CHAPTER - IV

MATERIALS AND METHODS

This chapter describes the materials used and methods adopted for

carrying out the experimental work.

4.1 Materials

Different types of Materials like Samples, Chemicals, Equipment/

Instruments, Reagents and Reactors used described below.

4.1.1 Samples

The following phenolic compounds and chemicals were used for

preparing synthetic waste water, purchased from Sigma –Aldrich

Company Bangalore, India. They are a) 4-Chloro-2-nitrophenol (4C-2-NP)

b) 2-Chloro-4-nitrophenol (2C-4-NP) c) 2-Chloro-5-methylphenol (2C-5-

MP). Real Effluent samples (Bulk drug pharmaceutical wastewater and

dye intermediate wastewater) were collected from Jeedimetla Effluent

Treatment Plant in Hyderabad.

4.1.2 Water

Water used in all experiments was laboratory distilled water pH (7.2-8.0),

alkalinity (40-120mg/l), chlorides (20-30mg/l), for preparation of the

synthetic wastewater and raw effluent dilution.

4.2 Equipment/Instruments

The instruments used for carrying out the present study included Digital

pH Meters (MKVI Systronics) for pH measurement and Conductivity

meters (Systronics 304) for conductivity measurements, centrifuge(R24

Remi, India), UV –Visible Spectrophotometer (analytic jena SPEKOL

1200), Microscope (Olympus, USA), Kjeldhal Assembly (kjel plus, India),

hot air oven, muffle furnace, colony counter, Incubator rotary

shakers(Neolab, India), Hach – COD Reactor for COD measurement,

Fourier Transmittance Infrared Spectro Photometer (FTIR), Gas

Chromatography (GC-MS) Agilent Technologies, Laminar Air Flow (Clas,

India) etc.

4.3 Chemicals

Name of the Molecular Sl.No. Chemical Formula Vendor Purity

1. Ferrous Sulphate FeSO4 Merck 99%

2. Sulfuric Acid H2SO4 Finar 98.08%

3. Sodium Hydroxide NaOH Merck 97%

4. Sodium thio Sulphate Na2S2O3 S.D.Fine Chem 99%

5. Ammonium Chloride NH4Cl S.D.Fine Chem 99%

6. Potassium Chloroplatinate K2PtCl6 Merck 99%

7. Ammonia NH4 Sisco 25%

8. Ferrous Ammonium Sulphate (FeNH4 )2 (SO4)2 Merck 98%

9. Potassium dichromate K2Cr2O7 Merck 99%

10. Mercuric Sulphate HgSO4 S.D.Fine Chem 99%

11. Silver Sulphate AgSO4 Merck 98.5%

12. Glucose C6H12O6 S.D.Fine Chem 99%

13. Sodium acetate CH3COONa S.D.Fine Chem 99%

14. Silver nitrate AgNO3 S.D.Fine Chem 99%

15. Potasium chromate K2CrO7 Merck 99%

16. Iso propyl alcohol Merck 99% 17. Hydrochloric acid HCl S.D.Fine Chem 99%

4.4 Filtration

After Photo Catalytic treatment samples were filtered through 0.02 m

(mdi, India type SY25NN) membrane syringe filters are used.

4.5 Sample collection and storage of wastewater sample

Wastewater samples were collected from the equalization tank in the

effluent treatment plant. The sampling bottles were cleaned and rinsed

carefully with distilled water and then with the effluent. About 2.5 cm air

space is left in the bottles to facilitate mixing by shaking. Then samples

were stored at 40C within one to two hours of sample collection.

4.6 Waste Water Analysis

Table: List of continuously monitored parameters along with reagents used in the study

S.No PARAMETER REAGENTS METHOD

1 pH 7.0, 4.0 and 9.2 buffer 4500 – H+ B

2 Electrical

Conductivity

0.01 KCl 2510 A

3 Total alkalinity Methyl orange indicator,

phenolphthalein indicator, 0.02N

Sulfuric acid, 0.02N sodium

carbonate

2320 B

4 Volatile fatty

acids

Concentrated sulfuric acid, 0.1N

sodium hydroxide

5560 C

5 Sulphates Conditioning reagent, Barium

chloride crystals

4500–SO4 2-

6 Nitrates 1N Hydrochloric acid 4500-NO3- B

7 Phosphates Reagent A as prescribed

Reagent B as prescribed,

Ascorbic acid

Colorimetric

Ammonium

Molybdate-

Ascorbic

acid

method(P.K.

