Professional Documents
Culture Documents
Bio Hydrogen Production
Bio Hydrogen Production
fikret.kargi@deu.edu.tr, ilgi.karapinar@deu.edu.tr
ABSTRACT
The major biological processes utilized for hydrogen gas production are bio-photolysis of water by
bacteria. Sequential dark and photo-fermentation process is a rather new approach for biohydrogen
production. One of the major problems in dark and photo-fermentative hydrogen
production is the raw material cost. Carbohydrate rich, nitrogen deficient solid wastes such as
agricultural and food industry wastes and some food industry effluents such as cheese whey, olive
mill and baker’s yeast industry wastewaters can be used for hydrogen production by using suitable
inexpensive energy generation with simultaneous waste treatment. This review article summarizes
bio-hydrogen production from some waste materials. Types of potential waste materials,
bioprocessing strategies, microbial cultures to be used, bio-processing conditions and the recent
1. INTRODUCTION
Hydrogen is considered as a viable alternative fuel and “energy carrier” of future. It is a clean fuel
with no CO2 emissions and can easily be used in fuel cells for generation of electricity. Demand on
hydrogen is not limited to utilization as a source of energy which may be used as raw material for
industry. Recent reviews on hydrogen indicated that the worldwide need on hydrogen is increasing
with a growth rate of nearly 10% per year for the time being (Winter, 2005) and contribution of
hydrogen to total energy market will be 8-10% by 2025 (Armor, 1999). Conventional hydrogen
gas production methods are energy intensive processes requiring high temperatures (> 850
C).
Electrolysis of water may be the cleanest technology for hydrogen gas production. However,
electrolysis should be used in areas where electricity is inexpensive since electricity costs account
for 80% of the operating cost of H2 production (Armor, 1999). Due to increasing trend of
hydrogen demand, development of cost effective and efficient hydrogen production technologies
In accordance with sustainable development and waste minimization issues, bio-hydrogen gas
production from renewable sources, also known as “green technology” has received considerable
during conversion of organic wastes into organic acids. Photosynthetic processes include algae
which use CO2 and H2O for hydrogen gas production. Some photo-heterotrophic bacteria utilize
organic acids such as acetic, lactic and butyric acids to produce H2 and CO2.
Production of clean energy source and utilization of waste materials make biological hydrogen
production a novel and promising approach to meet the increasing energy needs as a substitute for
fossil fuels. On the basis of these facts, this review focuses on potential use of carbohydrate rich
wastes as the raw material, microbial cultures, bioprocessing strategies and the recent
The major criteria for the selection of waste materials to be used in bio-hydrogen production are
the availability, cost, carbohydrate content and biodegradability. Simple sugars such as glucose,
sucrose and lactose are readily biodegradable and preferred substrates for hydrogen production.
However pure carbohydrate sources are expensive raw materials for hydrogen production. Major
waste materials which can be used for hydrogen gas production may be summarized as follows.
Many agricultural and food industry wastes contain starch and/or cellulose which are rich in terms
of carbohydrate contents. Complex nature of these wastes may adversely affect the
fermentation may be required depending on the nature of the agricultural waste. Some
biodegradable carbohydrate containing and non-toxic industrial effluents such as dairy industry,
olive mill, baker’s yeast, and brewery wastewaters can be used as raw material for bio-hydrogen
required for industrial effluents. The waste sludge generated in wastewater treatment plants also
contains large quantities of carbohydrate and proteins which can be used for energy production
Starch, cellulose or hemicellulose content of wastes, carbohydrate rich food industry effluents or
waste biological sludge can be further processed to convert the carbohydrates to organic acids and
then to hydrogen gas by using proper bioprocessing technologies. Figure 1 shows schematic
diagram for bio-hydrogen production from food industry wastewaters and agricultural wastes by
Grinding
Delignification
Hydrolysis
Glucose syrup
Wastewater Pretreatment
Dark fermentation
Organic acids
Photo-fermentation
CO2,
H2
CO2, H2
H2
separation
containing wastes
Treated wastewater
Pretreatment
CO2
H2
Major bioprocesses utilized for hydrogen gas production can be classified as bio-photolysis of
combined dark and photo-fermentations are rather new concepts in bio-hydrogen production.
