C H A P T E R

23
Thermophilic Biohydrogen Production
Dimitar Karakashev*, Irini Angelidaki
Department of Environmental Engineering, Technical University of Denmark, Lyngby, Denmark
*Corresponding author: E-mail: dbka@env.dtu.dk

1 BACKGROUND
In recent years, hydrogen gas is attracting widespread attention as a clean and environmentfriendly fuel that produces water when combusted. The future energy economy will have an
important role for hydrogen as a clean, CO2-neutral energy carrier.
Hydrogen can be produced by both biological and nonbiological methods. The main
industrial process to produce H2 consists in steam reforming from natural gas and petroleum,
a process which depends on fossil fuels and thus is not CO2 neutral. Another source is electrolysis of seawater, which could be sustainable if electricity is generated from renewable
resources, such as from windmill electricity. An alternative way to circumvent the dependence of H2 production from fossil fuels is to utilize the potential of H2-producing
microorganisms to derive hydrogen from widely available biomass as a renewable energy
source (Lee et al., 2010). Currently, hydrogen is applicable in fuel cells.
Biohydrogen production can be realized by microorganisms using carbohydrate-rich and
nontoxic raw materials (Kapdan and Kargi, 2006). Among the various processes leading to
biohydrogen production, direct and indirect biophotolysis, hemoheterotrophic (dark) fermentation, photoheterotrophic (light-driven) fermentation, and in vitro enzymatic conversion
of biomass are important. Currently, dark fermentation is the most feasible process for
biohydrogen production from renewable biomass due to its higher rate of hydrogen evolution
in the absence of any light sources as well as the versatility of the substrates used. However,
the key issue that still needs to be addressed includes the much lower hydrogen yields (up
to 2.5-2.9 mol H2/mol glucose) compared to the theoretical yield of 4 mol H2/mol glucose
for fermentation with only acetate as liquid end fermentation product. One of the reasons
for the low hydrogen yield is that in many microorganisms the actual yields are reduced by
hydrogen recycling mechanisms due to the presence of one or more uptake hydrogenases,
which consume part of the produced hydrogen (Hallenbeck and Benemann, 2002).
To reach higher rates and yields in biohydrogen production, fermentation under high
temperatures (50-80
C) with application of thermophilic and extreme thermophilic

Biofuels: Alternative Feedstocks and Conversion Processes

525

2011 Elsevier Inc. All rights reserved.

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23. THERMOPHILIC BIOHYDROGEN PRODUCTION

microorganisms is a very promising alternative. Higher temperatures thermodynamically

favor hydrogen production. Besides, elevated temperatures contribute to better pathogenic
destruction and limit hydrogen consumption by hydrogen consumers (methanogens,
homoacetogens, sulfate reducers). Furthermore, certain thermophilic bacteria have the ability
to produce ethanol simultaneously with hydrogen (Zhao et al., 2009). Ethanol is also a renewable biofuel, and its coproduction can increase the benefits from future commercial exploitation of those thermophilic/extreme thermophilic organisms.
This review describes the thermophilic biohydrogen production process, focusing on thermodynamics, metabolic pathways, and the microorganisms involved. The effect of operational
conditions (pH, loading rate, retention time, concentrations of dissolved hydrogen and carbon
dioxide, soluble metabolic profile, and SMP) on process performance as well as some practical
aspects such as feedstock used and reactor technologies applied, is also given, with respect to
challenges, possibilities, and future perspectives of the process.

2 THERMODYNAMIC ASPECTS
Stoichiometrically, the maximum hydrogen yield for complete conversion of glucose to
H2 and CO2 is 12 mol H2/mol glucose (Equation 1). However, according to standard
(25
C) Gibbs free energy of reactions (Equations 14), production of 12 mol of hydrogen
(Equation 1) is thermodynamically unfavorable. From the thermodynamic point of view,
the most favorable conversion is with butyrate as end fermentation product (Equation 3),
followed by mixed propionate-acetate-type fermentation (reaction 4) and acetate fermentation (Equation 2). Meanwhile, from the practical point of view, the most desirable is acetate fermentation (Equation 2), whereby 4 mol H2/mol glucose can be obtained. However,
this stoichiometric yield is only attainable under near equilibrium conditions, which
implies very slow rates and/or at very low partial pressure of hydrogen (Hallenbeck
and Benemann, 2002).
C6 H12 O6 12H2 O ! 6HCO3  12H2 6H DGo 0 241 kJ mol1 ;

