ARTICLE IN PRESS

Bioresource Technology xxx (2007) xxxxxx

Biofuels generation from sweet sorghum: Fermentative hydrogen

production and anaerobic digestion of the remaining biomass
Georgia Antonopoulou a,b, Hariklia N. Gavala a,b,*, Ioannis V. Skiadas
K. Angelopoulos c, Gerasimos Lyberatos a,b
a
b

a,b

Department of Chemical Engineering, University of Patras, Karatheodori 1 st., 26500 Patras, Greece
Institute of Chemical Engineering and High Temperature Chemical Processes, 26504 Patras, Greece
c
Department of Biology, University of Patras, 26500 Patras, Greece
Received 10 July 2006; received in revised form 26 November 2006; accepted 27 November 2006

Abstract
The present study focuses on the exploitation of sweet sorghum biomass as a source for hydrogen and methane. Fermentative hydrogen production from the sugars of sweet sorghum extract was investigated at dierent hydraulic retention times (HRT). The subsequent
methane production from the euent of the hydrogenogenic process and the methane potential of the remaining solids after the extraction process were assessed as well. The highest hydrogen production rate (2550 ml H2/d) was obtained at the HRT of 6 h while the highest yield of hydrogen produced per kg of sorghum biomass was achieved at the HRT of 12 h (10.4 l H2/kg sweet sorghum). It has been
proved that the euent from the hydrogenogenic reactor is an ideal substrate for methane production with approximately 29 l CH4/kg of
sweet sorghum. Anaerobic digestion of the solid residues after the extraction process yielded 78 l CH4/kg of sweet sorghum. This work
demonstrated that biohydrogen production can be very eciently coupled with a subsequent step of methane production and that sweet
sorghum could be an ideal substrate for a combined gaseous biofuels production.
 2006 Elsevier Ltd. All rights reserved.
Keywords: Biofuels; Fermentation; Hydrogen; Methane; Sweet sorghum

1. Introduction
Renewable energy sources have received great interest
from the international community during the last decades.
Biomass is one of the oldest and the most promising energy
sources and includes organic and animal wastes, wastewater, energy crops, agricultural and industrial residues that
can be used for the production of biofuels. Nowadays, biomass provides approximately 14% of the total world-wide
energy needs (International Energy Agency, 1998) and rep*

Corresponding author. Address: Department of Chemical Engineering, University of Patras, Karatheodori 1 st., 26500 Patras, Greece.
Tel.: +30 2610997858; fax: +30 2610993070.
E-mail addresses: gavala@chemeng.upatras.gr, hng@biocentrum.
dtu.dk (H.N. Gavala).

resents an important contributor to the world economy

(Chum and Overend, 2001; Parikka, 2004). There are several conversion technologies for the energy exploitation
of biomass, that is, direct combustion (which is responsible
for over 97% of the worlds bioenergy production),
gasication,
pyrolysis,
and
biological
treatment
(Demirbas, 2004). The latter has economical and environmental advantages compared to the other technologies.
Biomass can be biologically converted to liquid or gaseous
fuels (Claassen et al., 1999), such as ethanol, methanol,
methane and hydrogen, which was recently characterized
as the fuel of the future.
Hydrogen is a clean and environmentally friendly fuel,
which produces water instead of greenhouse gases when
combusted. It can be produced by renewable raw materials,
such as organic wastes, and possesses a high-energy yield

0960-8524/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.11.048

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G. Antonopoulou et al. / Bioresource Technology xxx (2007) xxxxxx

