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CCHHEEMMIICCAALL  EENNGGIINNEEEERRIINNGG  TTRRAANNSSAACCTTIIOONNSS 

VOL. 38, 2014

A publication of 

The Italian Association 
of Chemical Engineering 

www.aidic.it/cet
Guest Editors: Enrico Bardone, Marco Bravi, Taj Keshavarz
Copyright © 2014, AIDIC Servizi S.r.l., 
ISBN 978-88-95608-29-7; ISSN 2283-9216       

Effect of Starting pH on the Produced Methane from Dairy 
Wastewater in Thermophilic Phase 

Bani.Kheiredine, Kerroum. Derbal, Mossaab. Bencheikh-Lehocine 
 Université Constantine 3, Laboratoire de l’ingénierie des procédés de l’environnement, Constantine,

Algeria
 envi_dz@yahoo.fr 

The experimental study was undertaken in order to evaluate the effects of the initial pH on the anaerobic 
digestion of dairy waste.  The biodegradability tests were carried out in a series of reactor of 400ml of 
volume with four arrangements of the initial pH (pH= 4; 5.5; 7; and 9.5) in thermophilic phase (T = 55 °C). 
The dairy wastewater was the only source of organic carbon. 
After the incubation period (50 days), the result show that the height efficiency of removal COD (about de 
90.8 %) was obtained for initial pH = 7, allowed by Reactor of pH=9.5 79.64 %; pH=5.5 63.75 % and finally 
pH=4 (49.11%). Concerning the produced biogas it volume was: 163ml, 1000ml, 2000ml and 1500ml for 
pH= 4; 5.5; 7 and 9.5 respectively. 
However the produced of CH4 for pH=4 is negligible, for the pH=5.5 and  pH=9.5 is slightly over 50 % and 
75 % respectively compared to the production of the test pH=7  
It can be concluded that dairy waste degradation in anaerobic process can happen optimum on range 
neutral pH. 

1. Introduction
Energy production from the organic matter  by means of anaerobic Digestion processes allow better waste 
management, preservation of the environment, development and diversification of energy resources.. 
Because of the important methane fraction in the produced biogas, between 50 to75 %, different research-
works were undertaken last years with the objective of assessing produced biogas quantities during the 
degradation of a given waste.  
One a very important factor affecting the anaerobic digestion of organic waste is the temperature 
(Angelidaki and Sanders 2004). Generally, anaerobic digestion process is operated under mesophilic or 
thermophilic condition, in which thermophilic digestion is reported to be the more efficient 
 Many researches in this field were presented, and a wide range of waste can be used as substrate for 
anaerobic digestion( Nallathambi 1997); such as: (Angelidaki et al 2009), proposed a protocols for the 
determination of the bio-methane potential of organic solid wastes,( Moller et al 2004) used 
biodegradability test to determine Methane productivity of manure, straw , solid fractions of manure and 
waste of factory of production of olive oil ( Fezzani and Bencheikh  2007), (Neves et al, 2006), used the 
biodegradability test to determine the bio-methane produced from codigestionof coffee waste and sewage 
sludge. 
Other researchers have studied the influence of different parameters on biogas production; Such as the 
influence of the used inoculums activity on the biodegradation of the substrate as well as the substrate to 
inoculums ratio (Chen et al, 1996), Influence of inoculum to substrate ratio on the biochemical methane 
potential of maize in batch tests (Raposo et al, 2006) the influence of the granulometry of waste 
(Palmowski  and Muller 2000). 
In this paper the biodegradability test is used to determine the effects of the initial pH of dairy wastewater 
in thermophilic temperature (55 °C). 

 

 

  DOI: 10.3303/CET1438086 
 

 
 

 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Please cite this article as: Kheireddine B., Derbal K., Bencheikh-Lehocine M., 2014, Effect of starting ph on the produced methane from 
dairy wastewater in thermophilic phase, Chemical Engineering Transactions, 38, 511-516  DOI: 10.3303/CET1438086

511



2. Materials and methods

2.1 Methodology 
Measures of methanogenic potential ((Biochemical Potential methanogenic or BMP) are performed 
following (Vedrenne et al 2005). Concretely, 400 mL of a substrate mixture inoculums and nutrient solution 
are introduced into a 570mL serum bottle.The quantity in substrate is calculated in order to obtain a ratio 
S/I equaling to 1.There are 4 anaerobic batch reactors with pH variation arrangement which are: Reactor 
of pH = 4, pH = 5.5, pH = 7, pH = 9.5 To adjust these pH use added NaOH  and HCL 0.1M.   
All the reactors batch are distributed between witnesses containing the inoculum only (the white), and tests 
which contain the mixture of the inoculum and of the substrate. These tests are doubled and the results 
are averaged over the two experimental measurements. After filling, the flasks are sealed with a rubber 
septum, and their atmosphere is purged with N2. Measuring the volume of biogas produced during the 
times is performed by a column of water (pH = 2). 
During all the period of digestion, the quantity of produced biogas was given the every day. The 
experiment continues until observation of a production of null biogas (50 days). 