Gupta)

8 Chemical

oxygen demand

1N Ferrous ammonium sulphate,

0.025N potassium dichromate,

Ferroin indicator, Concentrated

sulfuric acid, Mercuric sulphate,

5220 C;

5220 B

9 Total hardness Ammonia buffer, Muroxide

indicator, Eriochrome black-T

indicator, 0.01 Ethylene di amino

tertra acetic acid, sodium

hydroxide.

2340 C

10 Chlorides Potassium chromate indicator,

Silvaer nitrate solution (0.0141N),

Sodium chloride (0.0141N).

4500- Cl-1 B

11 Ammonical

Nitrogen

Std sulphuric acid(0.02N), Borate

buffer, Boric acid, Mixed

indicator, Sodium hydroxide.

4500- NH3 B

12 Kjedhal

Nitrogen

0.01N Sulfuric acid, 0.01N

Sodium hydroxide, Concentrated

sulfuric acid.

4500- NorgB

13 Biochemical

Oxygen demand

Std potasium dichromate,

Concentrated sulfuric acid,

Starch indicator, Sodium thio

sulphate, Phosphate buffer,

Magnesium sulphate, Alkali

iodide azide reagent

5210 B

4.6.1 Preparation of Stock and working compound solution

Required amount of accurately weighed dry powder of compound was

dissolved in double distilled water using a magnetic stirrer at 20°C to

ensure complete dissolution. Desire standards were prepared from the

stock solution. The stock is stored at 4°C in a refrigerator.

4.6.2 Estimation of Methane percentage

The methane percentage was estimated in the biogas by using Gas

chromatography (Agilent technologies 6590). 1ml of head space sample

was injected in to the GC. Thermal conductivity detector and SS

Molecular sieve M16, 3 × 1/8‖ column were used in GC. The

temperatures of oven, injector and detector was 40˚C, 100˚C and 180˚C

respectively ((Khursheed Karim and Gupta, 2006).

\

4.6.3. Estimation of compound concentration

The concentrations of compounds like 4C-2-NP, 2C-4-NP and 2C-5-MP

was measured at 234, 240, 350 nm respectively by using UV-Visible

Spectrophotometer. These spectrophotometric values are further

confirmed with HPLC. The methanol and water was used as a mobile

phase in the ratio of 80:20 at a flow rate of 1ml/minute at 254, 240, 350

nm.

4.6.4 Morphological Characterization by using Scanning Electron

Microscope (SEM):

The treated anaerobic sludge initially washed with 0.1 M phosphate

buffer and incubate it in a 6% glutaraldehyde for over night. After these

samples are again further washed with 0.2 M sodiumcacodylate buffer

six times. Then these samples are further washed with acetone- distilled

water including 30%, 50%, 70%, 90%, 95% and 100%. A gold coating

was applued on the upper side of the sample and finally scanned in a

SEM at different magnifications.

4.7 Experimental setup

4.7.1. Experimental setup

The laboratory experiments were performed in 4 UASB rectors namely

(R1, R2, R3, and R4) with 7 liters capacity (fig 1). In these experiments

R1 was used as a control, where as R2, R3 and R4 was used 4C-2-NP,

2C-4-NP and 2C-5-MP fed reactors. The length of the reactor was 90 cm

with a circular column, The internal diameter and wall thickness was

10cm amd 2mm respectively. The reactor was having four sampling ports

along its length at equal distance. The gas solid separator was provided

at the top of the reactor which is used for the seperte biogas and solids

from the reactor. The outlet of gas tube was connected to the liquid

displacement system for measuring the biogas production. A peristallic

pump is used to give the feed to the reactor.

