International Journal of Hydrogen Energy 27 (2002) 1359–1365www.elsevier.com/locate/ijhydene
Hydrogen production from rice winery wastewater in anup'ow anaerobic reactor by using mixed anaerobic cultures
Hanqing Yua ; ∗, Zhenhu Zhua, Wenrong Hub, Haisheng ZhangcaLaboratory of Clean Energy, School of Chemistry & Materials, The University of Science & Technology of China, 230026 Hefei,
Anhui, ChinabSchool of Resources and Environmental Engineering, Shandong University, Jinan, 250100, China
cJingzi Wine Distillery Company, Jingzi, Shandong 253400, China
Abstract
Continuous production of hydrogen from the anaerobic acidogenesis of a high-strength rice winery wastewater by a mixedbacterial 'ora was demonstrated. The experiment was conducted in a 3.0-l up'ow reactor to investigate individual e2ects ofhydraulic retention time (HRT) (2–24 h), chemical oxygen demand (COD) concentration in wastewater (14–36 g COD=l),pH (4.5–6.0) and temperature (20–55
◦C) on bio-hydrogen production from the wastewater. The biogas produced under all
test conditions was composed of mostly hydrogen (53–61%) and carbon dioxide (37–45%), but contained no detectablemethane. Speci9c hydrogen production rate increased with wastewater concentration and temperature, but with a decrease inHRT. An optimum hydrogen production rate of 9:33 l H2=g VSS d was achieved at an HRT of 2 h, COD of 34 g=l, pH 5.5and 55
◦C. The hydrogen yield was in the range of 1.37–2:14 mol=mol-hexose. In addition to acetate, propionate and butyrate,
ethanol was also present in the e<uent as an aqueous product. The distribution of these compounds in the e<uent was moresensitive to wastewater concentration, pH and temperature, but was less sensitive to HRT. This up'ow reactor was shown tobe a promising biosystem for hydrogen production from high-strength wastewaters by mixed anaerobic cultures.? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
Hydrogen is a promising candidate as a clean energycarrier in the future. Microbial hydrogen production usingfermentative, photosynthetic bacteria, or algae is an envi-ronmentally friendly and energy saving process, and it hasrecently attracted considerable attention as a way of con-verting organic wastes to hydrogen e2ectively [1]. Duringthe acidogenesis of organic wastes, hydrogen, carbon diox-ide, volatile fatty acids (VFA), and sometimes alcohols, aresimultaneously produced [2]. The feasibility of applyingacidogenesis of organic wastes to produce hydrogen hasbeen widely demonstrated at various laboratories [1,3–8].Compared with photosynthetic bacteria, fermentative
∗ Corresponding author. Tel.: +86-551-360-7592; fax:+86-551-360-1592.
E-mail address: [email protected] (H. Yu).
bacteria produce hydrogen with a lower cost because theydo not need light provision and have simple requirementsfor microbial growth [1].
In all the studies concerning biohydrogen production, acontinuous stirred tank reactor (CSTR) is used for con-tinuous generation of hydrogen from organic wastes [1].Because of its intrinsic structure, the CSTR is unable tomaintain high levels of fermentative biomass for hydro-gen production, and its speci9c hydrogen-producing rate ishardly kept high [3]. To overcome this problem, an up-'ow anaerobic reactor, with a similar structure to the up'owanaerobic sludge blanket (UASB) reactor, was tested in thisstudy.
On the other hand, in most studies on microbial produc-tion of hydrogen glucose or other pure chemicals were usedas a substrate [1]. In this study, rice winery wastewater, acarbohydrate-rich waste, was selected as the substrate forhydrogen production. Throughout China there are over tenthousands of rice fermentation plants for production of ricewine, a traditional Chinese drink. The e<uent from a rice
0360-3199/02/$ 22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.PII: S 0360 -3199(02)00073 -3
1360 H. Yu et al. / International Journal of Hydrogen Energy 27 (2002) 1359–1365
Table 1Characteristics of rice winery wastewater
COD BOD5 TN TP SS pH
(g=l) (g=l) (g=l) (g=l) (g=l)
29.5–35.4 15.6–18.7 0.07–0.14 0.02–0.03 0.31–0.73 4.8–5.9
winery plant has an organic strength of about 20–40 g=lof chemical oxygen demand (COD) [9]. An anaerobic pro-cess is the appropriate method for the treatment and en-ergy recovery from high-strength wastewaters such as ricewinery wastes. This study was thus conducted to investi-gate the individual e2ects of operating parameters on thehydrogen production of rice winery wastewater by usingmixed acidogenic cultures, and to evaluate the feasibilityof employing an up'ow reactor for continuous hydrogenproduction.