(122 kJ/g) due to its light weight. Furthermore, hydrogen

could be directly used to produce electricity through fuel
cells (Lay et al., 1999; Benemann, 1996). One of the main
drawbacks of hydrogen use is the diculty of securing a
safe storage, especially in automobiles. However, this problem could be overcome by the use of metal hydrides and
carbon nanotubes, which reversibly adsorb hydrogen at
room temperature and low pressures (Ramachandran and
Menon, 1998; Noike and Mizuno, 2000; Ajayan and Zhou,
2001). There are still signicant technical barriers concerning these technologies but overcoming them is an area of
active research.
Biological hydrogen production, one of the several ways
to produce hydrogen, has received special attention during
the last decade. Biohydrogen may be produced by cyanobacteria and algae through biophotolysis of water (Asada and
Miyake, 1999) or by photosynthetic and chemosyntheticfermentative bacteria. Anaerobic fermentative bacteria produce hydrogen without photoenergy, and so the cost of
hydrogen production is 340 times lower than the photosynthetic process (Morimoto, 2002; Atif et al., 2005). In addition, it is well known that carbohydrates are the main
source of hydrogen during fermentative processes and therefore wastes/wastewater or agricultural residues rich in carbohydrates can be considered as potential sources of
hydrogen (Kapdan and Kargi, 2006). Glucose and sucrose
are the fermentation substrates most studied in the laboratory (Chen et al., 2001; Zoetemeyer et al., 1982a). Degradation of sugars is accompanied by the production of hydrogen
and various metabolic products, mainly volatile fatty acids
(acetic, propionic and butyric acids), lactic acid and ethanol,
during the fermentation process. The hydrogen yield varies
proportionally to the nal metabolic products. Production
of acetic and butyric acids favors production of hydrogen
(Nandi and Sengupta, 1998; Hawkes et al., 2002), with the
fermentation to acetic acid giving the highest theoretical
yield of 4 mol H2/mol hexose, while low H2 yields are associated with more reduced end products, i.e. propionic and
lactic acids and ethanol.
However, mixed acid fermentation producing butyrate
in excess of acetate occurs upon biological degradation of
glucose by clostridia-type microora (Chen et al., 2001;
Mizuno et al., 2000; Fang and Liu, 2002). This genus produces hydrogen using the activities of pyruvate ferrodoxin oxidoreductase and hydrogenase enzymes. Sporeformer clostridia are selected from natural environments
by heat treatment (Lin and Chang, 1999; Lay, 2000; Chen
and Lin, 2001; Sung et al., 2002). Boiled anaerobically
digested sludge has been shown to give successful start-up
of continuous laboratory-scale bioreactors fed with glucose
(Mu et al., 2006). Hydrogen production by fermentative
bacteria is highly dependent on the conditions of the process, such as pH, hydraulic retention time (HRT) and gas
partial pressure, which aect the microbial metabolic balance and subsequently the fermentation end-products. In
general, the dominant metabolism in a mixed acidogenic
culture depends strongly on the pH of the microbial culture

(Lay, 2000) and hydrogen production is suppressed by

both low and high pH (Afschar et al., 1986; Ueno et al.,
1996; Chen et al., 2002; Lee et al., 2002). It has been
reported that maximum hydrogen yields are obtained when
the pH of the culture medium is between 5 and 6 (Zoetemeyer et al., 1982b; Lay, 2000; Fang and Liu, 2002). On
the other hand, Ren et al. (1997) stated that optimum
hydrogen production would occur at pH less than 5. Thus,
it is important to control the pH in order to maintain
satisfactory hydrogen production.
Apparently, fermentative hydrogen production (acidogenesis) process does not signicantly reduce the organic
content of the feed. Usually, chemical oxygen demand
(COD) removal is below 20% during hydrogen production
process, which corresponds to a mean hydrogen production of 2.5 mol/mol glucose. This can be removed in a subsequent anaerobic digestion step with the conversion of
organic content to methane. The present study focuses on
the exploitation of sweet sorghum biomass as a source
for hydrogen and subsequent methane production and
organic matter stabilization from the hydrogen reactor
euent. In general, biomass from energy crops, such as
sweet sorghum, can be used as raw material for biohydrogen production. Sweet sorghum is an annual C4 plant characterized by high photosynthetic eciency. It is a high
biomass yielding and rich on carbohydrates crop. Its stalks
mainly consist of sucrose that amounts up to 55% of dry
matter and of glucose (3.2% of dry matter). They also contain cellulose (12.4%) and hemicellulose (10.2%). Sweet
sorghum biomass is rich in readily fermentable sugars
and thus it can be considered as an excellent raw material
for fermentative hydrogen production. Overall, out of
many new crops that are currently investigated as potential raw materials for energy and industry, sweet sorghum
seems to be the most promising one (Dalianis et al.,
1996; Gosse, 1996). To date, ethanol and methane are
among the best-known microbial products produced from
sweet sorghum (Jackman, 1987; Richards et al., 1991; Christakopoulos et al., 1993; Lezinou et al., 1994, 1995;
Mamma et al., 1995, 1996). Specically, the energy yield
from ethanol obtained from the above referenced studies
ranged between 65008900 kJ/kg dry and 14002700 kJ/
kg fresh sorghum biomass, respectively (assuming that
the energy yield from ethanol is 26500 kJ/kg).
The present study concerns: (a) the fermentative production of hydrogen from the sugars contained in a sorghum
extract in a continuous stirred tank type bioreactor
(H2-CSTR) at various hydraulic retention times using an
indigenous mixed microbial culture, and (b) the subsequent
anaerobic treatment of the euent of the H2-CSTR with the
simultaneous production of methane in a continuous stirred
tank type reactor as well (CH4-CSTR). Furthermore, the
methane potential of the solids remaining after the extraction process is determined and the overall potential of sweet
sorghum biomass for hydrogen and methane production is
assessed. The sweet sorghum biomass used in the present
study was produced through biological cultivation.

