Chromium toxicity and bioaccumulation by chromium tolerant bacillus and arthrobacter species 4

Chapter IV: Results and Discussion CHAPTER IV RESULTS AND DISCUSSION 4.1 Isolates screened from the solid waste and liquid effluent. Two isolates (one each from solid waste and the other from liquid effluent) were screened for microorganisms which most chromium tolerant. The two isolates were further investigated for the toxic effect of chromium by examining the morphological changes and metal-accumulation by the organisms. The isolate obtained from the solid waste, named S1, showed yellow colonies (Figure 4.1 (a)), whereas the one isolated from the liquid effluent, named L4, showed white smooth colonies (Figure 4.1 (b)) Figure 4.1: Chromium microorganism isolated from (a) solid waste (S1) (b) liquid effluent (L4) The biochemical characteristics of both the isolates were determined and listed in Table 4.1 and were cross-checked with reference from Bergey’s Manual of Systematic Bacteriology (Volume 2). The information provided is similar to that reported by Pal and 51 Chapter IV: Results and Discussion Paul (2004). These organisms were then sent for 16S RNA analysis to identify the species these isolates belong. Table 4.1 Biochemical characterization of the isolates from solid waste and liquid effluent. Characteristics S1 (solid waste) L4 (liquid effluent) Morphological characteristics: Colony morphology yellow, round white, small round Gram reaction +ve +ve Micro morphology Rod Cocoi Production of catalase +ve +ve Production of oxidase +ve +ve Hydrolysis of starch -ve +ve Gelatin hydrolysis -ve -ve Triple sugar iron (TSI) test ferments lactose neither glucose nor lactose and/or sucrose or sucrose is fermented Citrate utilization test -ve Biochemical characteristics: -ve 4.2 Effect of chromium toxicity on the growth of isolated organisms Several reports have been published on the effect of chromium on the growth of microorganisms. There are reports where the growth of organism is retarded when exposed to chromium. Here in this report, effect of chromium when added during the 52 Chapter IV: Results and Discussion exponential phase is reported. No paper has been reported so far that determine the cells’ behavior when metal is added in their exponential phase. Generally papers are published where authors have reported the addition of chromium at start of the experiment. Here, the cell growth during incomplete availability of the nutrients (during log phase) was examined. It was interesting to know the cell behavior in presence of enzymes produced during growth phase and then chromium was added. The cell adaptability against the chromium toxicity was determined when Cr is added from initial 0 ppm to 40 ppm (as K2Cr2O7). Figure 4.2 shows the growth of the organisms in a batch culture. 3 OD (600nm) 2.5 2 1.5 S1 1 L4 0.5 0 0 10 20 30 40 50 time (hrs) Figure 4.2: Batch growth of S1 and L4 (in the absence of chromium) From the Figure 4.2, it can be seen that the exponential phase occurs after 8 hours, with the stationary phase occurring after 24 hours. The following figures show the effect of increasing concentration of chromium on the growth of microorganisms. Figure 4.3(a) shows the effect of chromium (1 ppm) on the metal-unexposed cells, with chromium added during the exponential phase. An OD of 0.70 and 0.65 was attained by S1 and L4 respectively during the exponential phase. Figure 4.3 (a) showed that S1 couldn’t grow in the presence of chromium, whereas L4 was able to tolerate the chromium presence. 