ARTICLE IN PRESS

WAT E R R E S E A R C H 41 (2007) 683 – 689

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Hydrolysis and acidification of waste activated sludge at

different pHs

Yinguang Chen, Su Jiang, Hongying Yuan, Qi Zhou, Guowei Gu

State key laboratory of pollution control and Resources reuse, School of Environmental Science and Engineering, Tongji University,
1239 Siping Road, Shanghai 200092, PR China

art i cle info A B S T R A C T

Article history: The effect of pH from 4.0 to 11.0 on the hydrolysis and acidification of waste activated
Received 28 July 2006 sludge (WAS) was investigated. Experimental results showed that at room temperature the
Accepted 29 July 2006 sludge hydrolysis was in the following order: alkaline4acidic4(neutral and blank test), and
Available online 20 September 2006 between pH 6.0 and 11.0 the sludge hydrolysis increased with pH. The three main
Keywords: components, soluble protein, carbohydrate and volatile fatty acids (VFAs) in the hydrolytic
Waste activated sludge (WAS) product were analyzed. It was observed that both the soluble protein and carbohydrate
pH increased with pH in the pH range 7.0–11.0, but also increased to a smaller extent with pH
Volatile fatty acids (VFAS) from 7.0 to 4.0. The VFAs concentration was also affected by pH. Under alkaline conditions,
Protein the VFAs production was significantly higher than under other conditions. The concentra-
Carbohydrate tion of VFAs on the 8th day of fermentation at pH 4.0, 7.0 and 10.0 was, respectively, 354.49,
Hydrolysis 842.00 and 2708.02 mg/L, while VFAs in the blank test was only 633.59 mg/L. The VFAs
Acidification consisted of acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids, but
acetic, propionic and iso-valeric were the three main products. Also, the release of soluble
phosphorus and ammonia and the production of methane was studied during WAS
fermentation at different pHs.
& 2006 Elsevier Ltd. All rights reserved.

1. Introduction VFAs, in BNR facility (Moser-Engeler et al., 1998; Pitman et al.,

Soluble organic compounds are required for biological
nutrient removal (BNR) in wastewater treatment plants.
Denitrification needs readily biodegradable COD as the carbon
source, and volatile fatty acids (VFAs) are required by
phosphorous accumulating organisms (PAO) for enhanced
biological phosphorous removal (Abu-ghararah and Randall,
1991; Chen et al., 2004; Thomas et al., 2003). However, soluble
COD (including VFAs) in the influent is not sufficient for both
denitrification and biological phosphorus removal in some
cases, such as in the South China. In order to avoid costly
external carbon source addition, fermentation of primary
sludge or its mixture with waste activated sludge (WAS) has
been used as a means of increasing soluble COD, especially
1992; Skalsky and Daigger, 1995; Thomas et al., 2003).
It is believed that the initial hydrolysis of particulate
organic matter to soluble substances is the rate-limiting step
of anaerobic digestion (Eastman and Ferguson, 1981). If the
particulate organic matters contained in activated sludge
were not properly solubilized, it had been reported that only
30–50% of the total COD (TCOD) or volatile solids (VS) in WAS
were biodegraded in 30 days (Parkin and Owen, 1986). Thus,
several efforts have been made to enhance the efficiency of
anaerobic digestion by improving the rate of sludge hydro-
lysis, such as thermal (Hiraoka et al., 1985; Li and Noike, 1992),
thermo-chemical (Sakai et al., 1999; Vlyssides and Karlis,
2004), mechanical (Hwang et al., 1997), ultrasonic (Chiu et al.,
1997; Knapp and Howell, 1978) and microbiological methods
(Tiehm et al., 1997).

Corresponding author. Tel.: +86 21 65981263; fax: +86 21 65986313.