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2. Experimental
2.1. Analytical methods
Determinations of soluble (after centrifugation at 3000
rpm for 10 min and ltration of the supernatant liquid)
chemical oxygen demand (COD), total (TSS) and volatile
(VSS) suspended solids and Kjendahl nitrogen were carried
out according to Standard Methods (APHA, 1975). For
total P determination, the persulfate digestion method
and the ascorbic acid method (APHA, 1975) were
employed. For the quantication of volatile fatty acids
(VFA) and ethanol (EtOH), 1 ml of sample acidied with
30 ll of 20% H2SO4 was analyzed on a gas chromatograph
(VARIAN CP-30), equipped with a ame ionization detector. The oven was programmed from 105 C to 160 C at a
rate of 15 C/min, and subsequently to 235 C (held for
3 min) at a rate of 20 C/min for VFA analysis and from
60 C (held for 1 min) to 230 C (held for 0.5 min) at a rate
of 45 C/min for ethanol analysis. Helium was used as the
carrier gas at 15 ml/min, the injector temperature was set at
175 C and the detector at 225 C and 200 C, for VFA and
ethanol determinations, respectively. The concentration of
lactic acid was measured on a liquid chromatograph (DIONEX DX300), equipped with an electron conductivity
detector and a Dionex IonPac column (AS11-HC, 4
250 mm Analytical). The eluent (sodium hydroxide solution) ow rate was 1.5 ml/min and the analysis was carried
out at 30 C. The produced gas composition in hydrogen
and methane was quantied with a gas chromatograph
(VARIAN STAR 3600) equipped with a thermal conductivity detector and a packed column with nitrogen as carrier gas. The injector, column and detector temperatures
were set at 70 C, 80 C and 180 C, respectively. The measurement of the produced gas volume was based on the displacement of acidied water. For the determination of
carbohydrates, a colored sugar derivative was produced
through the addition of L-tryptophan and sulfuric and
boric acids and subsequently measured colorimetrically at
520 nm (Joseson, 1983).
2.2. Feedstock
Fossil fuel equipments and agrochemicals (e.g. fertilizers
and pesticides) are usually used when applying conventional
farming methods and techniques for energy crops cultivation resulting to increased emissions of CO2 and nitrogen
oxides (Cook and Beyea, 2000). Therefore, alternative cultivation methods, such as organic or biological farming, need
to be used so that the cultivation of energy crops is emissions
neutral. The sweet sorghum biomass (Sorghum bicolor L.
Moench) used in the present study was produced in eld
experiments through biological farming techniques according to European Regulation EC 2092/91. The experiments
were conducted at the University of Patras experimental station (latitude 3825 0 N, longitude 218 0 E). Sweet sorghum
var. Keller seeds were sown at mid of May in plot rows

and the stalks were harvested at mid of October. The size

of plots was 7 7 m2. The distance between rows was
0.70 m and between plants on the same row 0.20 m (about
7 plants per m2). An amount of 145 mm water for irrigation
was applied by a drip irrigation system. For soil fertility
green manure from vetch (Vicia sativa L.) biomass was
incorporated in soil at mid of April. Several microorganisms
preparations diluted in tap water (CompeteR Plus, MycorRTree Injectable, Plant health Care, Inc. Pittsburg, Austria)
and potassium/magnesium fertilizer (Patentkali, Veterin)
were also added in soil about a month before the seeds were
sowed. Ten plants were sampled per 15 days and fresh and
dry weights of leaf and stalks were measured for biomass
productivity estimation. The sugar content of stalks was
measured by a refractometer after juice extraction.
2.3. Sorghum extraction process
After the harvesting of sorghum stalks, the fresh stems
were stripped from the leaves, the stalks chopped to a size
of 20 cm and were stored in the freezer at 20 C. Subsequently, these were milled by a laboratory grinder to an
average particle size of 12 mm. Extraction of free sugars
of the sorghum biomass was done in batches, by mixing
5 kg of milled sorghum stalks with 30 l of tap water for
1 h, at 30 C. After the extraction process a liquid fraction,
rich in soluble carbohydrates and a solid fraction were
obtained. The liquid fraction (sorghum extract) was preserved at 20 C and used as substrate for the hydrogen
and subsequent methane production experiments in the
CSTR-type bioreactors. The methane potential of the solid
fraction was determined in batch experiments.
2.4. Continuous experiments for biohydrogen production
A 500 ml active volume mesophilic (35 C) CSTR-type
digester (H2-CSTR) was started-up and fed with sorghum
extract. The reactor was cylindrical in shape, made of stainless steel and stirred periodically for 15 min, 2 times per
hour. For start-up, the reactor was lled up with sorghum
extract, operated anaerobically at a batch mode for 24 h in
order to activate the indigenous microora and was subsequently switched to the continuous mode at the designated
HRT. The reactor was operated anaerobically at hydraulic
retention times (HRT) of 24, 12, 8, 6 and 4 h and fed intermittently with sorghum extract maintained at a temperature
below 4 C for 1, 2, 3, 4 and 6 min, respectively, every 3 h.
Feeding was programmed always with the stirring on. Simultaneous ow of the euent occurred during feeding by liquid
overow, in order to maintain constant reactor volume. The
initial concentration of carbohydrates was calculated for
every feeding cycle according to the following equation:
Q

