Waste Biomass Valor (2010) 1:21–39
DOI 10.1007/s12649-009-9001-2
Biohydrogen Production from Biomass and Wastes via Dark
Fermentation: A Review
I. Ntaikou • G. Antonopoulou • G. Lyberatos
Received: 15 November 2009 / Accepted: 14 December 2009 / Published online: 4 February 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The present review article aims to summarize
the microbiological and technological background of the
dark fermentation processes for hydrogen generation,
emphasising on the exploitation of biomass and wastes as
potential feedstocks. The basic principles, the microbiol-
ogy and the current technology of the processes are out-
lined. Subsequently, the use of different types of biomass
and wastes that have so far been tested as feedstocks is
analysed focusing on the advantages, possible limitations
and future prospects of their exploitation. Moreover, dif-
ferent types of so far suggested pretreatment methods for
better utilisation of the feedstocks are presented, pointing
out the advantages and disadvantages of each method.
Finally, methods for possible further utilisation of the
generated by-products are laid out as well as the present
status of the real scale applications.
Keywords Biohydrogen
Energy
Biomass
Wastes

Wastewater
Introduction
Woody biomass has been the major source of energy for
mankind throughout the ages. During the fist steps of man
on earth the combustion of wood was more than enough to
meet his energy requirements, which actually were limited
I. Ntaikou
G. Antonopoulou
G. Lyberatos (&)
Department of Chemical Engineering, University of Patras,
Karatheodori 1 st., 265 00 Patras, Greece
e-mail: lyberatos@chemeng.upatras.gr
I. Ntaikou
G. Antonopoulou
G. Lyberatos
Institute of Chemical Engineering and High Temperature
Chemical Processes, 265 04 Patras, Greece
to cooking, warming and protection. The idea of using
waste as a source of energy is not new either. As a matter
of fact the use of animal manure as fuel via combustion is a
quite ancient practice, too. However as the world popula-
tion started to grow, mankind started searching for more
effective means of energy production, turning thus initially
towards the use of coal, and later onto oil, an even more
energy efficient fossil fuel. Nowadays, the overexploitation
of fossil fuels has brought mankind to a dead end from both
an environmental and energy resource standpoint. It is
indeed true that the fossil fuel reserves are not endless.
Consequently, their heedless use in order to cover the
continuously increasing energy demands [1] has resulted to
the excessive accumulation of carbon dioxide in the atmo-
sphere on one hand [2], while bringing humanity to an
imminent world energy crisis on the other [1, 3]. As a result of
those facts, an exigent need for identifying and exploiting
alternative energy sources has emerged; energy sources that
would be renewable and environmental friendly.
Hydrogen, although not being readily available in its
molecular form, is quite abundant in nature and compared to
other fuels, it has the highest specific energy and energetic
density. Moreover it can be produced via a number of pro-
cesses from renewable sources (it is called in such cases
biohydrogen), and it generates zero emissions when burned
for energy recovery [4, 5]. Those characteristics make
hydrogen an appealing candidate for a future energy system
based on sustainability. Biological hydrogen production
methods include biophotolysis of water, photofermentation
and dark fermentation of organic matter [6]. Among them,
dark fermentative hydrogen production comprises the sim-
plest technology, resulting at the same time to highest
yields. For the scaling up of such a process, the raw material
cost is considered to be among the major limitations. As raw
materials some carbohydrate- and/or, starch-rich wastes/
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wastewaters as well as cellulose-rich biomass are consid-
ered the most suitable feedstocks.
Main Principles of Hydrogen Production via Dark
Fermentation
The generation of biohydrogen via dark fermentation is
achieved mainly by strictly anaerobic or facultative anaer-
obic bacteria under anaerobic conditions [7]. Although dif-
ferent organic substances, such as carbohydrates, sugars,
proteins and lipids can in principle be used as substrates, for
the estimation of the theoretical yields of fermentative
hydrogen the reaction of glucose biotransformation towards
acetate is widely accepted as reference. According to that
reaction (1) [8, 9] the maximum theoretical yield of biohy-
drogen from glucose fermentation is 4 mol H2 per mol of
consumed glucose:
C6 H12 O6 þ 4H2 O ! 2CH3 COO þ 2HCO þ
3 þ 4H þ 4H2
DGo ¼ 206:3 kJ mol1 ð1Þ
The maximum hydrogen theoretical yield of 4 mol H2
per mol of consumed glucose can also be achieved in two
steps via the fermentation of glucose towards acetate and
formate according to reactions (2a) and (2b) [10, 11]:
C6 H12 O6 þ 2H2 O ! 2CH3 COO þ 2HCOO þ 4Hþ þ 2H2
DGo ¼ 209:1 kJ mol1 ð2aÞ
2HCOOH ! 2CO2 þ 2H2
DGo ¼ 6 kJ mol1 ð2bÞ
In the case that instead of acetate, butyrate is the sole
organic byproduct, the maximum hydrogen theoretical yield
becomes 2 H2 per mol of consumed glucose according to
reaction (3) [8]:
C6 H12 O6 þ 2H2 O ! CH3 CH2 CH2 COO
þ 2HCO þ
3 þ 3H þ 2H2
DGo ¼ 254:8 kJ mol1 ð3Þ
The basic step in all the above reactions is the metabolism
of glucose towards pyruvate as shown in reaction 4, through
which 2 mol of hydrogen can theoretically be generated
during the subsequent regeneration of the produced NADH
(NADH ? H? ? NAD? ? H2). In most bacteria, the
metabolism of glucose to pyruvate is usually carried out
via the EMP metabolic path.
C6 H12 O6 þ 2NADþ ! 2CH3 COCOO þ 4Hþ þ 2NADH
DGo ¼ 112:1 kJ mol1 ð4Þ
Nevertheless the ‘key’ substance for hydrogen production
from organic matter is acetylCoA, which is further produced
by pyruvate. The subsequent fate of acetylCoA is the factor
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Waste Biomass Valor (2010) 1:21–39
that will actually define whether the maximum theoretical
hydrogen yield will be 4 or 2 mol H2 per mol of consumed
sugar, but also whether the observed final yield will reach its
maximum value. Depending on the enzymatic system that a
microorganism has, acetylCoA can be generated either via
reactions (5) or (6).
pyruvate þ CoA þ Fdox $ acetylCoA þ CO2 þ Fdred
DGo ¼ 19:2 kJ mol1 ð5Þ
pyruvate þ CoA $ acetylCoA þ formate
DGo ¼ 16:3 kJ mol1 ð6Þ
Reaction (5) is catalyzed via the enzyme pyruvate-
ferredoxin oxidoreductase, where ferredoxin is the
coenzyme that acts as electron receiver [12]. This enzyme
has been found in many strictly anaerobic [13] as well as in
facultative anaerobic bacteria [14] but also in cyanobacteria
[15]. AcetylCoA can further be metabolized either to acetate
(Fig. 1a), or to butyrate (Fig. 1b), and in both cases from the
re-oxidation of each mole of ferredoxin one mole of
hydrogen will be generated via a hydrogenase enzyme. In
the case that acetate is the final product, one extra mole of
hydrogen will also occur from the reduction of each mole of
NADH, that was produced during glycolysis, to NAD?,
leading thus to a total hydrogen yield of 4 moles/mol of
consumed glucose. In the case that butyrate is the final
product, the produced from glycolysis NADH is actually
used for the oxidation of acetoacetylCoA to butyrate, and
thus the total final hydrogen yield is 2 mol/mol of consumed
glucose. Depending on the culture conditions as well as the
type of microorganism, simultaneous generation of acetate
and butyrate can often be observed, leading thus to a
hydrogen yield with values between 2 and 4. Such reactions
are quite typical for some clostridia such as Clostridium
pasteurianum [16] and C. butyricum [17].
The second way of acetylCoA generation, as described
by reaction (6), leads to the simultaneous production of
formate [18]. This reaction, as shown in Fig. 2, is catalysed
by the enzyme pyruvate-formate lyase [19], and is typical
of the metabolism of enterobacteria, such as Enterobacter
aerogenes and Escherichia coli [20] under anaerobic con-
ditions. The function of pyruvate-formate lyase in entero-
becteria is regulated at the transcription level based on the
oxygen level in the environment [21]. Besides enterobac-
teria, the presence of pyruvate-formate lyase has also been
reported for some clostridia [22].
It is nevertheless true that glucose fermentation can also
lead to the generation of other products besides acetate,
butyrate and formate. Such products are propionate, suc-
cinate, lactate and 2,3 butanediol that emerge directly from
pyruvate, as well as ethanol, butanol and isopropanol that
emerge through a further step from acetylCoA metabolism
Waste Biomass Valor (2010) 1:21–39 23