53 Chapter IV: Results and Discussion Hence, the adaptability towards chromium was slower in case of S1 when compared to L4. 2.5 OD (600nm) 2 1.5 S1 L4 1 0.5 0 0 10 20 40 50 time (hrs) 30 Figure 4.3 (a): Toxicity effect of Cr (1 ppm) on metal unexposed organisms There are reports of decreased microbial activity due to exposure/stress of metals (Maliszewska et. al., 1985). Cr (VI) was found to inhibit soil biological properties, such as phosphatase and sulfatase activities and to decrease microbial biomass (Speir et. al., 1995). Although the extent of inhibition caused by Cr (VI) diminished with time, the differences were generally much smaller than the observed decline in extractability of Cr (VI) (Speir et. al., 1995). The increase in adaptability towards chromium is shown in this study. S1 initially when exposed to Cr (VI) showed decreased growth, but further it was observed when these 1 ppm exposed cells were exposed to higher concentration of Cr (VI), it show enhanced resistance. Figure 4.3(b) shows the behavior of these isolates when 2 ppm is added at exponential phase to freshly grown 1 ppm exposed cells. 54 Chapter IV: Results and Discussion 3.5 OD (600nm) 3 2.5 S1 2 L4 1.5 1 0 5 10 15 20 25 30 35 40 time (hrs) Figure 4.3 (b): Toxicity effect of Cr VI (2 ppm) on 1 ppm Cr exposed cells. From the above Figure 4.3 (b), it could be interpreted that the bacteria develop a mechanism where it is able to tolerate higher concentration of metal. S1 is able to grow better in the presence of toxic chromium when compared to previous batch i.e. unexposed batch in Figure 4.3(a). Also it was observed that L4 has also gain resistance when compared to previous batch (figure 4.3(b)). Turpeinen et. al., (2004) has demonstrated a clear relationship between bioavailability of arsenic and As (III) resistance, indicating that there had been a selection for As (III) resistant bacteria in the contaminated soils due to high toxicity of As (III). In this present study, step-wise adaptation of the microorganisms to gain resistance towards chromium is demonstrated. The effect of chromium on the growth characteristics and its slow resistance development when metal is added in exponential phase is reported. The following Figures 4.3 (c-f) show how these two isolates have developed resistance from Cr concentration ranging from 1 ppm concentration of Cr to 40 ppm concentration. 55 Chapter IV: Results and Discussion 4.5 OD (600nm) 4 3.5 3 S1 2.5 L4 2 1.5 0 5 10 15 20 25 30 35 time (hrs) Figure 4.3 (c): Toxicity effect of Cr VI (4 ppm) on 2 ppm Cr exposed cells. From Figure 4.3(c), it can be seen that the cells have adapted resistance towards chromium very rapidly. From the absorbance, it can be seen that cells growing in 5 hours is more when compared to Figure (4.3-b) where cells were only exposed to 2 ppm chromium. This clearly shows that the cells are developing resistance towards chromium subsequently. 5 4.5 OD (600nm) 4 3.5 3 2.5 S1 2 L4 1.5 1 0.5 0 0 10 20 30 40 50 60 70 time (hrs) Figure 4.3 (d): Toxicity effect of Cr VI (10 ppm) on 4 ppm Cr exposed cells. 56 Chapter IV: Results and Discussion When increasing concentration from 4 ppm to 10 ppm, a slow growth pattern of the cells was evident (Figure 4.3-d). L4 shows adaption at this concentration as well (though over a slightly longer period), but S1 had slow activity at this concentration (Figure 4.3-d). 