S S 0  S 0  S in  eV t

where S is the resulting concentration when feeding was

completed, S0 is the inuent concentration, Sin is the concen-

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2.6. Batch experiments for methane potential determination

Batch experiments were carried out in duplicates at
35 C in 160 ml serum vials, in order to determine the
methane potential of the solid fraction obtained after the
extraction process. Solids were dried at 105 C, pulverized
and diluted with tap water at a nal concentration of 4 g
total solids/l (solution A). 30 ml mixed liquor from the
methanogenic reactor was used as inoculum and 20 ml of
solution A were added as substrate. The content of the
vials was gassed with a gas mixture of N2/CO2 (80/20) in
order to secure anaerobic conditions. The vials were sealed
with butyl rubber stoppers and aluminum crimps and
methane production was monitored versus time.
3. Results and discussion
3.1. Sweet sorghum biomass
Biomass and stalk sugar productivity during crop development are shown in Fig. 1. The results showed that stalks

The average characteristics of sorghum extract obtained

from the dierent extraction batches are presented in Table
1. No ethanol or volatile fatty acids were detected. It is
noticeable that the liquid fraction obtained from the
extraction process consisted mainly of soluble carbohydrates, which are an ideal substrate for the fermentative
hydrogen production. The concentration of nitrogen and
phosphorus were too low to support microbial growth
and thus the sorghum extract was supplemented with urea
and KH2PO4 to make up for nutrient deciency, as mentioned in the previous section.
3.3. Results of the continuous experiments for hydrogen
production in the H2-CSTR
No traces of methane were detected during the operation of the hydrogenogenic reactor at any time. In Table
2 the main characteristics of the reactor at each steady state
are presented, while the corresponding biogas and hydrogen production rates and hydrogen yields are presented
in Table 3. The glucose conversion eciency was greater
than 95% at all HRTs. The biogas and hydrogen produc-

2000

dry biomass productivity (experimental)

dry biomass productivity (Boltzmann sigmoidal fit)
sugar productivity (experimental)
sugar productivity (Boltzmann sigmoidal fit)

200
-2

A 3 l active volume mesophilic (35 C) CSTR-type digester (CH4-CSTR) was started up using anaerobic sludge and
fed with the euent of the hydrogenogenic reactor. The
digester had the same structural characteristics with H2CSTR and was operated anaerobically at a hydraulic retention time of 20 days. The reactor was fed for 1 min every
8 h. It must be noted, however, that the hydrogenogenic
and the methanogenic reactor were not connected; the euent of the H2-CSTR was collected, homogenized and preserved at 20 C before use. The reactor performance
(biogas production and composition in CH4, pH, soluble
COD and VFA concentration) was monitored 34 times a
week and complete characterization of the reactor euent
was made when steady state was reached.

3.2. Characterization of sorghum extract

Sugar productivity (g m )

2.5. Continuous experiments for methane production

are mature for cropping early in October about 110 days

after plant emergence. In this period the fresh biomass
yield was 91 ton/ha.

