Biotechnol Lett (2007) 29:685695

DOI 10.1007/s10529-006-9299-9

REVIEW

Improving the yield from fermentative hydrogen production

Jeremy T. Kraemer David M. Bagley

Received: 4 October 2006 / Revised: 20 December 2006 / Accepted: 21 December 2006 /

Published online: 6 February 2007

Springer Science+Business Media B.V. 2007

Abstract Efforts to increase H2 yields from

fermentative H2 production include heat treatment of the inoculum, dissolved gas removal, and
varying the organic loading rate. Although heat
treatment kills methanogens and selects for
spore-forming bacteria, the available evidence
indicates H2 yields are not maximized compared
to bromoethanesulfonate, iodopropane, or perchloric acid pre-treatments and spore-forming
acetogens are not killed. Operational controls
(low pH, short solids retention time) can replace
heat treatment. Gas sparging increases H2 yields
compared to un-sparged reactors, but no relationship exists between the sparging rate and H2
yield. Lower sparging rates may improve the H2
yield with less energy input and product dilution.
The reasons why sparging improves H2 yields
are unknown, but recent measurements of dissolved H2 concentrations during sparging suggest
the assumption of decreased inhibition of the
H2producing enzymes is unlikely. Significant

J. T. Kraemer (&)
Department of Civil Engineering, University of
Toronto, 35 St. George Street, Toronto, ON, Canada
M5S 1A4
e-mail: jeremy.kraemer@utoronto.ca
D. M. Bagley
Department of Civil and Architectural Engineering,
University of Wyoming, 1000 E. University Ave.
Dept. 3295, Laramie, WY, USA 82071

disagreement exists over the effect of organic

loading rate (OLR); some studies show relatively
higher OLRs improve H2 yield while others show
the opposite. Discovering the reasons for higher
H2 yields during dissolved gas removal and
changes in OLR will help improve H2 yields.
Keywords Carbon dioxide
Dissolved gases

Heat treatment
Hydrogen
Organic loading
rate
Sparging

Introduction
Biological H2 production via dark fermentation of
organic wastes is being investigated as a potential
source of renewable energy (Hawkes et al. 2002).
Fermentative H2 production is carried out by
anaerobic bacteria that ferment organic compounds to volatile fatty acids (VFAs), alcohols,
CO2, and H2. Many different substrates can be
fermented to produce H2 (Kapdan and Kargi 2006;
Nishio and Nakashimada 2004), although carbohydrates (e.g. glucose) have been most commonly
used. The reactor environmental conditions capable of achieving H2 production are well understood, as demonstrated by several recent reviews
(Hallenbeck 2005; Hawkes et al. 2002; Nath and
Das 2004). For example, completely-mixed reactors with a pH of 5.5 and solids retention time of
612 h can achieve H2 production.

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The H2 yield is the moles of H2 produced per

mole of substrate. Care must be taken when
comparing studies because some authors report
the yield per mol of substrate converted whereas
others report per mol of substrate applied to the
reactor. Additionally, the H2 yield is not applicable to complex substrates where moles of substrate cannot be measured (e.g. sludge).
Kraemer and Bagley (2005) addressed these
issues by proposing the use of the H2 productivity
(HP), which they defined as the percent of
influent substrate electrons distributed to H2 gas
(gaseous + dissolved phases). The maximum H2
yield from fermentative H2 production is 4 mol
H2/mol glucose (HP = 33%), which occurs if
acetic acid is the only VFA produced and no
electrons are used for growth (Angenent et al.
2004). The H2 yield is lower if other metabolites
are produced, such as butyric acid (2 mol H2/mol
glucose, HP = 17%) or ethanol (0 mol H2/mol
glucose, HP = 0%). Thus, production of VFAs is
preferred over alcohols and acetate is preferred
over butyrate. Substrate electrons contained in
the residual fermentation products can be converted into methane in a second-stage reactor
(Kraemer and Bagley 2005). Current H2 productivities reported in the literature are 10-20%
(Angenent et al. 2004; Benemann 1996; Logan
2004), much less than the theoretical maximum of
33%. Higher H2 yields would be beneficial for
practical application of the technology.
This paper reviews the efficacy of three operational techniques for increasing the yield from

Fig. 1 Interactions
between fermenter,
acetogen, and
methanogen metabolic
groups in a fermentative
H2-producing system. KLa
is the overall masstransfer coefficient
assuming the liquid-phase
is mass-transfer limiting

fermentative H2 production: (1) inoculum heat

treatment, (2) dissolved gas removal, and (3)
varying the organic loading rate. Additionally,
this review identifies several recommendations
for practical application and research.