2.9 OD (600nm) 2.7 2.5 2.3 S1 2.1 L4 1.9 1.7 1.5 0 5 10 15 20 25 30 35 40 45 50 time (hrs) Figure 4.3 (e): Toxicity effect of Cr VI (20 ppm) on 10 ppm Cr exposed cells. From Figure 4.3(e), it was found that even at a concentration of 20 ppm (doubled the previous concentration) exposure of chromium, S1 was able to grow in Cr milieu and has shown maximum growth, though in a slightly longer time. L4, instead showed constant increasing growth kinetics. 3.5 OD (600nm) 3 2.5 S1 2 L4 1.5 1 0.5 0 10 20 30 40 50 60 time (hrs) Figure 4.3 (f): Toxicity effect of Cr VI (40 ppm) on 20 ppm Cr exposed cells. 57 Chapter IV: Results and Discussion From this Figure 4.3(f), it can be seen that both the isolates have almost reached a level where is not much further adaption taking place. It appears that the isolates have reached their maximum chromium tolerance capacity. From the Figure 4.3 (a-f), L4 was found to be marginally more tolerant to chromium. This has been shown in further experiments of bioaccumulation, in relation to metal resistance. 4.3 16S rDNA Analysis 4.3.1 16S rDNA analysis of isolate from solid waste (S1) Both the isolates S1 and L4 were analyzed for 16S rDNA for species identification. On the basis of nucleotide homology and phylogenetic analysis, S1 was found to be very similar to Bacillus marisflavi TF 11(AF483624) (Figure 4.4-a). Based on nucleotide sequence, the percentage homology of this strain was found to be 98% similar to Bacillus marisflavi TF 11(AF483624) and Bacillus aquimaris (DQ105971) (Table 4.2). The isolate exhibits catalyse and oxidase activities. DQ285074 (Bacillus sp.) AY373362 (Bacillus sp.) DQ105973 (Bacillus marisflavi) 55 DQ448746 (Bacillus sp.) AF483625 (Bacillus aquaemaris st. TF-12) 100 AJ315068 (Bacillus sp.) 100 AJ244686 (Bacillus sp.) AY505499 (Bacillus aquimaris st. GSP18) DQ105971 (Bacillus aquimaris) S1 46 AF483624 (Bacillus marisflavi st. TF-11) Figure 4.4 (a): Phylogenetic Tree of S1 (using neighbor joining method). 58 Chapter IV: Results and Discussion S. No. Isolates 1 S1 2 AF483624 3 DQ105971 4 AY505499 5 DQ105973 6 AY373362 7 DQ285074 8 DQ448746 9 AF483625 10 AJ315068 11 AJ244686 PERCENTAGE HOMOLOGY 1 2 3 4 5 6 7 8 9 10 11 * 98 98 98 98 98 98 97 97 97 96 * 100 100 100 99 99 98 98 98 98 100 100 99 99 98 98 98 98 100 99 99 98 98 98 98 * 99 99 98 98 98 98 * 99 98 97 97 97 * 98 98 98 97 * 99 99 98 * 99 99 * 99 * * * Table 4.2: Percentage homology of S1 based on nucleotide sequence Several Bacillus species are known to remove chromium from contaminated soil or effluent. However, there is no report on heavy metal removal by Bacillus marisflavi. Yoon et. al., (2003) isolated Bacillus marisflavi st. TF 11 (AF483624) from sea water of a tidal flat in Yellow Sea in Korea. Rafidinarivo et. al., (2007) isolated several bacillus species from marine sediments, out of which Bacillus marisflavi is one of them. Bacillus marisflavi has also been isolated from water pipeline in Gulf of Mexico (Lopez et. al., 2006). Narita et. al., (2004) has shown the presence of mercury-resistant transposon Tn5085 in Bacillus marisflavi. Riis et. al., (2003) has shown Bacillus marisflavi to be a 59 Chapter IV: Results and Discussion potential hydro-carbon degrader. To the best of this authors’ knowledge, Bacillus marisflavi is not known for chromium tolerance and bioaccumulation so far. S1 is a chromium-tolerant bacillus strain which is only 98% similar with the nearest neighbor (Bacillus marisflavi TF 11 (AF483624) and Bacillus aquimaris (DQ105971)) (Table 4.2). Hence, S1 is a novel strain, in accordance to 16S rDNA analysis. 