-2

tration when feeding started, namely the concentration measured at the end of each cycle, Q is the volumetric feeding
rate, V is the reactor volume and t is the duration of feeding.
2.24 g NaOH and 6.8025 g KH2PO4 per liter of sorghum
extract were added in order to maintain the pH of the
H2-CSTR at levels (4.75.5) allowing for hydrogen production. Also 2 g urea (NH2CONH2) per liter of sorghum
extract was added to make up for N deciency of the feed.
Gas and liquid samples were taken 1015 min before feeding started. The reactor performance (biogas production
and composition in H2, pH, carbohydrates, soluble COD,
ethanol, butanol, lactic acid and VFA concentration) was
monitored daily throughout the experimental period. Gas
samples were analyzed for methane daily, in order to monitor whether methane production was taking place. Complete characterization of the reactor euent was made
once a steady state was reached. Steady-state here meant
constant conditions between successive feeding cycles.

Dry biomass productivity (g m )

1500
150
1000
100

500

50

0
0
160 170 180 190 200 210 220 230 240 250 260 270 280 290
Julian days

Fig. 1. Dry biomass and sugar productivity during crop development.

Table 1
The main characteristics of sorghum extract
Characteristics

Value

pH
TSS (g/l)
VSS (g/l)
Total COD (g/l)
Soluble COD (g/l)
Soluble carbohydrates (g/l)
Total Kjendhal nitrogen (g/l)
Total phosphorus (g/l)

7.5 0.5
1.98 0.27
1.87 0.35
18.5 2.5
17.5 2.0
17.0 2.0
0.025
0.035

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Table 2
The characteristics of the H2-CSTR at steady state
HRT
(h)

pH

TSS
(g/l)

VSS
(g/l)

Glucose
(mg/l)

Eciency of glucose
consumption (%)

24
12
8
6
4

5.5
5.3
4.8
4.9
4.8

8.5
5.5
10.7
12.8
22.0

6.4
4.1
8.8
10.2
16.5

110 20
470 70
680 110
450 20
290 70

99.4
97.4
95.3
97.6
97.9

tion rate systematically increased, when the HRT was

decreased from 24 to 6 h, where the higher hydrogen production rate was obtained (2550 90 ml H2/d). Further
decrease of the HRT to 4 h led to a reduction of the overall
biogas and the hydrogen production rates. In general, the
biogas composition in hydrogen always lied between 30%
and 40% and was higher (40%) for the HRTs of 12, 8,
and 6 h compared to those at 24 and 4 h (30% and 35%,
respectively). Moreover, the yield of hydrogen produced
per mole of glucose consumed and, consequently, that of
hydrogen produced per kg of sweet sorghum as well (Table
3), reached the highest value at the HRT of 8 h, amounting
up to 0.86 0.04 mol H2/mol glucose consumed and 10.4 l
H2/kg sweet sorghum, respectively. That corresponds to an
energy yield of 104 kJ/kg fresh and 400 kJ/kg dry sorghum
biomass, respectively (assuming that the energy yield from
hydrogen is 122 000 kJ/kg). In general, the hydrogen yield
was within the range of 0.370.86 mol H2/mol glucose consumed, signicantly lower than the theoretical yield of
4 and 2 mol H2/mol glucose for acetic and butyric acid
production, respectively. In general, the decreased

hydrogen yield is due to the competing reactions shown

in Eqs. (4)(10) and the obtained values are comparable
to those reported in relevant studies with other feed materials, as shown in Table 4. At this point it should be emphasized that the start-up of the reactor was made with
indigenous sweet sorghum microora without heat treatment, which generally favors the spore-forming, hydrogen-producing clostridia. The fact that the sorghum
microora can be used for the production of hydrogen is
important for the operation of a full-scale plant suggesting
that no extra energy will be required neither for the startup (heat treatment) of the reactor nor for the pasteurization or sterilization of the inuent. Based on the above
observations on the hydrogen yield and the production
rate, the optimal HRT seems to be between 12 and 6 h.
The concentrations of the main metabolic products measured at each steady state are presented in Fig. 2. The dominant metabolic products were butyric and acetic acids,
with the production of butyric acid being 1.53 times
higher than that of acetic acid. The concentration of butyric acid was the highest (5780 mg/l) at the HRT of 12 h
while at the HRT of 8 h, the highest concentration of acetic
acid was obtained (3480 mg/l). The concentration of propionic acid increased with the HRT, while ethanol production was favored at the HRT of 6 and 4 h, reaching the
highest concentration of 634 mg/l at the HRT of 4 h. The
concentration of lactic acid decreased with increased
HRT until no lactic acid was detected at the HRT of
24 h. This could be attributed to either a dierent distribution of metabolic products at dierent HRT, or the accumulation of lactic acid due to kinetic limitation of its