E-mail address: yg2chen@yahoo.com (Y. Chen).
0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2006.07.030
 ARTICLE IN PRESS
684 WA T E R R E S E A R C H
Although it has been observed in the literature that sludge
solubilization can be significantly increased by alkaline
treatment (Lin et al., 1998; Vlyssides and Karlis, 2004), the
effects of pH on VFAs production and phosphorus release
during sludge fermentation under alkaline conditions have
seldom been investigated. Some researchers reported that
the VFAs formation during sludge acidification was affected
by pH, but the information was obtained in the case of
pH less than 7.0 and used primary sludge or its mixture with
WAS as the fermentation substrate (Elefsiniotis and Oldham,
1994; Yu et al., 2003). pH was an important parameter
observed in our investigation which influenced the hydrolysis
and acidification of single WAS in the range of pH 4.0–11.0,
but it has not been explored in the literature. The purpose of
this work was therefore to investigate the effects of pH on
WAS hydrolysis, protein and carbohydrate solubilization,
nitrogen and phosphorus release, and methane and VFAs
production.
2. Materials and methods
2.1. Source of WAS and operation
The WAS used in this study was obtained from the secondary
sedimentation tank of a municipal wastewater treatment
plant in Shanghai, China. The sludge was concentrated by
settling at 4 1C for 24 h, and its characteristics are shown in
Table 1. As seen in Table 1, the protein and carbohydrate are
the two predominant organic compounds of WAS, which
account for approximately 90% of the volatile suspended
solids.
Nine identical reactors, with working volume of 1.5 L
each were maintained at room temperature (2171 1C).
13.5 L of WAS were divided equally into 9 reactors. Each
reactor was mechanically stirred at 80 rpm (rotations per
minute). The pH value in reactor 1–8 was controlled at 4.0, 5.0,
6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, respectively, by adding 2 M
sodium hydroxide (NaOH) or 2 M hydrochloric (HCl). The
reactor 9, in which the pH was not adjusted, was set as the
blank test.
Table 1 – Characterization of the WAS after settlement
Parameter Valuea
pH 6.8
TSS (total suspended solids) 13808
VSS (volatile suspended solids) 10815
SCOD (soluble chemical oxygen demand) 41
TCOD (total chemical oxygen demand) 13407
BOD5 5417
Total carbohydrate (as COD) 1522
Total protein (as COD) 8180
Lipid and oil (as COD) 131
a
All values are expressed in mg/L except pH.
41 (2007) 683– 689
2.2. Analysis
Sludge samples from the reactors were immediately filtered
through a Whatmann GF/C glass microfiber filter (1.2 mm pore
size). The filtrate was immediately analyzed for VFAs, SCOD,
PO3 +
4 -P, NH4 -N, carbohydrate and protein, and the filter
was assayed for TSS and VSS. The analyses of SCOD, PO3 4 -P,
NH+4 -N, TSS and VSS were conducted in accordance with
Standard Methods (APHA, 1998). Carbohydrate was measured
by the phenol-sulfuric method with glucose as standard
(Herbert et al., 1971). Soluble protein was determined by the
Lowry–Folin method with BSA as standard (Lowry et al., 1951).
Sludge lipid was extracted by the Bligh–Dyer method (Bligh
and Dyer, 1959) from the acidified sample, and was then
measured gravimetrically after the solvent was evaporated at
80 1C (APHA, 1998). The total protein content of sludge was
calculated from the corresponding TKN concentration by
subtracting the inorganic nitrogen concentration and dividing
the difference by 0.16, and then multiplying the result by 1.5
(Miron et al., 2000).
To analyze VFAs, the filtrate was collected in a 1.5 mL gas
chromatography (GC) vial, and 3% H3PO4 was added to adjust
the pH to approximately 4.0. An HP5890 GC with flame
ionization detector and equipped with a 30 m  0.32 mm
 0.25 mm CPWAX52CB column was utilized to analyze the
composition of VFAs. Nitrogen was the carrier gas and the
flux was 50 mL/min. The injection port and the detector was
maintained at 200 and 220 1C, respectively. The oven of GC
was programmed to begin at 110 1C and to remain there 2 min,
then to increase at a rate of 10 1C/min to 200 1C, and to hold at
200 1C for an additional 2 min. The sample injection volume
was 1.0 mL.
Methane was measured by a GC (14B, Shimadzu, Japan)
equipped with a thermal conductivity detector (TCD) and a
3-m stainless column packed with Porapak Q (80/10 mesh).
The temperature of the injection, column, and detector was
set at 40, 50 and 90 1C, respectively. Nitrogen was used as the
carrier gas at a flow rate of 30 mL/min.
3. Results and discussion
3.1. WAS hydrolysis
Sludge hydrolysis can be expressed by the changes of SCOD
concentrations (Andreasen et al., 1997; Hatziconstantinou
et al., 1996). Fig. 1 shows the effect of pH on SCOD at different
fermentation times. It was obvious that the SCOD concentra-
tion in each reactor increased gradually with time, which
indicated that more and more particulate organics in WAS
became soluble substrates. However, the amount of SCOD
generated at different pH values was quite different. The
reason was that the produced soluble protein and carbohy-
drate were different at different pHs, which will be studied in
the following text. The SCOD at alkaline pH (pH ¼ 9.0, 10.0
and 11.0) was significantly higher than those at near neutral
pH (pH ¼ 6.0, 7.0 and 8.0) or acidic pH (pH ¼ 4.0 and 5.0) or in
the blank test. On the 8th day of fermentation, SCODs were
7939 mg/L at pH 11.0, 1870 mg/L at pH 4.0, 1685 mg/L at pH 7.0,
and 1242 mg/L in the blank test. As the initial WAS concen-
 ARTICLE IN PRESS
WAT E R R E S E A R C H 41 (20 07) 68 3 – 689 685

