Ph Effect on Vsv (A): For Improved Ebb, (B) Control Sample The Study Also Aims to Select the Appropriate Ph Range for Growth

Experimental diagram

H 2 SO 4

NaOH

Test box

test

EBB improvement

progress

H 2 SO 4

NaOH

(a) (b)

Figure 2.5. Effect of pH on VSV (a): for improved EBB, (b) control sample The study also aimed to select the appropriate pH range for the growth

and adhesion of microorganisms in improved EBB material. Improved EBB blocks were put into a tank with a capacity of 50 liters. Sagi Bio2 biological product was put into the experimental tank at 50 ml with a density of 10 8 CFU /ml of microorganisms and supplemented with nutrients for microorganisms at a BOD:N:P ratio of 100:5:1.

During 10 days, the number of aerobic and anaerobic microorganisms in the improved EBB blocks was measured in density and compared with the number before the experiment.

In this experiment, there was also a control experimental system ( Figure 2.4 ) (a tank without improved EBB placed inside), which was operated in parallel with the tank with improved EBB inside to compare the COD treatment efficiency of improved EBB.

Experimental stages

The experiment was divided into three phases as shown in Table 2.4 . Water samples were taken from two tanks and analyzed for COD for each phase on days 1, 5 and 10.

Table 2.4. Experimental stages at different pH ranges.

Stage

Phase 1	Phase 2	Phase 3
pH range	4 ÷ 5	7 ÷ 8	9 ÷ 10
Number of days of experiment	10	10	10

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The pH ranges (4÷5, 7÷8 and 9÷10) were strictly controlled by the BL981411 pH controller. Four improved EBB tablets weighing 0.7 kg/tablet were put into the 50-liter experimental tank ( Figure 2.5 ); NaOH (5%) and H 2 SO 4 (5%) solutions were used to adjust the pH. The experiment was conducted under normal temperature conditions. At each new stage, the wastewater in the experimental tank was replaced with new water. The COD index was measured by the Dichromate method using K 2 Cr 2 O 7 as the oxidizing agent (ISO 6060:1989 standard).

2.6.3. Method for determining the Ammonium adsorption efficiency of improved EBB material

Experiment

NH 4 Cl solution was prepared in distilled water saturated with Ar gas. The modified EBB material was surface treated in an ultrasonic bath for 15 minutes before adsorbing ammonium ions. The adsorption process was carried out with initial solution concentrations, pH and determined amount of adsorbent, depending on the experiment, 0.1M NaOH solution and 0.1M HCl to adjust pH. The samples were shaken on a vibrating shaker at 120 rpm. Then centrifuged, filtered to get the solution to analyze the remaining amount of ammonium ions.

To study the material's ability to adsorb ammonium ions, perform the following series of experiments:

- Effect of pH on the material's ability to adsorb Ammonium ions:

Prepare 9 solution samples with NH 4 Cl ion concentration of 30 mg/L, adsorbent material mass of 150 g/L. The pH values of the experimental samples were adjusted to 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively.

- Ammonium ion adsorption kinetics and the effect of initial concentration on the adsorption efficiency of the material:

NH 4 Cl solution has initial NH 4 + concentrations of 10, 30, 45 mg/L, solid mass of 150 g/L, maintaining pH 6. Sampling times are: 30, 60, 90, 120,150, 180,

240, 360, 480 and 720 minutes.

- Adsorption kinetics:

To study the adsorption kinetics of N ions on improved EBB materials, two kinetic models were examined:

First-order apparent kinetic model:

ln (q e – q t ) = lnq e – K 1 .t (2.1)

Apparent second-order kinetic model:

In there:

𝑡

𝑞 𝑡

= 1

K 2 .𝑞 𝑒 2

+𝑡

𝑞 𝑒

(2.2)

q e (mg/g): amount of NH 4 + adsorbed at equilibrium q t (mg/g): amount of NH 4 + adsorbed at time t (minute) K 1 (minute -1 ): apparent first-order rate constant

K 2 (mg/g. min 1/2 ): apparent second-order rate constant.