4.3.2 16S rDNA analysis of isolate from liquid effluent (L4) The isolate from liquid effluent was found to be only 96% similar to one Arthrobacter sp. with accession no. AB248532. From the phylogenetic tree (Figure 4.4(b)) and percent homology (Table 4.3), this isolate showed 94% and 93% similarity to another Arthrobacter sp. (accession no. AB248526) and Arthrobacter nicotinovorans (X80743) respectively. AM409362 (Arthrobacter sp. st. 4C1-b) AF408952 (Arthrobacter sp.) AB248529 (Arthrobacter sp.) AM409361 (Arthrobacter sp. St. 4C1-a) AY651318 (Arthrobacter sp. st. ADG1) 54 65 AJ785761 (Arthrobacter sp. st. KA4-2) 95 AF102267 (Arthrobacter chlorophenolicus) 100 X80743 (A. nicotinovorans) 99 AB248526 (Arthrobacter sp.) 100 L4 100 AB248532 (Arthrobacter sp.) Figure 4.4 (b): Phylogenetic Tree of L4 (using neighbor joining method). 60 Chapter IV: Results and Discussion S. No. Isolates 1 L4 2 AB248532 3 AB248526 4 X80743 5 AF102267 6 AJ785761 7 AY651318 8 AM409361 9 AB248529 10 AF408952 11 AM409362 PERCENTAGE HOMOLOGY 1 2 3 4 5 6 7 8 9 10 11 * 96 94 93 94 94 94 94 94 94 94 * 96 95 96 96 96 96 96 96 96 * 97 97 98 98 99 99 99 99 * 97 97 96 97 97 97 97 * 98 98 98 98 98 98 * 98 98 98 98 98 * 99 98 98 99 * 99 99 99 * 99 99 * 98 * Table 4.3: Percentage homology of L4 based on nucleotide sequence There are several reports on Arthrobacter species on removal or resistance towards heavy metals, which include chromium. Ren et. al., (2004) isolated Arthrobacter nicotinovorans that secrete a protease which released cadmium from scallop hepatopancreas (the main residue after removing the edible parts of scallop) into liquid medium. Similarly, Horton et al., (2006) has shown reduction of hexavalent chromium by Arthrobacter aurescens. Arthrobacter viscosus supported on GAC (granulated activated carbon) has been reported for treatment of chromium (Quintelas et. al., 2009; Quintelas and Tavares, 2001). Toxicity of hexavalent chromium and its reduction by Arthrobacter 61 Chapter IV: Results and Discussion sp. isolated from soil contaminated with tannery waste is also known (Megharaj et. al., 2003). Edgehill (1996) has shown the effects of copper-chrome-arsenate (CCA) components on pentachlorophenol (PCP) degradation by Arthrobacter strain ATCC 33790. The isolate that is reported in this report is only 96% similar to Arthrobactersp.(accession no. AB248532). It is only 94% and 93% similar to another Arthrobacter sp. (accession no. AB248526) and Arthrobacter nicotinovorans (X80743) respectively. Hence, the strain L4 is novel according to 16S rDNA analysis. 4.4 Chromium removal assay 4.4.1 Chromium removal from synthetic effluent inoculated with isolate from liquid effluent (L4): Chromium removal capacity of Arthrobacter sp. (L4) for different initial concentration of chromium added in logarithmic phase is shown in Figure 4.5(a). In this experiment, the total chromium (Cr (VI) and Cr (III)) removal from the synthetic effluent containing different initial concentration of Cr (added during log phase) in the form of K2Cr2O7 was analyzed. From the Figure 4.5(a), it can be seen that more than 50% of the chromium is removed during first 3-5 hours and more than 95% is removed from the synthetic effluent within 24 hours. Several papers have reported the removal of chromium by Arthrobacter sp. (Megharaj et al., 2003; Horton et al., 2006) but not on the removal of chromium when added at log phase. 