3 Materials and Methods 80
3.2 Methodology 90
3.2.1
Water Quality Analytical Methods
Liquid samples were collected from the feed tank, anaerobic and anoxic column top and the effluent. The analyses were either done the day of sampling or the samples were refrigerated at 4 °C prior to analysis. Total suspended solids (TSS), volatile suspended solids (VSS) and biochemical oxygen demand (BOD) were analyzed in accordance with
Standard Methods 2540D, 2540E and 5210 [1] respectively. DO and ORP were measured
onsite using an Oakton DO 6 meter, and an Oakton ORPTestr 10 (Oakton, Singapore). HACH methods and testing kits (HACH Odyssey DR/2800) were used to analyze total and soluble chemical oxygen demand (TCOD and SCOD), total and soluble nitrogen (TN
and STN) and total phosphorus (TP), NH4-N, NO2-N, NO3-N, and PO4. Alkalinity was
measured by titration with 0.01 N H2SO4 in accordance with the Standard Method no
2320. [1] Sulfate (SO
42-) was measured using the ion chromatography (IC, Dionex 600,
USA) equipped with CS16-HC and AS9-HC columns. Sodium carbonate solution, 9 mM, was applied as an eluent at a flow rate of 1 mL/min for 30 minutes, with sulfate detected 20 minutes following injection.
Both dissolved and total metals were measured following the standard method 3120
using ICP (Vista-Pro, VARIAN). [1] The soluble metals were measured by doing the
analysis on the filtered sample (0.45µm filter paper) and the total was obtained by digesting the sludge samples followed by filtration through a 0.45µm filter paper prior to analysis using ICP. For major metals (Ca, Na, K, and Mg) and most of the trace metals (Fe, Cu, Cr, Al, Co, Ni, Zi, and Mn) the digestion method was followed according to
method 3030D of Standard Methods. [1]
The rate of biogas produced in the anaerobic methanogenic column was measured by a gas wet tip gas meter (Rebel wet-tip gas meter company, Nashville, TN, USA) connected to the top of anaerobic column. Methane, nitrogen gas, hydrogen gas were determined by injecting 0.5 mL of the biogas composition into a gas chromatograph (Model 310, SRI Instruments, Torrance, CA) equipped with a thermal conductivity detector (TCD) and a molecular sieve column (Molesieve 5A, mesh 80/100, 182.88 × 0.3175 cm). The
temperatures of the column and the TCD detector were 90 and 105oC, respectively. Argon was used as carrier gas at a flow rate of 30 mL/min.
3.2.2
Fed-Batch Experiments
Batch tests were carried out to test the maximum specific nitrification rate (SNR), specific denitrification rate (SDNR) and specific methanogenic activities (SMA) of the attached biomass in the aerobic, anoxic and anaerobic aforementioned systems. Batch reactors (0.5 L working volume) equipped with magnetic stirrers were used for nitrification by injecting air or for denitrification by avoiding intrusion of air. To reduce the effect of substrate mass transfer limitation into the biofilm, the biofilm was removed from 30-40 g of media using sonication and then placed into the reactors. The biomass in the SDNR and the SNR tests were in the range of 1500-4000 mg VSS/L and 240-500 mg VSS/L respectively, considering the amount of biofilm in the anoxic and aerobic column, 25-50 mg VSS/g media and 4-6 mg VSS/g media. The initial acetate COD in the denitrification batch tests was set at 350-450 mg/L while the initial alkalinity used in the
nitrification test was 250-350 mg/L as CaCO3. For the SNR tests, the initial ammonia
concentrations were 35-55 mg/L, added as ammonium chloride.
The biofim-coated particles from anaerobic column were used for specific methanogenic activity (SMA) at 37°C, using 250 mL bottle capped with Teflon septum. Approximately 10 g anaerobic biofilm coated particles and 0.3-0.6 mL acetic acid were added together into the 125 mL-bottles containing 0.2 mL of nutrients
(2000 mg/L FeCl2⋅4H2O, 50 mg/L H3BO3, 50 mg/L ZnCl2, 30 mg/L CuCl2,
500 mg/L MnCl2⋅4H2O, 50 mg/L AlCl3, 50 mg/L CoCl2⋅6H2O) and 3000 mg alkalinity
per litre as CaCO3. All the bottles were sealed after purging the headspace with nitrogen
to eliminate the present of oxygen/air. The experiment was continued until the bottles stopped producing biogas. Daily biogas was measured by inserting needle attached to a 100-ml syringe (Hamilton, Nevada, USA). Methane composition was measured using
3.2.3
Bacterial Community Analysis
Samples were taken from bottom and top of anoxic, anaerobic and aerobic columns. The total genomic DNA was extracted from each sample using the UltraClean Soil DNA Isolation Kit (MO BIO Labratories, Carlsbad, CA, USA). PCR amplification of a region of the 16S rRNA gene was performed with universal the primer set 349f-GC (5'-CGCC CGCC GCGC GCGG CGGG CGGG GCGG GGGC ACGG GGGG CCTA CGGG AGGC AGCA G-3') and 907rM (5'-CCGT CAAT TCMT TTGA GTTT-3', where
M=A+C) [2] using a MyCycler thermal cycler (BioRad, Hercules, CA, USA). The PCR
products were applied directly to a 6% (w/v) polyacrylamide gel with 20-50% denaturing gradient (100% denaturing gradient corresponds to 70M urea and 40% (v/v) formamide). Electrophoresis was run at a constant voltage of 130V at 58 °C for 6 h. The DNA templates from the bands of interest were re-amplified and the PCR products were purified with the QIAquick PCR purification Kit (QIAGEN Sciences, MD, USA). The fragments were sequenced at the Sequencing Facility at the Robarts Research Institute (The University of Western Ontario, London, Ontario, Canada) and compared with available sequences from the GenBank database using the Basic Local Alignment Search
Tool (BLAST) program. [9]
3.2.4
Attached Biomass
The attached biomass on the carrier media was measured and expressed as mg VSS/g
clean particles, based on Standard Method no 2540. [1] Approximately 10-20 g biofilm-
coated particles were taken from columns and suspended in a 100 mL vial and sonicated for 3 h at 30°C in an Aquasonic sonicator (SK 1200H Kupos, China) with a rated power of 45 Watts. After sonication, the TSS and VSS content of the detached biomass was
determined following Standard Methods no 2540D and 2540E [1] and the values were
divide by the weight of the dry clean particles.
