Biochemical Methane Potential Test to the Biosludge Pretreated by

Ozonation and Sonication

Desiana and T. Setiadi
Department of Chemical Engineering, Faculty of Industrial Technology,
Institut Teknologi Bandung
Jl. Ganesa 10, Bandung 40132, Indonesia
Fax: +62-22-2501438, Email: tjandra@che.itb.ac.id
Anaerobic digestion is a basic technique in reducing bio-sludge. In anaerobic digestions
of solid feed, the hydrolysis step was reported as a limiting rate because it can reduce the
production of biogas. Bio-sludge pretreatment was developed to increase biogas production
by accelerating the hydrolysis step. Ozonation and sonication are pretreatment methods that
relatively simple in breaking biomass cell walls. A study on BMP (biochemical methane
potential) and ATA (anaerobic toxicity assay) on textile-mill bio-sludge were carried out in
this research. The effect of pretreatment to its biodegradability and toxicity were also studied.
The experiments showed that the fresh biosludge, ozonated and sonicated biosludge
have no inhibition effect to the production rate of gas at low concentration (around 4%). But it
has been a significant inhibition at higher concentration. The measurement of methane
potential production that was developed by Owen, et al. (1979) was not accurate enough to be
applied on biosludges. The extended ATA measurements gave a more accurate result
especially on low concentration (around 4%) or on COD level around 1.500 mg/L. Based on
the extended ATA measurement, the biochemical methane production of textile biosludge was
relatively high as biodegradability valued around 62%.
The pretreatment of ozonation and sonication could break cells on the bio-sludge that
was identified by the increase in soluble COD values, TVFA (total volatile fatty acids) and the
decrease in total volatile solid (TVS). These pretreatments also decrease the toxicity on low
concentration (measured concentration around 4%). Otherwise at a higher concentration (1040%), the toxicity was not decrease. The COD degradation of bio-sludge was satisfactorily
increased by both of ozonation and sonication. The use of ozone doze 0.005 g O3/g COD and
0.01 O3/g COD could raise the biodegradability from 62% to 69% and 76% respectively.
While for sonication on frequency 51 kHz 6% 120 watt for 30 minutes and 60 minutes could
increase the biodegradability from 62% to 68 and 73% respectively
Keywords : Anaerobic toxicity assay, biochemical methane potential, ozonation, sonication,
textile mill biosludge.
Introduction
It has been well known that the domestic and industrial wastewater treatment systems
will usually produce a large amount of biosludge, namely it is about 20-40 kg of dry sludge
for every population-served per annum (Kroiss, 2004)). This amount presents an
environmental issue for itself. The conventional methods of sludge treatment, such as landfill,
incineration, and composting are still having some limitations. In the developing countries,
including Indonesia, the primary treatment of biosludge is used landfill systems. This system
in large scale creates environmental problems, especially due to (1) gas production from
fermentations and (2) surface water and ground water pollution from the leachate (Heredia
and Garcia, 2005)
In developed countries, direct landfilling of biosludges has been forbidden and stopped.
An alternative for treating biosludge that is currently intensively-studied is the anaerobic
digestion. The anaerobic digestion has many advantages compare to other methods, namely