- Effect of adsorbent amount:

The initial concentration of NH 4 Cl solution was 30 mg/L, the adsorption concentrations used were 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 g/L, respectively, pH at 6. Sampling time was after 240 minutes of contact.

- Ammonium ion adsorption isotherm of the material:

The experiment was conducted under the following conditions: Solid content was 150 g/L, initial NH 4 Cl concentrations were: 10, 16, 20, 25, 30, 45, 50 and 60 mg/L respectively; pH was controlled at a value of 6. Contact time was 240 minutes.

Determine the constants of the Langmuir adsorption isotherm equation:

(2.3)

In there:

q – Adsorption capacity at equilibrium time (mg/g) q max – Maximum adsorption capacity (mg/g)

C f – Equilibrium concentration (mg/L)

b - Constant characterizing the interaction between the adsorbent and the adsorbate.

This equation can be transformed into the form:

C f 

q max

. C f

 1

b . q max

(2.4)

This is a straight line equation representing the linear dependence of C f /q on C f .

In there:

qe Adsorption capacity at equilibrium (mg/g).

q max Maximum adsorption capacity (mg/g).

C f Equilibrium concentration (mg/L).

b Constant characterizing the interaction between the adsorbent and the adsorbate.

- Analysis to determine the content of Ammonium ion

Ammonium ion concentration in water is determined by colorimetric method with Nessler reagent.

In alkaline environment, NH 4 + reacts with Nessler reagent to form a complex with color ranging from yellow to brown, depending on the concentration of Ammonium in the solution.

Interfering factors: Iron interferes with the determination, which is removed with complexone (III) salt. Organic compounds, alcohols, aldehydes, aliphatic and aromatic amines, chloramines react with Nessler's reagent, and when present in water, must be distilled to separate ammonia before determination. In case of turbid water, it must be treated with 25% zinc sulfate solution.

Determination method: take 5 ml of sample, add 0.2 ml of benzetic and 0.5 ml of Nessler solution respectively. Leave for 10 minutes, then measure the optical absorption at 420 nm wavelength.

(2.5)

In there:

R: removal efficiency (%)

Co: initial concentration of adsorbed substance (mg/L)

Ce: concentration of adsorbed substance at the time of sampling (mg/L).

2.6.4. Method for determining the effectiveness of microbial activity in improved EBB using molecular biology techniques

The experimental system for monitoring the density of microorganisms in the improved EBB material using molecular biology techniques is arranged in Figure 2.6 .

Figure 2.6. Experimental system to evaluate the activity of microorganisms in improved EBB materials. In which:

(i) Water trough A1: Has improved EBB material; is inoculated with microorganisms from Sagi-Bio 2 preparation; has air supply.

(ii) Water trough A2: With improved EBB material; no air supply; inoculated with microorganisms from Sagi-Bio 2 preparation

(iii) Water trough A3: Has control foam padding material, is inoculated with microorganisms from Sagi-Bio 2 preparation, and has air supply.

(iv) Water trough A4: no improved EBB material, no bacterial culture, no air supply,

(v) Water trough A5: No EBB material, no bacteria, air supply

Domestic wastewater was collected from a sewer in Nghia Do ward, Cau Giay district, Hanoi and brought back for testing. Domestic wastewater was stored in a 200-liter tank. Here, there were 05 dosing pumps with the same flow rate. Wastewater from 05 dosing pumps was divided equally into 05 water troughs of the experimental system. Wastewater was analyzed and determined for indicators such as COD, Amoni daily and after 10 days, samples were taken to analyze and evaluate the microbial community in the test samples using DGGE denaturing electrophoresis biological technique.

- DNA separation technique

The aim was to analyze the diversity of bacterial communities in improved EBB materials before and after inoculation with the strains of microorganisms in Sagi-Bio 2 preparation.

+ Sample processing

 Each filter material sample is soaked in sterile MQ water, using strong mechanical impact to dissolve the microbial cells attached to the filter material surface into water to obtain the maximum amount of microbial cells attached to the filter material surface.