10000
4.0

5.0
8000
6.0

SCOD (mg/L)
6000 7.0

8.0
4000 9.0

10.0
2000
11.0

blank test
0
4 8 12 16 20
Time (d)

Fig. 1 – SCOD concentration at different pHs.

1800 4.0

5.0
Protein concentration (mg/L)

1500
6.0
1200
7.0

900 8.0

9.0
600
10.0
300 11.0

0 blank test
2 6 10 14
Time (d)

Fig. 2 – Effect of pH on soluble protein.

tration, i.e. TCOD was identical in all batch reactors, the ratio
of SCOD/TCOD at alkaline pH was therefore much higher
than other pH conditions. After 20 days, the SCOD/TCOD at
pH 11.0 was 68.3%, while it was only 22.1% at pH 4.0, 13.2% at
pH 7.0, and 13.8% in the blank test. At any other fermentation
time, the same observation could be made (see Fig. 1).
Apparently, the WAS hydrolysis rate was accelerated under
alkaline conditions, which has also been observed by other
researchers (Vlyssides and Karlis, 2004).
3.2. Protein and carbohydrate solubilization
It has been reported that protein, carbohydrate and lipid are
the main constituents of domestic sludge (Tanaka et al.,
1997). In this study, the WAS consisted of 61% protein, 11%
carbohydrate, less than 1% lipid, and over 27% unknown
components on the basis of sludge TCOD. Thus, protein was
the largest constituent of the WAS, and the lipid content
could be neglected in this study. The SCOD composition was
therefore further analyzed by focusing on soluble protein and
carbohydrate.
As shown in Figs. 2 and 3, pH had almost the same effect on
soluble protein and carbohydrate concentrations as on SCOD,
i.e. concentrations of these two substrates under alkaline
conditions were greater than other conditions. It is well
known that both protein and carbohydrate are the main
compositions of extracellular polymeric substances (EPS) of
sludge (Liu and Fang, 2002). The alkaline pH resulted in the
dissociation of acidic groups in EPS and repulsions between
the negatively charged EPS, and the solubility of protein and
carbohydrate in water therefore increased (Wingender et al.,
1999). Protein and carbohydrate concentrations on the
second day were 312.27 and 55.93 mg/L at pH 4.0, 112.25 and
30.11 mg/L at pH 7.0, and 938.33 and 119.29 mg/L at pH 11.0,
respectively. However, instead of increasing gradually as
SCOD during fermentation, their concentrations, especially
the carbohydrate, fluctuated with the increase of time. The
reason was that soluble protein and carbohydrate were the
result of a net balance between competing rates of release
and degradation. When the degradation rate exceeded the
release, the concentration was observed to decrease.
3.3. PO3 +
4 -P and NH4-N release
Fermentation of WAS resulted in significant increases of
soluble phosphorus (PO3 +
4 -P) (Fig. 4) and NH4 -N (Fig. 5). The
levels of P or N in the mixed liquor appeared to depend on
sludge retention time and pH values. As seen in Figs. 4 and 5,
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686 WA T E R R E S E A R C H 41 (2007) 683– 689