(1) a relatively low cost, (2) consumes little energy and nutrition, and (3) anaerobic bacteria
ability to transform almost all organic substances into methane (Heredia and Garcia, 2005).
Goel, et al. (2003) states that the anaerobic process is the most fundamental method to reduce
the amount of biosludge in solid waste treatment. Various researches on anaerobic process is
currently focused on increasing biosludge reduction through pretreatment (Dohanyos et al.,
2000; Goel et al., 2003; Heredia and Gracia, 2005; Bougrier et al., 2006).
Anaerobic process efficiency tests are usually done based on the ratio of BOD and COD
values. This method is less effective because it does not illustrate an anaerobic process. Owen,
et al. (1979) succeeded in developing a simple and affordable methane formation potential
test or BMP (biochemical methane potential) and anaerobic toxicity assay (ATA).
In anaerobic digestions of solid feed such as biosludges, the hydrolysis step was
reported as a limiting rate because it can reduce the production of biogas (Bougrier et al.,
2006). Bio-sludge pretreatment was developed to increase biogas production by accelerating
the hydrolysis step. Ozonation and sonication are pretreatment methods that relatively simple
in breaking biomass cell walls. A study on BMP (biochemical methane potential) and ATA
(anaerobic toxicity assay) on textile-mill bio-sludges were carried out in this study. The effect
of pretreatment, i.e ozonation and sonication, to its biodegradability and toxicity were also
studied
Material and Methods
Biosludge and sample characterization
The fresh biosludges were collected from the biological wastewater system treating
textile mill effluent located in the east of Bandung, Indonesia. The fresh biosludge and
pretreated biosludge was characterized based on COD (chemical oxygen demand), BOD
(biochemical oxygen demand), TKN (total kjedahl nitrogen), TVFA (total volatile fatty acid),
TVS (total volatile solid), and pH. The parameters were measured in accord with the Standard
Method (1989).
The ozonated biosludge was treated by a lab-scale ozonator (BL-2000 series) for 30 and
60 minutes with the ozone dose of 0.005 and 0.01 g O3/g COD, respectively. While the
sonicated biosludge was exposed by a Branson 5200 sonicator on frequency 51 kHz 6%, 120
watt for 30 and 60 minutes.
Inoculum Preparation and Acclimatization
Cow dung was collected from a farm and screened through a sieve to remove coarse
material. Then 1 litre of the sludge was transfered to a 2 litre flask containing 100 ml of
synthetic waste (Chen et al., 1985) and purge with N2. The flask was placed in a 35 oC water
bath. Synthetic wastewater (100 ml) was added to the flask each day to finally obtain 2 litres
in the flask. After the 2 litre volume was obtained, 100 ml supernatant was removed each day
from the flask and replaced with 100 ml synthetic wastewter to maintain a constant volume.
This practice was continued for about 1 months, then the the textile biosludge replaced the
synthetic wastewater. The acclimatization with the biosludge was conducted for about 1
month.
ATA and BMP Test
Anaerobic toxicity assay (ATA) dan biochemical methane potential (BMP) tests have
been developed by Owen et al. (1979). The tests were carried out in parallel using test-bottles
in batch sequence operations. The 160 ml test bottles were used, to which were inserted 30 ml
media solution (Owen et al., 1979), 30 ml acclimatized seed and biosludge samples (4, 10, 20,
40 ml), and filled with aquadest to a working volume of 100 ml. In the ATA test, 2 ml of

acetic-propionic acid solution containing 75 mg acetic acid and 26.5 mg propionic acid is
added. Then, the nitrogen gas was sparged into the bottle containing the sample, media
solution, and seed for 3 minutes, closed tightly, and followed by incubation at 35oC in a water
bath.
Gas accumulation produced during the incubation was collected using a syringe. The
syringe piston was pushed up due to the increase of pressure inside the bottle when the gas
was produced. Then, the volume gas was measured and the gas composition was analysed by
the Gas Chromatography (GC). The diagram of BMP and ATA test was presented in Figure 1.
In the ATA test, measurements of gas production were done every day for the first 5
days, then the gas production rate (k, as ml/hour) was measured for each sample and also for
the control (without biosludge). The toxicity percentage was calculated according to equation
1.
ke kc
Toxicity (%) =
(1)
x 100%
kc
where ke = gas production rate of biosludge sample (ml/h), kc = gas production rate of control
(ml/h)

Figure 1. BMP/ATA test diagram

In the BMP test, incubation was carried out up to 60 days with the gas collection was
done every 5 days. The COD to methane conversion is calculated using equation 2.

g Vgs Vgk %CH 4

COD consumption
Vs 395
l

(2)

where, Vgs = gas volume accumulation produced by biosludge sample (ml), Vgk = gas volume accumulation
produced by control (mL), Vs = sample volume added to the test bottle (l), % CH4 = CH4 composition in the gas
(analyzed by the GC). The 395 value was the volume of CH4 produced for every g of COD consumed at 35oC, 1
atm.