Fig. 1 Generation of hydrogen

with simultaneous production of
acetate (a) and butyrate (b) via
glycolysis. Extra molecular
hydrogen can be generated in
the case of acetate production
by NADH that is generated
during glycolysis

undesired byproducts, the generation of which leads to

lower overall hydrogen yield. Those products can often
emerge when fermentations are carried out via mixed
metabolism bacteria or via mixed cultures. The distribution
of metabolites seems to be highly dependent on the pre-
vailing culture conditions. It is reported that for E. coli, for
example, for pH values below 7 the generation of lactate is
favored [23], leading thus to reduced hydrogen yields. In
such cases, a strict selection of proper culture conditions
has to be made so as to lead the metabolism towards
hydrogen generation and/or towards the dominance of
hydrogen producing microorganisms when mixed cultures
are used.

Microorganisms

Fermentative hydrogen production can be carried out via a

Fig. 2 Generation of hydrogen with simultaneous production of
acetate, ethanol, lactate and formate (mixed acid fermentation) via
glycolysis. Extra molecular hydrogen can be generated by NADH that
is generated during glycolysis
[21]. The generation of all abovementioned metabolites is
not accompanied by hydrogen generation, and thus during
dark fermentative production, these are characterized as
wide range of microorganisms, with quite diverse require-
ments in terms of substrate preference, pH and temperatures
[24]. Those parameters do not only determine the growth of
the microorganisms, but also have a crucial role on the
metabolic path that the microorganisms will follow,
affecting thus severely the final observed hydrogen yield
from the whole process. Hydrogen production can be
achieved either through mixed acidogenic microbial