2. Materials and methods
2.1. Wastewater
Rice winery wastewater, obtained from a local wine dis-tillery company, was used in this investigation. The char-acteristics of the raw wastewater are given in Table 1. Thein'uent was prepared by using the raw wastewater as thesole carbon source, supplemented with balanced nutrientsand bu2ering chemicals [10]. For in'uent of a di2erent con-centration, the amounts of all organic and inorganic con-stituents were adjusted pro rata. The pH of the mixed liquorin the reactor was adjusted by using 6 N NaOH and 6 N HClsolutions.
2.2. Reactor and inoculum
The plexiglass-made up'ow reactor had a workingvolume of 3:0 l with an internal diameter of 90 mmand a height of 480 mm. This volume, excluding theliquid in the gas–liquid–solid separator, was used todetermine biomass concentration and hydraulic reten-tion time (HRT). Four evenly distributed samplingports were installed over the height of the column.Total biomass in the reactor was estimated based onthe pro9le of the volatile suspended solids (VSS) ofthe samples taken from the sampling ports. Underthe cap of the reactor was a gas–liquid–solid separa-tor with an internal diameter of 125 mm and a heightof 260 mm making a 9lled volume of 2:9 l. The re-actor was placed in a temperature-controlled woodenbox.
The inoculum was obtained from the secondary settlingtank in a local municipal wastewater treatment plant. Theinoculum was acclimated with glucose (10 g COD=l) in a
5-l CSTR for 21 days. In order to wash out the methanogens,the CSTR was then operated by feeding winery wastewaterat a concentration equivalent to 10 g=l of COD, at 20–25
◦C,
pH 5.0 and 16 h of HRT over 30 days. Near the end, VFAproduction became steady and no methane was detected inthe biogas. The up'ow reactor was seeded with this enrichedacidogenic inoculum equivalent to 32:4 g of VSS.
2.3. Operation
Four series of experiments were conducted to investigatethe individual e2ect of four operational parameters. In Se-ries I, the HRT was decreased stepwise from 24 to 2 h whilekeeping substrate concentration, pH and temperature con-stant at around 34 g-COD=l, pH 5.5 and 35
◦C, respectively;
in Series II, the wastewater concentration was increasedfrom 14 to 36 g COD=l, keeping HRT, pH and temperatureat 2 h, 5.5 and 35
◦C, respectively; in Series III, the pH of
the mixed liquor was lowered stepwise from 6.0 to 4.0 whilekeeping wastewater concentration, HRT and temperature at34 g COD=l, 2 h and 35
◦C, respectively; and lastly in Series
IV, the temperature was increased stepwise from 20 to 55◦C
while keeping substrate concentration, HRT and pH at 34 gCOD=l, 2 h and 5.5, respectively. Each series consisted of3–4 runs. Each run lasted over 3 weeks to ensure reactorreaching steady state before changing to the next condition.E<uent and biogas compositions were continuously moni-tored. Only those obtained under steady-state conditions arereported.
2.4. Analysis
The amount of biogas produced in the reactor wasrecorded daily using the water replacement method. Thecontent of the biogas was analyzed by a gas chromatograph(GC-8A, Shimadzu) equipped with a thermal conductivitydetector and a 1:5 m× 2 mm stainless-steel column packedwith Porapak T (50–80 mesh). Injector, column and detec-tor temperatures were kept at 120
◦C, 100
◦C and 180
◦C,
respectively.The e<uent concentrations of VFA and alcohols were an-
alyzed by a second gas chromatograph (Hewlett Packard,Model 4890) equipped with a 'ame ionization detector anda 2 m glass column packed with Gaskuropack 68 (80–100mesh). E<uent samples were 9ltered through a 0:45 �m 9l-ter, acidi9ed by formic acid, and measured for free acidsand alcohols. The temperature of the column was 50
◦C
for 3 min and then 110◦C for 3 min, and 9nally 145
◦C
for 5 min. The temperatures of the injector and detectorwere 100
◦C and 180
◦C, respectively. Nitrogen was used as
the carrier gas at a 'ow rate of 20 ml=min. Carbohydratewas measured using the calorimetric ferric-cyanide method[11]. Measurements of COD, pH and VSS (volatile sus-pended solids) were performed according to the standardmethods [12].
