Among various processes of biological hydrogen production, anaerobic fermentation,
commonly termed dark fermentation, has several advantages over other methods.
Apart from its simple technology, it can utilize a wide range of substrates,
both pure as well as waste products and does not rely on light availability.
However, the major bottleneck of the dark fermentation process stems from a
lower achievable yield of hydrogen per mole of substrate (Das
and Iu, 2001). The pathways and experimental evidence cited in the literature
reveal that at most four moles of hydrogen are produced from each mol of glucose
consumed during acetate fermentation, as shown in Eq. 1 (Hawkes
et al., 2002; Nath and Das, 2004). The theoretical
maximum hydrogen yield from dark fermentation of glucose is 4 moles H2
(mol glucose)-1 if acetic acid was only Volatile Fatty Acid (VFA)
formed (Yokoi et al., 2001; Liu
et al., 2003; Liu and Shen, 2004; Lay,
A combination of dark and light fermentations, as shown in Eq. 1 and 2, could be expected to reach as close to the theoretical maximum production of 12 moles of H2 (mol glucose)-1 equivalent as possible, according to the following reactions:
Stage 1: Dark fermentation by CSN1-4 (anaerobes)
II: Photo-fermentation by RS8253 (photosynthetic bacteria)
Some studies have combined dark fermentation with photofermentation to enhance
hydrogen productivity from food processing waste water and sewage sludge. One
way to overcome the economic restriction of biological hydrogen production is
to associate this process with waste treatment, as practical applications of
hydrogen production cannot utilize expensive synthetic culture media (Yokoi
et al., 2002; Han and Shin, 2004).
Renewable resources, such as biomass and agricultural and domestic wastes,
are attractive raw materials for bio-hydrogen production on a large scale. Sequential
dark and light anaerobic fermentations are used for bio-hydrogen production
from carbohydrate-rich biomasses or waste materials. The first step is the enzymatic
hydrolysis of the biomass to a highly concentrated sugar solution, followed
by dark fermentation by acetogenic-anaerobic organisms to produce (VFA), hydrogen
and CO2, the organic acids produced by dark fermentation can be further
fermented by the photo-heterotrophic bacteria such as Rhodobacter spp.
to produce CO2 and H2 (Kapdan and
Karig, 2006; Manish and Banerjee, 2008; Kotay
and Das, 2008; Ni et al., 2006).
In present study, Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564; CSN1-4) and Rhodobacter sphaeroides NCIMB 8253 (RS8253) were used in a two-stage fermentation process for maximization of hydrogen yield via combined dark and photo-fermentation processes, respectively. Glucose was fermented by CSN1-4 in the first stage to produce acetate and then consumed by RS8253 to produce hydrogen.
MATERIALS AND METHODS
Microorganisms and culture conditions: A stock culture of CSN1-4 was
obtained from the culture collection maintained at the Biotechnology Laboratory,
Chemical and process Engineering Department, UKM (Cirilo
et al., 2008). The stock culture was grown on TYA agar medium, that
containing 40 g glucose, 2 g yeast extract, 6 g Bacto-Tryptone, 3 g ammonium
acetate, 10 mg FeSO4.7H2O, 0.5 g KH2PO4,
0.3 g MgSO4•7H2O and added 15 g agar Bacteriological
(No.1) per litre of distilled water. Single colonies (three to five) of CSN1-4
from TYA agar culture were transferred anaerobically into 10 mL 15% potato-glucose
(PG) medium (PG containing; 150 g fresh mashed potato, 0.5 g (NH4)2SO4,
10 g glucose and 3 g CaCO3) and these were incubated anaerobically
at 37°C for 30 h. A 1 mL aliquot of this culture was transferred into 9
mL of fresh PG medium, which was incubated in the same conditions at 37°C
for 18 h and used as inoculum (Kalil et al., 2003).
