Biochemical Engineering Journal 21 (2004) 193197

Short communication
Two-phases anaerobic digestion of fruit and
vegetable wastes: bioreactors performance
H. Bouallagui
a,
, M. Torrijos
c
, J.J. Godon
c
, R. Moletta
c
,
R. Ben Cheikh
b
, Y. Touhami
a
, J.P. Delgenes
c
, M. Hamdi
a
a
UR-Procds Microbiologiques et Alimentaires, Institut National des Sciences Appliques et de Technologie (INSAT),
B.P 676, 1080 Tunis, Tunisia
b
Ecole Nationale dIngnieurs de Tunis (ENIT), B.P, 37, 1002 Tunis, Tunisia
c
Laboratoire de Biotechnologie de lEnvironnement, INRA, Avenue des Etangs, 11100 Narbonne, France
Received 8 December 2003; received in revised form 6 May 2004; accepted 14 May 2004
Abstract
The two-phase anaerobic digestion of a mixture of fruit and vegetable wastes (FVW) was studied, using two coupled anaerobic sequencing
batch reactors (ASBR) operated at mesophilic temperature. The effect of increasing loading rates on the acidication step was investigated.
Results indicated that the hydrolysis yield (81%) stabilized at an OLR of 7.5 g COD/L.d. The volatile fatty acids concentration increased
when the loading rate was increased and reached its maximumvalue (13.3 g/L) at higher loading rate tested (10.1 g COD/L.d). Methanogenic
fermentation of the liquefaction acidication products was efciently performed in the ASBR reactor and high methane productivity was
obtained (320 L CH
4
per kg of input COD). Total COD in the nal efuent from the methanizer was usually below 1500 mg/L, and soluble
CODbelow400 mg/L. Overall CODremoval in the treatment systemwas 96%. Phase separation with conventional ASBRreactors resulted
in high process stability, signicant biogas productivity and better efuent quality from fruit and vegetable wastes anaerobic digestion.
2004 Elsevier B.V. All rights reserved.
Keywords: Waste treatment; Anaerobic processes; Acidication; Two-phases; Anaerobic sequencing batch reactor; Biogas
1. Introduction
Fruit and vegetable wastes (FVW) are produced in large
quantities in markets, and constitute a source of nuisance
in municipal landlls because of their high biodegradabil-
ity [1,2]. A possible way to dispose of these wastes is using
the anaerobic digestion process [3,4]. The successful ap-
plication of anaerobic technology to the treatment of solid
wastes is critically dependent on the development and the
use of high rate anaerobic bioreactors [5,6]. In recent years
a number of novel reactor designs have been adapted and
developed. These processes differ especially in the way mi-
croorganisms are retained in the bioreactor and the separa-
tion between the acidogenic and the methanogenic bacte-
ria and then to reduce the anaerobic digestion limitations
[79].
Given the very large biodegradable organic content of
FVW, a major limitation of anaerobic digestion of these
wastes in one stage system is a rapidly acidication de-

