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3.1 Chemicals and Reagents
This section describes different techniques and methods followed in the present research.
All chemicals and reagents used in this study were of analytical grade and supplied by Hi- Media Pvt. Ltd., India, LOBA Chemie Pvt. Ltd., India, SRL Chemicals Pvt. Ltd., India, Merck India Ltd., and CDH Pvt. Ltd., India. Membrane filtered water (reverse osmosis (RO)) was used for carrying out all experiments in the study (Sartorious, Arium 61316RO
& 611UF, Germany).
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sulfate reduction. The biomass sources include WWTP, UFAR and APR. Simultaneous sulfate reduction and heavy metal removal experiments were carried out using 100 mL serum bottles fitted with a rubber stopper and aluminum crimp seal. Individual metal stock solutions of Cu(II), Cd(II), Ni(II), Fe(III), Pb(II) and Zn(II) of 10,000 mg/L concentration each were prepared using CuCl2∙2H2O, Cd(NO3)2∙4H2O, NiCl2∙6H2O, FeCl3∙6H2O, PbNO3
and Zn(NO3)2∙6H2O, respectively.
Serum bottles containing the medium as mentioned earlier were added with the individual metal containing stock solution yielding 10 and 50 mg/L initial concentration of the respective metals. These bottles were purged with nitrogen gas before and after inoculation with 10% v/v biomass, as mentioned earlier. During the heavy metal removal experiments both under batch and continuous mode of operation, sulfate and COD concentration in the influent were adjusted to obtain a COD/SO42- ratio of 0.67 ± 0.08 as this is the ideal stoichiometric proportion for complete sulfate reduction and degradation of organic substrates (Rinzema and Lettinga, 1988). The bottles were then agitated on an orbital shaking incubator set at 30 °C temperature and 120 rpm shaking speed (Lab Tech, LSI- 3016R, Korea). Bottles with medium containing only the carbon source and biomass, but without any added metal, served as the control in these experiments. Samples were taken at regular intervals during the experiments to determine conductivity, pH, sulfate, COD, metal and sulfide concentrations. Mixed liquor volatile suspended solids (MLVSS) value in the samples was quantified using muffle furnace (Lab Tech, LEF-115P-2, Korea) (APHA, 2005). All these batch experiments were conducted in duplicate and the results presented are average of duplicate sample analysis.
3.3.1.1 Characterization of the metal precipitates
Characterization of the metal precipitates formed due to the anaerobic biomass collected from the UFAR which proved to be the best for metal removal among the three sources of biomass was carried out using Fourier transforms infrared (FTIR) spectrometer, transmission electron microscopy equipped with energy dispersive spectroscopy (TEM- EDS) and field emission scanning electron microscopy equipped with energy dispersive X- ray spectroscopy (FESEM-EDX). Fourier transforms infrared spectra were obtained to explain the changes in the functional groups of the biomass due to sulfate reduction for heavy metal removal. For FTIR analysis, control and metal loaded biomass were centrifuged (8000×g) for 5 min (Sigma, Sigma 1-15, Germany), washed twice with RO water and the pellets obtained were vacuum dried and analyzed using a FTIR spectroscope
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(IR Affinity-1, SHIMADZU, Japan) (Singh et al., 2011). Similarly, for TEM analysis, copper loaded biomass was used as it showed the best removal efficiency among all the metals in the batch study. The pellet obtained was loaded on copper grid coated with carbon (Pacifi-Tech Grid, Cu-300CK, USA) for observation under TEM (JOEL, JEM2100, Japan) at 200 kV integrated with EDS (Jalali and Susan, 2000).
For FESEM analysis, the precipitates obtained by centrifuging the samples were oven-dried at 80 °C (Tanco, OVEN PLT-125, India) for 2 h and gold-coated in a sputter coater (Quorum, SC7620, UK and Edwards, RV3, Czech Republic) (Cao et al., 2013). The precipitates were then analyzed for morphology and elemental composition using FESEM- EDX (Zeiss, Sigma, Germany). Metal removal results obtained using the SRB were compared with the metal removal by chemical precipitation using sodium sulfide. To determine the influence of externally sulfide on heavy metal removal, different concentrations of sulfide (5, 10, 15 and 25 mg/L) were tested with two different concentrations (10 and 50 mg/L) of the heavy metals (Cd(II), Cu(II), Ni(II), Fe(III), Pb(II) and Zn(II) (The deatils of this study are presented in Appendix B).
