Bio Hydrogen Production

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Proceedings International Hydrogen Energy Congress and Exhibition IHEC 2005

Istanbul, Turkey, 13-15 July 2005

Biohydrogen production from waste materials

Fikret Karg and Ilgi Karapnar Kapdan

Dokuz Eylül University, Department of Environmental Engineering,

Tinaztepe , Buca Izmir, Turkey

[email protected], [email protected]

ABSTRACT

The major biological processes utilized for hydrogen gas production are bio-photolysis of water by

algae, dark fermentation and photofermentation of organic materials, usually carbohydrates 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 bakers yeast industry wastewaters can be used for hydrogen production by using suitable

bio-process technologies. Utilization of aforementioned wastes for hydrogen production provides

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

developments are discussed

Keywords: bio-hydrogen, waste, bio-processing, dark fermentation, photo-fermentations.

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

o

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

has gained significant attention.

In accordance with sustainable development and waste minimization issues, bio-hydrogen gas

production from renewable sources, also known as green technology has received considerable

attention in recent years. Bio-hydrogen production can be realized by anaerobic and

photosynthetic microorganisms. Under anaerobic conditions, hydrogen is producedas a by product

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

1 Kargi and Kapdan

wastes as the raw material, microbial cultures, bioprocessing strategies and the recent

developments in bio-hydrogen production.

2. TYPES OF WASTE MATERIALS

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

biodegradability. Grinding or delignification by mechanical or chemical means before

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, bakers yeast, and brewery wastewaters can be used as raw material for bio-hydrogen

production. Pre-treatment to remove undesirable components and nutritional balancing may be

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

such as methane or hydrogen gas.

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

two stage, anaerobic dark and photo-fermentations.

Grinding

Delignification

Hydrolysis

Glucose syrup

Wastewater Pretreatment

Dark fermentation

Organic acids

Photo-fermentation

CO2,

H2

CO2, H2

H2



separation

Cellulose and starch

containing wastes

Treated wastewater

Pretreatment

CO2

H2

Figure 1. A schematic diagram for bio-hydrogen production from cellulose/starch containing

agricultural wastes and food industry wastewaters.

3. Bioprocesses for hydrogen gas production

Major bioprocesses utilized for hydrogen gas production can be classified as bio-photolysis of

water by algae, dark-fermentative hydrogen production in the acidogenic phase of anaerobic

digestion and photo-fermentation of organic acids by photosynthetic bacteria. Sequential or

combined dark and photo-fermentations are rather new concepts in bio-hydrogen production.

2Kargi and Kapdan.

3.1. Hydrogen gas production from water by algae and cyanobacteria

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

considerably lower than that obtained by dark or photo-fermentations.

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 oneof 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.

Therefore, dark fermentation and photo-fermentations are considered to be more advantageous

due

to simultaneous waste treatment and hydrogen gas production.

3.2. Hydrogen gas production by dark fermentation

3.2.1. Type of organisms and conditions

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

hot springs with maximal hydrogen production rate at 35

o

C (Minnan, 2005). T.

thermosaccharolyticum, (Shin, 2004), Thermococcus kodakaraensis KOD1 (Kanai, 2005) and

Clostridium thermolacticum (Collet, 2004) are the thermophilic hydrogen producers.

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

major products in hydrogen production by anaerobic dark fermentation of carbohydrates are

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).

3.2.2. Type of substrates

3.2.2.1. Use of simple sugars

Glucose is an easily biodegradable carbon source, present in most of the industrial effluents and

can be obtained abundantly from agricultural wastes. Theoretically bioconversion of 1 mol of

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

3Kargi and Kapdan

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

sucrose depending on bioprocessing conditions, microbial culture and concentration of sucrose

(Table 1).

Table 1. Rates and yields of bio-hydrogen production from glucose by batch dark-fermentations.