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cultures, derived from natural environments such as soil,
wastewater sludge, and compost, or through pure cultures
of selected hydrogen producing bacteria. Such bacteria
can be mesophilic (25–40°C), thermophilic (40–65°C),
extreme thermophilic (65–80°C), or even hyperthermo-
philic ([80°C). In any case, the final selection of the type of
culture to be used (mixed, pure, co-culture) as well as the
specific microorganism, has to be based on the specific
requirements of each possess.
Mixed Cultures
In general, for a full-scale application the selection of
mixed cultures is considered to be favourable, at least
from an engineering standpoint. This is due to the fact
that the control and operation of the process is facilitated
when no medium sterilisation is required, reducing thus
the overall cost, whereas it also allows for a broader
choice of feedstocks selection [25]. The mixed consortia
can be derived from a variety of different natural sour-
ces, such as sewage sludge [26–28], anaerobically
digested sludge [29–32], acclimated sludge [33], com-
post [34–37], animal manure [35] and soil [38, 39] or
even from the indigenous microorganisms found in cer-
tain wastes [40–43].
Despite the advantages of mixed cultures in terms of the
economical viability of a process, their use always lurks the
possible predominance of non-hydrogen producing species
such as methanogens, homoacetogens and lactic acid bac-
teria; a case which could eventually lead to the dramatic
failure of the viability of process. The strategy followed in
order to minimise such a possibility includes on the one
hand an initial pretreatment of the seed so as to remove to
the highest possible degree the undesired microorganisms,
and on the other, the maintenance of such environmental
and operational conditions that favour the predominance of
the desired hydrogen producing species. In terms of the
seed pretreatment, heat treatment is generally the most
common practice [40, 44]. By subjecting seed cultures to
high temperatures, only the spore-forming acidogenic
microorganisms survive the thermal shock, whereas the
methanogenic non-spore-forming bacteria die. The use of
temperatures reported are 75°C [45], 100°C [46–49] or
even 121°C [42] whereas the duration of the process has
been reported to vary between 15 min and 2 h [42, 45–49].
Alternatively to heat treatment, an acid/base treatment of
the seed has also been suggested as a possible way for
ensuring the dominance of hydrogen producing bacteria
[26, 50]. The principle of this method is to maintain the
seed microorganisms for a prolonged period of time at very
acidic or very basic conditions, which would eventually
lead to the removal of methanogens that cannot survive
such extreme pH values.
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Waste Biomass Valor (2010) 1:21–39
Pure Cultures of Wild Strains
Alternatively to mixed cultures, many researchers have
focused on the use of pure cultures of selected hydrogen
producing species. The main arguments for their advanta-
geous use are, the selectivity of substrates, the easiest
manipulation of the metabolism by altering growth condi-
tions, the higher observed hydrogen yields as an effect of
the reduction of undesired by-products as well as the
repeatability of the process. On the other side of the coin,
pure cultures can be quite sensitive to contaminations and
thus their use demands, in most cases, the presence of
aseptic conditions, which significantly increases the overall
cost of the process.
There is a wide range of microorganisms that are
capable of producing hydrogen via dark fermentation. This
includes strict anaerobes (Clostridia, methylotrophs, rumen
bacteria, methanogenic bacteria, archaea), facultative
anaerobes (E. coli, Enterobacter, Citrobacter), and even
aerobes (Alcaligenes, Bacillus). Among the hydrogen-
producing bacteria, Clostridium sp. and Enterobacter, are
the most widely studied. Species of genus Clostridium such
as C. butyricum [51], C. acetobutyricum and C. beijerinckii
[52], C. thermolacticum [53], C. tyrobutyricum [54], C.
thermocellum [55] and C. paraputrificum [56] are exam-
ples of strict anaerobic and spore forming microorganisms,
generating hydrogen gas during the exponential growth
phase. In parallel, facultative anaerobes such as E. coli and
species of genus Enterobacter, such as E. aerogenes [57,
58] and E. cloacae [59, 60] have also been used for
hydrogen production.
In the recent years, extensive research has also been
carried out in hydrogen production at high temperature,
using thermophilic or hyperthermophilic bacteria, since the
increase of temperature in principle improves the reaction
kinetics. The thermophiles that have been studied include
Caldicellulosiruptor saccharolyticus [61], Thermoanaero-
bacterium sp. such as T. thermosaccharolyticum [62] and
Thermotoga sp. such as T. maritima [63] and T. elfii [64].
Among them, the use of hyperthermophilic bacteria has
lately attracted increasing interest due to their many out-
standing properties. Since, they can grow successfully at
very high temperatures (reaching even 110°C) hyper-
thermophiles make hydrogen fermentation less sensitive to
contaminations by undesirable intruders. Moreover, hyper-
thermophiles show a better resistance to high hydrogen
partial pressures [65], one of most inhibitory factors in
fermentative hydrogen production processes.
Genetically Modified Microorganisms
Fermentative hydrogen production processes, either con-
ducted via mixed or pure cultures, still have certain
Waste Biomass Valor (2010) 1:21–39
limitations that could be crucial for scaling up. Inefficient
substrate conversion, formation of by-products, low resis-
tance to high H2 partial pressures and oxygen are only
some of them. On top of all those, comes the limiting upper
value of hydrogen yields which is 4 mol of H2/mol hexose
consumed, a limit that could be theoretically highly
increased taking into account that one mol of hexose
contains 12 gram-atoms of hydrogen. As an answer to the
abovementioned limitations several researchers have pro-
posed the use of genetically modified microorganisms with
advanced selected properties, constructed either via
mutagenesis or through genetic engineering. The main
areas of modification can be summarized as follows: (a)
introduction or manipulation of genes responsible for the
overexpression of enzymes with cellulolytic activities
such as cellulases, hemicellulases and lignases, or
enzymes for uptake of different types of sugars, both
aiming at the maximization of substrate availability and
conversion, (b) elimination of hydrogen-consuming
hydrogenases and (c) overexpression of hydrogen-pro-
ducing hydrogenases, that have also been modified so as
to be hydrogen tolerant [66]. E. coli, one of mostly
studied facultative anaerobes for hydrogen generation has
also been widely used as the carrier of different genes
through mutagenesis and genetic engineering. So far, the
construction of many different genetically modified
strains has been reported including strains capable of
consuming pentoses (case a) [67], strains with suppressed
lactate and succinate dehydrogenases activities (case b)
[68, 69] and strains showing overexpression of formate
hydrogenlyase [70–72]. Increase of cellulolytic activity
(case a) for enhancing hydrogen production from ligno-
cellulosic biomass has been achieved for Clostridium
beijerinckii [73].
Types of Feedstocks
Theoretically any organic substrate rich in carbohydrates,
fats and proteins could be considered as possible substrate
for biohydrogen production. However, as reported by
numerous studies, carbohydrates are the main source of
hydrogen during fermentative processes and therefore
wastes and biomass rich in sugars and/or complex carbo-
hydrates turn out to be most suitable feedstocks for bio-
hydrogen generation [6]. According to a comparative study
by Lay et al. [35], using substrates of different chemical
composition treated with the same mixed consortium, it
was shown that the hydrogen-producing potential of car-
bohydrate-rich waste (rice and potato) was approximately
20 times higher than that of fat-rich waste (fat meat and
chicken skin) and of protein-rich waste (egg and lean
meat).
25
The major criteria that have to be met for the selection
of substrates suitable for fermentative bio-hydrogen pro-
duction are availability, cost, carbohydrate content and
biodegradability [74]. Simple sugars such as glucose,
sucrose and lactose are readily biodegradable and thus
preferred as model substrates for hydrogen production
[75–78]. However, pure carbohydrate sources are expen-
sive raw materials for real scale hydrogen production,
which can only be viable when based on renewable and
low cost sources [79, 80]. Different types of classifications
have been reported so far for the description of biomass
and wastes that have been used for fermentative hydrogen
productions. Most often, the classification criteria used are
either the chemical composition of the major present sub-
strate [6, 51] and/or the origin of the biomass/waste [6, 81–83].
The most frequently exploited types of biomass/wastes
used as feedstocks for hydrogen production are analysed
below.
Energy Crops
The idea of exploiting whole crops for energy production
had already emerged by the early 1980s [84, 85]. By def-
inition ‘energy crops’ refer to certain plants that are cul-
tivated solely for the further exploitation of their biomass
(either whole or part of it) as feedstock for energy pro-
duction, which can be directly exploited for its energy
content via combustion or be biotransformed to biofuels. In
general, the sustainability of such processes can only be
assured if (a) the crops are produced at low cost, thus with
minimum nutrient and water requirements, (b) they are
resistant to environmental stresses, and (c) they are highly
biomass yielding [74]. Furthermore, such plans in order to
be suitable for hydrogen production via dark fermentation
should also have high sugar and/or carbohydrates’ content
and low lignin content [74]. According to their chemical
composition, energy crops used for fermentative hydrogen
production can be divided in sugar based crops (e.g. sweet
sorghum, sugar cane and sugar beet), starch based crops
(e.g. corn and wheat), and lignocellulose based crops
including herbaceous (e.g. switch grass and fodder grass)
and woody (e.g. Miscanthus and poplar). The results from
studies conducted for hydrogen production via dark fer-
mentation with different types of energy crops are sum-
marised in Table 1. As shown, energy crops can be quite
sufficient for hydrogen generation, especially when the
latter is based on cultures of pure microorganisms.
Lately however, the continuously rising food prices, the
sustainability doubts and the energy-equation challenges
have led to a backlash against the use of energy crops as
feedstocks for biofuels generation. This backlash was
centred on the food-vs.-fuel debate [95, 96], since in many
countries huge agricultural areas have been turned into
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Table 1 Fermentative hydrogen production from energy crops

Crop Microorganism Operation Maximum H2 Maximum H2 yield References
mode production (mol H2/mol cons.
rate (LH2/l/day) hexose)

Miscanthus (pretreatment: Thermotoga elfii Batch – 1.1a [64]

mechanical and NaOH)
Wheat starch Mixed mesophilic cultures Continuous 3 1.26 [86]
Sugarbeet juice Mixed mesophilic cultures Continuous 2.2b 1.9 [87]
Corn starch Mixed mesophilic cultures Continuous 2.57 0.51 [88]
Sweet sorghum extract Indigenous microbial Continuous 8.52 0.86 [89]
mesophilic culture
Sweet sorghum stalks Rumicococcus albus Batch – 3.15 (59 l/kg wet [90]
biomass)
Sweet sorghum extract Rumicococcus albus Batch – 2.61 [90]
Ryegrass Mixed mesophilic cultures Continuous 6 82c [91]
Sweet sorghum Caldicellulosiruptor saccharolyticus Batch – 1.75 (30.17 l/kg dry [92]
biomass)
Sugar beet extract Caldicellulosiruptor saccharolyticus Batch – – [93]
Barley grains Caldicellulosiruptor saccharolyticus Batch – – [93]
Corn grains Caldicellulosiruptor saccharolyticus Batch – – [93]
Miscanthus (pretreatment: Thermotoga neapolitana Batch 13.1d 3.2 [94]
NaOH, Ca(OH)2)
Miscanthus (pretreatment: Caldicellulosiruptor saccharolyticus Batch 12.6d 3.4 [94]
NaOH, Ca(OH)2)
a
mol H2/mol consumed sugars
b
ml/min l
c
ml H2/g dry mass
d
mmol H2/l h