Inoculum heat treatment

In a reactor operated for fermentative H2 production, three groups of H2 metabolising bacteria
appear to be important (Fig. 1): H2 producers
(fermenters), H2-consuming methanogens, and
H2-consuming acetogens. Using molecular phylogenetic techniques, researchers have identified
Clostridium spp. as being the most common in
continuous-flow bioreactors engineered for fermentative H2 production (Table 1). In addition,
Bacillus, Enterobacter, and Thermoanaerobacterium spp. have also been regularly observed, but
less frequently than Clostridium spp. Methanogenesis (Kraemer and Bagley 2005; Shizas and
Bagley 2005) and acetogenesis (Park et al. 2005)
have also been observed. The key to high H2
yields is to optimize the conditions for the H2
fermenters while preventing the methanogens
and acetogens from consuming the produced H2.
Heat treatment has been a common practice
for killing methanogens contained in inocula
(Ahn et al. 2005; Kim et al. 2006a, b; Oh et al.
2003a). Heat treatment at 80104C and exposure
times of 15120 min have been used (Zhu and
Beland 2006). Heat treatment selects for bacteria

H2,gas

CO2,gas

CH4,gas

headspace
liquid

KLaH2
glucose

H2
HCO3

H2,dissolved
CO2,dissolved
HCO3

acetate, butyrate
ethanol, butanol
acetogen

KLaCH4

KLaCO2

fermenter

CH4,dissolved
H2
HCO3
methanogen

H2 HCO3

acetate

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687

Table 1 Microbial community assessments using molecular techniques for continuous-flow mixed-culture biohydrogen
systems
Heat
Treatment

Inoculum

Observed Organisms

Study

no

secondary sludge

Fang et al. (2002a)

no

secondary sludge

no
no
no

digester sludge
thermophilic digester
sludge
compost

yes

digester sludge

yes

soil

yes

digester sludge

yes
yes

digester sludge
soil

yes

sludge

Clones: 69.1% 4 Clostridium spp., 13.5%

Sporolactobacillus racemicus, 5.8% no close
relative, 11.5% phylogeny not checked
Clones: 64.6% 3 Clostridium spp., 18.8%
Enterobacteriaceae, 3.1% Streptococcus bovis,
13.5% 8 unidentified OTUs
Methanogens observed
Glucose: mostly Bacillus; cellulose: mostly
Clostridia
10 of 15 bands either Clostridium or
Thermoanaerobacterium, 1 Symbiobacterium,
1 Bacillus, 1 Sulfobacillus, 1 Ruminococcus,
1 Selenomonas
Clostridia, Bacilli, Bacteroides; primarily
Thermoanaerobacterium
thermosaccharolyticum
HRT = 30 h: Bacillaceae, Clostridiaceae,
Enterobacteriaceae; HRT = 10 h:
Clostridiaceae only
15 of 17 bands were Clostridium spp.,
1 Gluconacetobacter, 1 Lactobacillus
9 of 10 bands were Clostridia, 1 Bacillus
Most related to Clostridiaceae and
Flexibacteraceae
Prominent band was similar
to Clostridium pasteurianum

that can form endospores, such as Clostridium,

Bacillus and Thermoanaerobacterium (Table 1).
The microbial community after heat treatment is
relatively homogeneous, whereas microbial diversity is higher in reactors using non-heat-treated
inocula; reactors without heat treatment contain
more enteric and other non-spore-forming bacteria (Table 1). However, depending on whether
the inoculum is dry or wet and the time and
temperature of exposure, not all vegetative cells
will be killed by heat treatment. For example,
non-spore-forming bacteria such as Bacteroides,
Lactobacillus and Enterobacter have been observed even though heat treatment was employed
(Table 1) (Ahn et al. 2005; Iyer et al. 2004; Kim
et al. 2006a).
Although the intent has primarily been prevention of methanogenesis, the use of heat
treatment does not select exclusively for
H2-producing bacteria. This is because the characteristic of H2 production is not directly associated with the ability to form endospores. For

Fang et al. (2002b)

Shizas and Bagley (2005)

Ueno et al. (2001a)
Ueno et al. (2001b)

Ahn et al. (2005)

Iyer et al. (2004)

Kim et al. (2006a)

Kim et al. (2006b)
Oh et al. (2004a)
Wu et al. (2006)

instance, non-spore-forming H2-producers include enteric bacteria like Enterobacter spp.

(Nakashimada et al. 2002) and Citrobacter spp.
(Oh et al. 2003b). There are also many H2consuming groups of bacteria that can form
spores and therefore survive heat treatment,
including acetogens (Acetobacterium, some Clostridium spp., Sporomusa), certain propionate and
lactate producers (Propionibacterium, Sporolactobacillus), and a sulphate-reducer (Desulfotomaculum) (Madigan et al. 2000). For example,
Kim et al. (2006b) observed the acetogen Clostridium scatologenes in a heat-treated H2-producing sludge.
Current evidence does not prove that heat
treatment actually increases the H2 yield compared to non-heat-treated systems. Only a few
reports have assessed heat-treated versus nonheat-treated inocula in the same experiment, and
all of these were batch studies. Oh et al. (2003a)
found the H2 yield was higher with heat treatment
compared to non-heat-treated or pH 6.2.

