MATERIALS AND METHODS
3.3 Analytical Methods .1 Wastewater parameter
In general, standard techniques as detailed in standard methods (APHA, 1998) has been followed unless otherwise specified. Samples of reactor effluents were collected and centrifuged at 7500 rpm prior analysis as per respective methods. Influent and effluent pH was measured using a digital pH meter with a sensitivity of 0.01 with temperature correction facility. The instrument was calibrated periodically with standard buffer solutions. Temperature was measured with thermometer. Dissolved oxygen concentration was measured using a digital DO meter with a sensitivity of 0.01. Conductivity, oxidation reduction potential etc intermittently measured using a multi-parameter water quality measurement kit (Model 6920-M Yellow Springs, Ohio-USA).
FMBR
CMBR
Phenol was estimated by colorimetric method using 4-aminoantipyrene measuring absorbance at 500 nm. Chemical oxygen demand (COD) was estimated by closed refluxed method and titrating samples by ferrous ammonium sulphate. Thiocyanate was measured by colorimetric method using ferric nitrate in acidic pH and absorbance was measured at 460 nm. Ammonia-nitrogen was measured by phenate method at 640 nm using sodium nitroprusside. Volatile fatty acid was measured by titrimetric method.
Nitrate-nitrogen concentration was determined by using ultra violet screening method measuring absorbance at 220 and 275 nm in UV-spectrophotometer and Ion chromatograph. Wavelength of 220 nm was used to obtain NO3-N reading and a wavelength of 275 nm to determine interference due to dissolved organic matter. Nitrate ions were also measured using ion Chromatograph equipped with a Dual 3 column (250 mm×4 mm), a RP guard column, and a conductivity detector. Samples taken during the experiments were centrifuged at 8000 rpm for 10 min and were filtered through a C-18 reverse-phase cartridge and then through 0.45µm filter for analysis. NaOH (5mM) served as the eluent and sulfuric acid (2.0mM) as the regenerant in the chromatogram analysis.
Nitrite-nitrogen (NO2--N) was estimated by colorimetric method using N (1-Naphthyl) ethylene-diamine dichloride) at 543 nm. Sulfate was analyzed by turbidity method using barium chloride.
Concentration of pyridine was estimated using reverse phase HPLC equipped with a UV- VIS detector at wavelength 254 nm and an Onsphere C-18 column (Varian, particle size 5 µm, length 15 cm, diameter 4.6 mm) using acitonitrile: water (80%:20%) as the mobile phase. About 300 µl of sample was filtered through sterile syringe filter (PTFE filter media of 13 mm diameter, pore size 0.20 µm, Pall, USA) to remove biomass or any other particulate matters. The retention time of pyridine was found to be 1.4 min at flow rate of 0.8 mL/min.
For the SEM (scanning electron microscope) photograph, fragments of sponge cube containing the microbial bio-film were sampled at various sampling points of the moving bed reactor and cut into small thin pieces. The sample was mounted on aluminum stubs and then coated with gold using sputter coater (Edward, USA). The stubs were then introduced into the specimen chamber of LEO 1430vp for scanning. Figure 3.4 (a) and (b)
shows the SEM image of clean sponge cube before use and sponge cube with biomass after use in reactor.
Figure 3.4 SEM image of sponge cube (a) before use (b) with biomass (a)
(b)
Concentrations of heavy metals, such as cobalt, strontium, lead and Nickel etc, in the real wastewater were measured using atomic absorption spectrometer (AAS) (Varian, USA, Model No. Spectra AA 240 FS) at their respective absorbance maxima.
3.3.2 Biomass concentration
For measuring suspended biomass, known amount of mixed liquor was taken. Total volatile solid (VS) were measured after drying mixed liquor and sponge cubes at 103oC and ignited at 550oC, respectively.
For attached biomass randomly 3-4 sponge cubes were taken. Rinsed with distilled water thoroughly and dried in 105oC and then ignited at 550oC according to APHA 1998. During the initial days operation four sponge cubes were collected from top (near effluent port) and middle (one foot below the effluent point) of anaerobic, anoxic and aerobic reactors and rinsed with distilled water. Biomass in sponge was determined from weight difference of the rinsed liquor after drying in oven (110 C) and muffle furnace (550 C) and calculated based in sponge cube number.