feedstock ‘industries’ for the production of biofuels. The
main arguments against the use of energy plants is that
crops that could support human dietary needs—either
directly or indirectly through farmed animals—are diverted
to the production of biofuels. Even in the case of non-food
crops cultivation, the sustainability issue is getting under
question. As an answer to those issues, the production of
second generation biofuels is proposed, i.e. biofuels pro-
duced by feedstocks that are not competitive to edible
crops such as wastes and residues.
Wastes and Residues
Lignocellulosic Residues
Different types of lignocellulosic residues have been
studied as potential renewable feedstocks for fermentative
biohydrogen production. These include agricultural resi-
dues such as sugar cane and sweet sorghum bagasse, corn
stalks and stover, fodder maize, wheat straw, etc. and
forestry residues such as wood trimmings. Among them
agricultural residues seem to be the most abundant as
estimated by the Food and Agriculture Organization.
Around 2.9 9 103 million tons from cereal crops and
1.6 9 102 million tons from pulse crops, 1.4 9 10 million
tons from oil seed crops and 5.4 9 102 million tons from
plantation crops are produced annually worldwide [97],
being thus a voluminous source of lignocellulose that could
be potentially utilised via bioconversions. Compared to the
use of energy crops, the exploitation of residues, remaining
after the harvesting and processing of the starch or sugar
crops that cannot be further exploited in the food industry
chain, is more likely to yield a solution with far better
overall prospects for economic and environmental sus-
tainability [98]. Hydrogen generated from such feedstocks
can be characterised as ‘2nd generation hydrogen’ since its
production is not competitive to food.
Although being an abundant and almost zero cost
feedstock, agricultural and forestry residues do not contain
easily fermentable free sugars, but complex carbohydrate
polymers, i.e. cellulose and hemicellulose, which are
tightly bonded to lignin [99, 100]. Thus, their biotransfor-
mation to hydrogen is not an easy task in most cases. Even
in the case that cellulolytic microorganisms are used for
fermentative hydrogen production, residues have to be
subjected to some kind of pretreatment, so that their

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Waste Biomass Valor (2010) 1:21–39 27

Table 2 Fermentative hydrogen production from lignocellulosic residues

Lignocellulosic Pretreatment Microorganism Operation H2 production Maximum H2 References
residue mode rate yield (mol/mol
cons. hexose)

Wood fibers Mechanical Clostridium Batch – 1.47 [55]

thermocellum
Corn stover Steam explosion Mixed mesophilic Continuous 10.56 mmol/h 3 [101]
(90–220°C, 3–5 min) cultures
Sugarcane bagasse Acid-thermal Clostridium Batch 1.611 l/l/day 1.73b [102]
hydrolysate hydrolysis butyricum
H2SO4 0.27–7(v/v),
?121°C, 60 min
Fobber maize Mechanical Mixed mesophilic Continuous – 69.4c [91]
juice cultures
Sweet sorghum Mechanical Rumicococcus albus Batch – 2.59 [90]
residues
Wheat straw Mechanical Caldicellulosiruptor Batch – 3.8 (44.7 l/kg [92]
saccharolyticus dry biomass)
Maize leaves Mechanical Caldicellulosiruptor Batch – 3.6 (81.5 l/kg [92]
saccharolyticus dry biomass)
Barley straw Mild acid 1.8% Caldicellulosiruptor Batch – – [93]
H2SO4w/w saccharolyticus
Corn stalks Mild acid 1.8% Caldicellulosiruptor Batch – – [93]
H2SO4w/w saccharolyticus
Bagasse Alkali-thermal Mixed thermophilic Batch 0.28 mmol/h/g 13.39d [103]
0.2–4 g/l NaOH, cultures TVS
100°C, 2 h
Corn stover Acid-thermal hydrolysis Thermoanaerobacterium Batch 3.305 l/day 2.24 [104]
H2SO4 0.25–4(v/v), thermosaccharolyticum
?121°C, 30–180 min
a
l/kg TVS
b
mol/mol total sugar
c
ml H2/g dry mass
d
mmol H2/g TVS

delignification and the subsequent loosening of the struc-
ture of cellulose and hemicellulose can be achieved,
facilitating thus the subsequent liberation and uptake of
sugars [93]. In Table 2 different types of residues used as
feedstocks for hydrogen production are presented, as along
with the achieved hydrogen yields and rates.
Wastes and Wastewaters
The biotransformation of wastes and wastewater towards
hydrogen can be considered quite appealing from both the
environmental (pollution control, renewable energy) and
the economical (resources recovery, low total cost waste
management) standpoint. The criteria according to which a
waste/wastewater would be characterised as efficient
feedstock for hydrogen generation are a high concentration
of degradable organic compounds, high proportion of
readily fermentable compounds such as sugars and
carbohydrates and low concentration of inhibitory to
microbiological activity compounds.
Many types of food processing industrial wastewaters
can be considered suitable feedstocks for hydrogen pro-
duction via dark fermentation. Rice winery, noodle, sugar,
and molasses manufacturing, olive mill wastewater, olive
pulp and cheese whey are some of the wastewaters that
have been successfully tested for hydrogen production at
laboratory scale (Table 3). Coming from food industry,
such wastewaters theoretically meet all three abovemen-
tioned criteria, which can make them ideal candidates for
hydrogen production via microbial processes. Indeed, quite
high yields of hydrogen have been achieved from different
food industry wastes without any pretreatment. However, it
has to be mentioned that in most cases dilution of the raw
waste has to be performed so as to lower the organic
loading which otherwise can prove to be inhibitory for the
success of the process [32, 43, 49, 114, 117]. Moreover,

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Table 3 Fermentative hydrogen production from different types of waste and wastewaters
Type of waste/ Microorganism Operation H2 production Maximum H2 yield References
wastewater mode rate

Sugar factory Mixed thermophilic culture Continuous 4.4 l/l/day 2.6 mol/mol hexose [105]
wastewater
OFMSW Mixed mesophilic culture Batch 0.4 l/g VSS/day 0.15 l/g OFMSW [40]
Rice winery Mixed culture Continuous 9.33 l/g VSS/day 2.14 mol/mol hexose [106]
wastewater 3.81 l/l/day
Food waste—sewage Mixed mesophilic culture Batch 2.67 l/g VSS/day 122.9 ml/g COD [107]
sludge carbohydrate
Food waste Mixed thermophilic culture Batch 0.288 l/g VSS/day 1.8 mol/mol hexose [108]
Cheese whey Clostridium saccharoperbutylacetonicum Batch 28.3 ml/h 7.89 mmol/g lactose [109]
Potato processing Mixed mesophilic culture Batch – 2.8 l/l wastewater [110]
wastewater
Cheese whey Mixed mesophilic culture Batch – 10 mM/g COD [111]
Dairy wastewater Mixed mesophilic culture Continuous 1.59 mmol H2/l/day – [112]
Molasses Mixed mesophilic culture Continuous 4.8 l/l/day – [113]
Cheese whey Mixed mesophilic culture Batch 8.1 mmol/l/h 5.9 mol/mol lactose [114]
Cheese whey Mixed mesophilic indigenous Continuous 2.51 l/l/day 0.9 mol/mol hexose [43]
microbial culture
Olive pulp Mixed mesophilic culture Continuous 0.26 l/l/day 0.19 mol/kg TS [115]
Olive oil mill Mixed mesophilic culture Continuous 201.6 ml/day 196.2 ml/g hexose [49]
wastewater
Wastepaper Ruminococcus albus Batch – 2.29 mol/mol hexose [116]
(282.76 l/kg dry biomass)

