3.8 Analytical Methods
4.1.2 Batch heavy metal removal from multi-component system
4.1.2.3 Characterization of metal bioprecipitates
Horizontal bars in these charts represent the effect due to the individual metals, and those extending past the reference line (vertical line on the chart) represent the significant ones (α=0.05). In summary, an increase in Cu(II), Ni(II) and Zn(II) concentration level in the mixture was inhibitory to their own removal (Fig. 4.11a, 4.11b and 4.11e). Fe(III) and Ni(II) inhibited Fe(III) removal (Fig. 4.11c), whereas Pb(II), Ni(II) and Cu(II) showed inhibitory effect on Pb(II) removal in the multi-component system (Fig. 4.11d). Pareto chart results matched well with the student t test and all these effects of heavy metals on each other removal from mixture can be attributed to the solubility product constant value of the corresponding metal sulfide salts (Hill et al., 2005). Among the different metals, Cu(II) inhibited both sulfate and COD reduction by SRB (Figs. 4.11f and 4.11g). This can be attributed to the formation of high sulfide precipitates which may result in diffusion limitation of the substrates (COD and sulfate) (Kieu et al., 2011).
The TEM images (Figs. 4.12a and 4.13a) confirmed the ability of these SRB to grow in the presence of metals in mixture which is also confirmed by color change of the input medium from colorless to black due to the formation of FeS and generation of hydrogen sulfide owing to SRB growth and activity (Herbert and Gilbert, 1984; Hamilton, 1994; Singh et al., 2011). The images also reveal the presence of a layer or shell like structure on cell wall of the bacteria, confirming that metal sulfide is associated with outer layer of the bacterial cell surface (Figs. 4.12b and 4.13b).
Figure 4. 13 (a) TEM image of a metal loaded bacterial cell from experimental run #1 showing intact metal precipitate on SRB cell surface, (b) EDS spectrum from a spot on the bacterial cell surface.
Elemental composition of the control biomass was confirmed by EDX spectrum shown in Fig. 4.14a; insert to Fig. 4.14a represents the FESEM image of the control biomass which appears as a coalescent material. Similarly, elemental composition of the biomass taken from experimental run 1 was confirmed by EDX spectrum (Fig. 4.14b); insert to Fig. 4.14b represents the FESEM image of the same biomass. A comparison between these figures confirms metal sulfide precipitation by SRB together with the presence of other elements that constituted the modified Postgate medium, which is similar to the findings obtained in the previous study (Fig. 4.9). The presence of sulfur peak in the spectra (Figs. 4.12b-4.14b) is attributed to the metal sulfide precipitation as a result of SRB activity. The precipitates formed are predominantly amorphous form of sulfide salts corresponding to different metals added in mixture (Fig. 4.14). Sulfate and COD reduction along with metal sulfide formation confirmed that sulfidogenesis is the governing mechanism for heavy metal removal by SRB (Jin et al., 2007).
Figure 4. 14 EDX spectrum of (a) control biomass, (b) metal loaded biomass from experimental run #1. Insert to these figures show the image of the respective biomass.
Overall, the results from TEM and FESEM revealed that the metals were mainly removed by sulfide precipitation. From FESEM-EDX spectra of the precipitates (Fig. 4.14), it is clear that the metals were precipitated as metal sulfides (Azabou et al., 2007). Different metals precipitated in the experimental run 1 are highlighted with circle in Fig. 4.14b. Among the peaks due to the different elements, only the peak due to sulfide is significant indicating that the metals were present as sulfides in the precipitate (Figs. 4.12-4.14). All other forms, such as M(OH)x, MCO3, etc., were not significant.
Figure 4.15 shows the FTIR spectra of the heavy metal laden anaerobic biomass from the experiments which was obtained to verify the interaction between the metal ions and the
functional groups present on the bacterial surface. The major stretching in the spectrum was in the range 468-3696 cm-1 for the control biomass and 467-3468 cm-1 for biomass obtained from experimental run 1. The spectra show characteristic sharp peaks in the different wave regions 599-618 cm-1, 1018-1043 cm-1, 1535-1544 cm-1 and 1638-1642 cm-1, respectively.
Occurrence of neutral C=O complex was indicated by the stretching at 1644 cm-1 in control biomass and a minor shift to 1642 cm-1 in the heavy metal laden biomass.
Bands in the range 1199-800 cm-1 are associated with C–O–C and C–O–P stretching. These stretching vibrations involve oligo and polysaccharides present in the bacteria (Rubio et al., 2006). Bands corresponding to the stretching from 1525 to 1558 cm-1 are mainly due to -NH stretching and can be attributed to protein amide I and amide II bands (Quan et al., 2013).
Bands corresponding to the range from 3748 to 3764 cm-1 indicate either N-H stretching of amine group or O-H stretching of hydroxyl group. An earlier study on the FTIR spectra of SRB has reported that sulfate ions exhibit five bands centered around 1230 cm-1, 1130 cm-1, 1070 cm-1, 1000 cm-1 and 610 cm-1 (Nakamoto, 1970). Similar observation was made in the results obtained from single component system (Fig. 4.7).
Figure 4. 15 FTIR spectra of control biomass and heavy metal loaded biomass from experimental run # 1 in the study.
The FTIR spectra (Fig. 4.15) of the biomass used in this multi-component system showed bands corresponding to the sulfate ions, which clearly indicate sulfate reduction for its growth and metabolism (Singh et al., 2011; Zaina et al., 2011). FTIR analysis confirmed a
high degree of similarity of the functional groups corresponding to SRB in the present study with several other reported species of SRB, such as Desulfovibrio vietnamensis DSM 10520, Desulfovibrio gigas ATCC 19364, Desulfovibrio gabonensis DSM 10636 and Desulfovibrio indonesiensis NCIMB 13468 (Feio Maria et al., 2004).