H. Yu et al. / International Journal of Hydrogen Energy 27 (2002) 1359–1365 1361
30
40
50
60
70
0 4 8 12 16 20
Par
tial p
ress
ure
(kP
a)
H2CO2
(a)1
2
3
4
5
6
7
0 8 12 16 20 24
Con
cent
ratio
n(g
/l))
acetate propionatebutyrate ethanol
(b)
1
1.5
2
2.5
3
0 4 8 12 16
HRT (h)
H2
yiel
d(m
ol-H
2/m
ol−h
exos
e)
(c)
0
2
4
6
8
10
0 8 12 16 20
HRT (h)
H2
prod
uctio
n ra
te(l/
g-V
SS
-d)
(d)
20 24
24 4
4 24
Fig. 1. In'uence of HRT on the performance of the hydrogen-producing reactor: (a) partial pressures of H2 and CO2, (b) concentrations ofaqueous products, (c) hydrogen yield, and (d) speci9c hydrogen production rate.
3. Results and discussion
3.1. E9ect of HRT
Acidi9cation of winery wastewater produced a biogascontaining hydrogen and carbon dioxide, but without de-tectable methane, for HRT ranging 2–24 h. Fig. 1a illustratesthat partial pressures of both hydrogen and carbon dioxide'uctuated within the narrow range of 55–59 kPa for hydro-gen and 40–45 kPa for carbon dioxide.
Fig. 1b illustrates that the fermentation process was notsensitive to HRT. On an average, the e<uent was com-posed of 30% acetate, 28% propionate, 21% butyrate and21% ethanol. Acetate accounted for 29–33% of the totalVFA=alcohol in the e<uent, whereas propionate, butyrateand ethanol ranged between 26% and 30%, 20% and 26%and 18% and 22%, respectively. Results of previous studiesshowed that product composition in acidi9cation of wastedsludge [13] and dairy wastewater [14] was strongly af-fected by HRT. This is contrary to the results of this studywith winery wastewater, which were similar to the acidi-9cation of other readily biodegradable substrates, such asglucose [15], sucrose [16] and gelatin [17]. It seems thatHRT has more in'uence on the product composition in theacidi9cation of substrates that are more recalcitrant to bio-degradation.
As shown in Fig. 1c, the hydrogen yield increasedwith HRT, from 1:74 mol H2=mol hexose at 2 h to2:14 mol H2=mol hexose at 24 h. This suggests that morecarbohydrates in the wastewater were converted intohydrogen at longer HRTs. On the other hand, Fig. 1dillustrates that the speci9c hydrogen production rate
decreased as HRT increased, from 8:02 l H2=g VSS d at 2 hto 1:40 l H2=g VSS d at 24 h. The biomass in the reactorhad the maximum hydrogen-producing capacity at 2 h, theshortest HRT.
3.2. E9ect of substrate concentration
Fig. 2 illustrates the e2ect of COD concentration inwastewater on acidi9cation for Series-II experiments con-ducted at pH 5.5, 35
◦C and 2 h of HRT. Fig. 2a shows that
the partial pressure of hydrogen slightly increased with thewastewater concentration, from 53 kPa at 14 g COD=l to59 kPa at 36 g COD=l, whereas carbon dioxide decreasedfrom 43 to 40 kPa, correspondingly.
Acidi9cation at pH 5.5, 35◦C, 2 h of HRT and wastew-
ater equivalent to 14–36 g COD=l produced 76–83% VFAand 17–24% ethanol. At 13 g COD=l, acetate accountedfor 37% of total VFA=alcohol in the e<uent, propionate18%, butyrate 28% and ethanol 17%, respectively. At36 g COD=l, acetate represented 26% of total VFA=alcoholwhereas propionate, butyrate and ethanol changed to 30%,20% and 24%, respectively. As shown in Fig. 2b, acetateand butyrate both decreased as wastewater concentrationincreased, whereas the opposite was true for propionate andethanol.
Fig. 2c illustrates that the hydrogen yield slightlydecreased as wastewater concentration increased, from1:89 mol H2=mol hexose at 13 g COD=l to 1:79 mol H2=molhexose at 36 g COD=l. This suggests that the wastewaterconcentration had a slightly negative e2ect on the hydrogenyield. However, Fig. 2d illustrates that the speci9c hydro-gen production rate substantially increased with wastewater
1362 H. Yu et al. / International Journal of Hydrogen Energy 27 (2002) 1359–1365
30
40
50
60
70
10 20 30 40
Par
tial p
ress
ure
(kP
a)
H2CO2
(a)0
1
2
3
4
5
10 20 30 40
Con
cent
ratio
n(g
/l)
acetate propionatebutyrate ethanol
(b)
1.6
1.7
1.8
1.9
2
10 20 30 40
Substrate COD (g/l)
H2
yiel
d
(mol
−H2/m
ol−h
exos
e)
(c)
0
2
4
6
8
10
10 20 30 40Substrate COD (g/l)
H2
prod
uctio
n ra
te
(l/g-
VS
S-d
)
(d)
Fig. 2. In'uence of substrate concentration on the performance of the hydrogen-producing reactor: (a) partial pressures of H2 and CO2, (b)concentrations of aqueous products, (c) hydrogen yield, and (d) speci9c hydrogen production rate.
concentration, from 3:62 l H2=g VSS d at 13 g COD=l to8:12 l H2=g VSS d at 36 g COD=l.