C6 H12 O6 4H2 O ! 2CH3 COO 2HCO3  4H2 4H DGo 0 48 kJ mol1 ;

C6 H12 O6 2H2 O ! CH3 CH2 CH2 COO 2HCO3  2H2 3H DGo 0

137 kJ mol1 ;
C6 H12 O6 3H2 O ! CH3 CH2 OH CH3 COO 2HCO3  2H2 3H DGo 0
97 kJ mol1 :

A possible strategy to increase the H2 yield is to increase the cultivation temperature and
hence decrease the Gibbs free energy of the conversion process according to the second law of
thermodynamics (Equation 5):
DG DH  TDS;

where DG is the change in Gibbs free energy, DH is the change in enthalpy, T is the absolute
temperature, and DS is the change in entropy.

527

3 BIOCHEMICAL PATHWAYS AND MICROBIOLOGY

3 BIOCHEMICAL PATHWAYS AND MICROBIOLOGY

The total hydrogen yields from the different anaerobic glucose degradation pathways are
shown in Table 1. The biochemistry of the anaerobic glucose catabolism shows that the production of propionate, ethanol, and lactate is associated with absence of hydrogen generation. In
practice, highest hydrogen yields are associated with production of acetate, or a mixture of acetate and butyrate, and low hydrogen yieldswith production of different combinations of
butyrate, acetate, ethanol, propionate, and lactate as the main liquid fermentation end products.
The main biochemical pathways involved in dark fermentative conversion of simplest
sugar-glucose to hydrogen and different end products are presented in Figure 1.
Fermentative hydrogen production is usually carried out under mesophilic or thermophilic
conditions by a wide variety of microorganisms such as strict anaerobes (clostridia, ruminococci,
and archae), facultative anaerobes (Escherichia coli and Enterobacter aerogenes), and facultative
bacteria (Alcaligenes eutrophus and Bacillus licheniformis), when kept under anoxic conditions.
Recently, attempts for improving biohydrogen production using thermophilic (50-60
C) and
extreme thermophilic (60-80
C) bacteria and archae have been made. Among pure cultures
of thermophiles and extreme thermophilesCaldicellulosiruptor sp., Thermoanaerobacter sp.,
Thermoanaerobacterium sp., Thermotoga sp., Pyrococcus sp., Desulfotomaculum geothermicum, Clostridium thermocellum (Bartacek et al., 2007; Kapdan and Kargi, 2006; O-Thong et al., 2008a,b,c;
Van Niel et al., 2002; Willquist et al., 2009)studied for dark fermentative hydrogen production, the extreme thermophiles (70
C) Caldicellulosiruptor saccharolyticus and Thermotoga elfii
(Van Niel et al., 2002) appear to be most promising as they display superior hydrogen yields

TABLE 1
Pathways

Fermentation End Products and Hydrogen Yields from the Main Anaerobic Glucose Degradation

Liquid Fermentation
End Product(s)

Equation for Anaerobic Glucose Degradation Pathway

Theoretical
Hydrogen Yield
(mol H2/mol glucose)