 2 mL of each water sample obtained above was centrifuged at 6000 rpm for 10 minutes at room temperature to collect the cell residue.

 Add 1 ml of sterile MQ water to each tube, stir to dissolve cell residue, then continue centrifuging at 6000 rpm for 10 minutes at room temperature (to wash the sample).

 Discard the liquid. Add 300 µl of sterile MQ water to the sample, stir to re-dissolve the residue.

+ Total DNA separation

 Add 300 µL of Phenol-Chloroform-isoamyl alcohol mixture (25:24:1) and stir thoroughly by vortex or by hand for about 30 seconds.

 Centrifuge at 14000 rpm, RT, for 12÷15 minutes.

 Use a light pipette to remove the top supernatant and transfer to another eppendor tube (about 200 µL).

 Add in order: 1 µL Glycogen, 100µl 7.5M ammonium acetate and 750µl 100% Ethanol.

 Incubate in freezer (- 20 o C) overnight

 Centrifuge at 14000 rpm, 4 o C for 30 minutes to precipitate DNA.

 Decant the liquid, add 150µl of 70% ethanol, mix gently with a pipette.

 Centrifuge at 14000 rpm, 4 o C for 10 minutes to wash DNA. Repeat this step 2 times.

 Decant the liquid and let it dry.

 Add 20 µl of PCR water to dissolve the DNA.

 Prepare the solution

- DGGE electrophoresis technique

Prepare denaturing solution

Table 2.5. Proportions of denaturing solutions for conducting experiments.

Stock solutions

45% denatured	60% denatured	0% denaturation
60% - solution	7.5 mL	10 mL	0 mL
0% - solution	2.5 mL	/	5 ml
TEMED	8 µL	8 µL	5µL
APS 10%	40 L	40µL	22L

Steps to follow:

+ Install gel mold.

+ Pipette 2 ml of denaturing solution (45% and 60%) into the gel pouring device, 60% on the right (with outlet), 45% on the left, open the valves, place the needle in the middle of the glass plate (so that the solutions run evenly inside).

+ Turn on the pumper switch, set to the lowest pump speed. After finishing the 60% and 45% solutions, leave for about 30 minutes to dry.

+ Place the comb between 2 glass plates, use a pipette to gently add 0% solution and avoid creating air bubbles.

+ Let the gel stabilize for 3 hours.

+ Turn on the electrophoresis device for about 1 hour, reaching 60 o C before proceeding.

+ Remove the comb from the gel, wash each well with a pipette, check the quality of the wells by dropping diluted Loading dye into the wells. Place the gel in the electrophoresis chamber.

+ Mix Loading dye with samples (7µl +10 µl sample). Use a pipette to drop each sample into the wells from left to right.

+ Turn on the power source, run 120 V for 30 minutes to let the sample-loading dye mixture enter the gel, then electrophoresis at a voltage of 38 V/16 hours (from 5:30 p.m. to 9:30 a.m. the next day)

+ Take out the gel, remove the glass plate and mark by cutting a small corner below the gel.

+ Stain the gel for 30 minutes in GreenSafe staining solution, rinse with distilled water for 10 minutes.

+ Place the gel on the gel viewing table, observe and take pictures.

2.6.5. Method for evaluating COD and Ammonium treatment performance of improved EBB material in the laboratory.

The improved EBB experimental system is shown in Figure 2.7 . This model is mainly made of PVC plastic, the size of the improved EBB reaction trough is 1.4 meters long, 10 cm wide, and 13 cm high. Inside the trough are placed 4 EBBs with each weighing 2.5 kg. The distance between the improved EBBs is 5 cm.

Figure 2.7. Improved EBB experimental model.

The treatment tank has a total volume of 18.2 liters with a useful volume of 12 liters (tank number 1). Domestic wastewater is taken from a wastewater outlet in Nghia Do ward, Cau Giay district, Hanoi. The input wastewater is determined through the parameters of COD and Ammonium concentration.

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