CHAPTER 3: Screening of anaerobic biomass collected from different sources for heavy
3.2 Materials and methods
3.2.1 Screening of anaerobic biomass for heavy metal removal and recovery
Anaerobic biomass collected from three different sources were screened for heavy metal removal and recovery as nanopowder from aqueous solution. These sources were: (1) large scale upflow anaerobic sludge blanket (UASB) reactor (located in Kavoor, Mangalore, Karnataka, India) treating sewage and wastewater from small scale industries, (2) a wastewater treatment plant (WWTP) situated in IIT Guwahati, Guwahati, Assam, India, and (3) a laboratory scale anaerobic rotating biological contactor (An-RBC) reactor treating heavy metal containing wastewater. The collected anaerobic biomass was stored at 4 °C in a refrigerator until further use.
For initial activation of microorganisms present in the biomass, 10% (v/v) of the respective anaerobic biomass from different sources was added to 1 L aspirator bottle containing modified Postgate media, which is commonly used for cultivating SRB, such as Desulfovibrio and Desulfotomaculum species. After purging with nitrogen, the bottles were incubated in an orbital shaker set at 30 °C and 150 rpm for one week. 60% (v/v) of sodium lactate was used as the carbon source and electron donor for culturing these biomass types. Sulfate was added as Na2SO4 at COD/SO42- ratio of 0.67. The active biomass was subsequently tested for heavy metal removal and recovery experiments, as detailed later in the next section.
Composition of the modified Postgate medium for anaerobic growth of the biomass was as follows (g/L): ammonium chloride (1.0), potassium dihydrogen phosphate(0.5), sodium sulfate(1.47),
calcium chloride dihydrate (0.1), ascorbic acid (0.1), sodium citrate (0.3), ethylene diamine tetraacetic acid (0.3), ferrous sulfate heptahydrate (0.289), and yeast extract (1.0) (Postgate 1984).
The pH of the media was adjusted to 7.0 using 2 N NaOH.
3.2.2 Heavy metal removal and recovery experiments
Batch experiments for heavy metal removal by sulfate reduction were performed using serum bottles of volume 120 mL each and sealed with polytetrafluoroethylene (PTFE) septum. The bottles were incubated in orbital shaker set at 30 °C and 150 rpm for five days. The bottles were purged with nitrogen gas prior to the experiment with 10% (v/v) biomass as the inoculum. Bottles containing biomass and carbon source but without any added metal served as the control in these batch experiments.
Individual metal stock solutions of Cu(II), Cd(II), Ni(II), Fe(II), Pb(II), Mn(II) and Zn(II) of 10,000 mg/L concentration each were prepared using copper chloride dihydrate, cadmium nitrate tetrahydrate, nickel chloride hexahydrate, iron chloride tetrahydrate, lead nitrate, manganese chloride tetrahydrate and zinc chloride, respectively. Serum bottles containing the Postgate medium as mentioned earlier were added with the individual metal-containing stock solution to obtain desired metal concentrations. Initial metal concentration in these experiments was chosen based on the composition of acid mine drainage from Makum Coalfield area located in North-East India (Equeenuddin et al., 2010). Table 3.1 provides the metal concentration tested in this study.
Table 3.1 Metal concentration tested in this study (pH 7.0)
S.No. Metal Concentration (mg/L)
1 Fe 50 - 150
2 Cu 5 - 15
3 Mn 5 - 15
4 Cd 1 - 5
5 Ni 1 - 5
6 Pb 1 - 5
7 Zn 1 - 5
Samples were taken at regular intervals during the experiments for analysis of mixed liquor volatile suspended solids (MLVSS), chemical oxygen demand (COD), pH, metal concentration, volatile fatty acid (VFA), sulfate and dissolve sulfide contents. All these batch experiments were conducted in triplicate and results reported are average of triplicate sample analysis. Among the biomass collected from different sources, it was found that the biomass obtained from An-RBC reactor was very efficient in terms of metal removal and sulfate reduction. Hence, metal sulfide precipitates obtained using this biomass type were subsequently used for recovering the metals present. The following equation was used to evaluate the metal recovery efficiency:
Metal recovery efficiency (%) = Mb
Mtotal× 100 (3.4)
Where Mb is metal (mg) recovered in the form of precipitates and Mtotal is the total metal (mg) added prior to the experiment.
3.2.3 Metal nanoparticle characterization
To verify potential application of metal nanopowders obtained in this study, its composition analysis and detailed characterization were carried out using techniques such as Fourier-transform infrared spectroscopy (FTIR), field emission scanning electron microscopes-energy dispersive X- ray (FESEM-EDX), field emission transmission electron microscopy (FETEM) and rotating anode X-ray diffractometer (XRD).
For FTIR analysis, control biomass and metal loaded biomass were centrifuged at 10,000 rpm for 10 min followed by washing of pellet obtained with distilled water. The samples were finally dried under vacuum for FTIR analysis (IR affinity-1S, Shimadzu, Japan). Samples treated similar to that for the FTIR analysis were used for FESEM-EDX (Zeiss, Sigma, Germany) analysis to check the morphology of the metal precipitates. In order to check the internal structure and morphology of the metal precipitates by FETEM analysis, control and metal loaded biomass was centrifuged thrice at 5,600×g for 10 min each followed by washing with distilled water after every run. The final pellet obtained was placed on copper grid and analyzed using EDS integrated FETEM (JOEL, JEM2100, Japan). Structural characterization of the samples were carried out by drying the sample at 120 °C and then grinding it to a powdered form, followed by analysis using a rotating anode based powder X-ray diffractometer (XRD) (Rigaku TTRAX III, 18 kW) with Cu Kα radiation (λ = 0.1542 nm).
3.2.4 Analytical methods
Biomass was estimated as mixed liquor volatile suspended solids (MLVSS) as per the method defined in the American Public Health Association (APHA, 2005). Sulfate concentration was determined using the standard barium chloride based turbidimetric method (APHA, 2005),
whereas COD in the samples was determined by the closed reflux method (APHA, 2005). Sulfide was measured based on a method described by Cord-Ruwisch (1985). Acetate was measured by high pressure liquid chromatography (Biorad, Shimadzu) after sample filtration through 0.45 µm nitrocellulose filter (Millipore). The HPLC was fitted with aminex hpx 87 h column and the mobile phase was 5 mmol H2SO4 at 0.6 mL/min flow rate.
Metal concentration in the samples was determined by atomic absorption spectroscopy (Varian, AA240, Netherlands) as per the APHA (2005) after passing the sample through 0.45 µm nitrocellulose filter (Millipore). To determine the metal recovery percentage in this study, the metal precipitates were first collected by withdrawing the liquid above and then the precipitates were acidified using 20% HNO3 to ensure complete dissolution of the metal precipitates. The soluble metals were determined as mentioned earlier. All chemicals used in this study were of analytical grade (AR).
3.3 Results and discussion