3.2.5
Biofilm Thickness Measurement
Biofilm coated particles were periodically taken from sampling ports along the columns for the purpose of measuring the biofilm thickness. The sampling took place by a syringe at the same pressure inside each column to minimize disturbances to the biofilm
structure. Each particle was then transferred to a small container filled with water. Using a microscope (MITUTOYO, Sakada, Japan) with 50X magnification coupled with a camera (LEICA DC300, Germany) each particle was photographed and then transferred
to its container. The volumetric equivalent particle diameter (dp) and the volumetric
equivalent media diameter (dm) were measured with the VISIONGAUGE software
(Flexbar Machine Co, New York, USA) synchronized to the camera. In order to maximize the accuracy of the measurements, all the measurements were periodically checked with the microscope’s Standard Measurement Ruler.
3.2.6
Dry and Wet Biofilm Densities
In order to measure biofilm dry density, Equation (3.1), Proposed by Ro and
Neethling (1990), was used. [3] Samples were taken and photographed to measure dp and
dm and then sonicated (Aquasonic SK 1200H Kupos, China) for three hours at 30°C to
remove the biofilm from the media. Since the biofilm sizes were not completely identical in each sample, average values for diameters were considered. Each sampling took place at a different stage of biofilm development and also hydrodynamic conditions such as superficial liquid velocity. As a result, different biofilm thicknesses were obtained at different times but the thicknesses of the biofilms were relatively equal in each sampling.
1 3 − = γ ρ ρd m X (3.1)
where ρd is the biofilm dry density, ρm is the media true density, X is the ratio of dry mass
of biofilm to dry mass of media, and γ is dp/dm
A hydrostatic method was developed to measure the wet biofilm density accurately. The experimental data verified the most accurate equation to calculate this value. Samples were taken, photographed for measuring the biofilm thickness and then sonicated to remove the biofilm from the media. Different concentrations of sodium acetate were dissolved in deionised water in order to make liquids with different densities
of 1060, 1065, 1070, 1075, 1080, 1085, 1090 and 1095 kg/m3. The density of liquid in
filled with the provided liquids. The biofilm without carrier media was placed inside the vials and then well shaken to lessen the size of bio-particles in order to make the effect of buoyancy force uniform. After two hours bio-particles in one vial did neither float nor settle. Since the gravity force equaled to the buoyancy force, the density of bio-particles, wet density, was considered equal to the density of the liquid.
3.2.7
Carrier Media Size Determination
The size of the bare and biofilm coated particles was measured using a Mastersizer 2000 laser analyzer (Malvern Instruments Inc., UK).
3.2.8
Pressure Gradient and Axial Distribution of Solids
Eight manometers (4 for each aerobic and anoxic column) connected to an air collector were used to measure the pressure difference along columns. Thereafter, axial distribution of solids in a three-phase fluidized bed with heavy particles was determined
from the pressure gradient along the column. Axial void fraction (ε), solid hold-up (εS),
and solids concentrations were calculated using on-line pressure transducers data along
the columns and following Equations: [4]
g L pS = × − × P − × Δ − (1
ε
) (ρ
ρ
) (3.2)ε
ε
S =1− (3.3) ) 1 ( ε ρ × − = P AL M (3.4)where, L, A, g, ΔpS, ρ, and ρp are length of the section (m), cross sectional area (m2),
acceleration of gravity (m/s2), additional pressure drop (kPa) due to the presence of
solids, liquid density (kg/m3), and particle density (kg/m3) respectively.
3.2.9
Statistical Analysis
The student t-test was used to test the hypothesis of equality at a 95% confidence level. The null hypothesis was defined to be no difference between the two groups tested versus the alternative hypothesis that there is a statistical difference between the two
groups. Non-linear regression was done by Matlab (MathWorks, Massachusetts USA) and all other data was analyzed using SigmaPlot and Excel 2007.