Corresponding author. Tel.: +216 22 524 406; fax: +216 71 704 329.
E-mail address: hassibbouallagui@yahoo.fr (H. Bouallagui).
creasing the pH in the reactor, and a larger volatile fatty
acids production, which stressed and inhibited the activity
of methanogenic bacteria. The two-phase systems appear
as higher efcient technologies for anaerobic digestion of
FVW. Their greatest advantage lies in the buffering of the
organic loading rate taking place in the rst stage, allowing
a more constant feeding rate of the methanogenic second
stage [1012].
Application of sequencing batch reactor (SBR) technol-
ogy to anaerobic treatment of FVW is of interest because of
its inherent operational exibility, characterised by a high
degree of process exibility in terms of cycle time and se-
quence, no requirement for separate clariers and can retain
a higher concentration of slow-growing anaerobic bacteria
within the reactor. Research into the anaerobic sequencing
batch reactors (ASBR) process has been carried out by sev-
eral investigators [1315]. Satisfactory high-solid-content
waste degradation and suspended solid removal (9093.5%)
using the ASBR were reported [16,17].
The aimof the present work was to assess the performance
of a two-phases anaerobic digestion of a mixture of fruit and
vegetable wastes in an ASBR.
1369-703X/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2004.05.001
194 H. Bouallagui et al. / Biochemical Engineering Journal 21 (2004) 193197
Nomenclature
FVW fruit and vegetable waste
ASBR anaerobic sequencing batch reactor
COD chemical oxygen demand (g L
1
)
HRT hydraulic retention time (day)
SRT solid retention time (day)
TS total solid (%)
TVS total volatile solid (%)
TSS total supended solid (g L
1
)
VSS volatile suspended solid (g L
1
)
TNK total nitrogen Kjeldahl (g L
1
)
OLR organic loading rate (g L
-1
d
1
)
VFA volatile fatty acid (g L
1
)
2. Material and methods
2.1. Reactors design and operational conditions
Two laboratory-scale reactors of different volumes were
used. The hydrolysisacidication step was carried out in
a glass reactor of 1.5 L effective volume. The reactor was
stirred by an inox stirrer and operated with 1 cycle per day
and 21 h of reaction, 3 h of settling, draw off and ll during
the last hour of the settling period. The pH was controlled
by automatic addition of 2 N NaOH.
The methane fermentation was performed in a double-
walled glass reactor of 5 L effective volume. Mixing in the
reactor was done by a system of magnetic stirring. The
methanogenic ASBR was operated with cycles including
the following four discrete steps: (i) ll (30 min), (ii) react
(21 h), (iii) settle (2 h), (iv) draw off (30 min). The reactors
were maintained at 35

C and inoculated with an anaerobic

ecosystem obtained from the settled output of an anaerobic
xed bed reactor treating winery efuent.
2.2. Analysis and fermentation parameters
Total solids (TS), total volatile solids (TVS), total sus-
pended solids (TSS), pH, total nitrogen Kjeldahl (TNK)
and chemical oxygen demand (COD) were determined ac-
cording to the standard methods [18]. Volatile fatty acids
(VFA) concentration was determined by semi-capillary
gas chromatography (Chrompack CP 9000) with a Econo-
fap FFAP (Altech) column. The maximum temperature
of injection and the regeneration temperature are 250 and
200

C, respectively. The volume of biogas produced was

measured by an Aalborg mass ow meter 020 mL/min
and analysed by gas chromatography (Shimadzu GC 8A)
[14].
Hydrolysis yield (HY)(%): it was calculated as following:
HY =
100 [(total input COD soluble input COD)
(total output COD soluble output COD)]
(total input COD soluble input COD)
Acidication yield: total VFA in the reactor expressed in
mg COD over total COD of crude input.
Pollutant removal yield (%): difference between the total
COD at inlet and total COD at outlet 100 over total COD
at inlet.
3. Results and discussion
3.1. Substrate characteristics
The putrescible FVW used in this study was collected
from the group market of Narbonne (South France). After
shredding to small particles and homogenizing, it was stored
in 1 litre tins at 4