2.2 Analytical methods 
Liquid phase characterization was undertaken before and after anaerobic digestion period through the 
determination  of  pH, total solids (TS), total volatile solids (TVS), Alkalinity (TA ) and total alkalinity (TAC ), 
volatile fatty acids (AFG), chemical oxygen demand (COD), ammonia nitrogen (NH4+ ), total Nitrogen 
(NTK) and total phosphorus (Pt) according to Standard Methods ( APHA, et al , 1998 ) pH was determined 
using a pH-meter (Jenway 3510 PH meter) and methane in the biogas was analyzed by a gas 
chromatograph (Arlo Erba strumentazione 4300 (fugueur,120 DFL)  with a flame ionization, equipped with 
stainless steel column (4m long, 3mm outer diameter). The injector, detector and oven temperatures were 
40, 80 and 120 ◦ C, respectively, where a 1mL gas sample was injected into the chromatograph using 
Helium as a gas carrier. 

2.3 Characteristic of the liquid phase before incubation 
The physicochemical main features of the mixture (mud and dairy waste) in each reactors are deferred in  
table .1 

Table 1: Characterization of the liquid phase of different reactors before incubation 

Parametrs  Unit Initial characterization of each bottle 
TA mg CaCO3/L 184 
TAC mg CaCO3/L 689.6 
ST gTS/L 33.5 
SVT gTS/L 20.20 
%SVT % 60.2 
CDO mgO2/L 8888.4 
NH4+ mgN/L 57.7 
NTK mgN/L 121.7 
Pt mgP/L 1.68 

Table 1, above presents the preliminary characterization of substrate (dairy waste and inoculums (sludge) 
mixture in different reactors. shows that it is rich in volatile matter (60.2 %). Which encourages the 
treatment of the latter by anaerobic biological process? 
Similarly, the analysis results of alkalinity, ammonia nitrogen, organic nitrogen and phosphorus, are below 
the values that can inhibited the anaerobic digestion process. 

3. Results and discussion

3.1 Characteristic of the liquid phase after incubation 

pH Reactor  
From Table (2), the pH value obtained after the incubation period in the reactor pH = 4 is between 5.35 
and 5.41, this is an acid pH not favouring the anaerobic digestion. For reactors pH = 5.5, the pH is about 
de7.30 is a pH slightly higher than neutral, it means that there was ascended of pH made the environment 
favourable to digestion. 

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Table 2: characterization of the liquid phase after incubation 

reactors 
parametrs 

unit pH=4 pH=5.5 pH=7 pH=9.5 

pH / 5.38 7.52 7.66 7.70 
TA Mg caco3/l 0,00 4110 5780 7120 
TAC Mg caco3/l 3100 4740 6260 7830 
AFG Mg caco3/l 3100 630 4,800 710 
AFG/TAC / /  0.15 0.09 0.08 
ST g(TS)/L 37.87 30.57 29.13 27.13 
TVS g(TVS)/L 21.06 12.24 11.06 6.84 
TVS% / 55,64 40,04 37.96 25.21 
TKN mg/L 190.66 124.85 118.8 96.74 
NH4+ mg/L 39.82 75.22 9292 61.95 
Pt 
COD 

mg/L 
mg/L 

1.13 
4482.44 

1.32 
3238.05 

115 
817.74 

0.36 
1808.91 

than neutral, it means that there was ascended of pH made the environment favourable to digestion. 
Regarding the reactor pH = 7 and pH = 9.5, pH values obtained after the incubation period in thermophilic 
phase are around 7.75 is a pH slightly above neutral.It is usually considered that the optimum pH range for 
anaerobic digestion is between 6.7 and 7.3. 

Total solids and volatile solids removal efficiency 

-15

-10

-5

0

5

10

15

20

25

pH=4 pH=5,5 pH=7 pH=9,5

Yield of TS (%)

Yield of  TS

-10

0

10

20

30

40

50

60

70

ph=4 ph=5.5 ph=7 ph=9.5

y
ie

ld
 o

f 
 T

V
S

 (
%

)

Figure 1: removal efficiency of TS   Figure 2: removal efficiency of TVS 

The figures represent yields of elimination of TS and TVS. The best performance of volatils solid (TVS) is 
62 % corresponding to the test pH= 9.5. For a pH = 4, we note that the total solids were increased 
compared to those obtained before incubation which gave a negative return. This increase can be justified 
by the conversion of microorganism’s endogenous phase. 

Chemical oxygen demand removal efficiency 
Figure 3 shown that there has been a considerable reduction in COD compared to those obtained prior to 
digestion for pH = 7and pH=9.5 with a yield 90.80 % and 79.64 respectively. 