Algae split water molecules to hydrogen ion and oxygen via photosynthesis. The generated
hydrogen ions are converted into hydrogen gas by hydrogenase enzyme. Chlamydomonas
reinhardtii is one of the well known hydrogen producing algae (Ghirardi, 2000). Hydrogenase
activity has been detected in green algae, Scenedesmus obliquus (Florin, 2001), in marine green
algae Chlorococcum littorale ( Ueno, 1999) and in Chlorella fusca (Winkler, 2002).
Cyanobacterial hydrogen gas evolution involves nitrogen fixing cultures such heterocystous
filamentous Anabaena cylindrica (Pinto, 2002). However, A. variabilis has received more
attention in recent years because of higher hydrogen production capacity (Tsygankov, 2002,
Masukawa, 2002). At the present time the rate of hydrogen production by Anabaena sp is
The algal hydrogen production could be considered as an economical and sustainable method in
terms of water utilization as a renewable resource and CO2 consumption as one of the air
pollutants. However, strong inhibition effect of generated oxygen on hydrogenase enzyme, low
hydrogen production potential and no waste utilization are the limitations of the process.
Many anaerobic organisms can produce hydrogen from carbohydrate containing organic wastes.
The organisms belonging to the genus Clostridium such as C. buytricum (Yokoi, 2001), C.
pasteurianum (Liu, 2004, Lin and Lay, 2004), C. bifermentants (Wang, 2003-a) are obligate
anaerobes and spore forming organisms. Hydrogen production capacity of anaerobic facultative
bacterial culture Enterobacter aerogenes has been widely studied (Tanisho and Ishiwata, 1994,
1995). Recently a hydrogen producing bacterial strain Klebisalle oxytoca HP1 was isolated from
C (Minnan, 2005). T.
The optimum pH for the maximum hydrogen yield or specific hydrogen production rate could
range from pH=4.5 to pH=8.0 (Liu, 2004, Collet, 2004,Shin, 2004, Fang ,2002). The final pH in
anaerobic hydrogen production is reported as around 4.0-4.8 regardless of initial pH (Yokoi, 2001,
Liu, 2004). Reducing agents such as argon are used to remove trace amounts of oxygen present in
the medium. Utilization of such reducing agents is relatively expensive and uneconomical for
industrial production of hydrogen gas. Mixed culture of facultative anaerobe E. aerogenes and
Clostridum sp have been suggested to overcome use of reducing agents (Yokoi, 1998-a,). The
acetic, butyric, lactic and propionic acids (Collet, 2004, Lay, 2001, Lin and Lay, 2004). Ethanol
may be produced depending on the environmental conditions (Collet 2004, Lin and Lay, 2004).
Glucose is an easily biodegradable carbon source, present in most of the industrial effluents and
glucose yields 12 moles of hydrogen gas (H2). According to reaction stoichiometry, bioconversion
of 1 mole of glucose into acetate yields 4 mol H2 /mol glucose, but only 2 mol H2 /mol glucose is
formed when butyrate is the end product. The highest hydrogen yield obtained from glucose is
around 2.0-2.2 mol/mol (Table 1). Production of butyrate rather than acetate or partial
biodegradation of glucose may be one of the reasons for deviations from the theoretical yield
(Fang and Liu, 2002). Batch and continuous hydrogen gas production from sucrose has been
widely studied. The yield from sucrose varies between 1.5 mol/mol sucrose and 6.0 mol/mol
(Table 1).
Table 1. Rates and yields of bio-hydrogen production from glucose by batch dark-fermentations.
Clostridia sp 14.2 mmol/g VSS h 359 mmol/Ld 1.7 Lin and Chang 2004
C. pasteurium 4.58 mmol/g VSS h 270 mmol/L d 4.8 Lin and Lay 2004
E. cloacae IIT-BT 08 29.5 mmol/g DW h 660 ml/ L h 6 Kumar et al. 2000
Mixed culture 340 ml/g VSS h 5.10 L/h L 2.1 Lee et al. 2004
Klebsiella oxytocaHP1 15.2 mmol/g DW h 350 ml/L h 3.6 Minnan et al. 2005
Starch containing materials are abundant in nature and have great potential to be used as a
carbohydrate source for hydrogen production. According to reaction stoichiometry, each gram of
starch produces a maximum of 553 ml of hydrogen with acetate as a by-product (Zhang, 2003).