Table 3
Biogas and hydrogen production rates and hydrogen yields in the H2-CSTR at steady state accompanied by their standard deviation
HRT
(h)

Biogas composition
in H2 (%)

Biogas production
rate (ml/d)

Hydrogen production
rate (ml/d)

Hydrogen yield (mol H2/mol

glucose (consumed))

Hydrogen yield (l H2/kg sweet

sorghum biomass)

24
12
8
6
4

30.4 1.2
39.9 1.2
40.5 1.9
39.2 0.5
35.0 1.5

1490 130
4260 180
5160 120
6510 200
6180 400

410 40
1740 100
2070 120
2550 90
2180 150

0.37 0.02
0.86 0.04
0.75 0.05
0.70 0.02
0.41 0.02

4.9
10.4
8.4
7.6
4.3

Table 4
Hydrogen productivity of dierent mesophilic mixed cultures fed with sugar-based medium in continuous suspended growth systems
Study

Fang and Liu (2002)

Lin and Chang (1999)
Lin and Chang (2004)
Ueno et al. (1996)
Mizuno et al. (2000)
(with sparging)
Mizuno et al. (2000)
(non-sparging)
Gavala et al. (2006)
Present

Operating conditions

Reactor characteristics and eciency regarding hydrogen production

HRT
(h)

Organic loading rate

(mmol glucose/l/d)

pH

VSS (mg/l)

% H2

l H2/l/d

mmol H2/mmol
glucose

6
6
6
12
8.5

155.5
416
416
109.45
150.11

5.5
5.7
6.2
6.8
6

780
1270
1670
1590
1060

64
43.1
42.6
64
22.9

4.6
15.9
8.0
4.4
4.8

2.1
1.7
1.4
2.6
1.4

8.5

150.11

1450

53.4

3.0

0.9

233
188.89

5.1
5.3

680
4140

42.2
39.9

4.4
3.6

1.6
0.9

6
12

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acetic acid
propionic acid
butyric acid
ethanol
lactic acid

6000
5500
5000

mg products /L

4500

In general, production of acetic and butyric acid favors

the production of hydrogen, according to Eqs. (2) and (3)
while production of propionic acid consumes hydrogen
(Eq. (4)):
Acetic acid production

4000

C6 H12 O6 + 2H2 O ! 2CH3 COOH + 2CO2 + 4H2

3500
3000

Butyric acid production

2500

C6 H12 O6 ! CH3 CH2 CH2 COOH + 2CO2 + 2H2

2000
1500

Propionic acid production

1000

C6 H12 O6 + 2H2 ! 2CH3 CH2 COOH + 2H2 O

500
0
24h

12h

8h

6h

4h

Fig. 2. The main metabolic products in the hydrogenogenic reactor,

H2-CSTR, at the dierent steady states.

consumption to acetic and/or propionic acids. Butanol,

valeric, isovaleric and isobutyric acids were not detected
at all during fermentative hydrogen production from sweet
sorghum extract in the CSTR at all steady states reached.
In Fig. 3, the measured and calculated COD concentrations of the reactor content at each steady state are presented. Calculated COD concentration represents the sum
of COD of dierent products (hydrogen, ethanol, lactic,
butyric, propionic and acetic acids) as well as the COD
of non-consumed carbohydrates measured as glucose units.
The remaining COD value after subtracting the calculated
COD from the measured COD concentration corresponds
to non-identied metabolic products during glucose fermentation. The remaining COD represented only a very
small percentage of the COD measured (Fig. 3) at HRTs
in the range of 412 h. Therefore, it can be assumed that
the main metabolic products of sweet sorghum sugars fermentation were the compounds already measured. On the
other hand, at the HRT of 24 h, the remaining COD
amounted up to 15% of the COD measured, suggesting
that there was another metabolic product not detected at
that HRT.

Lactic acid is produced from glucose via three metabolic

pathways, the homofermentative (Eq. (5)), the heterofermentative (Eq. (6)) and the bidum pathway (Eq. (7)).
In all three pathways the hydrogen balance is zero, i.e.
no hydrogen is consumed nor produced. The same is for
ethanol production, where the hydrogen balance is zero
as well (Eq. (8)).
Homofermentative pathway
C6 H12 O6 ! 2CH3 CHOHCOOH

Heterofermentative pathway
C6 H12 O6 ! CH3 CHOHCOOH + CH3 CH2 OH + CO2
6
Bifidum pathway
2C6 H12 O6 ! 3CH3 COOH + 2CH3 CHOHCOOH