3.3.3 Chemical characteristics of sludge
Sludge was collected from reactors R1, R2 and R3 and digested using concentrated nitric acid and analysed for various parameters.
3.3.4 Specific methanogenic activity (SMA)
SMA analysis was carried out with sludge from R1/B1 in a procedure similar to Isa et al.(1997). A known amount of sludge with VSS of 1-2 g/L was taken from anaerobic reactor and poured into serum bottles. Feed was added to each of serum bottle identical to synthetic feed purged with nitrogen gas used in the study varying thiocyanate concentration. Each test was carried out for three times. Supernatant was decanted, sludge was washed and fresh feed was given after the completion of each cycle. Data of third feeding was considered as data for actual specific methanogenic activity of the anaerobic reactor sludge. Biogas generation in R1 was measured by using water displacement method.
3.3.5 Enrichment, isolation and identification of microorganisms
Isolation of pure culture was carried out using pour-plate method. From each reactor randomly two sponge cubes were collected, dipped in distilled water separately for 5 minutes and scraped using a spatula. Then this water was serially diluted with distilled water upto105 times by serial dilution method. One mL of diluted culture was poured to Petri dish containing peptone beef extract and agar and incubated at 35C for 24 hours.
Discrete and well separated single colony was collected by sterile inoculating loop and transferred separately to conical flask containing peptone (5 g/L), beef extract (3 g/L), synthetic feed, phosphate buffer (1 mL/L), trace metal solution (1 mL/L) in a total volume of 75 mL. Synthetic feed of anaerobic culture was prepared by adding phenol (1000 mg/L), SCN- (500 mg/L) and NH4+-N (500 mg/L). For anoxic culture synthetic feed contained phenol (500 mg/L), SCN- (250 mg/L), NH4+-N (250 mg/L) and NO3--N (500 mg/L). For aerobic reactor culture, synthetic feed contained phenol (100 mg/L), SCN- (100 mg/L) and NH4+-N (250 mg/L).
All conical flasks were incubated for two days. After two days 1 mL of culture was taken from conical flask and used for each biochemical test separately. All the dilution, inoculation and transfer of culture were carried out in sterile condition. The identification of microorganisms was carried out by Morphological and biochemical tests along with microscopic observations. Biochemical tests were conducted according to Cappuccino and Sherman (1998). In biochemical test for identification of strain, gram reaction test, fermentation test (lactose, dextrose, sucrose and inulin), H2S production, nitrate reduction, indole production, Methyl red test, Vogues Proskauer test, catalase, oxidase and citrate tests were carried out.
Various instruments used during the study is mentioned with their make etc. in Table 3.6
Table 3.6 Instruments and equipments used in the present investigation Instruments/
Equipments
Parameters tested/
Measured
Model/Manufacturer Specification
Digital pH meter pH Orion 3 star –QY-14478, Thermo
Scientific, Singapore
Dissolved oxygen meter DO Orion 3 star –QY-14478, Thermo Scientific, Singapore
Electronic balance Weight Sartorius, BT-224S (4 decimal) Centrifuge Separation of suspended solids Remi C-24-BL Mumbai, India
COD digester COD HACH, DRB-200, USA
UV-visible spectrophotometer
Phenol, Thiocyanate, Ammonia- nitrogen, Nitrate, Nitrite-N
Model lambda-45, Perkin Elmer, USA,
Ion Chromatograph Nitrate-N, Sulfate, Chloride (Metrohm 792 AG, Herisau, Switzerland
Nephelo metric turbidity meter
Sulfate, turbidity Systronics 132
Oven MLSS, Drying other materials Tonco-PLT-125, India
Autoclave Sterilization Equitron-7407PAD
India Laminar Air flow hood To transfer pure culture in aseptic
manner
Clean air system, CAH -1800 Chennai, India
BOD incubator To prepare culture in agar media Delux model- IK-120 Delhi, India
High performance liquid Chromatography
Phenol, pyridine, m, o, p- cresol Varian Prostar 210
Atomic Absorption spectrophotometer
Pb, Co, Cr and Cd Varian, USA, Model No.Spectra AA 240FS
Air compressor Aeration in aerobic reactors Sonee SSY-8 Water purification system To provide distilled water and
Millipore water
Sartorius –AG, arium -611uv, 61315. Germany
Peristaltic Pump Feeding to reactors Miclin India limited PP10 Hot air blower Maintain temperature Philips