3.3. E9ect of pH
Series-III experiments were conducted at 35◦C, 2 h
of HRT, 34 g COD=l, and pH ranging 4.0–6.0. Fig. 3aillustrates that the biogas composition was slightly in'u-enced by pH. The biogas was composed of 43% carbondioxide and 54% hydrogen at pH 4.0. At pH 6.0, thecorresponding compositions became 45% and 53%. Thebiogas was free of methane at all pH levels. The partialpressure of hydrogen reached the highest at pH 5.5. Thisresult is in accordance with the results of several stud-ies about the in'uence of pH on hydrogen production[3,4,8].
The e<uent composition was strongly dependent on pH.In general, lower pH favored the production of propionateand ethanol, accounting for 40% and 26% of acidi9cationproducts in the e<uent at pH 4.0; both decreased with theincrease of pH, accounting for 20% and 13% of the ef-'uent products at pH 6.0. On the other hand, as shownin Fig. 3b, the percentages of acetate and butyrate bothincreased with pH, from 18% and 15%, respectively, atpH 4.0 to 38% and 29% at pH 6.0. The change of prod-uct distribution was probably due to the shift of micro-bial population in the reactor. Product distribution was alsofound sensitive to the variation of pH for the acidi9cation ofglucose [16].
Fig. 3c illustrates that the hydrogen yield reached its max-imum, 1:74 mol H2=mol hexose, at pH 5.5. The hydrogen
yields were 1.45 and 1:46 mol H2=mol hexose at pH 5.0 and6.0. Fig. 3d shows that the speci9c hydrogen production rateranged between 4.72 and 8:02 l H2=g VSS d at pH 4.5–6.0.At pH 5.5, the reactor reached the highest speci9c hydrogenproduction rate of 8:02 l H2=g VSS d. It suggested that theoptimum pH level was 5.5 for hydrogen production in thisstudy.
3.4. E9ect of temperature
Series-IV experiments were conducted at 34 g COD=l,2 h of HRT, pH 5.5 and temperature ranging 20–55
◦C.
Fig. 4a illustrates that the partial pressure of hydrogen in-creased with temperature, from 53 kPa at 20
◦C to 61 kPa at
55◦C, whereas that of carbon dioxide changed correspond-
ingly from 45 to 37 kPa. No methane was detected in allruns.
As shown in Fig. 4b, percentages of acetate and butyrateslightly decreased with the increase of temperature, from35% and 24% at 20
◦C, respectively, to 26% and 17% at
55◦C, whereas the percentages of propionate and ethanol
both increased with temperature. This concurs with the 9nd-ings of Zoetemeyer et al. [18] in which propionate andethanol became the predominant products in the acidi9ca-tion of glucose at 55–65
◦C.
Figs. 4c and d illustrate that the hydrogen yield and spe-ci9c hydrogen production rate both increased with temper-ature. This suggests that higher temperature was bene9cialfor biohydrogen production from rice winery wastewaterand that the optimal temperature for the hydrogen produc-tion was 55
◦C in this study. Conventionally, single-stage
H. Yu et al. / International Journal of Hydrogen Energy 27 (2002) 1359–1365 1363
30
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50
60
70
4 4.5 5 5.5 6
Par
tial p
ress
ure
(kP
a)
H2CO2
(a)1
2
3
4
5
4 4.5 5 5.5 6
Con
cent
ratio
n(g
/l)
acetate propionatebutyrate ethanol
(b)
1.2
1.4
1.6
1.8
2
4 4.5 5 5.5 6
pH
H2
yiel
d(m
ol-H
2/m
ol-h
exos
e)
(c)
0
2
4
6
8
10
4 4.5 5 5.5 6
pHH
2 pr
oduc
tion
rate
(l/g-
VS
S-d
)
(d)
Fig. 3. In'uence of pH on the performance of the hydrogen-producing reactor: (a) partial pressures of H2 and CO2, (b) concentrations ofaqueous products, (c) hydrogen yield, and (d) speci9c hydrogen production rate.