3.3.2 Metal removal from multi-component system
Based on the results obtained from the previous study on metal removal from single component system using anaerobic biomass from three different sources, biomass that showed the best metal removal was chosen for studying the metal removal from multi- component system. In this study, different combinations of high and low concentration levels of six heavy metals, viz. Cd(II), Cu(II), Ni(II), Fe(III), Pb(II) and Zn(II), were chosen using the Plackett-Burman screening design. Analysis of variance (ANOVA) and student’s t test were then applied for statistical analysis of the results to interpret the significance and effect of these metals on each other removal as well as on sulfate and COD removal in the study. The Plackett-Burman design consisted of twelve experimental runs with different combination levels of Cd(II), Cu(II), Ni(II), Fe(III), Pb(II) and Zn(II) (Table 3.1). The low and high concentration levels of each of Cd(II), Ni(II), Pb(II) and Zn(II) were chosen as 5 and 10 mg/L, respectively. Whereas, for Cu(II), these were 25 and 50 mg/L; in the case of Fe(III), 10 and 25 mg/L were chosen as the low and high initial levels, respectively.
All these initial levels of the heavy metals were based on the results obtained from the results of previous single component study using the anaerobic biomass collected from
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UFAR. Input metal concentration levels used in Table 3.1, i.e., +1 and -1 indicate the high and low level of the metals, whereas 0 indicate centre point level of the metals. Individual metal stock solutions of different heavy metals used in this study were prepared as described earlier in Section 3.3.1. The modified Postgate medium as mentioned before was added with the corresponding metal stock solution so as to obtain a desired concentration of the heavy metals in each of the experimental runs. The statistical software Minitab (Version 16, PA, USA) was used for statistical analysis of the results obtained.
Table 3. 1 Plackett-Burman experimental design matrix showing different combination levels of the heavy metals in the multi-component study
Exp Run Different combination levels of the heavy metals (mg/L)
Cd Cu Ni Fe Pb Zn
1 -1(5) -1(25) -1(5) -1(10) -1(5) -1(5)
2 +1(10) -1(25) +1(10) -1(10) -1(5) -1(5)
3 -1(5) -1(25) +1(10) +1(25) +1(10) -1(5)
4 -1(5) +1(50) -1(5) -1(10) -1(5) +1(10)
5 -1(5) +1(50) +1(10) +1(25) -1(5) +1(10)
6 -1(5) +1(50) +1(10) -1(10) +1(10) -1(5)
7 +1(10) +1(50) -1(5) +1(25) +1(10) -1(5)
8 +1(10) -1(25) -1(5) -1(10) +1(10) +1(10)
9 +1(10) -1(25) +1(10) +1(25) -1(5) +1(10)
10 +1(10) +1(50) -1(5) +1(25) -1(5) -1(5)
11 -1(5) -1(25) -1(5) +1(25) +1(10) +1(10)
12 +1(10) +1(50) +1(10) -1(10) +1(10) +1(10)
All the experiments in this study were performed using 100 mL serum bottles. These bottles were purged with nitrogen gas before and after inoculation with 10% v/v anaerobic biomass (measured as MLVSS). The bottles were then incubated on an orbital shaker set at 30 °C temperature and 120 rpm agitation speed (Lab Tech, LSI-3016R, Korea). Bottles without any added metals but containing only the media, carbon source and the biomass, served as the control in these experiments. Liquid samples were taken from the bottles at regular intervals during the experiments to determine conductivity, pH, sulfate, COD, MLVSS, metal and sulfide concentrations in the samples. All these experiments were conducted in duplicate and the results presented are average of duplicate sample analysis.
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3.3.2.1 Characterization of the metal bio-precipitates
Characterization of the metal bio-precipitates formed due to the SRB was carried out by FTIR spectroscopy, TEM-EDS and FESEM-EDX. For FTIR, TEM-EDS and FESEM-EDX analyses, samples from control experiment and experimental run 1 were prepared as described earlier in Section 3.3.1.1. Biomass sample from the experimental run 1 (Table 3.1) was chosen as it yielded a very high metal removal efficiency in the study. The precipitates were then analyzed for morphology and elemental composition using TEM and FESEM-EDX.
3.4 Batch Heavy Metal Removal Using Immobilized SRB Beads