such wastes can have a quite complex chemical com-
position, including different organic and inorganic sub-
stances with varying concentrations that may limit the
reproducibility of a proposed process. Consequently,
defining the exact conditions for the efficient treatment
of a certain waste is not always possible. For example,
in the case of cheese whey, a rich in readily ferment-
able sugars wastewater, Davila-Vazquez et al. [114]
suggest that a pH between 6 and 7 is optimum for
obtaining the highest hydrogen yield and production
rate, respectively, whereas Yang et al. [118] suggest
that a pH in the range 4–5 is the best in terms of both
hydrogen yield and rate. In both cases mixed mesophilic
cultures where used.
Complex solid wastes, such as wastes from kitchen,
food processing, mixed wastes, and municipal wastes have
also been tested as feedstocks for fermentative hydrogen
production. Such wastes apart from carbohydrates usually
have quite high contents of proteins and fats, and thus their
conversion efficiencies to hydrogen are comparatively
lower than those obtained from carbohydrate based
wastewaters. Indeed, according to previous studies, the
hydrogen production potential of carbohydrate-based
wastes, was 20 times higher than that of fat-based and
protein-based wastes [35]. This observation was partially
attributed to the consumption of hydrogen towards
ammonium using nitrogen generated from protein biodeg-
radation. Among different types of wastes, the organic
fraction of municipal solid waste (OFMSW) could be
considered quite promising as a potential feedstock for
hydrogen production, since it can represent up to 70% of
the total MSW produced, consisting of paper (up to 40%),
garden residues, food wastes and wood [99]. It has to be
noted though that such exploitation would require an initial
selection/separation of the suitable substrates, which will
lead to an additional cost in the overall process.
A special type of waste that is attracting increased
attention lately is crude glycerol, coming from the bio-
diesel production industry. Glycerol is a valuable chemical
that is widely used in the cosmetics industry. The recent
increase of the biodiesel production from vegetable oils
and fats nowadays has lead to the generation of large
quantities that have to be disposed somehow. In addition,
the potential of glycerol utilisation would help improve the
economics of biodiesel production. Although it is well
established that the maximum theoretical hydrogen yield
that can be obtained from carbohydrates is 4 [7], the
maximum possible theoretical hydrogen yield from glyc-
erol is still under question. Akutsu et al. [119] based on the
study of Thauer et al. [8] on microbial metabolism, assume