Organism SHPR VHPR H2 Yield References

H2 production from glucose*

Klebsielle oxytoca HP1 9.6mmol /g DW h 87.5 ml/L h 1 Minnan et.al. 2005

E. cloacae IIT-BT 08 447 ml/L h 2.2 Kumar et al.2000

Mixed culture 20 mmol/g VSS h 1.1 Chen et al. 2000

Clostridia sp 14.2 mmol/g VSS h 359 mmol/Ld 1.7 Lin and Chang 2004

Mixed culture 191 ml /g VSS h 2.1 Fang et al. 2002

H2 production from sucrose

+

Klebsielle oxytoca HP1 8.0 mmol/g DW h 1.5 Minnan et al. 2005

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

H2 yield *mol/mol glucose; H2 yield

+

mol/mol sucrose; SHPR: specific hydrogen production rate;

VHPR: volumetric hydrogen production rate.

3.2.2.2. Use of complex charbohydrates

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, eachgram 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.

Organism SHPR VHPR H2 Yield References

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

Mixed culture 97.5 ml/g VSS h 1497 L/m

3

d 1.29 L/g starch COD Lay 2000

C. butyricum+ 1300 ml/L h 2.6 mol/mol glucose Yokoi et al.1998-a



E. aerogenes

T. kodakaraensis 14.0mmol/g DW h 9.46 mmol/L h 3.33 mol/mol starch Kanai et al.2005

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

4Kargi and Kapdan.

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

explained as partial hydrolysis of cellulose. Utilization of cellulose hydrolysate provided 4.10

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).

3.2.2.4. Use of food industry wastes and wastewater

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

hydrogen gas production from different wastewaters and solid wastes.

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.

Carbon Source SHPR VHPR YP/S References

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

Potato Ind. WW 2.8 L/L WW Ginkel et.al. 2005

Apple Ind. WW 0.9 L/LWW Ginkel et.al. 2005

Molasses 36 mmol/l culture h 138 ml /L h 1.5 mol/mol sucrose Tanisho et al.1994

Rice winery WW 389 ml/g VSS h 159 ml/L h 2.14 mol/mol hexose Yu et al. 2002

Sludge Filtrate 15 mg/ g COD Wang et al. 2003-b

Starch residue 2.4 mol/mol glucose Yokoi et al. 2001

Starch residue 2.7 mol/mol glucose Yokoi et al. 2002

OFMSW: Organic fraction of solid waste

3.3. Hydrogen gas production by photo-fermentations

3.3.1. Types of organisms and the conditions

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.

5Kargi and Kapdan

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, Erolu, 1999), Rhodobacter capsulatus (He, 2005, Fang,

2005), Rhodovulum sulfidophilum W-1S (Maeda, 2003) and Rhodopseudomonas palustris

(Barbosa, 2001) have been investigated to some extent. Photoproduction of hydrogen from CO or

other organic acids by carbon-monoxide dependent dehydrogenase (CODH) enzyme containing

cultures such as Rhodospirillum rubrum (Najafpour, 2004) and Rodopseudomonos palsutris P4

has also been reported (Oh, 2004).

The hydrogen production takes place under anaerobic conditions with light illumination at

pHopt=7.0 and T=30-35

0

C (Federov 1998, Erolu 1999). The organisms prefer organic acids as

carbon source such as acetic (Fang, 2005), butyric (Fang, 2005), propionic (Shi and Yu, 2004),

lactic (He, 2005) and malic acid (Erolu, 1999). Table 5 summarizes the yields and the rates of

hydrogen production from different organic acids by the photo-fermentative organisms.

Table 4. Rates and yields of bio-hydrogen production from organic acids by photo-fermentations.

Organic Organism Conv Eff. LCE

a

SHPR VHPR References

Acid

Acetate Rhodopseudomonas 72.8% 0.9% 25.2 ml H2/L h Barbosa et al.2001

R. palustris 14.8% 0.1% 2.2 ml H2/L h Barbosa et al.2001

R. capsulata 76.5% 22 ml/g VSS h 0.88 ml/h Fang et al. 2005

Lactate R. palustris 12.6% 0.5% 9.1 ml H2/L h Barbosa et al.2001

R. sphaeroides RV 80% 75 ml/g DW h 1.5 L/L d Fascetti et al. 1995

R.sphaeroides GL-1 86% 0.2 ml/ml PU matrix h Federov et al.1998

Butyrate Rhodopseudomonas 8.4% 0.3% 7.6 ml H2/L h Barbosa et al.2001



R. capsulata 67.6% 32 ml/g VSS h 1.28 ml/h Fang et al. 2005

Malate R. palustris 36% 0.3% 5.8 ml H2/L h Barbosa et al.2001

R. sphaeroides 2.4ml/ g DW h 12 ml/L h Erolu et al. 1999]

R. sphaeroides 35-45% 18 ml/ g DW h 5 ml H2/L h Koku et al. 2002

a

: Light conversion efficiency;

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

rates and cell concentrations as compared to continuous illumination (Koku, 2003).