30
40
50
60
70
20 30 40 50 60
Par
tial p
ress
ure
(kP
a)
H2CO2
(a)1
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cent
ratio
n(g
/l)
acetate propionatebutyrate ethanol
(b)
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1.2
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1.8
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20 30 40 50 60
Temperature (oC)
H2
yiel
d(m
ol-H
2/m
ol-h
exos
e)
(c)
0
2
4
6
8
10
20 30 40 50 60
Temperature (oC)
H2
prod
uctio
n ra
te(l/
-VS
S-d
)
(d)
Fig. 4. In'uence of temperature on the performance of the hydrogen-producing reactor: (a) partial pressures of H2 and CO2, (b) concentrationsof aqueous products, (c) hydrogen yield, and (d) speci9c hydrogen production rate.
methanogenic reactors are operated either at the mesophilictemperature of 35–40
◦C or the thermophilic temperature
of 55–60◦C [19]. No experiment was performed beyond
55◦C. However, both the hydrogen yield and produc-
tion rate were still increasing after reaching 55◦C, sug-
gesting that the optimum temperature might lie beyond55
◦C.
3.5. Evaluation of the up:ow reactor
The above experimental results demonstrated that the up-'ow reactor used in this study was of high-eOciency forhydrogen production. Biogas was composed of mostly hy-drogen (53–61%) and carbon dioxide (37–45%), but wasfree of methane. The optimum hydrogen production rate
1364 H. Yu et al. / International Journal of Hydrogen Energy 27 (2002) 1359–1365
Table 2Comparison of hydrogen production rates and yields obtained in this study to those cited in the literature
Substrate Maximum hydrogen yield Maximum speci9c Maximum volumetric Reference(mol H2=mol hexose) production rate production rate
(l H2=g VSS d) (l H2=l d)
Winery wastewater 2.14 9.33 3.81 This studyGlucose 1.70 6.76 0.54 [5]Glucose 0.70 2.18 0.23 [6]Sugar wastewater 2.59 = 0.69 [7]Glucose 2.30 5.70 0.78 [8]
of 9:33 l H2=g VSS d was achieved at HRT of 2 h, CODof 34 g=l, pH 5.5 and 55
◦C. The hydrogen yield was in
the range of 1.37–2:14 mol H2=mol hexose contained in thewastewater.
Table 2 compares the hydrogen yields, speci9c hydrogenproduction rates and volumetric hydrogen production ratesof this study with those found in the literature. In the otherstudies, a CSTR was employed as a hydrogen-producingreactor. The maximum hydrogen yield of 2:14 mol H2=molhexose obtained in this study was comparable to the yieldsreported. However, the maximum speci9c hydrogen pro-duction rate, 9:33 l H2=g VSS d, observed in this study, wasconsiderably higher than those of other studies. Because ofits intrinsic structure, especially the installation of a gas–solids–liquid separator [20], this up'ow reactor retaineda high level of biomass ranging 9.4–11:7 g VSS=l. Theaverage biomass concentrations in CSTRs were generallyless than 4 g VSS=l. Hence, because of its higher hydro-gen productivity of the biomass and higher concentrationof biomass, the up'ow reactor possessed much higher vol-umetric hydrogen production rates than the CSTRs. Con-sidering the above results and its stable operation for atleast 29 weeks, it might be concluded that this up'ow re-actor is a more promising biosystem for hydrogen pro-duction from high-strength wastewaters by mixed anaer-obic cultures, compared with a CSTR, which is widelyused for continuous generation of hydrogen from organicwastes.
4. Conclusions
The experimental results showed that anaerobic acidoge-nesis from rice winery wastewater in an up'ow reactor pro-duced hydrogen continuously. Biogas produced under alltest conditions was composed of mostly hydrogen and car-bon dioxide, but contained no detectable methane. Speci9chydrogen production rate increased with organic concentra-tion in wastewater and temperature, but with the decreaseof HRT. Higher hydrogen production rate was achieved atlower HRT, concentrated substrate, slightly acidic pH andhigher temperature. In addition to acetate, propionate, andbutyrate, ethanol was also present in the e<uent as the
aqueous product. The distribution of these compounds inthe e<uent was more sensitive to wastewater concentra-tion, pH and temperature, but was less sensitive to HRT.Compared with a CSTR, this up'ow reactor was shownto be a more promising biosystem for hydrogen produc-tion from high-strength wastewaters by mixed anaerobiccultures.
Acknowledgements
The authors wish to thank the Natural Science Foun-dation of China (Grant No. NSFC-GM 20122203) andthe Ministry of Science & Technology, China (Grant No.2001AA515050) for the partial 9nancial support of thisstudy, and also appreciate the invaluable and kind helpprovided by Prof. Herbert H.P. Fang when the 9rst authorworked at the University of Hong Kong.
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