Acetic acid CH3COOH

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

Butyric acid
CH3CH2CH2COOH

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

Butyric acid
CH3CH2CH2COOH
acetic acid CH3COOH

4C6 H12 O6 2H2 O ! 3CH3 CH2 CH2 COOH 2CH3 COOH

2.5

Ethanol CH3CH2OH
acetic acid CH3COOH

C6 H12 O6 2H2 O ! CH3 CH2 OH CH3 COOH 2H2 2CO2

Propionic acid
CH3CH2COOH

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

Ethanol CH3CH2OH

C6 H12 O6 ! 2CH3 CH2 OH 2CO2

Lactic acid C3H3O3

C6 H12 O6 ! C3 H3 O3

10H2 8CO2

528

23. THERMOPHILIC BIOHYDROGEN PRODUCTION

Glucose

Glyceraldehyde-3P

Dihydroxyacetone-P

2 ADP

2 NAD

2 ATP

2 NADH
2 NADH

2 NAD+

2 Pyruvate

2 Lactate
2 H2

2 ATP

4 NADH

2 ADP

2 Acetate

4 NAD+

2 Ethanol

2 Acetyl-CoA
2 NADH
2 NAD+
ATP

Butyrate

ADP

2 NADH

Butyryl-CoA

2 NAD+

Butanol

FIGURE 1 Schematic pathways for glucose conversion to hydrogen/other products via dark fermentation
(modified from Nath and Das, 2004).

up to 3.3 mol H2/mol hexose. In addition, these microorganisms can utilize a wide spectrum of
carbon sources ranging from simple sugars (pentoses and hexoses) to more complex
carbohydrates such as starch and cellulose. However, the major drawback of using these
microorganisms is their large dependence on growth factor sources such as yeast extract.
Some hyperthermophiles (optimal growth at 85
C) such as the archaeon Thermococcus
kodakaraensis were also demonstrated to produce hydrogen (Kanai et al., 2005). Recently,
mixed thermophilic cultures were also investigated with respect to their hydrogen-producing
potential (Karakashev et al., 2009; Kongjan et al., 2010; Zhao et al., 2009, 2010). However, the
results obtained with those mixed cultures are not comparable with those obtained by C.
saccharolyticus or T. elfii (with respect to hydrogen yield). Additional attempts are required
to optimize the dark fermentative thermophilic biohydrogen process with mixed cultures
to make them commercially attractive.

4 EFFECT OF PROCESS CONDITIONS

The major factors influencing dark fermentative biohydrogen production are organic loading rate (OLR), pH, hydraulic retention time (HRT), dissolved hydrogen and dissolved carbon dioxide concentrations, and SMP (Table 2).
pH is a factor affecting fermentative hydrogen production to a great extent. There are many
contradictory reports regarding the optimal pH for the biohydrogen production process.
In general, the optimal pH for growth of fermentative hydrogen producers is around 7.
However, due to fact that hydrogen producers face strong competition from hydrogen
consumers (mainly methanogens) growing best at pH 7, the optimal pH of fermentative

529

4 EFFECT OF PROCESS CONDITIONS

TABLE 2

Main Factors Affecting Thermophilic Biohydrogen Production

Factor

Effect(s)

References

OLR

Affects cell metabolism

Kraemer and Bagley (2007)

and Ren et al. (2007)

High OLR results in substrate inhibition

pH

Affects cell metabolism-cell membrane charge, membrane

transport, enzymatic activities; extreme pH stimulates spore
formation

Lee et al. (2002) and

Ren et al. (2007)

HRT

Too low HRT-microbial washout and substrate inhibition

Zhang et al. (2008a,b) and

Hawkes et al. (2002)

Partial pressure
of H2 (pH2)

Affects fermentation metabolism; increased pH2 inhibits

hydrogen production and leads to formation of reduced end
products (ethanol, lactate)

Kengen et al. (2009) and

Soboh et al. (2004)

Partial pressure
of CO2 (pCO2)

Affects the activity of homoacetogens and methanogens

Willquist et al. (2009) and

Kim et al. (2006)

Soluble metabolic
profile (SMP)