For photo-fermentation, RS8253 culture stock was obtained from the culture collection maintained at the Biotechnology Laboratory, Chemical and process Engineering Department, UKM. The organism was cultured in modified Biebl and Pfennig growth medium for the hydrogen production experiment. The amount of inoculum used in the hydrogen production medium was 10% by volume and the bacterium was anaerobically grown in liquid medium at 32°C under a light intensity of 5.2±0.5 klux with a tungsten lamp (100 W) at a distance of 25 cm as the light source from one face. The initial pH of the growth medium was maintained to 6.8±0.2 after was dropped from first stage. Argon gas was used to create anaerobic conditions.
Experimental procedure: Dark fermentation was carried out at 37°C
in a 500 mL Duran bottle. At the top of the bottle, there was an inlet for the
medium and outlets for gases. CSN1-4 was grown in PG for 30h and used as the
inoculum. The inoculum (10% v/v) was transferred into fermentation medium (TYA)
containing 40 g glucose, 2 g yeast extract, 6 g Bacto-Tryptone, 3 g ammonium
acetate, 10 mg FeSO4.7H2O, 0.5 g KH2PO4
and 0.3 g MgSO4.7H2O per litre of distilled water. The
culture pH was not controlled during fermentation. The initial pH of the TYA
medium was adjusted to 6.0±0.2 with 5 M NaOH and sterilized at 121°C
for 15 min (Alalayah et al., 2008).
The fermentation temperature was maintained at 37°C using a water bath.
The evolved gas mixture from the reactor, containing both hydrogen and carbon
dioxide (Kaushik et al., 2006), was passed through
a 5 M KOH solution to absorb most of the carbon dioxide (Wooshin
at al., 2006) and then analyzed by Gas Chromatography (GC).
Photo-fermentation was performed in another vessel of the same dimensions with a light source as the photo-bioreactor. The fermentation temperature was maintained at 32°C. Illumination was provided by 100 W tungsten lamps, with a distance of 25 cm from one face. The initial pH of the growth medium was maintained to 6.8±0.2. For the hydrogen production study, the spent medium from the first stage was centrifuged in some run and not centrifuged in others, before inoculation with 10% (v/v) RS8253 to study the effect of bacteria removed on fermentation processes. The evolved gas from the headspace of each of the reactors was channeled through a 5 M KOH solution, which stripped most of CO2 from the gas stream.
Analytical method: The composition of the gas liberated from fermentation processes was analysed using gas chromatography (SRI 8610C, USA) with a helium ionization detector. The temperatures of the oven, injector and detector were 50, 100 and 150°C, respectively. The cell biomass concentration was estimated as the Dry Cell Weight (DCW) by measuring the optical density at 660 nm and then related the optical density to the DCW. The cells were centrifuged at 10,000 (rpm) for a period of 15 min in a refrigerated centrifuge (Kubota-5220, Japan).
The reducing-sugar (glucose) content of the medium was estimated using the
Miller method (Miller, 1959). The glucose concentration
in the medium was measured using the 3, 5-dinitrosalicylic acid (DNS) assay
for total reducing sugars. A 1 mL aliquot of the sample and 2 mL aliquot of
the DNS reagent mixture were combined in a test tube. The mixtures were placed
in a boiling water bath for 5 min and then diluted with 10 mL of distilled water.
The absorbance at 550 nm was recorded for all samples and the glucose concentration
was calculated from a standard calibration curve.
Dark fermentation using CSN1-4: Results showed the highest yield of
hydrogen was observed when the initial glucose concentration was 10 g L-1
and then decreased with increasing glucose concentration as indicated in (Table
1). The value of hydrogen yield was based on a glucose utilization of 81.8%.
Effect of initial glucose concentration on hydrogen production
by CS N1-4
effects of light on growth of CSN1-4 in fermentation
effects of light on H2 production by CSN1-4
Effect of light on H2 production in the first stage with CSN1-4:
The results show the maximum yield of hydrogen production by CSN1-4 was at the
initial pH of 6±0.2, 10 g L-1 initial glucose and 37°C
in the dark stage. The yield of hydrogen was 3.10 mol H2 (mol glucose)-1
in the dark fermentation Fig. 1 and 2, while
at the same conditions of temperature, initial pH and initial glucose concentration,
but in the presence of light, the maximum yield of hydrogen was 1.40 mol H2
(mol glucose)-1, the higher specific growth rate and doubling time,
as determined by the biomass concentration measured by OD660 nm. This indicates
that the light reduced the growth and therefore the hydrogen production. This
reduction in growth is likely due to the increased temperature, as previously
reported (Alalayah et al., 2009).