Equation 3 calculated COD to methane conversion efficiency.

g
COD consumed l
x 100%
Converted COD efficiency =
Total COD g

(3)

Results and Discussion

Biosludge Characterization
Biosludge characterization is given in table 1. The table shows that the fresh biosludge
has a relatively high content of COD, BOD, and TKN with the average value of 22,215; 4,945
and 887.3 mg/l, respectively. The BOD/COD ratio was 0.17 showing that it has a low
biodegradability (Henze et al., 1995).
Table 1. Biosludge characterization
Parameter

Biosludge sample
P1
P2
P3

COD
25,215
21,823
18,858
(mg/L)*
BOD
4,945
3,449
2,836
(mg/L)**
TKN
887.3
803.2
765.9
(mg/L)**
TVFA
14.7
22.1
1,573.5
(mg/L)**
*
14.98
15.32
13.60
TS (g/L)
TVS*
77%
73%
73%
(% of TS)
pH*
8.7
8.6
8.5
(average)
*average of 12 data:; **average of 4 data
P1 = fresh biosludge; P2 = 30 min. ozonated biosludge; P3 = 60 min. sonicated biosludge

The fresh biosludge contained a high percentage of liquid, around 98.38% with volatile
solids (TVS) amounting to 77% of total solids (TS). This shows that the non-volatile material
fraction in biosludge is relatively low, around 33% of total solids. The high level of liquids
and organic (volatile) fractions in biosludge would be amenable to the anaerobic treatment
(Juanga, 2005), since the hydrolysis stage that usually limits the anaerobic process will not
inhibite the process in the early stage due to the high content of liquids.
The table shows that the ozonated biosludge has a relatively lower value of COD and
BOD than that of fresh biosludge. However, the sonicated one has a rather significant
decrease of COD and BOD, and increase of TVA than that of fresh biosludge.
Biosludge toxicity level
Biosludge toxicity percentage after 5 days incubation is given in figure 2. The
percentage obtained was generally lower than that of control, which shows a presence of
toxics in the biosludge that disturbed methanogenic bacteria activities.
The toxicity level was calculated in the first 5 days of incubation based on the ratio of
sample and control gas production. For the 4% concentration test, P2 biosludge has a higher

gas production, P3 slightly lower and P1 lower than that of control. The values were 38%, 4%, and -35% respectively. This shows that P2 biosludge was non-toxic, P3 was slightly
inhibiting, and P1 was significantly inhibit to the methanogenic bacteria. However, in all the
higher concentration of biosludge, the inhibition was occured.

The first, second, third and fourth series: 4, 10, 20 and 40 % of biosludge sample

Figure 2. Toxicity level on day 5

Biosludge methane production potential
The methane production potential was conducted in two method tests, i.e. BMP and
extented ATA. The ATA was usually done only until the fifth day, however in this study the
test was extended till the 60th day. Thus, both experiments were carried out for 60 days. There
were six samples used in 10 % v/v biosludge concentration test. The first two runs were
without any pretreatment, and the third and fourth runs were pretreated by 30 and 60 minutes
ozonation, respectively. Then, the last two runs were pretreated by 30 and 60 minutes
sonication, respectively.
The percentage of converted COD based on BMP and extended ATA test is given in
figure 3. There was a significant biodegradability difference between extended ATA and BMP
tests. At 10%v/v concentration test, the ATA results gave an increase in biodegradability as
pretreatment time increases, while BMP exhibited an opposite result. The control
biodegradability test (0 minutes in the figure 3) showed an interesting value, namely at a value
of 30 % for ATA and at a value of 10.58 % for BMP. This distinction value showed a
condition in the system that inhibit gas production in the BMP tests, on the other hands there
was no inhibition in gas production at the ATA tests. These significant ATA and BMP test
results also show that the BMP tests were not appropiate to determine methane formation
potential for biosludge samples.