Affects fermentation metabolism-end products inhibition,

high organic acid concentration can result in cell lysis

Chin et al. (2003) and van

Ginkel and Logan (2005)

hydrogen production process was considered to be between 5.0 and 6.0 (Cai et al., 2004).
However, other studies (Lee et al., 2002; Pikuta and Hooevr, 2004) reported an unusual optimal pH for the fermentation process (around 9.00-9.5). Those findings suggest that optimal pH
value largely depends on the community composition of the original microbial consortium
used as inoculumwhether it is enriched by acidophilic/acidotolerant or alkalophylic/
alkalotolerant hydrogen producers.
HRT and OLR are the main optimization parameters for thermophilic biohydrogen
production (Hawkes et al., 2002; Kraemer and Bagley, 2007). Generally, short HRT was
considered to facilitate fermentative biohydrogen production with optimal HRT around
0.25 d-1 due to the fact that some hydrogen consumers (slowly growing methanogens)
are washed out under those conditions. On the other hand, high OLR (low HRT) can result
in substrate inhibition. Shock loading reduces hydrogen production through accumulation of organic acids (pH decrease), metabolic inhibition, and/or increased dissolved
hydrogen concentration.
Dissolved hydrogen concentration is another factor influencing thermophilic biohydrogen
production. Since dissolved hydrogen concentration is difficult to monitor, often H2 partial
pressure (pH2) is used as a parameter to approximate the dissolved hydrogen concentration.
This is, however, often inaccurate, as the process is usually not in equilibrium (Van Niel et al.,
2003). Since hydrogen is known to have an inhibitory effect on growth and its own production
in a variety of thermophiles (Kengen et al., 2009; Soboh et al., 2004), maximizing fermentative
hydrogen yield is only possible when pH2 is kept sufficiently low in the closed fermentation
system. Normally, this can be achieved by flushing off the produced hydrogen with an inert
gas such as N2 or He (Kraemer and Bagley, 2007). As the inert gases are expensive, use of CO2
might be a cheaper alternative since it is a by-product from the fermentation process.
However, stripping with CO2 has some disadvantages as discussed later.

530

23. THERMOPHILIC BIOHYDROGEN PRODUCTION

Carbon dioxide may also affect thermophilic hydrogen production (Willquist et al., 2009).
Although stripping with carbon dioxide was employed for hydrogen removal from gas phase
and subsequent increase of hydrogen yields (Kraemer and Bagley, 2007), there is one major
complication with this approach. Elevated carbon dioxide partial pressure (pCO2) might
inhibit hydrogen production as it triggers homoacetogenic reaction, resulting in increased
acetate levels (Equation 6) and finally acidification:
4H2 2CO2 ! CH3 COOH 2H2 O:

SMP and more specifically organic acid (acetate and butyrate) levels could have a significant effect on the fermentative metabolism. Those acids in their undissociated form can pass
the cell membrane, dissociate within a cell, release a proton, and finally uncouple the proton
motive force across the cell membrane, which can result in metabolic inhibition and cell lysis.
At high concentrations, organic acids can decrease the cell growth rate (Chin et al., 2003) and
cause a metabolic shift, from hydrogen production (acetate or butyrate pathway) to propionate or solvent (ethanol, butanol) synthesis (Van Niel et al., 2003).

5 PRACTICAL APPLICATIONS
Practical application of thermophilic biohydrogen production depends on the microorganisms employed, feedstock (substrates), and process technology (operational conditions
such as temperature and pH, fermentation mode, and reactor type applied) utilized (Table 3).
In comparison to mesophilic pure cultures, utilization of pure thermophilic (f.ex. Thermoanaerobacterium thermosaccharolyticum) or extreme thermophilic (T. elfii, C. saccharolyticus)
cultures for biohydrogen production is more sustainable as those cultures are more resistant
to contaminations by traditional mesophilic hydrogen scavengers such as methanogens and
sulfate reducers. However, application of pure cultures (although they have the highest
hydrogen yields) is still limited to fermentation of defined substrates, mainly sugars that
are usually sterilized. When nonsterile waste materials are used as feedstock, pure cultures
would face strong competition with the complex microflora of those wastes. In such cases,
application of mixed thermophilic or extreme thermophilic cultures (Table 3) will be a more
appropriate choice as, generally, mixed cultures are more robust to process imbalances and
stress situations inside the reactor.
The substrates (Table 3) utilized for thermophilic biohydrogen production can be divided
into the following groups:
Pure substrates (glucose, xylose, arabinose, cellulose, starch)
Industrial wastes and wastewaters from agriculture, food, sugar, pulp, and paperprocessing industry (cow waste slurry, palm oil mill effluent, rice winery wastewater,
wheat straw hydrolysate, paper sludge hydrolysate)
Among the different reactor technologies studied on lab scaleanaerobic sequencing
batch (ASBR), continuously stirred tank (CSTR), membrane bioreactor (MBR), and upflow
anaerobic sludge blanket (UASB) reactorsCSTR and UASB were the most widely used.
The highest thermophilic hydrogen production rate of 199 mmol H2/L/d was obtained with
mixed cultures in CSTR operating at 60
C and pH 5.0 (Ueno et al., 2006). With respect to