C. The composition of the raw shredded

FVW is shown in Table 1. Total initial COD was about
120 g/kg (humid weight), with soluble COD and particulate
COD of 79 g/kg and 41 g/kg, respectively. The COD/N ratio
was balanced, being around 120/3.8; therefore, no nitrogen
was added to the acidication reactor.
3.2. Hydrolysis and acidication stage
The acidication reactor was operated at a constant hy-
draulic retention time (HRT) of 3 days and fed with different
dilutions of FVW to change the organic loading rate (OLR).
The whole experiment was carried out over three runs (run
1: OLR = 3.7 g COD/L.d; run 2: OLR = 7.5 g COD/L.d and
run 3: OLR = 10.1 g COD/L.d).
3.2.1. Start-up and pH adjusment
The rst part of this study was carried out at a low
OLR (run 1) in order to choose the best pH for the
liquefaction-acidication step. When not controlled, pH
dropped rapidly to 4, especially just at the end of the feed
period, and an inhibition of hydrolysis was observed. Koster
[19] mentioned that butyric acid-producing bacteria are not
viable in a medium with a pH lower than 4.2. Several au-
thors suggested that optimal pH for better hydrolytic and
acidogenic bacteria activity is comprised between 5 and 6
[2,11,20,21]. After that, pH was maintained at 6. In these
conditions, the rate of volatile fatty acids production was
low (1.2 g COD/L.d). Then acidication was activated and
the rate of volatile fatty production was improved to 4.5 g
Table 1
Raw shredded FVW characteristics
Analysis Average values
pH 4.2
Total solids (g/kg) 100
Volatile solids (g/kg) 88
Total COD (g/kg) 120
Particulate COD (g/kg) 78.9
Total suspended matter (g/kg) 74.4
Total nitrogen Kjeldahl (g/kg) 3.8
H. Bouallagui et al. / Biochemical Engineering Journal 21 (2004) 193197 195
Fig. 1. Cumulative total VFA production (), C2 (), C3 (), C4 ()
and C5 () during a typical cycle of acidication reactor, operated at
different OLR: run 1 (a), run 2 (b) and run 3 (c).
COD/L.d by lowering pH to 5.5 by the addition of a 2 N
chlorhydric acid solution. The pH was maintained at this
level during the whole experiments.
3.2.2. Particulate organic carbon solubilization and VFA
production at different OLR
The results of the hydrolysis acidogenesis stage are shown
in Table 2. Variation in OLR indicated that the hydrolysis
yield (81%) stabilized at an OLR of 7.5 g COD/L.d. VFA
concentration increased dramatically with the increase of
the OLR. The highest VFA concentration (13.3 g/L) was
obtained in run 3, with the highest OLR. Fig. 1 shows the
VFA production and composition during a typical cycles of
reactor operation at different OLR. Butyric acid and valeric
acid were the major VFA produced at a lower OLR (run 1)
(Fig. 1a). When increasing the OLR, acetic acid and butyric
acid were the main VFA produced, with an average value of
40% for each at an OLR of 10.1 g COD/L.d. In agreement
with Sans et al. [22] and Raynal et al. [11], the VFA prole
showed that butyric acid concentrations were quite high for
FVW acidication. The percent of soluble COD in the form
of VFA in the outlet of the reactor increased as the OLR
rose, with 97% of soluble COD as VFA in the last run
(Table 2).
Total COD reduction results based on a COD balance
at steady-state conditions was about 45 for the highest
OLR (run 3) (Table 2). During the liquefaction phase,
the production of carbon dioxide and small quantities of
methane and hydrogen resulted from COD degradation.
The gas production in the acidogenic reactor (at 1 atm
and 0

C) was about 0.3 L/L.d (run 1), 0.41 L/L.d (run 2)

