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steady state values. For the assessment of individual and collective effect of the metals, the results obtained from the fractional factorial design were analyzed in the form of student’s t test and ANOVA. The statistical software, Minitab (Version16, PA, USA) was used for designing the fractional factorial experiments and for statistical analysis of the results obtained. The effect due to a particular factor was defined as either reduction (negative) or improvement (positive) in the responses.
3.6 Continuous Heavy Metal Removal Using The Anaerobic Rotating Biological
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Table 3. 5 Design specification of the An-RBC reactor Specifications
Number of stages 2
Number of disks in each stage 7 Diameter of each disk 16 cm Spacing between each stage 2 cm Total working volume 3 L
Disc Submergence 40%
Figure 3. 5 Schematic of the An-RBC reactor setup.
3.6.2 Biofilm development
Sulfate reducing bacteria present in the anaerobic biomass collected from UFAR, which showed maximum metal removal efficiency in the batch study was used in this continuous An-RBC reactor study. For SRB immobilization in the reactor, the culture was allowed to grow onto the reactor discs covered with polystyrene mesh and PUF, which served as the bio-support material. For SRB growth during the reactor startup phase, modified Postgate medium at pH 7 and with the SRB biomass, but without any added metal were supplied to the reactor. The An-RBC reactor was operated at an ambient temperature of 25 ± 2 °C in batch mode and the reactor was recharged with a fresh medium after every five days for over three months during this startup phase. Change in color of the input medium from colorless to black at the outlet due to the formation of FeS and generation of hydrogen sulfide during this startup phase confirmed SRB growth and activity in the reactor (Herbert and Gilbert, 1984; Hamilton, 1994; Singh et al., 2011). Photographs of the An-RBC reactor before and after immobilization with SRB are shown in Figs. 3.6 and 3.7. However, no efforts were made to measure the amount of biomass immobilized in the reactor. Later, the An-RBC reactor was operated under continuous mode by supplying the medium at a constant flow rate for performing the heavy metal removal experiments.
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Figure 3. 6 Photograph of the experimental setup showing the rotating biological contactor (RBC) reactor prior to immobilization with SRB.
Figure 3. 7 Photograph of experimental setup showing the rotating biological contactor (RBC) reactor with the immobilized SRB on its discs.
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3.6.3 Characterization of the bio-support material
Characterization of the bio-support material utilized for immobilizing the SRB anaerobic biomass was carried out by FESEM analysis, for which the SRB immobilized bio-support material was fixed using 3% glutaraldehyde for 2 h followed by dehydration using different concentrations of ethanol and by drying at 30 °C (Scigenics, ORBITEKR LETTO, India) (Utgikar et al., 2002). The finally prepared sample was then observed using a FESEM.
3.6.4 Heavy metal removal experiments 3.6.4.1 Single component system
Experiments for studying heavy metal removal from single component system using the An-RBC reactor were carried out under continuous mode of operation. Individual metal stock solutions were prepared as per the procedure described in Section 3.3.1. The modified Postgate medium as described earlier was added with the corresponding metal stock solution so as to obtain a desired concentration of the heavy metals in each of the experimental phases. The initial concentration for each of the metals Fe(III), Pb(II), Ni(II), Zn(II) and Cd(II) were chosen as 50, 75 and 90 mg/L. Whereas, in case of Cu(II), these values were 100, 150 and 175 mg/L. All these initial levels of the heavy metals were chosen based on the results of batch heavy metal removal using the SRB (Section 3.3.1). Phase wise operational conditions followed with the An-RBC reactor are presented in Table 3.6.
Table 3. 6 Operational conditions followed with the An-RBC reactor for continuous metal removal experiments
Parameter Different experimental phases
I II III
HRT (h) 48 24 48 24 48 24
Inlet metal
concentration (mg/L) Cd(II), Ni(II), Fe(III), Pb(II) and Zn(II)
50 50 75 75 90 90
Cu(II) 100 100 150 150 175 175
The reactor performance in terms of heavy metal removal efficiency was evaluated at two different HRT values (24 h and 48 h) as described earlier (Table 3.6). Each experiment was carried out for a period until three steady state values of effluent heavy metal concentration at the respective HRT were obtained (Villa-Gomez et al., 2015).
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Effect of metal loading on the performance of the An-RBC reactor
The combined effect of inlet metal concentration and HRT on metal removal was examined by calculating the ILR (mg/L∙h) and the corresponding metal removal rate (mg/L∙h) as per the equations 3.1 - 3.3 mentioned earlier (Section 3.5.2.1). Samples collected at regular time intervals were centrifuged at 8000 × g for 5 min (Remi, C24-L or R-24, India), and the supernatant obtained was analyzed for metal, sulfate, sulfide and COD concentrations. Each sample analysis was carried out in duplicate and the results presented are average of duplicate sample analysis.
3.6.4.3 Identification and characterization of SRB
Sample preparation for V3-V4 Metagenomics Sequencing and Analysis
For SRB characterization, the bacterial culture from the An-RBC reactor was collected after completing the three phases of continuous operation of the reactor (Table 3.6) and grown in the presence of modified Postgate medium using serum bottles at 30 °C temperature under continuous agitation (120 rpm) (Scigenics, ORBITEKR LETTO, India) for seven days. The culture was then sent to AgriGenome Labs Pvt Ltd, India for V3-V4 metagenomics sequencing and analysis for its detailed characterization. Complete details of V3-V4 metagenomics sequencing and analysis are presented in Appendix C.