Ethanol production
C6 H12 O6 ! 2CH3 CH2 OH + 2CO2

A ow chart showing the dierent reaction pathways discussed in the present study is presented in Fig. 4. Based
on the above equations and on the metabolic products
measured the anticipated hydrogen production rate was
calculated in the H2-CSTR, i.e. 2 mmol of hydrogen per
1 mmol of acetic acid produced, 2 mmol of hydrogen per
1 mmol of butyric acid produced and minus 1 mmol of
hydrogen per 1 mmol of propionic acid produced. The proCOD - products
COD- calculated

18000
16000

COD (mg/L)

14000
12000
10000
8000
6000
4000
2000
0
24

12

8
HRT (h)

Fig. 3. COD measured (COD-products) and calculated at dierent steady states of H2-CSTR.

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GLUCOSE

(eq. 4)
C6H12O6 + 2H2 2CH3CH2COOH + 2H2O
GLYCERALDEYDE
PHOSPHATE

PROPIONIC ACID

[PYRUVIC ACID]

[ACETYL-CoA]

(eq. 8)
C6H12O6 2CH3CH2OH + 2CO2
ETHANOL

(eqs. 5, 6, 7)

(eq. 3)

C6H12O6 2CH3CHOHCOOH
C6H12O6 CH3CHOHCOOH + CH3CH2OH + CO2
2C6H12O6 2CH3CHOHCOOH + 3CH3COOH

C6H12O6
CH3CH2CH2COOH + 2CO2 + 2H2
BUTYRIC ACID

LACTIC ACID

H2 + CO2

(eq. 9)
3CH3CHOHCOOH
2CH3CH2COOH + CH3COOH + CO2 + H2O

(eq. 10)

(eq. 2)
C6H12O6 + 2H2O
2CH3COOH + 2CO2 + 4H2

PROPIONIC ACID +
ACETIC ACID

4H2 + 2CO2
CH3COOH + 2H2O

ACETIC ACID

Fig. 4. Flow chart showing the dierent reaction pathways of glucose.

duction rates of acetic, propionic and butyric acids and the

theoretically calculated hydrogen production rate, rHcalc,
compared to the experimentally measured hydrogen production rate, rHexp, are shown in Table 7. It was observed
that the rHexp is much lower than the rHcalc. Subsequently
and assuming that the production of acetic and propionic
acids proceeded through lactic acid fermentation according
to Eq. (9), the anticipated hydrogen production rate was
calculated taking into account that 2 mmol of hydrogen
per 1 mmol of butyric acid are produced. Again, the rHexp
is lower than the rHcalc. An explanation could be that
microorganisms producing acetic acid with hydrogen consumption (Eq. (10)) have been established in the system
and thus consuming a considerable amount of the produced hydrogen:
3CH3 CHOHCOOH
! 2CH3 CH2 COOH + CH3 COOH + CO2 + H2 O
4H2 + 2CO2 ! CH3 COOH + 2H2 O

9
10

3.4. Results of continuous experiments for methane

production in the CH4-CSTR
The mean values of the main characteristics of the inuent of the anaerobic digester after collection of the euent
of the H2-CSTR, homogenization and preservation at
20 C are presented in Table 5. The inuent of the methanogenic reactor was rich in volatile fatty acids, as it was
anticipated, with the concentration of soluble carbohydrates being almost negligible compared with the soluble
COD concentration. The digester was operated at an

Table 5
The main characteristics of the inuent of the CH4-CSTR
Characteristic

Value

pH
TSS (g/l)
VSS (g/l)
Soluble COD (g/l)
Soluble carbohydrates (g COD/l)
Volatile fatty acids (g COD/l)

4.7 0.5
1.9 0.1
1.5 0.1
15.9 1.5
0.9 0.3
12.2 2.0

HRT of 20 days for more than 120 days (which correspond

to six hydraulic retention times), with an inuent ow rate
of 150 ml/d. The characteristics of the CH4-CSTR at
steady state are presented in Table 6. Fig. 5 shows the evolution of biogas and methane throughout the experimental
period. The reactor performance was very stable with
acetic and butyric acids at low levels, while ethanol and
propionic and lactic acids were not detected at all. The percentage of COD removal was approximately 97%, implying
that the performance of the CSTR is not kinetically limited. This implies that it should be possible to reduce someTable 6
The characteristics of the CH4-CSTR at steady state accompanied by their
standard deviation
Characteristic

Value

pH
Alkalinity, mg CaCO3/l
Biogas production (l/d)
% in CH4
Soluble COD (mg/l)
Acetic acid (mg/l)
Butyric acid (mg/l)

