72h incubation, SH-1 showed 95%, 63% and 45% reduction in LB media, tannery effluent, and pond water, respectively. Although, we have found that Cr(VI) reduction also occurred in the control, which was cell-free ( Figure 2C). It can be suggested that components may have caused the reduction in the LB medium since no cells were inoculated into the medium. A study by Shanab et al. found that medium components along with carbon sources are able to interact with toxic metals such as arsenate, cadmium, chromium, cobalt, copper, lead, nickel, mercury and zinc to give misleading results [38]. Indigenous organism present in the pond water and tannery effluent has also reduced 8% and 36% in pond water and tannery effluent, respectively. We assume that such high reduction of Cr(VI) in the tannery effluent probably due to the presence of organic and inorganic compounds as well as the probable presence of Cr resistant and reducing organism [14].
Enzymatic reduction of Cr(VI) has been described as one of the main mechanism that microorganism uses to survive in the chrome-polluted wastewater [27]. Zahoor et al. reported that cell-free extracts of Bacillus sp. JDM-2-1 reduced 83%, and S. capitis reduced 70% of 10 mg/L of toxic hexavalent chromium [14]. Previous study also showed the reduction of Cr(VI) caused by soluble type enzymes present in the cell-free extracts collected from Bacillus sp. [39]. In the present study, the cell-free extracts of SH-1 and SH-2 showed the reduction of Cr(VI) which indicates the production or the activity of the Cr reducing enzyme in the presence of Cr(VI).
Biosorption of heavy metals is considered an eco-friendly technique for the removal of toxic metals from the environment [13]. The current study revealed high sorption capacity of live biomass in response to different experimental conditions. At low pH, the biomass of isolates showed high biosorption rate as compared to high pH. Similar results were observed for the Cr(VI) biosorption by Ochrobactrum anthropic and green algae of which the maximal biosorption capacity was found at pH 2.0 [40,41]. These results suggest that the negatively charged Cr species (Cr 2 O 7 2 ) bind through electrostatic attraction force to the positively charged ions on the biosorbents' surface. So, functional groups of biosorbent at low pH become protonated and thus negatively charged Cr is attracted and binds in the surface. On the other hand, within pH 3.0 to 8.0 biosorption showed no statistically significant differences since at high pH deprotonation of biosorbent takes place and functional groups become negatively charged which prevents negatively charged Cr to bind with it [42].
The biosorption capacities of both the live and dead biomass were found to have increased with the increasing concentration of Cr ion. Similar trends were found in the biosorption study of the biomass of Pantoea sp. TEM18 [43]. This might be due to the increase in the competition for the close-fitted binding sites amount on the surface of biosorbents [44]; which indicates the properties of biosorbents (e.g., functional groups, surface area, etc.) and the properties of metal sorbates (e.g., atomic weight, ionic size, etc.) may play an important role in the biosorption process [43].
Biosorption rate increased during the first 60 minutes and slowed down after 90 minutes due to reaching the equilibrium phase. Biosorption rate might be depended on initial metal ion concentration as reported by others [44]. Adsorption isotherm models suggested by Freundlich and Langmuir were found suitable for describing the short-term biosorption of Cr(VI) in this study. The Langmuir isotherm describes monolayer sorption where the finite number of identical sites is present onto a surface and assumes constant energies of adsorption on the surface of the adsorbent. To represent the experimental data, the Freundlich model is appropriate where it is assumed that if the absorbing surface is heterogeneous the metal biosorbs to form monolayers [45].
The previous study showed that pea seeds germination was not significantly affected at high concentration of Cr, though the growth of radicle and plumule was suppressed at concentrations of 147 mg/L Cr(VI) [46]. Similar trends were also observed during our study of the effect of Cr(VI) on the seed germination. The data suggest that seed germination and successive seedling growth were interrupted by high levels of chromium in which more toxic effect was observed on roots rather than on shoots. But after using the bacterial treated chrome water, we observed the amelioration of the phytotoxicity. This evidence carries significant environmental benefits of using eco-friendly bacteria to reduce the toxicity of Cr(VI) by either reduction or biosorption.
In a nutshell, Figure 5 illustrates the overview of the work. The schematic diagram shows how toxic Cr is being leached from tannery to the effluent and then to the natural water body such as rivers and canals. Next, Cr resistant bacteria were isolated from that tannery effluent, and then significant biosorption and enzymatic reduction were found. Furthermore, a beneficial environmental effect was found by the chickpea seed germination and seedlings growth test.

Conclusion
Both the isolates studied here showed strong potential for the removal of hexavalent chromium in LB Zahoor and Rehman [14]. Another screening was performed using laboratory isolates to find out the lowest resistant bacteria to use as a negative control.