TABLE 3
Materials)

Thermophilic Biohydrogen Production Processes with Different Feedstocks: Defined and Nondefined Carbon Sources (Organic Waste
Conditions

Hydrogen Production

Feedstock

Rate
(mmol H2/L/h)

Yield (mol
H2/mol
hexose)

5.5

Food waste

NA

1.8

Shin et al.
(2004)

70

7.0

Glucose

NA

2.4

Zhao et al.
(2009)

Batch

51

6.5

Glucose

NA

1.52

Karadag
et al. (2009)

Caldicellulosiruptor
saccharolyticus,
Thermotoga elfii

Batch

70
7
Sucrose
NA
(C. saccharolyticus), (C. saccharolyticus), (C. saccharolyticus),
65 (T. elfii)
7.4 (T. elfii)
glucose (T. elfii)

3.3

Van Niel
et al. (2002)

Mixed culture

Batch

55

NA

Palm oil mill

effluent

98

1.72

Ismail et al.
(2010)

Thermotoga elfii,
Caldicellulosiruptor
saccharolyticus

Batch

65 (T. elfii), 70
NA
(C. saccharolyticus)

Paper sludge
hydrolysate

0.3 (T. elfii), 0.25

NA
(C. saccharolyticus)

Kadar et al.
(2003)

Mixed culture

Batch

60, 75

7.0

Cow waste slurry 30.0

NA

Yokoyama
et al. (2007)

Mixed culture

Continuous
(UASB)

55

5.5

Rice winery
wastewater

2.14a

Yu et al.
(2002)

Initial pH

Mixed culture

Batch

55

Mixed culture

Batch

Mixed culture

6.56

Reference

5 PRACTICAL APPLICATIONS

Microorganism(s)

Fermentation
Mode
T (
C)

Continued

531

532
TABLE 3 Thermophilic Biohydrogen Production Processes with Different Feedstocks: Defined and Nondefined Carbon Sources (Organic Waste
Materials)Contd
Conditions

Thermoanaerobacterium
thermosaccharolyticum

Continuous
(UASB)

Mixed culture

Yield (mol
H2/mol
hexose)

Initial pH

Feedstock

Rate
(mmol H2/L/h)

60

5.5

Sucrose

152

1.7

O-Thong
et al. (2008b)

Continuous
(CSTR)

70

NA

Wheat straw
hydrolysate

8.2

NA

Kongjan
et al. (2010)

Mixed culture

Continuous
(UASB)

70

4.5

Glucose

2.3

2.5

Kotsopoulos
et al. (2006)

Mixed culture

Continuous
(ASBR)

55

5.5

Palm oil mill

effluent

2.6

2.24

O-Thong
et al. (2007)

Mixed culture

Continuous
(CSTR)

60

5.0

Artificial garbage
slurry containing
paper (AGSP)

199

NA

Ueno et al.
(2006)

Mixed culture

Continuous
(CSTR)

55

NA

Olive pulp

0.58

NA

Gavala et al.
(2005)

Mixed culture

Continuous
(CSTR)

55

5.5

Food waste

1.7

2.2

Shin and
Youn (2005)

Mixed culture

Continuous
(MBR)

Thermophilic

5.5

Glucose

48

NA

Oh et al.
(2004)

NA, not available.

a
Calculated based on the information provided.