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0

10

20

30

40

50

60

70

80

90

100

pH=4 pH=5,5 pH=7 pH=9,5

Yield  COD(%)

Figure 3: Removal efficiency concentration of COD 

3.2 Cumulated biogas volume obtained from substrate 
The cumulated biogas produced from organic fraction of dairy wastewater, in thermophilic phase  was 
presented in figure 4 

0

500

1000

1500

2000

2500

0 4 7 11 14 18 21 25 28 32 35 40 45 48

C
u

m
u

la
te

d
 b

io
g

a
z
 v

o
lu

m
e
 (

m
L

)

Time (day)

pH=4 pH=5,5 pH=7 pH=9,5

Figure 4: Cumulated biogas volume of dairy waste (T=55 ° C)   

The cumulated produced biogas from organic fraction of dairy waste, in thermophilic phase (T = 55 ° C) 
was presented in Figure 4. It should be noted that the biogas production of dairy waste was calculated 
after eliminating the inoculum effect. The production is maintained until the 50th day. 
We notice that the biogas production is negligible for test pH = 4, it is important for a test pH = 7 and 
represents twice the production test of pH = 5.5.  
The final values of biogas are 2000 mL, 1500 mL 1000 mL and 162 mL corresponding to the test pH = 7, 
pH = 9.5, pH = 5.5 and pH = 4 respectively.  
This result confirms the results of articles that address the pH of between 6.5 and 8.5 startup that gives the 
best efficiency degradation of organic matter. (Vedrenne et al 2005). 

3.3 Cumulated methane and carbon dioxide obtained from dairy waste 
The variations in cumulative volume of CH4 in Figure 5 for test pH = 5.5 and pH = 9.5 are small compared 
to the test pH = 7. Thus the total production of methane in the test pH = 5.5 represents half of the total 
production of methane compared to test pH = 7 and 2/3 compared to the test pH = 9.5 with respect to the 

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pH test = 4 the total méthane production is negligible. In the same way for the produced volume of carbon 
dioxide, see Figure 6.

0

200

400

600

800

1000

1200

1400

0 3 7 10 14 17 21 24 28 31 36 39 45 49

C
u
m

u
la

te
d
 M

e
th

a
n
e
 v

o
lu

m
e
 (

m
L
)

Time (Day)

Ph=4 pH=5.5 Ph=7 pH=9.5

0

100

200

300

400

500

600

700

800

900

0   3   7   10   14   17   21   24   28   31   36   39   45   49   
C

u
m

u
la

te
d
 C

a
rb

o
n
e
 D

io
x
id

e
  

v
o
lu

m
e
 (

m
L
)

Time (day)

pH=4 pH=5.5 pH=7 pH=9.5

Figure 5: Cumulated CH4 volume (T=55 ° C) Figure 6: Cumulated CO2 volume (T=55 ° C)

3.4 Biogas Composition  
Figure 7, represents the average composition of biogas expressed as a percentage of methane and 
carbon dioxide.  

0
10
20
30
40
50
60

pH=4 pH=5,5 pH=7 PH=9,5B
io

ga
z 

co
m

po
si

tio
n 

(%
)

% CH4 % CO2

Figure 7:  Biogas composition,         

The percentages of methane in reactors tested are 58.3%, 54.5%, 50.7% and 45.4%. Corresponding to 
the test pH =7, pH = 9.5, pH = 5.5 and pH = 4 respectively.   

4. Conclusion
Based on this research during 58 days, the maximum methane production is negligible for, pH=4 (50ml) 
and (500ml) for pH = 5.5. It is important for pH = 7 (1200ml) and pH 9.5 (800ml). 
Qualitatively, the percentages of methane in four reactors tested are 58.3 %, 54.5 %, 50.7 % and 45.4 %. 
Corresponding to the test pH =7, pH = 9.5, pH = 5.5 and pH = 4 respectively. 
It can be concluded that dairy waste degradation in anaerobic process can happen optimum on range 
neutral pH. 

515



References 

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Environ Sci Biotechnol 2004;3:117–29. 

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APHA, AWWA, WPCF, 1998, Standard methods for the examination of water and 
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Chen T., Hashimoto, A. G, 1996, Effects of pH and substrate:Inoculum ratio on batch methane. 
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Fezzani, B., Ben Cheikh, R., 2007, Thermophilic anaerobic co-digestion of olive mill wastewater with olive 
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Moller HB, Sommer SG, Ahring B, 2004,Methane productivity of manure, straw and solid fractions of 
manure Biomass Bioenergy;26:485–95. 

Nallathambi Gunaseelan V. 1997 Anaerobic digestion of biomass for methane production: a review. 
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Neves, L., Oliveira, R., Alves, M. M. 2004, Influence of inoculum activity on the bio-methanization of a 
kitchen waste under different waste/inoculum ratios. Process Biochem. 39(12), 2019–2024. 

Palmowski, L. M., Muller, J. A. 2000, Influence of the size reduction of organic waste on their anaerobic  
digestion. Water Sci. Technol. 47(12), 231–238. 

Raposo, F., Banks, C. J., Siegert, I., Heaven, S., & Borja, R. 2006, Influence of inoculum to substrate ratio  
on the biochemical methane potential of maize in batch tests. Process Biochemistry, 41(6), 1444-1450.  

Vedrenne, F, Béline F, Bernet N.2005, Evaluation of the methane production of livestock wastes: Ultimate   
productivity and organic matter characterization, 4th international symposium on anaerobic digestion of 
solid waste, Copenhague, 218-225. 

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