SBV3023 Issues in Biology and Environment

ANAEROBIC FERMENTION OF KITCHEN WASTE FOR

ORGANIC REMOVAL AND BIOGAS PRODUCTION
Objectives:
After completing this experiment students should be able to:
1. Understand basic principles required for strict and facultative fermentation
process.
2. Carry out anaerobic digestion process for organic waste (kitchen waste) at a
laboratory scale.
3. Determine organic reduction (BOD and COD) and biogas production
throughout anaerobic fermentation process.
Introduction:
Anaerobic fermentation process is used primarily for the treatment of waste sludge
and high-strength organic wastes. However, applications for dilute waste stream
have also been demonstrated and are becoming more common. Anaerobic
fermentation process is advantageous because of the lower biomass yields and
production of methane from the biological conversion of organic substrates. Three
basic steps are involved in the overall anaerobic oxidation process; (1) hydrolysis,
(2) acidogenesis and (3) methanogenesis. The three steps are illustrated in Figure 1
below. Through anaerobic digestion, the complex organic compounds i.e
carbohydrate is hydrolyzed into sugars which are converted to organic acids and
finally to biogas. Biogas, the gas generated from organic digestion under anaerobic
condition by mixed population of microorganisms, is an alternative energy source
which has been commenced to be utilized both in rural and industrial areas. The gas
generally composes of methane (55-65%), carbon dioxide (35-45%), nitrogen (03%), hydrogen (0-1%), and hydrogen sulfide (0-1%). The composition of biogas
depends on feed materials.

SBV3023 Issues in Biology and Environment

Figure 1: Anaerobic process schematic hydrolysis, acetogenesis and

methanogenesis
Materials:
Kitchen waste
Activated sludge
COD reagents
pH 2-3 H2SO4 solution
5M NaOH
5M HCl
Tryptone glucose yeast extract plate agar
Sterile water
Serum bottles or shake flasks (100 ml or 250 ml with cotton gauge) (all autoclaved)
Rubber stopper
Aluminium seal
Hand crimper
Hungate tubes
10 ml syringe and needle
Container fill with pH 2-3 H2SO4 solution
Nitrogen gas
Blender
Centrifuge tubes
Micropipette and tips
Test tubes
Shaker (room temperature)
HACH Spectrophotometer
Centrifuge
pH meter
Gas Chromatography
2

SBV3023 Issues in Biology and Environment

Methane standard
1L volumetric flask
DO meter
Procedure
Inoculum Preparation
1. The inoculum used is activated sludge obtained from biological wastewater
treatment plant.
2. Centrifuge 50 ml of sludge at 6000 rpm for 10 minutes. Discard the
supernatant, add distilled water to the and repeat centrifugation process.
3. Analyse the sludge for its viable bacterial enumeration using heterotrophic
plate count.
Kitchen Waste Preparation
1. Kitchen wastes used in this study is collected from restaurant/houses and
consists of rice and noodles mixture with a ratio of 1:1.
2. Weigh 250 g of each rice and noodles waste and add distilled water at 1:0.5
ratios before grind using a blender to obtain a mixture solution.
3. Determine the concentration of COD, BOD, pH and heterotrophic plate count
prior to the fermentation process.
Fermentation Conditions
1. Carry out fermentation of kitchen waste under non sterile and strict or
facultative anaerobic conditions.
2. Add 45 ml of kitchen waste mixture into three 100 ml serum bottles.
3. Add 5 ml (10%) of inoculums which give a total working volume of 50 ml.
4. Adjust pH of initial mixed solution to 7.0-7.5 using 5M NaOH or 5M HCl.
5. Sparge serum bottles containing mixed kitchen waste and inoculum using
nitrogen gas for 10-15 minutes.
6. Seal serum bottle with rubber stopper and crimp with aluminum seal using
hand crimper.
7. Prepare another three serum bottles with kitchen waste mixture but without
inoculums which served as a control.
8. Run the fermentation at temperature of 30oC with an agitation speed of 50
rpm.
Sampling
1. Collect gas produced using 10 ml syringe and needle.
2. Quickly add into hungate tube which is filled with H2SO4 solution, pH 2-3. Gas
will replace the acidic solution.
3. Cap the tube and keep it in inverse condition until further gas analysis.
4. Collect 2 ml samples using syringe and needle at regular interval (every day)
within 7-10 days of fermentation period.
5. Centrifuge samples at 6000 rpm for 15 minutes and analyse the supernatant
for BOD, COD and pH.
3