% COD degradation

10%
45
35

S BMP
O BMP

25

S ATA
O ATA

15
5
0

20

40
Time (min)

60

80

Figure 3. The COD converion (%) in extended ATA and BMP tests at 10% concentration test

pH

The difference between these two tests was caused by the addition of 2 ml of aceticpropionic acid containing 75 mg acetate and 26.5 mg propionate in the ATA test, and none in
the BMP test. These two chemicals are nutrition easily consumed by methanogenetic bacterias,
and also known as the most important precursor for methane formation (Speece, et al.,1986)
stated in their study that acetic acid as a methanogenesis precursor contribute in increasing
methane production. After 60 days of incubation, the system pH data show that BMP tests pH
is relatively higher than that of ATA tests. The comparison is given in figure 4.
8
7.9
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7
6.9

P1 ATA
P1 BMP
P2 ATA
P2 BMP
P3 ATA
P3 BMP

10%

20%

Concentration Test (v/v)

Figure 4. Comparison of pH values after ATA and BMP tests (60 days incubation)
An anaerobic system is microbe cultures consisting of 3 interconnected groups of
bacteria that convert the product of hydrolysis into acetate and hydrogen or formate; acetate
into methane; and hydrogen or formate into methane. An initial condition already containing
acetic-propionic acids (at the ATA test) facilitated methanogenic bacteria activities in forming
methane, while a hydrolysis stage being aided by ozonation or sonication pretreatment
accelerated acetogenic bacteria activities in forming acids. There was a natural buffer activity
system between methanogenic bacteria used acids and acetogenic bacteria produced acids
rapidly, so that a pH equillibrium was obtained. This condition was advantageous for
methanogenic bacterias work that consume acids but does not favor an acidic environment.
Acid accumulation during early incubation does not happen due to the presence of this buffer
system, resulting in a condusive environment for the methanogenic growth in the early stages
of the anaerobic process. This phenomenon promoted the gas accumulation in the ATA tests.
In early incubation of BMP tests, although there was yet enough nutrition for
methanogenic bacterias, the acetogenic bacterias produced acids rapidly causing a decrease in
the pH system. This might cause an inhibition to methanogenic bacterias growth in early
incubation, causing the failure of the natural buffer system. The high total nitrogen content of
biosludge also contributes to the inhibition of gas production in BMP tests. Biosludge
consisted of live and dead cells. Ericson, et al. (2004) states that chemicals originating from
dead cells have a large potential for methanogenic processes, but in reality this condition was
hindered by protein deamination that prohibit methanogenic bacteria growth.
Biochemical methane production potential results based on the extended ATA tests are
given in table 2. Methane production potential decreased with increasing biosludge
concentrations. The highest biochemical methane potential was given by the lowest
concentration (4%) or COD loads ranging from 1,424-1,040 mg/L. It was from 62 to 73%. At
10% concentration (COD load of 2,600-3,560 mg/L); 20% concentration with COD load of
5,200-7,120 mg/L; and 40% concentration with COD load of 10,400-14,240 mg/L, the
biochemical methane potentials range from 30-48%; 7-26%; and 4-8% respectively.

This finding suggested that the test developed by Owen et al. (1979) should be applied
carefully for the biosludge test. The biochemical methane production potential test for the
biosludge is proposed to use the extended ATA with the concentration of the biosludge
sample should be maintained at the lower level, namely for the current sample, it should be
not more than 4 % or at a COD load of 1,500 mg/L.
Effect of Pretreatment towards Biochemical Methane Formation Potential
Biochemical methane formation potential from pretreated biosludge based on extended
ATA tests up to 60 days at 4% test concentration is given in table 3. Biochemical methane
formation potential (as shown in the percentage of COD converted) increased after
pretreatment was applied, either for ozonation or sonication. Percentage of converted COD by
ozonation increased from 62% to 69-76%, while biosludge for sonication pretreatment had a
COD conversion percentage increased from 62% to 68-73%. Pretreatments also increased
specific gas production from 246 L CH4/kg COD in biosludge without pretreatment to 271302 L CH4/kg COD with ozonation and 270-287 L CH4/kg COD after sonication.
Table 2. Biochemical methane formation potential based on extended ATA tests
Gas
% COD
Vol.*)
Conversion
(ml)
4
106
62
P1
10
117
30
30
60.05
20
111
14
40
104
6
4
103
69
P2
10
110
31
26
60.58
20
144
26
40
111
8
4
108
73
10
141
48
P3
26.8
60.37
20
80
7
40
83
4
*)difference between sample and control gas volume
P1 = fresh biosludge, P2 = 30 min. ozonated biosludge, P3 = 60 min. sonicated biosludge
Sample