62 Chapter IV: Results and Discussion 45.00 40.00 Chromium in solution (ppm) 35.00 2 ppm 30.00 4 ppm 25.00 10 ppm 20.00 20 ppm 15.00 40 ppm 10.00 5.00 0.00 0 20 40 60 80 100 120 140 Time (hrs) Figure 4.5(a): Removal of chromium by L4 at various initial concentrations (2-40 ppm) 4.4.2 Chromium removal from synthetic effluent inoculated with isolate from solid waste (S1): Chromium removal capacity of Bacillus sp. (S1) isolated from solid waste at different initial concentration of chromium added in logarithmic phase is shown in Figure 4.5(b). In this experiment, total chromium (Cr (VI) and Cr (III)) removal from the synthetic effluent containing different initial concentration of Cr (added during log phase) in the form of K2Cr2O7 was analyzed. From the Figure 4.5(b), it can seen that more than 63 Chapter IV: Results and Discussion 50% of the chromium is removed during first 3-5 hours and more than 95% is removed from the synthetic effluent in 24 hours. Several papers report for the removal of chromium by Bacillus sp. Here, in this report a novel isolate which is 98% similar to Bacillus marisflavi has been reported. Bacillus marisflavi is only known for degrading hydrocarbon (Riis et. al., 2003). In this report, chromium tolerance and bioaccumulation capacity by S1 is reported. 45 40 Chromium in solution (ppm) 35 30 25 2 ppm 4 ppm 20 10 ppm 20 ppm 40 ppm 15 10 5 0 0 20 40 60 80 100 120 140 Time (hrs) Figure 4.5(b): Removal of chromium by S1 at various initial concentrations (2-40 ppm) 64 Chapter IV: Results and Discussion 4.4.3 Comparative study between S1 and L4 for removal of Chromium A comparison between the growth pattern of S1 and L4 at different initial concentration of Chromium in solution (ppm) chromium added during log phase is shown in Figures 4.6(a-e). 2.5 2 L4 1.5 S1 1 0.5 0 0 5 10 15 20 25 30 35 40 Time (hrs) Chromium in solution (ppm) Figure 4.6(a): Chromium removal at 2 ppm initial concentration of Cr for S1 and L4 4.5 4 3.5 3 L4 2.5 S1 2 1.5 1 0.5 0 0 10 20 30 40 50 60 Time (hrs) Figure 4.6(b): Chromium removal at 4 ppm initial concentration of Cr for S1 and L4 65 Chapter IV: Results and Discussion Chromium in solution (ppm) 12 10 8 L4 6 S1 4 2 0 0 10 20 30 40 50 60 Time (hrs) Chromium in solution (ppm) Figure 4.6(c): Chromium removal at 10 ppm initial concentration of Cr for S1 and L4 25 20 L4 S1 15 10 5 0 0 5 10 15 20 25 30 35 40 45 50 Time (hrs) Chromium in solution (ppm) Figure 4.6(d): Chromium removal at 20 ppm initial concentration of Cr for S1 and L4 45 40 35 30 25 20 15 10 5 0 L4 S1 0 10 20 30 40 50 60 Time (hrs) Figure 4.6(e): Chromium removal at 40 ppm initial concentration of Cr for S1 and L4 66 Chapter IV: Results and Discussion From all the above Figures 4.6(a-e), it can be observed that S1 (isolate from solid waste: Bacillus sp.) was able to sequester more chromium when compared to L4 (isolate from liquid effluent: Arthrobacter sp.). These results corroborate our earlier toxicity studies, which shows that Arthrobacter sp. (L4) was more tolerant towards Cr, and was unable to sequester more Cr compared to Bacillus sp. (S1). S1 was less tolerant to chromium due to high amount of sequestration when compared to L4. The toxic effect of Cr, as manifested in cell morphology, is reported later in section 4.10. Scott and Palmer (1988) have shown that Arthrobacter sp. (A. viscosus) was able to produce more exopolysaccharides which accounts for the higher tolerance of the cells towards heavy metal. The polysaccharides bind the heavy metal outside the cells, thereby protecting the cell from lysis. In section 4.9 of this report, it has been shown that L4 produce more polysaccharides when compared to S1. Hence it can be related that L4, being more tolerant to Cr (as a result of more secretion of exo-polysaccharide) was unable to sequester more Cr when compared to S1. 