123
Waste Biomass Valor (2010) 1:21–39
that the maximum theoretical yield is 3 mol H2/mol glyc-
erol, according to the reaction:
C3 H8 O3 þ 2H2 O ! CH3 COO þ HCO þ
3 þ 2H þ 3H2
In the study of Akutsu et al. [119] a mixed mesophilic
consortium was used as inoculum, in which it proven that
clostridia were the predominant species. The maximum
obtained yield was 38.1 ml H2/g-CODglycerol, with 1,3
propanediol being the main by-product, followed by acetate,
whereas no butyrate (the main by-product generated from
Clostridia during carbohydrates fermentation) was detected.
In contrast to the assumption of Akatsu et al., in the studies of
Ito et al. [120] and Sakai and Yagishita [121] the maximum
theoretical hydrogen yield from glycerol is assumed to have
the value of 1 mol H2/mol glycerol, based on the reaction of
glycerol conversion to H2, CO2, and ethanol as described by
the reaction:
C3 H8 O3 ! H2 þ CO2 þ C2 H5 OH
Both studies were conducted by pure cultures of the bac-
terium Enterobacter aerogenes which is known to follow
the enterobacteriaceae pattern for hydrogen production
from carbohydrates [21] The study of Ito et al. [120], has
shown that crude glycerol waste has to be diluted so as to
lower the organic loading and the salt concentrations, both
of which seemed to be inhibitory above a certain limit. It
was shown that batch cultures with suspended microbial
cells could lead to a hydrogen production rate of 30 mmol/
l/h, which though was doubled (63 mmol/l/h) when using
porous ceramics as a support material to fix cells in the
reactor. Another interesting finding from that study was
that the addition of nutrients, such as yeast extract and
peptone could enhance highly the hydrogen producing
capacity of the microorganism from the particular waste,
something though that could be expected to some degree,
since the waste is by definition poor in nitrogen, an element
which is necessary for microbial growth. The maximum
observed hydrogen yield observed by Sakai and Yagishita
[121] was 0.77 mol H2/mol glycerol with ethanol being the
dominant by-product, whereas no propanediol was detec-
ted. On the other hand, 1,3 propanediol was the main
by-product during fermentative hydrogen production by
pure cultures of Klebsiella pneumoniae [122]. The maxi-
mum obtained hydrogen yield from that study was reported
to be 0.53 mol H2/mol glycerol, whereas the maximum
hydrogen production rate was 17.8 mmol/l/h. Based on the
findings from those studies, it can be assumed that the
exploitation of crude glycerol towards hydrogen can be
more efficient when based on enterobacteria fermentations.
It has to be noted, however, that this is a rather new field
that needs to be further optimised.
29
Pretreatment of Lignocellulose-Based Biomass
and Wastes
Although starchy and sugar based biomass and wastes are
ð7Þ
readily fermentable by microorganisms for hydrogen gen-
eration, lignocellulosic biomass needs to be pretreated so as
to be exploitable. Pretreatment is commonly accepted to be
an essential prerequisite to make lignocellulosic biomass
accessible to enzymatic attack (either by microorganisms
or enzymes), by breaking the lignin seal, removing hemi-
cellulose, or disrupting the crystalline structure of cellulose
[123]. The limiting factors that affect enzymatic hydrolysis
of biomass have been traditionally divided into two groups:
biomass structural features and enzyme mechanism. Pre-
treatment is responsible for altering the structural features
of biomass which are classified as physical or chemical.
The chemical structural features involve the compositions
of cellulose, hemicellulose, lignin, and acetyl groups bound
to hemicellulose. The physical structural features include
ð8Þ
the accessible surface area, the crystallinity, the physical
distribution of lignin in the biomass matrix, the degree of
polymerization, the pore volume, and the biomass particle
size [124].
The correlation between structural features and
digestibility is reported to be of varying importance,
depending on the type of biomass to be treated [125,
126]. Consequently, because of the heterogeneity of
lignocellulosic biomass, the application of the same
pretreatment method can affect to a different extent each
of the above parameters, whereas different pretreatment
methods have a different effect on each of the above
parameters. In general, pretreatment methods of ligno-
cellulosic biomass can be divided into three main types,
according to the means used for altering its structural
features: mechanical, physicochemical and biological.
Mechanical pretreatment is almost always applied before
any other kind of pretreatment, and actually refers to
milling, though which reduction of particle size and
crystallinity of biomass is achieved. The reduction in
particle size leads to an increase of available specific
surface and a reduction of the degree of polymerization
[127]. Physicochemical pretreatment was the first to be
applied successfully in lignocellulosic biomass for sub-
sequent ethanol production [128–131] and this knowl-
edge has already been transferred to hydrogen production
field. However, though research on biological pretreat-
ment of lignocellulosic biomass for ethanol production
has also been performed [132, 133], the application of
biological pretreatment methods to the fermentative
production field has not been so far studied. The main
principles of different pretreatment methods are analysed
below.
123
30
Physicochemical Pretreatment
During physicochemical pretreatment, lignocellulosic bio-
mass is exposed to acidic, alkaline or oxidative conditions,
at ambient or elevated temperature. The use of high tem-
peratures without the addition of some chemical agent can
also be effectuated, leading to a process simply called
thermal pretreatment. In any of the above cases, the result
is that lignocellulosic biomass is fractionated since the
bonds that hold together cellulose, hemicellulose and lignin
are loosened and changes to the chemical structure of all
three polymers occur to a different degree, according to the
severity of each method. Among celluloses, hemicelluloses
and lignin, hemicelluloses are the most sensitive when
thermal-chemically treated and thus are the first to be
degraded [134]. Combinations of the methods have been
examined, combining two or more physical and chemical
pretreatment elements. For acid pretreatment of lignocel-
lulosic biomass, either dilute or concentrated acids, such as
H2SO4 and HCl, can be used. The main reaction that occurs
during acid pretreatment is the hydrolysis of hemicellulose,
especially xylan, since glucomannan is more stable. Under
such conditions furfural (FF) and hydroxymethylfurfural
(HMF) generation can occur because of dehydration of
xylose and galactose, mannose and glucose respectively,
whereas further production of formic and levulinic acids
can also be observed [99]. Lignin is hardly dissolved in
most cases, but it is disrupted to a high degree, leading to
increased cellulose susceptibility to enzymes [135]. Alka-
line pretreatment refers to the addition of dilute bases on
the biomass leading to an increase of internal surface by
swelling, a decrease of polymerization degree and
crystallinity, destruction of links between lignin and
other polymers, and lignin breakdown. The effective-
ness of this method depends on the lignin content of the
biomass [136]. Lignin is primarily affected by alkaline
pretreatment methods, causing depolymerisation and
cleavage of lignin-carbohydrate linkages [137]. Hemi-
cellulose solubilisation into its oligomers also takes
place whereas cellulose structure is affected to a less
degree. When thermally pretreated the lignocellulosic
biomass is actually heated at temperatures between 150
and 220°C. When treated at temperatures above 160–
180°C, part of the lignocellulosic biomass, first the
hemicelluloses and lignin shortly after, solubilise [138].
During thermal processes a part of the hemicellulose is
hydrolyzed, forming acids, which subsequently act as
catalysts for the further hydrolysis of hemicellulose.
However, it is concluded that other so far unknown
factors, apart from the formed acids, can play a role in
the solubilisation of hemicellulose [128, 139]. Thermal
pretreatment with temperatures of 160°C also lead to
the solubilisation of lignin. The produced compounds
123
Waste Biomass Valor (2010) 1:21–39
are almost always phenolic compounds and have in
many cases an inhibitory or toxic effect on bacteria,
yeast and methanogens/archae [140]. Temperatures of
250°C and higher should be avoided during pretreat-
ment, as unwanted pyrolysis reactions start to take
place. The application of different physicochemical
pretreatment methods on lignocellulosic biomass aiming
at facilitating hydrogen production, are summarized in
Tables 1 and 2.
Biological Pretreatment
Biological pretreatment of lignocellulosic biomass aims at
the removal of lignin so that the complex carbohydrates
cellulose and hemicellulose are liberated, becoming thus
accessible to enzymatic attack for their depolymerization
(saccharification). It can be performed either by whole cell
microorganisms or enzymes capable of degrading lignin
by oxidation [141]. Lignin is a polymeric substance of
coniferyl alcohol and sinapyl alcohol, with functional
groups such as hydroxyl, methoxyl and carbonyl. The
amorphous heteropolymer is non-water soluble and opti-
cally inactive, making thus its degradation very tough [99].
Consequently very few species are capable of performing
biological break down of lignin. Among them bacteria
symbiotic to termites [142] and white rot fungi are inclu-
ded. The enzymatic degradation of lignin proceeds through
the concerted action of specific enzymes, i.e. lignin per-
oxidase, manganese peroxidase, H2O2-generating enzymes
and laccase, which produce strong oxidants and thus
combust, the lignin framework [143]. On comparing the
efficiency of whole cell and enzymatic delignification, a
possible drawback of the first could be the degradation and
consumption of carbohydrates, resulting in lower yields
during the subsequent bioconversion to hydrogen. How-
ever, white-rot fungi can perform selective or non-selective
delignification of wood. In selective delignification, lignin
is removed without any marked loss of cellulose, whereas
in non-selective delignification all the major cell wall