Total
COD
(g/L)

%
CH4

Sample
(% v/v)

Table 3. Biochemical methane formation potential from pretreated biosludge

Specific Gas
Production
(L CH4/kg L CH4/kg
COD)
TS
246
492

Sample

Sample
Volume
(ml)

Gas
Volume*
(ml)

%
CH4**

Consumed
COD
g/L

Total
COD
g/L

%
Converted
COD

49

60

18.67

30

62

47

61

17.83

26

69

271

460

59

62

22.95

30

76

302

607

61

63

24.35

35,6

68

270

730

51

60

19.46

26,8

73

287

575

*sample gas volume after subtracted by control gas volume

** CH4 composition in gas using GC measurement on day 40th
Note:
A = fresh biosludge ; B = ozonated biosludge 0,005 g O3/g COD
C = ozonated biosludge 0,01 g O3/g COD
D = sonicated biosludge on 51kH6% frequency, 120 watts for 30 minutes
E = sonicated biosludge on 51kH6% frequency, 120 watts for 60 minutes

The increase of biodegradability with increasing ozone dosage was in accordance with
the finding of Goel, et al. (2003) and Bougrier et al. (2006). They found that CH4 formation
increased with increasing ozone dosage. Shceminski, et al. (2000) explained that the
biodegradibility increase was due to the ozone gas could disintegrate solid organic matter. At
the initial stages, ozone gas oxidizes dissolved organic substances, and with increased
concentration, the ozone broke the polimer bonds being the building blocks for the cell wall.
The cell would then be disintegrated, so that the entire cell contents came out. Enzymes and
cofactors contained within the cell helps to increase the biodegradability and anaerobic
bacteria activities.
On the sonication pretreatment study, Neis, et al. (2000) stated that domestic sludge
treatment with sonication caused cell disintegration, so that intracellular substances leak and
increases COD in the solution. If continued with anaerobic fermentation, the biogas amount
would rise significantly compared to control. This finding was also agree well with the result
of Bougrier et al. (2006).
Therefore, pretreatment to the biosludge, either ozonation or sonication, could increase
biochemical methane formation potential meanig a reduction in the biosludge residue. The
choice of the best pretreatment still needs to be reviewed based on the cost efectivity and
availability of operating energy for the equipment. This research is carried out in early stages,
therefore the results obtained from this study can only predict the biochemical methane
formation potential that can be achieved in anaerobic processes. Thus, better operating
conditions in pilot scale still need to be explored and further tests in larger scales are
necessary to achieve industrial applications.
Conclusions
The following conclusions were inferred based on the results of the study:
1. The textile-mill biosludge had a high liquid content (98%) with volatile solids comprising
77% of total solids.
2. Based on BOD/COD ratio value, the textile biosludge was considered to have a low
biodegradability (a ratio of 0.17)
3. Methane formation potential calculation by BMP tests developed by Owen et al. (1979),
was not accurate enough to be applied on biosludge. The extended ATA tests gave a more
accurate results, especially at a low test concentration (4%) or a COD concentration of
1,500 mg/L
4. Biochemical methane formation potential of the biosludge based on the extended ATA
tests have a relatively high biodegradability value of 62% .
5. The COD degradation of biosludge was satisfactorily increased by both of ozonation and
sonication pretreatment.
6. The use of ozone doze 0.005 g O3/g COD and 0.01 O3/g COD could raise the
biodegradability from 62% to 69% and 76% respectively. While for sonication on
frequency 51 kHz 6% 120 watt for 30 minutes and 60 minutes could increase the
biodegradability from 62% to 68 and 73% respectively.
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