4.5 Intracellular compartmentalization of the chromium uptake: Table 4.4 shows the relative distribution of chromium in the different compartments of S1 and L4, after metal uptake over 48 hours. Cellular fraction Periplasmic fraction Cytoplasmic fraction Membrane fraction Chromium Concentration (ppm) S1 L4 10.02 12.02 14.54 10.24 9.68 8.34 Table 4.4: Relative distribution of accumulated Cr (initial conc: 40 ppm) after 48 hours incubation. 67 Chapter IV: Results and Discussion The values in the table were calculated taking average values obtained from duplicate experiments. It can be inferred that out of 40 ppm, a total of 34.24 ppm and 32.60 ppm has been accumulated by S1 and L4 respectively. A mass balance obtained by summing up the metal uptake and the chromium remaining in the solution in Figure 4.6 (e) shows that almost all the 40 ppm chromium was accounted. From Table 4.4, it can be determined that periplasmic accumulation was more in the case of L4 when compared to S1. The total intracellular accumulation was higher for Bacillus sp. when compared to Arthrobacter one. The reason behind this less accumulation in the case of Arthrobacter sp. could be production of more amount of EPS. Scott and Palmer (1988) have showed that Arthrobacter is able to produce more amount of EPS and hence more resistant and less intracellular uptaking. 4.6 FTIR analysis The infrared spectra of the control biomass (i.e. in the absence of chromium) and treated biomass (at 40 ppm exposed chromium) of L4 and S1 are shown in Figures 4.7(a) and 4.7(b) respectively. The absorption peaks between 3400-3600cm-1 corresponds to -OH and -NH stretching of the protein and acetamido group (Bai and Abraham, 2002; Deng and Ting, 2005). The strong absorption peak ranging from 1640-1650 cm-1 is attributed to amide I band of amide bond in N-acetyl glucosamine polymer or of the protein peptide bond. It also relate to the C=O chelate stretching of -COOH functional group (Bai and Abraham, 2002; Yee et al., 2004; Park et al., 2005). Another characteristic peak found in 68 Chapter IV: Results and Discussion both the isolates relates to amide II band at the proximity of 1554 cm-1 (Kapoor and Viraraghavan, 1997; Yee et al., 2004; Park et al., 2005). The peak at the proximity of 1400 cm-1 corresponds to the symmetric vibrations of the C=O of COO- (Yee et al., 2004). The peaks at 1200-1250 cm-1 are related to the asymmetric stretching of phosphodiesters, free phosphates, or monoester phosphate functional groups (Yee et al., 2004). The absorption peak between 1050-1100 cm-1 corresponds to –CN stretching. 5 4 3 2 1 Absorbance 6 Treated Biomass with 40 ppm Cr Control 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Wave number (cm-1) Figure 4.7(a): FTIR analysis of L4 (Arthrobacter sp.) before and after chromium exposure. In above Figure 4.7 (a) after chromium treatment, six changes of the functional groups on the biomass have been found. The first change (1) corresponds to the formation of small peaks between 3200-3600 cm-1. These peaks may be due to overlapping of –OH and –NH stretching. It is in consistent with the result given by Deng and Ting (2005). However the hydroxyl group may not be involved in metal accumulation. The second 69 Chapter IV: Results and Discussion change (2) was the slight disappearance of the peak at 2926 cm-1 . It may be assigned to the –CH stretching of the functional groups (Kapoor and Viraraghavan, 1997; Park et al., 2005). The third