Fig1: Photographic representation of UASB reactor

Fig 2: Flow diagram of UASB reactor

4.8 Experimental methodology for the degradation of phenolic

compounds (4C-2-NP, 2C-4-NP, 2C-5-MP) by using UASB reactors

4.8.1. Seeding sludge

The sludge which is used for the degradation of phenolic compounds was

used flocculent sludge which we had taken from Slaughter house,

Rudraram, Hyderabad, India. The sludge was initially sieved with a mesh

size of 1.0 mm to remove the waste materials and other imputities. The

concentration of total suspended solids and volatile suspended solids

present in the sludge was 19.6 g/L and 48.5 g/L, respectively

4.8.2 Start up of the UASB reactors for the degradation of 4C-2-NP,

2C-4-NP, 2C-5-MP

2L of sludge was used for the inoculation in each UASB reactors. The

concentration of suspended solids and volatile suspended solids was

maintained in the reactor was 19.93kg VSS/m3 and 28 kg VSS/m3

respectively. The composition of the feed during the startup period in the

UASB reactor was given in table 4.3. 1 ml of the trace metal solution was

added. The composition of trace metal solution was given in table 4.4

Table 4.3: The composition of the feed during the startup period in the

UASB reactor

S.No Name of the chemical Concentration

1 glucose 1.0 g/l

2 NH4Cl 800 mg/L

3 KH2PO4 200 mg/L

4 CaCl2.H2O 48 mg/l

5 FeSO4.7H2O 40mg/l

6 H3BO3 0.1mg/l

7 ZnCl2 0.1mg/l

8 CuCl2 0.1mg/l

9 MnSo4. 4H20 0.1 mg/l

10 AlCl3 0.1mg/l

11 NiCl2 0.1mg/l

12 NaHCO3 3000mg/l

13 Trace metal solution 1ml/l

14 phenolic compounds variable

Table 4.4: The composition of trace metal solution

S.No Name of the chemical Concentration

1 Mg SO4. 7H2O, 5 g/l

2 FeCl2. 4H2O 6 g/l

3 CoCl2. 4H2O 0.88 g/l

4 H3BO3 0.1 g/l

5 ZnSO4 .7H2O 0.1 g/l

6 CuSO4.5H2O 0.05 g/l

7 NiSO4. 8H2O 1 g/l

8 MnCl2. 4H2O 5g/l

9 (NH4) 6Mo7O24. 4H2O 0.64g/l

4.8.3 Acclimation of UASB with 4C-2-NP, 2C-4-NP, 2C-5-MP

During the startup of the reactors when UASB rectors reached to steady

state conditions, Initially 2 mg/l of 4C-2-NP, 2C-4-NP and 2C-5-MP were

introduced in UASB rector at 20th day. After that the compound

concentration was gradually increased from 2 to 5, then 5-10, 10- 15,

15- 20, 20-25, 25 -30 and 30mg/l . 20 days was operated at each

increment of influent phenolic compound concentration. 24h HRT was

used for the acclimation of the reactor.

4.8.4 Substrate: co-substrate ratio study

Glucose was used as a co-substrate for the degradation of phenolic

compounds. In this study five different concentrations of co-substrate

was given. The substrate: co-substrate ratios was maintained in the

reactor were 1:33.3, 1:66.6, 1:100, 1:133.3, 1:166.6 by adding the

glucose concentration 1.0, 2.0. 3.0, 4.0, 5.0g/l respectively. At each

substrate: co-substrate ratio, the reactors were operated for 30-35 days.

4.8. 5 Hydraulic retention time (HRT) study:

The Hydraulic retention time (HRT) studies were carried out by changing

the flow rate of influent wastewater. Change in HRT (30-6hrs) produced

medium to high organic loading rate (OLR 1.0-5.0kg/m3/d) to the UASB

reactors. Reactors were operated at quasi-steady state for 30-35days at

every HRT study. In the biodegradation of phenolic compounds in

wastewater, the reactors were operated at five different HRTs:30, 24, 18,

12, and 8h.