and 0.52 L/L.d (run 3) (Table 2). Several analyses of gas
composition revealed that CO
2
was the predominant gas
generated (over than 70%), which is in agreement with the
results reported in the literature for the acedogenic step
[1,11].
3.3. Methanogenic ASBR digestor performance
This step in the experiments was carried out to evalu-
ate the degradation efciency and biogas productivity of
an ASBR digester treating the supernatant of the acido-
genic reactor efuent. The results are given in Table 3.
The hydraulic retention time was constant (10 days) so
the loading rate (from 0.72 to 1.65 g/L.d) depended on the
COD at the outlet of the acidogenic reactor. In these oper-
ating conditions, the maximum g COD total inlet/g volatile
suspended solid (VSS) was 0.18 and the SRT was about
50 days.
3.3.1. Biogas production and organic matter degradation
The rate of biogas production as well as the pH were
monitored continuously. An example of the evolution of the
biogas production rate and of the pH obtained at the highest
OLR, is presented in Fig. 2. The biogas production rate
was greatest at the start of the cycle, and then decreased
with time, reaching very low levels after about 8 h. At this
time, the VFA concentration in the reactor stabilized at its
lowest level and the react phase could then be considered as
nished. The duration of one cycle was about 10.5 h. The
highest biogas productivity (450 L per kg of added COD)
with high methane content (71%) was obtained at run 2.
Earlier, Raynal et al. [11] and Verrier et al. [1] obtained
similar productivity, using an upow anaerobic lters.
The average residual total COD and soluble COD in the
treated efuent were about 1300 and 270 mg/L at run 3, cor-
responding to a purication level of 93 and 97%, respec-
tively. These results show that the organic matter of FVW
after acidication, constituted mainly of VFA, is highly
biodegradable. TSS in the methanogenic reactor was be-
tween 13 and 15 g/L and settling was very good during the
whole experiment.
196 H. Bouallagui et al. / Biochemical Engineering Journal 21 (2004) 193197
Table 2
Average values of FVW at the liquefactionacidication stage
Run 1 Run 2 Run 3
Inlet total COD (g/L) 11.2 0.05 23 0.11 30 0.2
Inlet soluble COD (g/L) 6.8 0.09 14.2 0.3 15.7 0.4
Organic loading rate (g COD/L.d) 3.7 0.02 7.67 0.02 10 0.03
Retention time (days) 3 3 3
Outlet total COD (g/L) 7.16 0.18 12.9 0.78 16.5 0.36
Outlet soluble COD (g/L) 6 0.14 11.2 0.87 13.8 0.26
Acetic acid (g COD/L) 0.7 0.06 3.43 0.27 5.18 0.28
Propionic acid (g COD/L) 0.56 0.05 0.71 0.08 1.2 0.09
Butyric acid (g COD/L) 1.83 0.14 3.45 0.31 5.46 0.38
Valeric acid (g COD/L) 1.41 0.03 1.29 0.09 1.47 0.12
Total VFA (g COD/L) 4.5 0.16 8.8 0.64 13.3 0.43
TSS (g/L) 7.5 9.2 12.4
Hydrolysis yield (%) 74 1.5 81 3.5 81 3
Acidication yield 40.3 0.9 38.9 1.2 44.4 1.8
COD
VFA
/COD
solubleoutlet
(%) 75 1.2 80 2.5 97 4.2
Total COD removal (%) 36 0.9 44 1.2 45 1.3
Gas production (L/L.d) 0.3 0.02 0.41 0.01 0.52 0.01
Table 3
Average performances of the methanogenic reactor
Run 1 Run 2 Run 3
COD total input (g/L) 7.16 0.18 12.9 0.78 16.5 0.36
Organic loading rate (g COD/L.d) 0.72 0.03 1.29 0.02 1.65 0.02
Retention time (days) 10 10 10
Output total COD (g/L) 2.3 0.09 1 0.07 1.2 0.1
Output soluble COD (g/L) 0.35 0.02 0.32 0.01 0.27 0.01