400
4.0

Carbohydrate concentration (mg/L)

5.0
300 6.0

7.0

200 8.0

9.0

100 10.0

11.0

0 blank test
2 6 10 14
Time (d)

Fig. 3 – Effect of pH on soluble carbohydrate.

300
4.0

250 5.0

6.0
200
PO43--P (mg/L)

7.0

150 8.0

9.0
100
10.0
50 11.0

0 blank test
0 4 8 12 16 20 24
Time (d)

Fig. 4 – Variations of PO3

4 -P concentration during fermentation at different pHs.

400
4.0
350
5.0
300
6.0
NH4+-N (mg/L)

250 7.0
200 8.0
150 9.0

100 10.0

50 11.0
blank test
0
0 4 8 12 16 20 24
Time (d)

Fig. 5 – Variations of NH+4-N concentration during fermentation at different pHs.

concentrations of P and N in either pH controlled (pH 4.0–11.0) trations were different from that on SCOD, protein and
experiments or blank test all kept increasing during fermen- carbohydrate.
tation. Nevertheless, the concentrations of PO3 +
4 -P and NH4 -N One possible reason for very low NH+4 -N produced at
were the highest at acidic pH (pH 4.0 and 5.0). Apparently, pH 11.0 was due to the toxicity of stronger alkaline conditions
the influence of pH on observed PO3 +
4 -P and NH4 -N concen- decreasing the activities of sludge hydrolytic enzymes,
 ARTICLE IN PRESS
WAT E R R E S E A R C H 41 (20 07) 68 3 – 689 687

such as protease, peptidase, etc. Also, as shown in Figs. 4
and 5 the observed NH+4 -N release was greater than PO3 4 -P
in most cases. Banister et al. (1998) studied nitrogen and
phosphorus releases during primary sludge fermentation
under pH uncontrolled situations and observed the same
results. However, as seen in Figs. 4 and 5, the NH+4 -N
and PO3 4 -P had almost the same observed release levels
in the pH range 9.0–11.0. The reason is unclear at the
moment, and need to be further explored in the coming
investigations.
It is obvious that significant amounts of soluble phosphorus
and ammonia were released during sludge fermentation.
Thus, before the VFAs-rich effluent from fermented WAS can
be used to promote BNR, the released phosphorus and
ammonia should, ideally, be removed. Several methods can
be used for removal of high concentrations of phosphorus
and ammonia under alkaline conditions, such as phosphorus
precipitation and ammonia stripping, but simultaneous re-
cover of phosphorus and ammonia in the form of struvite
shows a distinct advantage over other methods because the
precipitate can be used as fertilizer.
3.4. Methane production
It was observed that the production of methane was also
affected by pH in the pH range 4.0–11.0 (Fig. 6). As seen in
Fig. 6 the methane production increased with the increase of
pH from 4.0 to 6.0, and decreased with further increasing pH
to 10.0. It is well known that most methanogenic bacteria
function in a pH range of 6.6–7.6 with an optimum near pH
7.0. When the pH decreased from 6.0 to 4.0, or increased from
7.0 to 10.0, the methane production was therefore decreased.
The influence of pH on methane production has also been
reported by Lay et al. (1997). Also, it can be seen in Fig. 6 that
there was no methane generated at pH 10.0 and 11.0. Thus,
the activity of methanogens decreased or was lost at alkaline
or acidic pH environments, which has also been observed by
other researchers (Cai et al., 2004; Lay et al., 1997).
3.5. VFAs production
Fig. 7 shows the effects of pH and fermentation time on total
VFAs production in terms of COD. It was observed that VFAs