4.9 Experimental methodology for the treatment of Bulk drug

pharmaceutical wastewater by using UASB reactors

4.9.1 Inoculum

The sludge used for the studies of the treatment of bulk drug

pharmaceutical wastewater was taken from Slaughter House, Rudraram,

Medak, Hyderabad. The concentration of VSS and SMA present in the

sludge were 28.86 g/l and 120.8 ml- CH4/g-VSS d respectively.

4.9.2. Wastewater Source and Characteristics:

The wastewater used for the degradation studies was procured from

Jeedimetla Effluent treatment plant, Hyderabad and Characterised by

using standard methods (APHA, 2000). The characteristics of the

wastewater was given in Table1

Table: Characteristics of Bulk drug pharmaceutical industrial

wastewater

S.NO parameter Concentration

(mg/l)

1 pH 7.0-7.5

2 Colour Orange

3 Electrical conductivity 40,000-40,500

3 Alkalinity 2,500-3,000

4 Chemical oxygen demand (COD) 8000-8,100

5 Biochemical oxygen demand (BOD) 3,900-4,000

6 Sulphates 300-450

7 Nitrates 120-170

8 phosphates 100-120

10 Total dissolved solids 6,500- 7000

11 Volatile fatty acids 600- 750

12 Total suspended solids 274-500

13 Phenol 25-30

14 2-methoxy phenol 20-25

15 2, 4, 6-tri chloro phenol 20-25

16 Dibutyl phthalate 30-40

17 1-bromo naphthalene 5-10

18 Antipyrine 5-10

*All the values are expressed as mg/l except pH , EC and colour

4.9.3 Analytical methods

GC-MS characterization for the analysis of hazardous compounds:

a gas chromatograph (6890N, Agilent) with a mass selective detector

(5973) was used for the identification of hazardous compounds present

in the wastewater. The compounds were identified by acidifying aqueous

samples to pH below 2, extracting with dichloromethane, filtering using a

MDI Teflon syringe filters (SY25NN, 0.2 m pore size) and injecting 1l of

the filtrate into a gas chromatograph with A HP 5 MS, capillary column,

(0.25mm, 30m, 0.25m), was used for sample elution.

4.9.4 Start-up of UASB reactor for the treatment of pharmaceutical

wastewater

Glucose was used for the startup of the reactor because, it is a readily

biodegradable substrate and it can produces the intermediate products.

3 L of the sludge was added in to the reactor. The

Table 4.3: The composition of the feed during the startup period in the

UASB reactor for the treatment of pharmaceutical wastewater

S.No Name of the chemical Concentration

1 C6H12O6 1.0 g/l

2 Sodium thio glycollate 0.05 mg/l

2 NH4Cl 800 mg/L

3 KH2PO4 200 mg/L

4 CaCl2.H2O 50 mg/l

5 FeSO4.7H2O 30mg/l

6 H3BO3 0.1mg/l

7 ZnCl2 0.1mg/l

8 CuCl2 0.1mg/l

9 MnSo4. 4H20 0.1 mg/l

10 AlCl3 0.1mg/l

11 NiCl2 0.1mg/l

12 NaHCO3 3000mg/l

13 Trace metal solution 1ml/l

14 phenolic compounds variable

One ml of the trace metal solution was added per litre of pharmaceutical

wastewater. The COD:N:P ratio was maintained around 300:5:1. The pH

of the reactor was maintained 5.6-7.5 which is favourable for

methanogenic bacteria. We had monitored the parameters like pH, COD,

BOD, Alkalinity, Sulphates, phosphates, etc. The parameters which we

has been monitored during the pharmaceutical wastewater was given in

Table 2

Table 2 : The parameters which we has been monitored during the

pharmaceutical wastewater

To study the reactors performance in anaerobic UASB reactor the

following parameters has been monitored.

S.No PARAMETERS FREQUENCY OF ANALYSIS

1 pH Once in every 6 hrs

2 Total alkalinity Once in a day

3 Sulphates Once in a day

4 Nitrates Once in a day

5 Phosphates Once in a day

6 Volatile fatty acid(VFA) Once in a day

7 Chemical Oxygen demand

(COD)

Once in a day

8 Biological Oxygen demand

(BOD)

Once in 3 days