However the yield may be lower than that value because of utilization of substrate for cell
synthesis. Table 2 summarizes the rates and yields of hydrogen production for batch and
continuous operations when starch was used as the substrate. The maximum specific hydrogen
production rate was 237 ml H2/g VSS.d when edible corn starch was used as the substrate by C.
pasteurianum (Liu and Shen, 2004). Zang et al. (2003) obtained higher specific yield of 480 ml
H2/gVSS.d with 4.6 g/L starch concentration by using a mixed sludge. Hydrogen yield obtained in
long term repeated batch operations was 2.4 mol H2/ mol glucose with 2 % starch residue
containing wastewater by the mixed culture of C. butyricum and E. aerogenes (Yokoi, 2001).
Table 2. The rates and yields of bio-hydrogen production from starch by dark-fermentations.
Thermophilic 15.2 ml/g VSS h 1.9 ml/h 92 ml/ g starch Zhang et al. 2003
C. pasteurianum 9.9 ml/gVSS h 4.2 ml/ h 106 ml/ g starch Liu et al. 2004
Cellulose is the major constitute of plant biomass and highly available in agricultural wastes and
industrial effluents such as pulp/paper and food industry. Hydrogen gas production potential from
microcrystalline cellulose was determined as 2.18 mol H2/mol cellulose with 12.5 g/l cellulose
concentration at mesophilic conditions (Lay, 2001). Liu et al. (2003) reported the maximum
hydrogen yield of 102 ml/g cellulose which is only 18% of the theoretical yield. Low yield was
mmol /h H2 production rate (Taguchi, 1996). Similarly, production of hydrogen gas was higher
from hydrolysate of Avicel cellulose or xylan than that of pure xylose or glucose (Taguchi, 1995).
Food industry wastes constitute a major fraction of the municipal solid wastes. High carbohydrate
content in form of simple sugars, starch and cellulose makes the solid food wastes a potential
feedstock for biological hydrogen production. The problem with the food waste is the variations in
carbohydrate and protein types and the contents in the mixture. Each component requires different
environmental and bioprocessing conditions for hydrogen gas production. Table 3 summarizes the
The biological hydrogen production from organic fraction of municipal solid wastes (OFMSW)
provided 43 ml/ g VSS. h specific production rate and 125 ml /g TVS. h production potential (Lay,
1999). Kim et al. (2004) obtained 111.2 ml H2/ g VSS. h when food waste was used as sole
substrate. Similarly, hydrogen production potential of carbohydrate-rich high solid organic waste
(HSOW) was 20 times larger than those of fat rich HSOW and protein rich HSOW (Lay, 2003).
The highest production yield was 0.21 L H2/ g COD from potato processing wastewater compared
to confectioners, apple industrial effluents and domestic wastewater (Ginkel, 2005). The maximum
rate of hydrogen production from molasses in continuous operation with E. aerogenes was 36
mmol /L h (Tanisho,1994). Immobilized cultures resulted in 2.2 mol H2/mol sugar (Tanisho,
1995).
Biosolids (sludges) from wastewater treatment plants contain large amounts of polysaccharides
and proteins. Hydrogen yields of 1.2 mg H2/g COD (Wang, 2003-b) and 0.6 mol/kg COD (Wang,
2004) were reported when sludge was used as the raw material. However, higher hydrogen yields
(15 mg H2/g COD) were obtained from the filtrate (Wang, 2003-b).
Table 3. The rates and yields of bio-hydrogen production from different waste materials by dark
fermentation.
Yield coefficient
OFMSW 16.8 ml/g VSS h 117 ml/g TVS h 150 ml/g OFMSW Lay et al. 1999
Food waste 12 ml//g VSS h 1.8 mol/mol hexose Shin et al. 2004
Rice winery WW 389 ml/g VSS h 159 ml/L h 2.14 mol/mol hexose Yu et al. 2002
Some photo-heterotrophic bacteria are capable of converting organic acids (acetic, lactic, butyric)
to hydrogen (H2) and carbon dioxide (CO2) under anaerobic conditions in the presence of light.