Reference

23. THERMOPHILIC BIOHYDROGEN PRODUCTION

Microorganism(s)

Fermentation
Mode
T (
C)

Hydrogen Production

533

6 CHALLENGES, POSSIBILITIES, AND FUTURE PERSPECTIVES

hydrogen yield from thermophilic mixed cultures, the highest value of 2.5 mol H2/mol glucose was obtained in UASB operating at 70
C and pH 4.5 (Kotsopoulos et al., 2006).
Recently, a very promising approach for enhancement of fermentative hydrogen production based on nanoparticle addition was developed (Zhang and Shen, 2007). However, this
investigation was performed only under mesophilic conditions. More detailed investigations
are required to clarify the technological and economical feasibility of this approach at higher
temperatures.
Although several efforts have been made to improve biohydrogen reactor performance,
full-scale biohydrogen production has not been developed yet. However, some pilot-scale
applications have emerged in the recent years (Ren et al., 2006). An anticipated disadvantage of large-scale hydrogen production that needs to be addressed during scale-up
is the escape of hydrogen through large plastic enclosures and thin metal sheets that might
occur due to the high diffusivity of hydrogen. However, as hydrogen becomes a more
important fuel, full-scale applications will emerge in the future.

6 CHALLENGES, POSSIBILITIES, AND FUTURE PERSPECTIVES

The ultimate goal, and challenge, for fermentative hydrogen research and development
focuses essentially on attaining higher yields of hydrogen. Significant improvement can be
expected through rapid gas removal and separation, optimized bioreactor design, and genetic
modifications in the microorganisms. Nonetheless, it is rather difficult to predict which of the
various approaches will ultimately succeed in substantial enhancement of hydrogen yields so
that the dark fermentation process of hydrogen generation becomes commercially competitive.
To increase the economic feasibility of the process, recent attempts have concentrated on
further biological processing (second stage) of the organic acids-rich effluents from dark
fermentation hydrogen production. Two possible second stages (Figure 2) were investigated:
photoheterotrophic hydrogen production (Hawkes et al., 2007) and methanogenesis

Sugar containing waste

CO2
H2 + CO2
Dark fermentation
H2
separation
Organic acids-rich effluent

Option I
Methanogenic anaerobic digestion

H2

Option II
Photofermentation
H2 + CO2

CH4 + CO2

FIGURE 2 Possible treatments of the effluent from dark fermentative biohydrogen production.

534

23. THERMOPHILIC BIOHYDROGEN PRODUCTION

(Liu et al., 2006). If the technologically effective and cost-effective photobioreactors were
available, the two-stage process combining dark and light-driven hydrogen fermentation
would be a very promising method as it has a theoretical maximal molar yield of 12 mol
H2/mol hexose converted in the two-stage process (Hawkes et al., 2007). However, some
studies indicate that photofermentation is a very inefficient and expensive process with
respect to high energy demands for light sources and requirement for elaborate
photobioreactors covering large areas (Hallenbeck and Benemann, 2002). There are good
indications that a two-stage process with an acidifying hydrogen-producing first stage and
a methanogenic second stage gives rise to more efficient waste treatment and energy recovery
than a single-stage methanogenic process (Liu et al., 2006). This process could easily be
implemented in existing and new biogas plants, where an additional reactor could be added
before the traditional biogas reactor for production of biohydrogen. As a powerful combustion stimulant for accelerating methane combustion, a mixture of 20% hydrogen and 80%
methane known as hythane (The Hythane System) will reduce the emission of CO, CO2,
and NOx of natural gas powered vehicles and increase the efficiency of internal combustion
engines.
It is likely that the food industry and kitchen wastes will initially prove most attractive as
substrates for biohydrogen production at elevated temperature conditions. Reactor capital
and operating costs are likely to be similar to those already well known for anaerobic digestion. The next major challenge is to determine whether the economics and reliability of dark
fermentative hydrogen production are sufficiently attractive for commercial production.

Acknowledgments
This study received support from Danish Agency for Science, Technology, and Innovation under Bio REF. Project
No. 2104-06-0004 and from Danish Council for Strategic Research under Project No. 2101-09-0135 High rate algal
biomass production for food, biochemicals and biofuels.

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