change (3) is the disappearance of the absorption peak at 1554 cm-1 attributed to the –NH bending after chromium adsorption. As amino group is the major constituent of the cell wall, its bending may be due to chromium binding (Bai and Abraham, 2002). Consistent with result given by Han et al. (2006), the fourth change (4) was interpreted as the disappearance or weakening of peak at1400 cm-1. It could be attributed to the complexation of carboxylate functional group by coordination with metal ions. The fifth change (5) is the disappearance of peak at 1238 cm-1, which corresponds to the involvement of –SO3 in metal removal (Lameiras et al. 2007). The last change (6) corresponds to the shift of peak at 1078-1060cm-1, that could be due to the involvement of C-O bond of polysaccharides in chromium attachment (Han et al., 2006) 5 4 3 2 1 Absorbance 6 Treated Biomass with 200mg/L Cr6+ Control 0 500 1000 1500 2000 Wave number 2500 3000 3500 4000 4500 (cm-1) 70 Chapter IV: Results and Discussion Figure 4.7(b): FTIR analysis of S1 (Bacillus sp.) before and after chromium exposure. In Figure 4.7 (b), after Cr (VI) treatment, six changes of the functional groups on the biomass have been found. The first change (1) was slight disappearance of the peak at 2926 cm-1. It may be assigned to the –CH stretching of the functional groups (Kapoor and Viraraghavan, 1997; Park et al., 2005). The second change (2) corresponds to strengthening of the peak at 1650 cm-1, that corresponds to the C=O chelate stretching. This change is in consistent by Park et al. (2005) and suggests that the carboxyl groups are involved in the binding of chromium. The third change (3) is the disappearance of the absorption peak at 1554 cm-1, which is attributed to the -NH bending after chromium adsorption. As amino group is the major constituent of the cell wall, its bending may be due to chromium binding (Bai and Abraham, 2002). The fourth change (4) is the formation of new peak at 1457 cm-1, which is attributed to the asymmetric bending of – CH3 of the acetyl moiety (Bai and Abraham, 2002). The fifth change (5) is the disappearance of peak at 1230 cm-1 which relates to the C-O stretching of COOH group and hence strengthens that the carboxyl group is involved in chromium binding. The last change (6) is the shift of peak from 1065 to 1110 cm-1, which could be due to involvement of the C-O of polysaccharides in chromium attachment. A similar study on C. miniata to adsorb chromium also suggested that C-O is involved in metal uptake (Han et al., 2006). 4.7 X-ray diffraction (XRD) analysis Both the isolates from solid waste and liquid effluent were analyzed using X- Ray diffractometer before and after exposure to 40 ppm chromium. The diffraction pattern 71 Chapter IV: Results and Discussion were compared with that obtained from JCPDS (Joint Committee for Powder Diffraction Studies) - International center for diffraction data for peak identification. The peaks were analyzed using PDF (Powder diffraction file) - version 4. Figures 4.8 (a) and (b) give an overall scan within the range of 2θ from 10° to 80° for isolate L4 with and without chromium respectively. Similarly Figure 4.8 (c) and (d) shows the results for isolate S1 with and without chromium treated respectively. 4500 Intensity (Counts) 4000 3500 3000 2500 2000 1500 1000 500 0 5 15 25 35 45 55 65 75 85 2θ Figure 4.8(a): XRD data for 10°[...]