Therefore, the organic acids produced during the acidogenic phase of anaerobic digestion of
organic wastes can be converted to H2 and CO2 by some photosynthetic anaerobic bacteria.
Hydrogen gas production capabilities of some purple photosynthetic bacteria such as Rhodobacter
spheroides (Koku, 2002, Federov, 1998, Eroğlu, 1999), Rhodobacter capsulatus (He, 2005, Fang,
(Barbosa, 2001) have been investigated to some extent. Photoproduction of hydrogen from CO or
The hydrogen production takes place under anaerobic conditions with light illumination at
carbon source such as acetic (Fang, 2005), butyric (Fang, 2005), propionic (Shi and Yu, 2004),
lactic (He, 2005) and malic acid (Eroğlu, 1999). Table 5 summarizes the yields and the rates of
Table 4. Rates and yields of bio-hydrogen production from organic acids by photo-fermentations.
Acid
Nitrogenase is the key enzyme that catalyzes hydrogen gas production by photosynthetic bacteria.
Since nitrogenase enzyme is inhibited in the presence of oxygen, ammonia or at high N/C ratios,
the process requires ammonium limited and oxygen free conditions (Koku, 2003). The metabolism
shifts to utilization of organic substance for cell synthesis rather than hydrogen production in the
presence of high nitrogen concentrations resulting in excess biomass growth and reduction in light
diffusion (Fascetti, 1995, Oh, 2004). Light intensity affects the performance of photo-fermentation
Increasing light intensity has a stimulatory affect on hydrogen yield and production rate, but has an
adverse effect on the light conversion efficiency (Barbosa, 2001). Hydrogen production under dark
conditions is usually lower than that of the illuminated conditions (Koku, 2003, Oh, 2004).
However alternating 14 h light and 10 h dark cycles yielded slightly higher hydrogen production
possible. The major problems in hydrogen gas production from industrial effluents are the color of
wastewaters, which could reduce the light penetration and high ammonia concentration which
reduces the hydrogen productivity. Moreover, high organic matter content (COD) and presence of
some toxic compounds in industrial effluents may require pre-treatment before hydrogen gas
production.
Table 5 summarizes hydrogen production studies from some food industry wastewaters by
photofermentation. Photo-production of hydrogen from pre-treated sugar refinery wastewater
(SRWW)
resulted in hydrogen production rate of 3.8 ml/l.h at 32
SRWW. Addition of malic acid (20 g/L) into SRWW enhanced the production rate to 5 ml/l.h.
(Yetiş, 2000). High dilutions (3-4%) of olive mill waste (OMW) were necessary to alter the
inhibitory effects of high organic content and dark color of OMW. 2% dilution resulted in the
highest hydrogen production of 13.9 L H2/L WW with R. sphaeroides OU 001 where around 35%
COD reduction was observed (Eroğlu, 2004). Tofu wastewater is a carbohydrate and protein-rich
C) was comparable to
the yield from glucose (3.6 L/ L wastewater) by immobilized R. sphaeroides RV. No ammonia
inhibition (2mM) was observed and 41% of TOC was removed (Zhu, 1999). 50% dilution of the
wastewater increased the yield to 4.32 L/L wastewater and TOC removal efficiency to 66% (Zhu,
2002). The hydrogen gas production from potato starch, sugar cane, juice and whey by using
Rhodopseudomonas sp.have been investigated and sugar cane juice yielded the maximum level of
Table 5. The rates and yields of bio-hydrogen production from food industry wastewaters by
photo-fermentations.
Sugar Refinery WW R.sphaeroides OU 001 13.44 L/mol C 5 ml/ L culture h Yetiş et al. 2000
Sugar Refinery WW R.sphaeroides OU 001 11.67 L/mol C 3 ml/ L culture h Yetiş et al. 2000
carbohydrate
Tofu WW R.sphaeroides 15.9 ml/L h Zhu et al. 1999
The major types of photo-bioreactors developed for hydrogen production are tubular, flat panel
and bubble column reactors. High illuminations cause lower light conversion yields, but higher
hydrogen production rates. 9.23% maximum energy conversion efficiency was obtained by using
the culture significantly affected the productivity (El-Shishtawy, 1997). Similar results were
along the light penetration axis. The efficiency of the conversion of light to hydrogen increased
with the depth in the reactor and 1 cm depth yielded the highest efficiency.