200
4d
160
8d
Methane (mL/L-WAS)

120

80

40

0
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
pH

Fig. 6 – Effect of pH on methane production on the 4th and 8th day of fermentation.

3000
4.0

2500 5.0

6.0
Total VFAs (mg/L)

2000
7.0

1500 8.0

9.0
1000
10.0
500
11.0

0 blank test
4 8 12 16 20
Time (d)

Fig. 7 – Variations of total VFAs concentration at different pHs.

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688 WA T E R R E S E A R C H 41 (2007) 683– 689

Table 2 – Percentage of individual VFA accounted for total VFAs at various pHs

pH HAc (%) HPr (%) i-HBu (%) HBu (%) i-HVa (%) HVa (%)

4.0 12.30 21.08 14.13 9.39 35.27 7.82

5.0 32.58 20.66 9.15 9.62 23.30 4.69
6.0 35.19 27.91 7.53 11.74 14.06 3.57
7.0 45.86 23.23 7.61 5.46 14.95 2.90
8.0 36.76 24.44 10.28 6.22 17.76 4.54
9.0 33.85 23.83 11.57 7.33 17.80 5.63
10.0 45.84 16.49 9.21 10.95 15.65 1.86
11.0 73.44 16.65 1.77 1.61 5.84 0.70
Blank test 47.94 21.82 8.08 2.62 15.99 3.56

Note: HAc ¼ acetic, HPr ¼ propionic, i-HBu ¼ iso-butyric, HBu ¼ n-butyric, i-HVa ¼ iso-valeric, HVa ¼ n-valeric. The fermentation time was 8
days.