Effects of initial nitrogen sparging on biomass concentration
and hydrogen production using CSN1-4 in batch reactor
of biomass and H2 production by removed CSN1-4 and no autoclave
Effect of nitrogen sparging on H2 production in the first stage: The effects of sparging the fermentation system with nitrogen were studied for dark and light fermentations to ensure a total removal of oxygen. It was observed that the biomass concentration and hydrogen production at an initial pH of 6±0.2, initial glucose concentration of 10 g L-1 and 37°C were increased by sparging with nitrogen (Table 2).
Combination of dark and photo-fermentation: Spent medium from the glucose-fed hydrogen fermentation by CSN1-4 exhibited a high total organic acid concentration, composed predominantly of acetic acid, together with a small amount of butyric acid and ethanol. The spent medium was used for photo-production of hydrogen in the second stage by RS8253 using different route, irrespective of its composition and with a separation of the accompanying metabolites. A volume of inoculum 10% (v/v) of light-grown RS8253 cells were transferred into the spent medium of the dark fermentation. The culture was incubated anaerobically at 32°C with light at 5.8 klux intensity.
Effect of removing CSN1-4: The yield of hydrogen in the second stage was 0.4 mol H2 (mol acetic acid)-1, as shown in Fig. 3. The spent medium used for photo-fermentation in a subsequent second stage was adjusted to 32°C and pH 6.8±0.2. Acetic acid was presumably the sole substrate for hydrogen production in the second stage.
Effect of co-existing CSN1-4 and RS8253: From the results in this experiment,
the yield of hydrogen in the second stage was 0.6 mol H2 (mol acetic
acid)-1 under the first stage conditions of 37°C, pH 6.0±0.2
(Fig. 4). Without adjusting the conditions, light was added
after 48 h to allow RS 8253 to grow and produce hydrogen. The overall yield
of hydrogen in the two-stage process, based on glucose as the initial substrate,
was higher than the single-stage process by 0.6 mol H2/spent media.
of biomass and H2 production with CSN1-4 and RS 8253 were co-fermentation
of H2 production without condition adjustments
Effect of adding RS 8253 to the CSN1-4 culture: In this experiment,
the spent medium from the first stage was used for photo-fermentation in the
second stage by adding RS8253 to the existing CSN1-4 fermentation without any
adjustment to the conditions. The overall yield increased by 0.5 mol. H2
(mol acetic acid)-1 as presented in Fig. 5.
Effect of autoclaving the CSN1-4 culture prior to photo-fermentation:
Here, the CSN1-4 dark fermentation effluent was autoclaved, followed by addition
of inoculum of RS 8253 culture. The yield of hydrogen in the second stage was
1.10-1.25 mol H2 (mol acetic acid)-1 (Fig.
6). The medium used for photo-fermentation in the second stage was first
adjusted to pH 6.8±0.2 and 32°C. The overall yield of H2from
glucose by the two-step process of dark and photo-fermentation increased two
folds compared to the dark process alone.
of biomass and H2 production by autoclaving CSN1-4 before the
addition of RS 8253
Dark fermentation in first stage was studied using TYA medium containing glucose
as the principal substrate. In dark fermentation, the initial medium pH of 6.0±0.2
and a temperature of 37°C were optimal for producing hydrogen by CSN1-4;
these parameters were maintained during second stage fermentation. Most of the
light fermentation studies were performed with pure volatile fatty acids (VFAs),
such as acetic or butyric acids, using different pure Rhodobacter species
(Kotay et al., 2008; Shi
et al., 2004). However, there have been a limited number of light
fermentation studies on dark fermentation effluents for bio-hydrogen production
(Shi and Yu, 2004; Oh et al.,
2004; Asada et al., 2006).