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Biotechnol Lett (2007) 29:685695

However, Zhu and Beland (2006) observed that

heat-treatment decreased H2 production compared to the use of bromoethanesulfonate (BES)
and iodopropane. Similarly, Cheong and Hansen
(2006) observed less H2 produced from wet- and
dry-heat-treated sludge compared to BES and
perchloric acid treatments. Kawagoshi et al.
(2005) observed the same H2 production from
non-heat-treated and heat-treated digester
sludge. Instead of increasing the H2 yield, heat
treatment may actually be detrimental to achieving maximum H2 production.
Controlling methanogens by heat treatment is
unnecessary because they can be easily controlled through operational means: operating at
a pH near 5.5 and employing a solids retention
time of 612 h can achieve methane-free biogas
production (Hussy et al. 2005; Kraemer and
Bagley 2006; Oh et al. 2003a). These conditions
wash out the methanogens because they grow
slower than the fermenting, H2-producing bacteria. One concern with untreated sludge inocula is
the possible establishment of a methanogenic
biofilm on the reactor walls (Kraemer and
Bagley 2005; Shizas and Bagley 2005), but this
can be avoided by transferring the sludge to a
new vessel after the initial start-up period (Zhu
and Beland 2006).

Fig. 2 Metabolic pathways in

Clostridium spp. fermenting glucose.
Solids lines indicate substrate
transformations, dotted lines
indicate ATP creation or utilization,
and dashed lines indicate electron
flow. ATP = adenosine
triphosphate;
G3P = glyceraldehyde-3-phosphate;
G3PDH = G3P dehydrogenase;
1,3-BPG = 1,3-bisphosphoglycerate;
NADH = nicotinamide-adenine
dinucleotide; Fd = ferredoxin;
PFOR = pyruvate: Fd
oxidoreductase;
NFOR = NADH:Fd
oxidoreductase;
H2ase = hydrogenase;
CoA = coenzyme A. Adapted from
Madigan et al. (2000)

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Dissolved gas removal

The metabolic pathways for the fermentation of
glucose by Clostridium spp. are well understood
(Fig. 2) (Madigan et al. 2000). Glucose is fermented via glycolysis to pyruvate with electrons
being transferred to nicotinamide-adenine dinucleotide (NADH). Pyruvateis oxidized by pyruvate:ferredoxin oxidoreductase (PFOR) to acetylCoA and CO2, with electrons being transferred to
ferredoxin (Fd). Several end-products are produced from acetyl-CoA, including acetate, butyrate, ethanol, and butanol. Production of acetate
or butyrate allows for ATP generation while
alcohols do not, although the more reduced
products utilize electrons from NADH thereby
maintaining redox balance. NADH can alternatively be re-oxidized by electron transfer to
ferredoxin
by
NADH:Fd
oxidoreductase
(NFOR).
H2 is produced by the hydrogenase enzyme,
which catalyzes proton reduction using electrons
from ferredoxin. The pool of reduced Fd is
generated from two sources: (1) pyruvate oxidation by PFOR and (2) NADH oxidation by
NFOR. These enzyme systems can be thermodynamically regulated by the H2 concentration
(Angenent et al. 2004): PFOR can function at

glucose
ATP
G3P
G3PDH

NADH

1,3-BPG
NFOR

ATP
pyruvate

PFOR

H2ase

Fd

acetyl -CoA

ATP
CO 2 acetate

H2

ethanol

butyryl- CoA

butyrate

butanol

Biotechnol Lett (2007) 29:685695

689

the H2 concentrations observed in fermentative

H2 systems (Angenent et al. 2004) so there will
always be some H2 produced, whereas NADH
oxidation by NFOR is inhibited for H2 >60
100 Pa (~0.50.8 lM) (Angenent et al. 2004; Hallenbeck 2005).
Various techniques have been used to remove
metabolic gases (H2, CO2) from the liquid phase.
Measuring the dissolved concentrations is important when assessing dissolved gas removal techniques because H2 and CO2 are supersaturated in
fermentative H2-producing systems and therefore
headspace concentrations are inaccurate (Kraemer and Bagley 2006).
Gas sparging
Gas sparging has been the most common method
used to decrease dissolved gas concentrations in
fermentative H2-producing reactors. In pure cultures, sparging has altered the relative amounts of
metabolic products. Argon and H2 sparging in a
culture of Enterobacter aerogenes decreased the
production of succinate while increasing acetate
(Tanisho et al. 1998). In a culture of Clostridium