SBV3023 Issues in Biology and Environment

Heterotrophic Plate Count

1. The heterotrophic plate count is used to estimate the number of viable
heterotrophic bacteria presents in the sludge and kitchen wastes samples.
Colonies may arise from pairs, chains, cluster or single cells and all were
determined in the term of colony forming units (CFU).
2. Dilute sample using serial dilution from 101 to 106 of dilution factor.
3. Transfer about 0.1 ml sample from each dilution tube using micropipette onto
surface of tryptone glucose yeast extract.
4. Incubate the plate for 48 hours at 35oC .
5. Count the number of bacterial colonies from plates have 30-300 of bacterial
colonies. The number of bacteria per ml is calculated as follows:
The number of bacteria/ml (CFU/ml) = Number of colonies/ volume of sample
Dilution factor.
Chemical Analyses
Biochemical Oxygen Demand (BOD)
1. Dilute sample with the dilution water to the required dilution factor in 1L
volumetric flask. Before that, the dilution water should be air saturated for at
least 15-20 minutes.
2. Mix the diluted sample and then poured into three clean BOD bottles so that
they overflow.
3. Insert a stopper into the bottle and make sure there are no air bubbles
adhering to the inside of the bottles by tapping the sides and then insert the
stoppers firmly.
4. Calibration of DO meter:
a. Saturate distilled water using air aeration of at least 15-20 mins.
b. Pour saturated water into BOD bottle, record the water temperature
and adjust the DO according to the temperature by referring to the
solubility of oxygen in water exposed to the water-saturated air (Table
1).
5. Place the DO probe in the sample, allow the meter to equilibrate and read DO
concentration directly.
6. Place the BOD bottles in the incubator at 20oC for five days and the DO is
tested again using the same procedure.
7. The BOD of a blank is determined by filling three bottles with dilution water
and measure its DO before and after five days.
8. BOD value is determined by the following formula:

SBV3023 Issues in Biology and Environment

BOD value in mg/l = [(a-b) (c-d)] /P

Where;
a= initial DO in a sample
b= final DO in a sample
c= initial DO in blank
d= final DO in blank
P= fraction of the BOD bottle that is represented by the sample or mL pipetted divided
by
300mL when a 300mL bottle is used.
Chemical Oxygen Demand (COD)
1.
2.

Dilute sample with distilled or deionized water.

Turn on the COD reactor. Preheat to 150 oC. CAREFULL the

reactor is hot.
3. Remove the caps from two COD Digestion Reagent Vials. (Be sure to use vials
for the appropriate range)
4. Hold one vial at a 45-degree angle. Use a volumetric pipette to add 2 ml of
sample to the vial prepared sample.
5. Hold another vial at a 45-degree angle. Use a clean volumetric pipette to add
2ml of deionized water to the vial (blank).
6. Cap the vial tightly. Rinse them with deionized water and wipe with a clean
paper towel.
7. Hold the vials by the cap over a sink. Invert gently several times to mix. Place
the vials in the preheated COD reactor. The sample vials will become very hot
during mixing.
8. Heat the vials for two hours.
9. Turn the reactor off. Wait about 20 minutes for the vials to cool to 120 oC or
less.
10.Invert each vial several times while still warm. Place the vials into a rack and
cool to room temperature.
11.Measure the COD at 420 nm (low range) or 620nm (high range). COD is
measured in a unit of mg/L
Biogas (methane)
Quantitative Measurement
1. Measure
biogas
concentration
(methane)
produced
Chromatography (GC) with thermal conductivity detector.
2. Prepare standard methane gas concentration (80% methane).
3. Inject sample from hungate tube to GC.
Qualitative Observation
1. Observe the presence of bubbles for biogas production.
5