TSS in the reactor (g/L) 13.1 13.7 14.8
pH in the reactor 6.9 0.1 7.2 0.1 7.49 0.15
Biogas productivity (L per L of reactor a day) 0.26 0.01 0.58 0.02 0.74 0.02
Biogas yield (L per kg of input COD) 363.1 16.5 450.3 22.3 448.5 19
Methane content (%) 69 2 71 2 70.6 1.5
COD removal (%) 67.9 3.2 92.2 2.1 92.7 2.3
COD removal of the total process (%) 79.46 2.8 95.65 2.1 96 2.6
Fig. 2. Biogas production rate and pH shift during a typical cycle of the methanogenic reactor operated at the higher loading rate (run 3).
H. Bouallagui et al. / Biochemical Engineering Journal 21 (2004) 193197 197
4. Conclusion
The study of two-stage anaerobic digestion of FVW was
undertaken. The acidication step was inuenced by the
variation in the OLR, especially the concentration of VFA
and the percent of VFA in the form of soluble COD. The
results obtained in this work show that the FVW is highly
biodegradable with a conventional two-phase reactor and
96% of the total COD was converted to biomass and bio-
gas. The total COD in the nal efuent from the methanizer
was usually below 1500 mg/L and the soluble COD below
400 mg/L. Compared with a previous study [4], phase sepa-
ration between the two groups of micro-organisms involved
in anaerobic digestion is necessary to improve the yield of
total process.
Acknowledgements
The authors wish to acknowledge the Ministry of Superior
Education and Scientic Research and Technology, which
has facilitated the carried work.
References
[1] D. Verrier, F. Roy, G. Albagnac, Biol. Wastes 22 (1987) 163.
[2] A.M. Vituria, J. Mata-Alvarez, F. Cecchi, G. Fazzini, Biol. Wastes
29 (1989) 189.
[3] J. Mata-Alvarez, F. Cecchi, P. Llabrs, P. Pavan, Bior. Technol. 39
(1992) 39.
[4] H. Bouallagui, R. BenCheikh, L. Marouani, M. Hamdi, Bior. Technol.
86 (2003) 85.
[5] G. Lissens, P. Vandevivere, L. De Baere, E.M. Biey, W. Verstrae,
Water. Sci. Technol. 44 (2001) 91.
[6] H.R. Srilatha, K. Nand, K. Sudhakar Bada, K. Madhukara, Proc.
Biochem. 30 (1995) 327.
[7] J. Mata-Alvarez, F. Cecchi, P. Pavan, P. Llabrs, Biol. Wastes 33
(1990) 181.
[8] A. Veeken, S. Kalyuzhnyi, H. Scharff, B. Hamelers, J. Environ.
Biotechnol. 126 (2000) 1076.
[9] A. Fernndez, S. Huang, S. Seston, J. Xing, R. Hickey, C. Criddle,
J. Tiedje, Appl. Environ. Microbiol. 65 (1999) 3697.
[10] R.C. Landine, G.J. Brown, A.A. Cocci, H. Viraraghavan, Agricult.
Wastes 7 (1983) 111.
[11] J. Raynal, J.P. Delgenes, R. Moletta, Bior. Technol. 65 (1998)
97.
[12] E. Panagiotis, K.O. William, J. Chem. Biotechnol. 60 (1994) 89.
[13] R.R. Dague, C.E. Habben, S.R. Pidaparti, Wat. Sci. Tech. 26 (1992)
2429.
[14] C. Ruiz, M. Torrijos, P. Sousbie, M.J. Lebrato, R. Moletta, Wat. Sci.
Tech. 43 (2000) 201.
[15] S. Suthaker, C. Polprasert, R.L. Droste, Wat. Sci. Tech. 23 (1991)
1249.
[16] S. Archana, U. Balag, S.h. Devendra, J. Biosci. Bioeng. 87 (1999)
678.
[17] J.M. Hur, D. Chang, T.H. Chung, J. Biosci. Bioeng. 87 (1999)
525.
[18] American Public Health Association, 18th ed., Washington, 1992.
[19] I.W. Koster, Agricult. Wastes 11 (1984) 241.
[20] K.V. Rajeshwari, D.C. Panth, K. Lata, V.N. Kishore, Waste. Manag.
Res. 1 (2001) 292.
[21] A. Converti, A. DelBorghi, M. Zilli, S. Arni, M. DelBorghi, Bioproc.
Eng. 21 (1999) 371.
[22] C. Sans, J. Mata-Alvarez, F. Cecchi, P. Pavan, A. Bastti, Biores.
Technol. 51 (1995) 89.