concentration came to the highest on the eighth or twelfth
day, and then decreased with further increasing time at all pH
values investigated except at pH 4.0 and 11.0. At pH 4.0, VFAs
decreased gradually from 375.93 on the fourth day to
119.54 mg/L on the 20th day, while at pH 11.0 VFAs increased
constantly from 628.98 to 2836.78 mg/L. However, during the
initial 16 days the greatest VFAs production was observed at
pH 10.0 instead of at pH 11.0. The reason for less initial VFAs
production at pH 11.0 might be attributed to the toxic effects
of stronger alkaline conditions on acidogenic bacteria, but
there was a remarkable increase in VFA production after 8
days when the acidogens adapted to the environment.
As also seen in Fig. 7, the amount of VFAs produced at
alkaline pH (pH ¼ 9.0, 10.0 and 11.0) were higher than those at
other pHs and in the blank test. For example, the concentra-
tion of VFAs on the 12th day at pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0,
10.0, 11.0 and in the blank test was, respectively, 251.57,
802.65, 553.06, 936.93, 1370.58, 1465.23, 2770.40, 1786.91 and
536.64 mg/L. Obviously, the production of VFAs from WAS can
be significantly improved by controlling fermentation at
alkaline pH. In the literature, the optimum pH values for
acid-phase bioreactors operation were usually set between
5.0 and 6.0 (Eastman and Ferguson, 1981; Zoetemeyer et al.,
1982), but it should be noted that those studies confined pH
below 7.0.
In this study, the higher VFAs production under alkaline
conditions was attributed to the following two reasons. One is
that the alkaline conditions increased the hydrolysis rate of
WAS to produce more soluble substrates for acidification (see
Figs. 1–3). The other is that alkaline pH decreased or
prevented the activity of methanogens (Fig. 6). As seen in
Fig. 6 an obvious VFAs consumption was observed in the
range of pH 4.0–9.0 and in blank test after 12-day’s fermenta-
tion due to the participation of VFAs consumers, such as
methanogens, but higher pH values, such as pH 10.0 and 11.0
could reduce or prevent these bacterial activities.
Short-chain fatty acids (C2 to C5) are usually the main
products of acidogenic digestion of sludge. Acetic, propionic,
butyric and iso-butyric acids can be formed directly from the
fermentation of carbohydrates, proteins and lipids, and the
higher molecular-weight VFAs, such as valeric and iso-valeric
acids are largely associated with the fermentation of proteins
(McInerney, 1988) because acidogensis of non-proteinaceous
substrates has been observed to produce little of these two
VFAs (Zoetemeyer et al., 1982). Both valeric and iso-valeric
acids can be formed through reductive deamination of single
amino acids, the product of protein hydrolysis, or by an
oxidation–reduction between pairs of amino acids via the
Stickland reaction (McInerney, 1988).
In this study the above six VFAs, acetic, propionic, n-butyric,
iso-butyric, n-valeric and iso-valeric acids were all detected in
both pH controlled (pH 4.0–11.0) and blank tests. Table 2
summarizes the percentage of individual VFA accounted for
total VFAs at different pHs when fermentation time was 8 days.
The data in Table 2 indicated that acetic, propionic and iso-
valeric acids were the three main products no matter what the
pH was. At pH 5.0, 7.0 and 10.0, these three VFAs accounted for
76.54%, 84.04% and 77.98% of total VFAs, respectively. Wang
et al. (1999) also found that the top three VFAs were acetic,
propionic and iso-valeric acids. In addition, their studies
showed that the individual VFA concentration was in the
following order: acetic4propionic4iso-valeric4iso-butyric4
(n-valeric, n-butyric), no matter whether the WAS was pre-
treated or not. In the current study, as shown in Table 2 the
order of individual VFA concentration in the case of pH greater
than 6.0 was also acetic4propionic4iso-valeric, but the
different observations were made at pH 4.0 and 5.0.
4. Conclusions
The performance of hydrolysis and acidification of WAS was
influenced by pH. Either acidic pH (pH ¼ 4.0, 5.0) or alkaline
pH (pH ¼ 9.0, 10.0 and 11.0) improved the SCOD concentra-
tion. However, the SCOD under alkaline conditions was
significantly higher than those at other pHs. It was observed
that the soluble proteins and carbohydrates were the most
important components of SCOD, and their concentrations
were also higher at alkaline pH. Further studies showed that
the production of VFAs during sludge hydrolysis and acid-
ification was also affected by pH, and the increase of pH to
8.0–11.0 could significantly improve the total VFAs concentra-
tion. At any pH value investigated, acetic, propionic and iso-
valeric acids were the three main VFAs. In addition, methane
 ARTICLE IN PRESS
WAT E R R E S E A R C H
production increased with pH in the range pH 4.0–6.0, but
decreased with pH increasing from 6.0 to 10.0. There was no
detectable methane production at pH 4.0, 10.0 and 11.0. The
data strongly implied that alkaline fermentation of WAS was
advantageous.
During fermentation of WAS, however, soluble phosphorus
and ammonia were released. Ideally, the released phosphorus
and ammonia should be removed or recovered before the
VFAs-rich effluent from fermented WAS can be used to
promote BNR. Fermentation of WAS under alkaline conditions
was also of benefit to the recovery or removal of phosphorus
and ammonia from the fermentation liquor by the methods
of phosphorus precipitation, ammonia stripping or struvite
formation.
Acknowledgements
This work was financially supported by the National Hi-Tech
Research and Development Program of China (863) (2004AA
649330) and Fok Ying Tong Education Foundation (101080). We
also thank the financial support from the National Science
Foundation of China (50678125), and the SRF for ROCS, SEM.
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