components are degraded [144]. Among the best studied
white-rot fungi are Phanerochaete chrysosporium and
Phlebia radiata which degrade lignin selectively, and
Pleurotus sp. which degrades lignin and hemicelluloses
selectively. The final delignification efficiency is affected
by several parameters such as the oxygen and moisture
level, the C:N ratio and concentration of specific ions like
Cu2?. The reactor system choice and development, solid
state or submerged fermentation system, is of crucial
importance as well. In overall, biological pretreatment,
using microorganisms and/or their enzyme systems to
breakdown the lignin present in lignocellulosic biomass, is
an environmentally friendly approach, and according to
some researchers with significant advantages over the
Waste Biomass Valor (2010) 1:21–39
physicochemical pretreatment technologies, such as
reduced energy and material costs and simplified processes
and equipment [145, 146]. Research results are very
encouraging so far; however, the development and appli-
cation of a fungi-based pretreatment step for efficient and
cost-effective lignocellulose break-down has a long way
to go.
Formation of Inhibitory Compounds
due to Pretreatment
Upon either physicochemical or biological pretreatment
of lignocellulosic biomass, various organics substances
are generated that are considered to be undesirable bio-
products since they may be inhibitory to the microbial
metabolism as has been demonstrated by several
researchers [102, 146–149]. The most commonly gener-
ated, being also the best studied are furfural, hydroxy-
furfural and phenolics. Furfural (FF, C5H4O2) and
5-hydroxymethylfurfural (HMF, C6H6O3) are aldehyde
and furan compounds, which are formed during the
thermal decomposition of sugars and carbohydrates,
whereas phenolics are chemical compounds containing a
hydroxyl group (–OH) bonded directly to an aromatic
hydrocarbon group and are produced during the degra-
dation of lignin. During thermochemical pretreatment, all
three inhibitory compounds are formed, whereas during
biological pretreatment only phenolics emerge.
The severity of the inhibitory effect of these com-
pounds depends on their concentrations and thus, in
order to achieve efficient fermentations, either mild
pretreatment methods should be selected, or the inhib-
itory compounds should be removed prior to feeding the
fermentation bioreactors [102, 147–149]. Such removal
is indeed possible and can be achieved by several
methods, such as extraction [150], ion exchange [151],
activated carbon [152], overliming [153] or laccase and
peroxidase treatment [154, 155]. Although the inhibi-
tory effect of FF, FHM and phenolic compounds on
hydrogen producing bacteria is not yet well defined, it
is expected that the results would be similar to those
observed during fermentations for ethanol or methane
production.
Summarizing, an ideal effective pretreatment process
would produce reactive fibre, yield pentoses in non-
degraded form, exhibit no significant inhibition of fer-
mentation, require little or no feedstock size reduction,
entail reactors of reasonable size (high solids loading), built
of materials with moderate cost, not produce solid residues,
and have a high degree of simplicity [155]. A process
meeting all those criteria at the same time unfortunately
has not yet been developed. Each type of pretreatment has
certain advantages in comparison to the others and certain
31
limitations too. One has to select the most efficient one
depending on the specific circumstances (feedstock type
and fermentation type to follow).
Types of Reactors
Reactor configuration is considered to be crucial for the
overall performance of fermentative hydrogen production.
It influences the reactor microenvironment, the prevailing
microbial population, the established hydrodynamic
behaviour, the contact between substrate and consortia, etc.
[156]. The most commonly used reactor type is the mixed
(continuously stirred) suspended growth reactor.
In general, mixed reactors for fermentative hydrogen
production can operate in either batch or continuous mode.
Batch mode fermentative hydrogen production has been
shown to be more suitable for research purposes [26, 157],
but any industrially feasible process would most likely
have to be performed on a continuous or at least semi-
continuous (fed or sequencing batch) basis.
The CSTR (Continuously Stirred Tank Reactor) is the
most commonly used continuous reactor system, offering
simple construction, ease of operation and effective
homogenous mixing as well as temperature and pH control.
In a conventional CSTR, biomass is well suspended in the
mixed liquor, which has the same composition as the
effluent.
The production of hydrogen in a CSTR may be influ-
enced by several operating parameters. The most important
parameter is the HRT (Hydraulic Retention Time). A small
enough SRT (Solids Retention Time) in a mixed culture
CSTR secures that no methanogenesis takes place, since
methanogens are slowly growing species. Typically a HRT
of 12–36 h, depending on the substrate, provides complete
conversion of carbohydrates and highest hydrogen yields,
avoiding the generation of methane [32, 49]. The temper-
ature and the pH are also important operating parameters as
outlined in section ‘‘Microorganisms’’. Finally, the partial
pressure of hydrogen seems to be a very important
parameter. High partial pressures of hydrogen have been
shown to inhibit hydrogen production. One way to avoid
high partial pressures is to sparge an inert gas through the
reactor [158, 159].
In some studies it has been found that batch hydrogen
production gives higher hydrogen yields than continuous
operation [32, 89, 90]. This could be attributed to the
substantially different microenvironment of the microbes
in a batch reactor (time-varying and consequently higher
substrate concentrations in the early part of the batch)
leading to a different metabolic flux than in the constant
low-substrate microenvironment in a CSTR. A batch bio-
reactor has the disadvantage of requiring long down-times
123
32
and startup procedures. An interesting alternative operation
of a mixed bioreactor is the sequencing-batch operation: In
a cycle, we distinguish ‘‘feed’’, ‘‘react’’, ‘‘settle’’, ‘‘decant’’
phases, so that the bioreactor is in essence operating in a
semi-continuous mode. The optimisation of a mixed bio-
reactor operation requires appropriate modelling. Rela-
tively few studies have been presented so far on modelling
fermentative hydrogen production [160, 161]. The most
promising framework is the ADM 1 (anaerobic digestion
model 1) framework of Batstone et al. [162], which needs
to be appropriately modified to properly describe fermen-
tative hydrogen production [52, 163–165].
In a CSTR, biomass has the same retention time (SRT)
as the HRT, and thus, its concentration in the mixed liquor
is limited and this limits the rate of hydrogen production.
Another category of reactors are systems characterized by
physical retention of the microbial biomass, which offer
several advantages compared to the conventional CSTRs.
In these systems, the SRT is independent of HRT due to
physical retention of the microbial biomass inside the
reactor, allowing high cell concentrations and thus high
hydrogen volumetric production rates with relatively small
reactor volumes. Physical retention of microbial biomass
could be accomplished by several different means,
including the use of naturally forming flocs or granules of
self immobilized microbes, microbial immobilization on
inert materials, microbial-based biofilms or retentive
membranes [166]. It has been recently found that hydrogen
producing biomass in a CSTR could be self-granulated or
flocculated under proper conditions [167]. Another
approach is to immobilize biomass on biofilms or artificial
granules made of various support materials such as cup-
rammonium rayon [168], polyvinyl alcohol, polyacryl-
amide and anionic silica sol [125, 169]. A potential
problem with attached growth bioreactors is the loss of
hydrogen through the formation of methane due to exten-
ded retention of the biomass inside the reactor permit-
ting the establishment of slow-growing methanogenic
populations.
Among the types of reactor used for continuous hydro-
gen production, are the CSTR [32, 89, 90, 117, 170] and
several variations of hybrid and attached growth reactors,
such as the Upflow Anaerobic Sludge Blanket reactor, the
UASB [171], the Packed Bed Reactor, PBR [172],
Anaerobic Sequencing Batch Reactor, ASBR [173], the
Fixed Bed Bioreactor with Activated Carbon, the FBBAC
[27], the Anaerobic Fluidized Bed Reactor, the AFBR
[174, 175], the Carrier-Induced Granular Sludge Bed, the
CIGSB [176], Membrane Bioreactor, the MBR [50] and the
Rhomboidal reactor [60]. A direct comparison of the per-
formance of the different types of reactor configurations
that have been studied in terms of hydrogen productivity is
not possible, since the operational parameters (feedstocks
123
Waste Biomass Valor (2010) 1:21–39
and operating conditions) along with the reactor configu-
ration, in all these studies, are quite different.
Exploitation of By-Products
Hydrogen yields of fermentative hydrogen production
processes are restricted by the existing metabolic pathways
to 2 or 4 mol H2/mol glucose consumed, for butyrate or
acetate fermentation, respectively. Moreover, regardless
the final hydrogen yield achieved, fermentative hydrogen
production processes are not generally expected to be
effective in terms of Chemical Oxygen Demand (COD)
removal. In contrast to the anaerobic digestion processes
during which organic carbon is finally converted to meth-
ane, thus diminishing the organic load, in the acidification
processes carbohydrates are transformed into more oxi-
dized soluble compounds, thus remaining in the liquid
phase in the form of various volatile fatty acids (VFAs) and
solvents. Consequently acidification is accompanied by
limited removal of COD [21]. Apart from the carbon that
remains trapped in the generated oxidative products, a
remarkable amount of hydrogen also remains trapped in
these compounds, resulting thus in final hydrogen yields in
the range of about 1–2 mol H2/mol of hexose. Even under
optimum conditions of 4 mol H2/mol glucose, about
60–70% of the organic matter of the feed remains in