Since present study carried out photo-fermentation on spent broth of CSN1-4.
Most of the reported studies on light fermentations employed single pure Rhodobacter
culture and no systematic investigations have been reported on the use of different
Rhodobacter species or their mixtures for bio-hydrogen production from
dark fermentation effluents. Its well known that glucose is the easiest
monosaccharide to be used as energy source. It has already been reported that
substrate inhibition gets predominant at higher glucose concentration because
this modifies the metabolic pathways (Fabiano and Perego,
2002; Oh et al., 2003).
The lower H2 production indicates that the carbon flux at high glucose
concentrations is more directed to the production of reduced by-products such
as ethanol and/or organic acids (Oh et al., 2003).
Since alcohol production involves the consumption of hydrogen in the form of
reducing equivalents such as NADH, it is inevitable that fermentation conditions
that favour the metabolism of sugar to alcohols reduce hydrogen production.
Niel et al. (2003), also studied substrate and
product inhibition of hydrogen production during sucrose fermentation by the
thermophilic bacteria Caldicellulosiruptor saccharolyticus. They found
hydrogen was a strong inhibitor, when allowed to accumulate in the culture.
However, the extent of inhibition by hydrogen was dependent on the density of
the culture. Without the initial nitrogen sparging, lower hydrogen production
and biomass concentration were observed. Since, CSN1-4 is an anaerobic bacterium,
precautions were taken to control the presence of oxygen in the culture after
medium sterilization and during inoculation (Kaushik et
Other investigators have used various strategies to enhance the anaerobic fermentation
environment, including using an anaerobic chamber for inoculation and maintaining
a constant stream of nitrogen gas over the culture headspace during fermentation,
or until the microorganism is capable of producing enough metabolic gases to
fill the culture headspace. It was described from the results that the spent
medium was used for photoproduction of hydrogen in the second stage light grown
cells of RS8253, were inoculated in the spent medium of dark fermentation (Nath
et al., 2005).
Acetic acid was presumably the sole substrate for hydrogen production in the second stage. However, estimation of total yield of hydrogen in the two stage process was done on the basis of initial glucose content, since glucose (present in TYA media of dark fermentation) was converted to acetic acid and no additional substrate was supplied therein.
The overall yield of hydrogen in the two-stage process, based on glucose as
the initial substrate, was higher compared to the single-stage process, as also
reported in a previous study with both dark and photo-fermentation using another
strain of Rhodobacter (Oh et al., 2004).
The two-step process of dark and photo-fermentation can increase the overall
yield from glucose to H2 by two fold compared to the dark process
alone. The estimation of the total hydrogen yield in the two-stage process was
done on the basis of the initial glucose content, since glucose (present in
TYA media of dark fermentation) was converted to acetic acid and no additional
substrate was supplied therein.
The overall hydrogen yield from RS8253 in a second, photo-fermentation stage
was increased by using spent medium from a dark fermentation by CSN1-4 in a
two-stage batch fermentation process. The yield of hydrogen in the first stage
was about 3.10 mol H2 (mol glucose)-1 at an initial glucose
concentration of 10 g L-1, an initial pH of 6±0.2 and a temperature
of 37°C and the trace of light and nitrogen sparging were cleared on hydrogen
yield and reported in this work. The highest yield of hydrogen in the second
stage was about 1.10-1.25 mol H2 (mol acetic acid)-1 when
the conditions were adjusted to pH 6.8±0.2 and 32°C, with the spent
CSN1-4 culture autoclaved prior to photo-fermentation. The overall yield of
hydrogen in two-stage process, based on glucose as the substrate, was higher
than the single-stage process.
The authors thank Prof. Dr. Yoshino Sadazo, Kyushu University, Japan, who provided us with CSN1-4. This research was supported by the UKM- GUP-KPB-08-32/128 grant.