7.5 0.1
6650 50
1.14 0.12
64
560 170
50 10
40 5

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G. Antonopoulou et al. / Bioresource Technology xxx (2007) xxxxxx

1600

biogas
methane

Production rate, ml/d

1400
1200
1000
800
600
400
200
0
0

20

40

60

80

100

120

140

160

180

Time, d

Fig. 5. The evolution of biogas and methane in the CH4-CSTR

throughout the experimental period.

what the HRT, without loss in performance. The methane

production rate reached 0.73 l CH4 per day giving a yield
of 4.9 l methane per l of inuent. This latter corresponded
to a yield of 29 l CH4/kg sweet sorghum, considering that
5 kg of sweet sorghum biomass were initially mixed with
30 l of water. The corresponding energy yield is 950 kJ/kg
fresh and 3660 kJ/kg dry sorghum biomass, respectively
(assuming that the energy yield from methane is
50120 kJ/kg).
3.5. Results of batch experiments for methane potential
determination
The methane production during the batch experiment for
the determination of the methane potential of the remaining
45

with solids
control without
solids

40

CH4, ml

35
30
25
20
15
10
5
0
0

10

12

14

16

Time, d

Fig. 6. Methane production during the batch experiment for the determination of the methane potential of the solid fraction after the extraction
process.

solids after the extraction process is shown in Fig. 6. The

calculated methane potential of the solid residues, after subtracting the control values is 11 mmol CH4/g TS. This yield
corresponds to the production of 78 l CH4/kg of sweet
sorghum and to an energy yield of 2560 kJ/kg fresh and
9845 kJ/kg dry sorghum biomass, respectively. The methanogenic potential of the solid residues after the extraction
process is much higher (more than double) than that of
the H2-CSTR euent. Moreover, a pre-treatment process
aiming at the increase of the bioavailability of the solid
organic matter could precede the anaerobic digestion of
the solid fraction resulting at an even higher methane recovery. This issue is currently under investigation.
4. Conclusions
The present study investigated the fermentative hydrogen production from the sugars of sweet sorghum extract
as well as the subsequent methane production from the
euent of the hydrogenogenic process and the methane
potential of the remaining solids after the extraction process. It has been proved that continuous fermentative
hydrogen production from sweet sorghum extract is possible and stable using the indigenous microora without a
pre-heating step. The highest biogas and hydrogen production rate (6500 ml biogas/d and 2550 ml H2/d, respectively)
was obtained at the HRT of 6 h while the highest yield of
hydrogen produced per kg of sorghum biomass was
achieved at the HRT of 12 h (10.4 l H2/kg sweet sorghum).
In order to decide for the optimal HRT, for a full-scale
plant, is necessary to take into account both economic
(based on hydrogen production rate) and technical (based
on hydrogen yield) aspects.
Moreover, the present study showed that sweet sorghum
extract could be used for hydrogen and methane production in a two-stage process. It has been proved that the
euent from the hydrogenogenic reactor is an ideal substrate for methane production with approximately 107 l
CH4/kg sweet sorghum, 78 l of which come from the solid
residues. Continuous methane production of the H2-CSTR
euent yielded 29 l CH4/kg of sweet sorghum, while the
methanogenic potential of the solid residues after the
extraction process yielded another 78 l CH4/kg sweet
sorghum.

Table 7
Production rate of acetic, propionic and butyric acids and the theoretically calculated hydrogen production rate compared with the experimentally
measured hydrogen production rate in the H2-CSTR
HRT
(h)

24
12
8
6
4

Production of acetic and butyric

acids (mmol/d)

Hydrogen production (mmol/d)

Acetic
acid

Propionic
acid

Butyric
acid

Theoretically calculated taking into

account all acids production

Theoretically calculated taking into

account only the butyric acid production

Experimentally
measured

19.93
30.98
86.99
68.27
95.36

4.04
3.34
1.70
2.05
4.91

26.87
65.66
93.52
127.68
186.38

89.55
189.94
359.31
389.85
558.56

53.73
131.31
187.03
255.35
372.76

18.35
77.59
92.37
113.75
97.37

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To sum up, this work demonstrated that biohydrogen

production can be very eciently coupled with a subsequent step of methane production and that sweet sorghum
could be an ideal substrate for a combined gaseous biofuels
production.
Acknowledgements
The authors thank the General Secretariat for Research
and Technology for the nancial support of this work
under PENED 2001, 01ED390.
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