16S rRNA identification of isolates
The bacterial genomic DNA was extracted according to the manufacturer's instruction using Maxwell

Determination of optimum growth condition of the isolates
Determination of the optimum temperature and pH for the growth of two isolates were performed according to the procedure described elsewhere [14]. The effect of Cr(VI) on the growth of bacteria was also investigated according to Zahoor and Rehman, 2009, with slight modifications [14]. Growth inoculum. The cultures were incubated at 37°C in a shaker at 120rpm. An aliquot of culture was taken out at regular intervals from 0h to 24h. Absorbance was measured at 600 nm.

Resistance to antibiotics and other heavy metals
Disk diffusion method was used to determine the antibiotic resistance properties of the isolated bacteria SH-1 and SH-2 [48]. After 24h of growth period, the inhibition zones were measured following the standard antibiotic disc sensitivity testing method [49]. Analysis of reduction capacity of Cr(VI) by the isolates in the culture medium, pond water, and tannery effluents The ability of bacterial isolates SH-1 & SH-2 to reduce Cr(VI) was examined in sterilized 125 mL Erlenmeyer conical flask. LB broth media (25 mL) with added Cr(VI) concentration of 100 mg/L as K 2 Cr 2 O 7 was inoculated with 20 μL of the selected young bacterial suspension. Cultures were incubated at 37°C with 120 rpm. Then 1 ml of the culture was taken after 24, 48 and 72 h, centrifuged at 1100 g for 5 min and the supernatant was analyzed for the residual Cr(VI) using 1,5diphenyl carbazide method [14].
The efficiency of the bacterial isolates SH-1 and SH-2 to reduce Cr(VI) in LB broth as well as in surface water containing Cr(VI) were tested according to the method described by Zahoor and Rehman [14].
In these experiments, 25 mL pond water and tannery effluents were taken separately in sterilized 125 mL Erlenmeyer conical flask with added Cr(VI) concentration of 100 mg/L. Then, 20 μL of an overnight culture of the selected isolates were inoculated into the respective pond water and effluents. After incubation of 24, 48 and 72 h in an orbital shaker at 37°C, samples (1mL) were taken for the measurement of residual Cr(VI) in the solution using above method [14].

Enzyme assay
The bacterial isolates (SH-1, SH-2, and V. cholerae) were grown in 10 mL LB broth medium for 48 h at cholerae acted as a negative control. After 12h of incubation at 37°C, Cr(VI) reduction was assessed according to the diphenylcarbazide method [14].

Batch biosorption experiments
The preparation of live and dead cell biomass was performed as described in other studies [43,50]. A batch equilibrium method [51] was used to determine the sorption of Cr(VI) by the biomass of the isolates (SH-1 live, SH-2 live, SH-1 dead, and SH-2 dead). All sets of experiments were done with 10 mg of the dried biomass in fixed volume (10 ml) of Cr(VI) solution in sterilized falcon tube (15 ml).
Bacterial biomass was exposed to Cr(VI) solution separately for 6h on a rotary shaker (140 rpm) at 30°C. The effects of pH, contact time, and initial Cr(VI) concentration on biosorption were recorded.
All pH values of the solutions were adjusted from 1.0 to 8.0 using 1N HCl and 1N NaOH. The samples were taken at different time intervals and centrifuged at 1100 g for 5 minutes afterward, the supernatant was analysed for Cr(VI) concentration by 1,5 diphenylcarbazide method [14].

Measurement of metal uptake
The formulae described by Vanderborght and Van Griekenm was used to calculate the amount of adsorbed Cr(VI) (mg/g) [52], which is as follows:

Adsorption isotherm analysis
The biosorption equilibrium study was carried out according to Langmuir and Freundlich isotherm models described by Dada et al. [45]. The Langmuir adsorption equation (i) To determine the Cr(VI) reducing effect, isolates (SH-1 & SH-2) were incubated with Cr(VI) and then added into the seed germination plate with or without 50 mg/L Cr(VI) as described above. The filtersterile pond water was used as control and pond water with 50 mg/L Cr(VI) was used as a negative control. Seeds were placed under the same conditions described above. Germination rate, as well as root and shoot length, were observed and measured after one week.

Statistical analysis
The    respectively. In figure 2C, D and E, paired two-tailed students t-test was performed between control and the study group and data are expressed as mean ± standard deviation (SD) (*P <0.05, **P < 0.01, ***P < 0.001).  The mean shoot and root length of the treated seeds of figure 4C. In figure B and D paired two-tailed students t-test was performed between control and the study group and data are expressed as mean ± standard deviation (SD). "*" denotes significant differences between control group and study group (*P <0.05, **P < 0.01, ***P < 0.001). "#" indicates significant differences between Cr(VI) 50mg/L and study group (#P <0.05, ##P < 0.01). Figure 5 The schematic diagram shows an overview of the work. Table legends   Table 2 a The amount of enzyme that converts 1.0 µM Cr(VI) per min at 37°C was defined as one unit of Cr(VI) reductase activity.
b Cr(VI) reduction was measured after 12 h of incubation.
All data in the table are expressed as mean ± standard deviation (SD).

Additional files
Supplementary data 1: Table S1: Analysis of Physicochemical properties of Tannery effluents.