MATERIALS AND METHODS
4.10 Characterization of Biosolids
The solids collected from the SmBRs and backwash solids from AGRs were characterized using FESEM-EDX and TEM-EDX, XRF, XRD and XAS.
4.10.1. Collection and Preservation of Biosolids
The biosolids from AGRs were collected from backwash suspension after a backwash. During backwash nitrogen gas was supplied along with distilled water to maintain anoxic condition. The backwash suspension was collected in anoxic condition in a container prefilled with nitrogen gas. The suspension was collected through the inlet port and the gas from the container was allowed to exit through the exit port. Schematic diagram of a container used to collect backwash suspension is shown in Figure 4.6. The backwash suspension, thus collected, was transferred to centrifuge tubes to separate solids for their various characterizations. The backwash solids (BWS) were centrifuged at 6000 rpm for 10 min to obtain wet paste, which was freeze dried and preserved at 4ºC in sealed specimen tubes in a double zip locked plastic bags. All necessary practical steps were taken to minimize contact between the solids and atmospheric air.
Figure 4.6 Schematic diagram of container used for collecting backwash suspension.
4.10.2 X-ray Fluorescence (XRF)
The qualitative elemental composition of biosolids generated in SmBRs was performed by AXIOS X-ray Fluorescence Spectrometer of PANalytical make (Model DY840). The XRF qualitative analysis is done by scanning the pressed pellet. 1g of finely ground sample is mixed with 0.5g of Boric acid (binder) and given a load of 50kN for 2 minutes. Pressed pellets made in the aforesaid method are scanned by qualitative XRF (SuperQ) software program in the XRF machine, which showed presence of As, Fe and S.
4.10.3 Microscopic Methods FESEM and EDX
Field emission scanning electron microscopy (FESEM) (Zeiss, Sigma, Germany) equipped with energy dispersive X-ray microanalysis system (EDX) (INCA 300, Oxford, UK) was used for topographical characterization and elemental confirmation of arsenic, iron and sulphur in the biosolids. Before the examination, the freeze dried backwash solids was lightly dusted onto the carbon tape of the SEM stub surface and coated with gold using a Scancoat Six SEM sputter coater system.
TEM and EDX
The detailed morphology, microstructure and chemical composition of BWS were examined by transmission electron microscopy (TEM), using a JEOL, JEM 2100 microscope operated at 200 kV, equipped with an energy dispersive X-ray spectrometry (EDX). Specimens for TEM observations were prepared using an ultrasonic vibration method. First, the powdered samples were immersed in acetone solution and subjected to ultrasound vibration (Vibra-Cell model VC 505, Sonics, USA) to disperse the sample homogeneously. Then, one drop of the suspension was dropped with a micropipette to holey carbon supporting film (TEM Grids). After being well dried under ambient conditions, the grid was mounted on the TEM specimen holder for examination. Samples were investigated thoroughly by TEM selected-area electron diffraction (SAED), high resolution imaging (HRTEM) and EDX. A series of Fast Fourier Transform (FFT) patterns calculated from the HRTEM images of samples was used to help identify their crystal structure.
4.10.4 X-Ray Diffraction (XRD)
X-ray powder diffraction was used to check the existence and crystalline form of the solid phase generated in the bioreactors. The analysis was carried out with a PANalytical X‘pert PRO-MPD diffractometer equipped with a monochromator in the diffracted beam. The X-ray powder diffraction spectra were recorded in the 2θ range from 10° to 80° using Cu Kα (wavelength = 1.54 Å, 40 mA, 40 kV) radiation with a step size of 0.05° and a step duration of 1 sec. The freeze dried backwash solids were used as a sample in XRD pattern recording.
4.10.5 X-ray Absorption Spectroscopy (XAS)
X-ray absorption spectroscopy (XANES and EXAFS) were used to determine the arsenic and iron oxidation state and local structure of the back wash solids. The characterization was performed at Raja Ramanna Centre for Advanced Technology (RRCAT), Indore, India. The samples for XAS analyses were filtered through 0.22 μm nylon filters and the filtered paste of back wash solids were transferred into airtight, crimp-sealed serum bottles without drying and then shipped to Indus Synchrotron Radiation Laboratory of RRCAT for XAS analysis. XAS samples were prepared for analysis by applying wet sample pastes onto a double layer of Kapton tape in anaerobic conditions. XAS spectra were collected at Indus-2 beamline (BL-9) (3 GeV, ~100 mA of maximum current) with an unfocused beam. The beam line mainly consists of Rh/Pt coated meridional cylindrical mirror for collimation and Si (111) based double crystal monochromator to select excitation energy. XANES and EXAFS measurements were carried out in transmission mode. The energy range of EXAFS was calibrated simultaneous measurements on a commercial Fe and Au foils. XAS spectra were also collected for Na2HAsO4·7H2O and NaAsO2 powders to use as As(V) and As(III) standards. Model compounds such as metallic iron foil (Fe(0)), amorphous FeS, and amorphous Fe2O3. The EXAFS data has been analysed using FEFF 6.0 code (Zabinsky et al., 1995), which includes background reduction and Fourier transform to derive the
( )R
versus R spectra from the absorption spectra (using ATHENA software) (Ravel &
Newville, 2005), generation of the theoretical EXAFS spectra starting from an assumed crystallographic structure and finally fitting of experimental data with the theoretical spectra using ARTEMIS software (Ravel & Newville, 2005).