3.3.2. Types of substrates

Utilization of industrial effluents for hydrogen gas production by photosynthetic bacteria is

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.

6Kargi and Kapdan.

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

o

C in batch operation with 20% diluted

SRWW. Addition of malic acid (20 g/L) into SRWW enhanced the production rate to 5 ml/l.h.

(Yeti, 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 (Erolu, 2004). Tofu wastewater is a carbohydrate and protein-rich

effluent. Hydrogen yield from tofu wastewater (1.9 L/L wastewater at 30

o

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

hydrogen production (45 ml/mg DW. h) (Singh, 1994).

Table 5. The rates and yields of bio-hydrogen production from food industry wastewaters by

photo-fermentations.

Wastewater Organism H2 Yield HPR Ref

Sugar Refinery WW R.sphaeroides OU 001 13.44 L/mol C 5 ml/ L culture h Yeti et al. 2000

Sugar Refinery WW R.sphaeroides OU 001 11.67 L/mol C 3 ml/ L culture h Yeti et al. 2000

Olive Mill WW R.sphaeroides OU 001 4ml/L culture h Erolu et al. 2004

Tofu WW R.sphaeroides 0.24 ml/ mg 2.1 L/ h m

2

gel Zhu et al. 1999

carbohydrate



Tofu WW R.sphaeroides 15.9 ml/L h Zhu et al. 1999

HPR: Hydrogen production rate

3.3.3. Photo-bioreactors for bio- hydrogen production

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

light-induced and diffused photo-bioreactor (IDPBR) at 300 W/m

2

light intensity and the width of

the culture significantly affected the productivity (El-Shishtawy, 1997). Similar results were

observed by Nakada et al. (1995) in a photo-bioreactor composed of four compartments aligned

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

reactor was developed by Modigell as a modular outdoor photo-bioreactor (Figure 2-b)

(Modigella, 1998). The hydrogen production rate from lactate reached 2 L/m

2

h with a light

conversion efficiency of 2% in outdoor experiments. A pneumatically agitated photobioreactor

(Figure 2-c) and a novel flat-panel airlift photo-bioreactor with baffles (Figure 2-d) have been

developed for cultivation of Rhodopseudomonas (Hoekaman, 2002) and Chlorella vulgaris

(Degen, 2001), respectively. Hydrogen gas production by using these photo-bioreactors has not

been examined yet.

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

7Kargi and Kapdan



light shade bands on photo-biorecators improved the light conversion efficiency (Wakayama,

2002).

a b

c d

Figure 2. Some configurations for photo-bioreactors used for hydrogen production. a.

Photo-bioreactor with gas recirculation (Hoekeman, 2002), (1); Membrane gas

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.

Flat panel airlift (FPA) photo-bioreactor; ( Degen 2001). c. Multi-tubular

(Tredici) photo-bioreactor ( Benemann 2000);d. A modular outdoor photobioreactor ( Modigella,

1998).

3.4. Hydrogen gas production by sequential dark and photo-fermentation

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

photo-fermentation processes. The effluent of dark fermentation in hydrogen production provides

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

the optimal performance of photosynthetic bacteria (Kawaguchi, 2001).

8Kargi and Kapdan.



Biohydrogen production by co-culture of anaerobic and photosynthetic bacteria in single stage has

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).

In addition, pH of the mixed fermentation remained around pH=7 which is considered as an

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

are the major problems.

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

production sequential or combined bioprocesses of dark and photo-fermentations seem to be the

most attractive approach resulting in high hydrogen yields for hydrogen production from

carbohydrate rich wastes and simultaneous waste treatment.

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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