... IV: Results and Discussion S No Isolates 1 L4 2 AB 248 532 3 AB 248 526 4 X80 743 5 AF102267 6 AJ785761 7 AY651318 8 AM409361 9 AB 248 529 10 AF408952 11 AM409362 PERCENTAGE HOMOLOGY 1 2 3 4 5 6 7 8 9 10 11 * 96 94 93 94 94 94 94 94 94 94 * 96 95 96 96 96 96 96 96 96 * 97 97 98 98 99 99 99 99 * 97 97 96 97 97 97 97 * 98 98 98 98 98 98 * 98 98 98 98 98 * 99 98 98 99 * 99 99 99 * 99 99 * 98 * Table 4. 3: Percentage... (ppm) 12 10 8 L4 6 S1 4 2 0 0 10 20 30 40 50 60 Time (hrs) Chromium in solution (ppm) Figure 4. 6(c): Chromium removal at 10 ppm initial concentration of Cr for S1 and L4 25 20 L4 S1 15 10 5 0 0 5 10 15 20 25 30 35 40 45 50 Time (hrs) Chromium in solution (ppm) Figure 4. 6(d): Chromium removal at 20 ppm initial concentration of Cr for S1 and L4 45 40 35 30 25 20 15 10 5 0 L4 S1 0 10 20 30 40 50 60 Time... shown in Figures 4. 6(a-e) 2.5 2 L4 1.5 S1 1 0.5 0 0 5 10 15 20 25 30 35 40 Time (hrs) Chromium in solution (ppm) Figure 4. 6(a): Chromium removal at 2 ppm initial concentration of Cr for S1 and L4 4. 5 4 3.5 3 L4 2.5 S1 2 1.5 1 0.5 0 0 10 20 30 40 50 60 Time (hrs) Figure 4. 6(b): Chromium removal at 4 ppm initial concentration of Cr for S1 and L4 65 Chapter IV: Results and Discussion Chromium in solution... (PCP) degradation by Arthrobacter strain ATCC 33790 The isolate that is reported in this report is only 96% similar to Arthrobactersp.(accession no AB 248 532) It is only 94% and 93% similar to another Arthrobacter sp (accession no AB 248 526) and Arthrobacter nicotinovorans (X80 743 ) respectively Hence, the strain L4 is novel according to 16S rDNA analysis 4. 4 Chromium removal assay 4. 4.1 Chromium removal... ppm 20 10 ppm 20 ppm 40 ppm 15 10 5 0 0 20 40 60 80 100 120 140 Time (hrs) Figure 4. 5(b): Removal of chromium by S1 at various initial concentrations (2 -40 ppm) 64 Chapter IV: Results and Discussion 4. 4.3 Comparative study between S1 and L4 for removal of Chromium A comparison between the growth pattern of S1 and L4 at different initial concentration of Chromium in solution (ppm) chromium added during... limitation and/ or by chemical reactions Figure 4. 11(a) and 4. 11(b) show the color difference between control and treated samples of S1 and L4 respectively for protein estimation Figure 4. 12(a) and 4. 12(b) summarize the EPS content for isolates S1 and L4 respectively in absence and presence of Cr (at 40 ppm) 80 Chapter IV: Results and Discussion S1 Cr treated S1 L4 Cr treated L4 Figure 4. 11:: Color difference:... L4 being more tolerant to chromium 4. 10 Atomic force microscopy (AFM) Analysis The morphological changes in the cells before and after Cr exposure were examined using AFM Figures 4. 13 (a, b and c) and Figure 4. 13 (d, e and f) show the cell morphology for L4 (Arthrobacter sp ) and S1 (Bacillus sp.) respectively Figure 4. 13(a): Cell morphology for L4 without chromium exposure 83 Chapter IV: Results and. .. for Arthrobacter sp (L4) without chromium 200 180 Intensity (Counts) 160 140 120 100 80 60 40 20 0 15 20 25 30 35 40 45 50 55 60 2θ Figure 4. 9(b): XRD data for 17° ... no AB 248 526) and Arthrobacter nicotinovorans (X80 743 ) respectively AM409362 (Arthrobacter sp st 4C1-b) AF408952 (Arthrobacter sp.) AB 248 529 (Arthrobacter sp.) AM409361 (Arthrobacter sp St 4C1-a)... (Bacillus sp.) DQ105973 (Bacillus marisflavi) 55 DQ 448 746 (Bacillus sp.) AF483625 (Bacillus aquaemaris st TF-12) 100 AJ315068 (Bacillus sp.) 100 AJ 244 686 (Bacillus sp.) AY50 549 9 (Bacillus aquimaris... AB 248 532 AB 248 526 X80 743 AF102267 AJ785761 AY651318 AM409361 AB 248 529 10 AF408952 11 AM409362 PERCENTAGE HOMOLOGY 10 11 * 96 94 93 94 94 94 94 94 94 94 * 96 95 96 96 96 96 96 96 96 * 97 97 98 98 99