solution [156]. Further utilization of the organic matter
contained in the effluent of a fermentative hydrogen pro-
ducing bioreactor, could increase the overall energy output
of the process and/or more products recovery. The devel-
opment of a two-stage process so far, involves the fer-
mentation of the substrate to hydrogen and organic acids in
the first stage and in a second stage either the additional
energy extraction or the generation of high added value
products by exploiting the effluent of the first stage reactor.
Biomethane
One approach in order to further utilize/reuse the remaining
organic matter is to produce a second useable form for
energy (an energy carrier) such as CH4 in a second stage.
Integration of an acidogenic process with a subsequent
methanogenic process for combined hydrogen and methane
generation, offers several advantages such as a higher
performance of the process in terms of waste stabilization
efficiency and net energy recovery [177]. Such a two-stage
system has been proposed so far for organic solid wastes,
rich in carbohydrates such as food wastes [31], cheese
whey [43, 117], olive mill wastewaters [115], household
solid waste [178], a mixture of pulverized garbage and
shredded paper wastes [179] and wastewater sludge [180].
A combined hydrogen and methane generation process has
Waste Biomass Valor (2010) 1:21–39
already been scaled up to the pilot plant stage, for organic
solid wastes [181]. The hydrogen and methane production
rates were 5.4 m3/m3/day and 6.1 m3/m3/day, respectively
while the process COD removal efficiency was 80%. The
overall efficiency of this combined process is demonstrated
by the fact that methane yields were twofold higher than a
comparable single-stage process [181].
Photoheterotrophic Hydrogen Production
A different approach for increasing the overall energy
extraction is to couple the fermentative hydrogen produc-
tion with photofermentation aiming to the recovery of
additional hydrogen. In such a two-stage process, the rich
in organic acids effluent produced in the first stage by
fermentative bacteria could be converted to hydrogen in the
second step by photosynthetic bacteria which capture and
use light energy. Light as energy source is necessary for the
conduction of such reactions since their Gibbs energy is
positive (e.g. Eq. 1) and thus they are not thermodynami-
cally favoured.
CH3 COOH þ 2H2 O þ ‘‘hv’’ ! 2CO2 þ 4H2
DGo ¼ þ75:2 kJ mol1 ð9Þ
This combination of both kinds of bacteria in a two
stage system not only reduces the light energy demand of
the photosynthetic bacteria but also enhances the hydrogen
yield as well [79]. Intensive research has been carried out
in this area [182, 183] in the recent years. However, there
are important factors limiting the practical application of
such a process. One of them is that the involved hydrogen
enzyme, nitrogenase, is potentially sensitive to the nitrogen
content of the medium/substrate, since nitrogen, especially
in the form of NH4?, not only inhibits the enzymatic
activity, but also represses the synthesis of the enzyme
[184, 185]. This limitation can be potentially overcome
either by genetic manipulation [186] or selection [187] to
remove nitrogenase regulation. In addition, one of the most
severe constraints is that photosynthetic efficiencies are
very low, since at even moderate light intensities, the main
part of captured light is dissipated as heat [188]. This
means that there will be a demand of large surface areas for
the production of hydrogen contributing to the total cost
and render the development of a two-stage process of
fermentation—photofermentation, far from practical
application.
Production of Polyhydroxyalkanoates
In both methods analysed above, the rich in acids effluent
from dark fermentation hydrogen producing systems is
used for extra energy generation. An alternative approach
is their exploitation for the production of other high added
33
value products such as polyhydroxyalkaonates (PHAs), via
selected microorganisms. The current sharp focus of
attention on the environmental pollution caused by dis-
carded petrochemical plastics, has given added impetus to
research on the production of plastics that can be fully
biodegraded in the natural environment, such as PHAs.
PHAs are biodegradable polyesters that can be produced by
bacteria [189], which they accumulate as intracellular
storage reserves of carbon and energy under stress condi-
tions in the form of inclusion bodies [190]. There are
several types of biopolyesters but poly(3-hydroxybutyrate)
and poly(3-hydroxyvalerate) are the most well-known
since they bear similar properties to conventional plastics
such as polypropylene and polyethylene [191]. The
copolymers of PHAs are less penetrable to oxygen than
conventional plastics, being thus ideal as packaging
material in food industry, but also in many other applica-
tions of the medical sector [192]. The production of PHAs
from acidified wastewaters had been previously investi-
gated in both lab [193] and pilot [194] scale, showing very
promising results. Combining dark hydrogen and PHAs
production in a two stage system has been proposed by
Ntaikou et al. [49] using three phase olive mill wastewater
as feedstock. Both processes were continuous; hydrogen
production was conducted anaerobically in a CSTR and the
effluent, consisting mainly of acetate, butyrate, propionate
and ethanol, was subsequently fed to an SBR where PHAs
were produced aerobically. Lately, the scale up of the
process has also been investigated [195], conducting the
two stage PHAs production at semi-pilot scale.
Real Scale Applications
Up to now, a continuous scaled-up process for sustainable
fermentative H2 production has not been reported in the
literature. Only very few studies on the fermentation of
sugars to hydrogen, at pilot scale, are available so far.
Ren et al. [196] performed a pilot scale study in a
continuous flow anaerobic fermentative reactor with an
active volume of 1.48 m3 using molasses as feedstock. The
reactor operated at organic loading rates of 3.11–85.57 kg
COD/m3 reactor/day and produced 5.57 m3 H2/m3 reactor/
day or 8240 l H2/day with a hydrogen yield of 26.13 mol/
kg COD removed. The effluent which was produced,
contained primarily acetate and ethanol and was as high as
3,000 l/day. This, rich in acetate, effluent could be further
exploited for hydrogen production through a subsequent
photoheterotrophic stage, which could increase hydrogen
production by 317%.
Vatsala et al. [197] evaluated the feasibility of hydrogen
production from a sugar cane distillery effluent using
co-cultures of Citrobacter freundii 01, Enterobacter
123
34
aerogenes E10 and Rhodopseudomonas palustris P2, at
100 m3 scale. The reactor operated in batch mode for 40 h,
and the hydrogen production was 21.38 kg with an average
yield of 2.76 mol H2/mol glucose and a rate of 0.53 kg/
100 m3/h. The results showed that distillery effluent could
be used as a source of hydrogen providing insights into
treatment for industrial exploitation.
Since data for real applications are not available so far,
designing of such a process will have to be based on the
respective lab scale experiments. The problem is that the
hydrogen productivity and yields depend significantly on
the prevailing conditions, the feedstocks as well as the
inoculum used. However, from laboratory-scale work on
continuous processes, it could be suggested that such a
process may operate at a mesophilic temperature, at a pH
around 5.5 and an HRT approximately 8–12 h, for simple
substrates. Higher HRTs are indicative for complex car-
bohydrate-rich feedstocks. Finally, such a process may use
as microbial inoculum, heat treated sludge form aerobic or
anaerobic process or the indigenous microbial species
available in the feedstock/waste, which has often proved to
work optimally [43, 89].
Conclusions and Outlook
It is widely believed that the future energy economy will be
based on hydrogen; this is due to the fact that hydrogen is a
clean and efficient energy carrier, with zero emissions
when burned, that can be produced by renewable sources
such as biomass and wastes. Fermentative hydrogen pro-
duction from biomass and wastes refers to the exploitation
of the latter via microorganisms which are capable of
converting organic matter to acids and alcohols with
simultaneous liberation of molecular hydrogen.
Different types of biomass and wastes, in terms of both
chemical composition and origin, have been tested as
potential feedstocks, leading in many cases to very prom-
ising results. Among them the use of energy crops,
although being very effective, has lately been put under
question due the arising food versus fuel debate. On the
other hand the lignocellulosic residues remaining from
such crops, together with other agricultural and forestal
residues remain an abundant source of biomass, on the use
of which, 2nd generation hydrogen production can be
based. Among the limiting steps for the efficient exploi-
tation of such biomass is identifying effective economical
viable and environmental friendly solutions for the libera-
tion of carbohydrates via pretreatment. On the other hand
readily fermentative carbohydrates are included to a high
degree in sugar and starch based wastes and wastewaters.
Their exploitation, not only leads to energy recovery but
can also be a cost reducing management method. The
123
Waste Biomass Valor (2010) 1:21–39
major limitation of hydrogen production from biomass and
wastes, however, remains the relatively low rates and
yields of hydrogen generation. A possible solution to that
could be combining the fermentative hydrogen producing
systems with a second stage process, via which the further use
of the emerging by-products would be feasible for extra
energy and/or materials recovery either via hydrogen, biogas
or other valuable products in a biorefinery framework.
Future progress leading to more efficient fermentative
hydrogen production will depend on research efforts to (a)
identify the most suitable feedstocks, (b) develop more
efficient pretreatment methods for saccharification of
lignocellulosics, (c) isolate and/or develop through genetic
engineering high hydrogen producing strains, (d) develop
optimal reactor configurations and operating strategies
through modelling and optimisation.
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