Tredici type multi-tubular photo-bioreactors (Figure 2-a) was used for hydrogen gas production in
the presence and absence of light by using Spirulina (Benemann, 2000). A modification of tubular
(Modigella, 1998). The hydrogen production rate from lactate reached 2 L/m
h with a light
(Figure 2-c) and a novel flat-panel airlift photo-bioreactor with baffles (Figure 2-d) have been
(Degen, 2001), respectively. Hydrogen gas production by using these photo-bioreactors has not
In relating to solar hydrogen production, the light conversion efficiency could be less during midday
because of high light intensity. In addition, a delay of 2-4 hours was observed in maximum
hydrogen production rate after the highest light intensity at noon (Miyake, 1999). Utilization of
2002).
ab
cd
pump; (2): gas bag for collection of produced gas; (3) two 1 L pressure vessels; (4)
pressure valve; (5) mass flow controller; (6) condenser; (7) pH/redox electrode; b.
Sequential dark and photo fermentation is a rather new approach in biological hydrogen gas
production. Two-stage system has certain advantages over single stage dark-fermentation or
sufficient amount of organic acids for the photo- fermentation. Therefore, the limitation by the
organic acid availability would be eliminated. Higher hydrogen production yields can be obtained
when two systems are combined (Yokoi, 1998-b, 2002). Further utilization of organic acids by
photo-fermentative bacteria could provide better effluent quality in terms of COD. However, the
system should be well controlled to provide optimum media composition and environmental
conditions for the two microbial components of the process (Yokoi, 2001, 2002). The ammonia
concentration in the effluent of anaerobic fermentation should not be at the inhibitory level for the
photosynthetic bacteria (Fascetti, 1998). Dilution and neutralization of dark fermentation effluents
are required before photo-fermentation to adjust the organic acid concentration and the pH to 7 for
also been studied. Yokoi obtained higher hydrogen production yield (4.5 mol/mol glucose) by
coculture of C. butryricum and Rhodobacter sp. as compared to single stage dark fermentation (1.9
mol/mol glucose) and sequential two step fermentation (3.7 mol/mol glucose) of starch ( Yokoi
1998-b). Similarly, higher hydrogen yields from different substrates were reported by co-cultures
of R. marinum and V. fluvialis as compared to R. marinum alone (Ike, 1999). Better hydrogen
yield (60%), was observed in combined fermentation by Lactobacillus amylovous and R. marinum
from algae biomass in comparison to sequential two-stage fermentation (45%) (Kawaguchi, 2001).
advantage over the two stage fermentation process. However, the differences in organic acid
production/consumption rates and therefore, potential accumulation of organic acids in the media
4. CONCLUSIONS
Hydrogen is considered as the ‘energy for future’ since it is a clean energy source with high
energy content as compared to hydrocarbon fuels and not readily available in nature as fossil fuels.
Therefore, new processes need to be developed for cost effective production of hydrogen.
Biological methods offer distinct advantages for hydrogen production over chemical methods such
as operation under mild conditions and specific conversions. However, the raw material cost is one
of the major limitations for bio-hydrogen production. Utilization of some carbohydrate rich, starch
or cellulose containing solid wastes and/or some food industry wastewaters is an attractive
approach for bio-hydrogen production. Among the various methods used for bio-hydrogen
most attractive approach resulting in high hydrogen yields for hydrogen production from
The major problems in bio-hydrogen production from wastes are the low rates and yields of
hydrogen formation and therefore large reactor volume requirements. This problem may be
overcome by selecting and using more effective organisms or mixed cultures, developing more
efficient processing schemes, optimizing the environmental conditions, improving the light
utilization efficiency and developing more efficient photo-bioreactors. Considerable research and
development studies are needed to improve the ‘state of the art’ in bio-hydrogen production.
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