using

Gas

SBV3023 Issues in Biology and Environment

2. In a case of serum bottle, the pressure exist when gas is collected using
syringe indicates biogas production.
pH
1. Measure pH using portable pH meter.
2. Calibrate pH probe with buffer solution of pH 7 and pH 4 prior to use.
Questions
1. Plot graphs of BOD and COD reduction and biogas production profile
throughout the fermentation process. Discuss the relationship between
organic removal and biogas production according to the results obtained from
the experiment.
2. Discuss how fermentation process you have learnt from this experiment can
be adopted to solve solid waste issue in Malaysia.

Table 1: Solubility of oxygen in water exposed to watersaturated air at atmospheric pressure (101.3kPA)

SBV3023 Issues in Biology and Environment

Chlorinity: 0
Temp
C
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
21.0
22.0
23.0
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
41.0
42.0
43.0
44.0
45.0

5.0

10.0

15.0

20.0

25.0

Salinity: 0

9.0

18.1

27.1

36.1

45.2

14.62
14.22
13.83
13.46
13.11
12.77
12.45
12.14
11.84
11.56
11.29
11.03
10.78
10.54
10.31
10.08
9.87
9.67
9.47
9.28
9.09
8.92
8.74
8.58
8.42
8.26
8.11
7.97
7.83
7.69
7.56
7.43
7.31
7.18
7.07
6.95
6.84
6.73
6.62
6.52
6.41
6.31
6.21
6.12
6.02
5.93

13.73
13.36
13.00
12.66
12.34
12.02
11.73
11.44
11.17
10.91
10.66
10.42
10.18
9.96
9.75
9.54
9.34
9.15
8.97
8.79
8.62
8.46
8.30
8.14
7.99
7.85
7.71
7.58
7.44
7.32
7.19
7.07
6.96
6.84
6.73
6.62
6.52
6.42
6.32
6.22
6.12
6.03
5.93
5.84
5.75
5.67

12.89
12.55
12.22
11.91
11.61
11.32
11.05
10.78
10.53
10.29
10.06
9.84
9.62
9.42
9.22
9.03
8.84
8.67
8.50
8.33
8.17
8.02
7.87
7.73
7.59
7.46
7.33
7.20
7.08
6.96
6.85
6.73
6.62
6.52
6.42
6.31
6.22
6.12
6.03
5.93
5.84
5.75
5.67
5.58
5.50
5.41

12.10
11.78
11.48
11.20
10.92
10.66
10.40
10.16
9.93
9.71
9.49
9.29
9.09
8.90
8.72
8.54
8.37
8.21
8.05
7.90
7.75
7.61
7.47
7.34
7.21
7.08
6.96
6.85
6.73
6.62
6.51
6.41
6.31
6.21
6.11
6.02
5.93
5.84
5.75
5.66
5.58
5.49
5.41
5.33
5.25
5.17

11.36
11.07
10.79
10.53
10.27
10.03
9.80
9.58
9.36
9.16
8.96
8.77
8.59
8.41
8.24
8.08
7.92
7.77
7.62
7.48
7.35
7.21
7.09
6.96
6.84
6.73
6.62
6.51
6.40
6.30
6.20
6.10
6.01
5.91
5.82
5.73
5.65
5.56
5.48
5.40
5.32
5.24
5.17
5.09
5.02
4.94

10.66
10.39
10.14
9.90
9.66
9.44
9.23
9.02
8.83
8.64
8.45
8.28
8.11
7.95
7.79
7.64
7.50
7.36
7.22
7.09
6.96
6.84
6.72
6.61
6.50
6.39
6.29
6.18
6.09
5.99
5.90
5.81
5.72
5.63
5.55
5.46
5.38
5.31
5.23
5.15
5.08
5.01
4.93
4.86
4.79
4.72