butyricum Crabbendam et al. (1985) observed a

lower acetate:butyrate ratio when N2 was passed
over the fermentation liquid instead of through
the liquid. These studies imply more H2 was
produced with sparging. Also, the H2 yield
increased by 47% when Rhodopseudomonas palustris P4 was intermittently purged with argon
(Oh et al. 2002).
In mixed cultures, gas sparging has increased
the H2 yield noticeably in comparison to unsparged conditions (Table 2). The H2 yield
improvement has been 2070% for N2 sparging,
80120% for CO2 sparging (discussed later), 88%
for methane sparging, and 012% for H2/CO2
(biogas) sparging. However, there does not
appear to be any relationship between the
amount of sparging and the increase in H2 yield
(Fig. 3). Even for the study by Kim et al. (2006a),
where multiple sparging rates were tested within
the same experiment, there was no meaningful
relationship between sparging rate and H2 yield,
although all sparging rates did provide higher H2
yields than the un-sparged case. These results
indicate higher H2 yields may occur at sparging
rates lower than those usually used in the liter-

Table 2 Comparison of gas sparging during fermentative H2 production in continuous-flow mixed-culture systems
Sparge
Gas

Study

Sparge flow, Qs
(ml min1)

Liquid volume, QS/V

Yield [mol H2
V (l)
(ml min1 l1) (mol hexose)1]

Yield
Increase (%)

No
With
Sparging Sparging
N2

CO2

Mizuno et al. (2000)

Hussy et al. (2003)
Hussy et al. (2005)
Kyazze et al. (2006)
Kim et al. (2006a)

Kraemer and
Bagley (2006)
Kim et al. (2006a)

methane Liu et al. (2006)

biogas
Kim et al. (2006a)

110
58
55
630
100
200
300
400
160

2.3
2.3
2.3
9.3
5
5
5
5
2

48
25
24
68
20
40
60
80
80

0.85
1.26
1.00
1.23
0.77
0.77
0.77
0.77
1.30

1.43
1.87
1.80
1.65
0.91
0.92
0.95
0.92
1.80

68
48
66
34
18
19
23
19
38

100
200
300
400
2
100
200
300
400

5
5
5
5
0.4
5
5
5
5

20
40
60
80
5
20
40
60
80

0.77
0.77
0.77
0.77
n/r a
0.77
0.77
0.77
0.77

1.40
1.65
1.68
1.54
n/r
0.86
0.83
0.77
0.82

82
114
118
100
88
12
8
0
6

n/r = not reported

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Biotechnol Lett (2007) 29:685695

Increase in H2 Yield (%)

75%

50%

25%

0%
0

15

30

45

60

75

90

Specific Sparging Rate (ml min-1 l-liquid-1)

Fig. 3 Increase in H2 yield versus sparging rate. Data are

from Table 2 for N2 sparging

ature. For example, Liu et al. (2006) used methane sparging at only 2 ml/min and observed an
88% increase in H2 yield.
Non-sparging techniques
Other techniques to decrease dissolved gas concentrations include increased stirring, decreasing
the reactor headspace pressure (i.e. applying a
vacuum), and using an immersed membrane to
directly remove dissolved gases.
Vigorous stirring of Clostridium thermocellum
cultures decreased the dissolved H2 content by a
factor of 3 and the ethanol/acetate ratio decreased compared to unstirred cultures (Lamed
et al. 1988). Lay (2000) stated that increased
mixing caused a release of H2. Thus, sufficient
mixing is important for aiding mass-transfer of
metabolic gases from the liquid to the reactor
headspace.
Kataoka et al. (1997) did not observe any
significant difference between continuous cultures
of Clostridium butyricum strain SC-E1 at
0.28 atm headspace pressure (vacuum operation)
compared to the control (non-vacuum operation).
In contrast, Mandal et al. (2006) observed double
the H2 yield during batch culture of Enterobacter
cloacae DM 11 at 0.5 atm headspace pressure
compared to non-vacuum operation. The H2 yield
during vacuum operation was 3.9 mol H2/mol
glucose, which was extremely high considering
enteric bacteria usually produce <1 mol H2/mol
glucose (Nishio and Nakashimada 2004), but
Mandal et al. (2006) used a chemically-selected

123

high H2-producing mutant strain. The authors

attributed the higher yield to lower concentrations of H2, but this was confounded with lower
CO2 concentrations than would be achieved with
lower headspace pressure alone because a CO2
trap (KOH) was used. Lower headspace
pressure may have different effects on each type
of bacteria and continuous-flow mixed culture
studies are needed to determine whether this
technique may be useful in practice.
Liang et al. (2002) observed a 15% increase in
H2 yield when a silicone rubber membrane was
used to remove dissolved gases directly from the
fermentation liquid of a mixed culture batch
reactor.
Effects of lower dissolved gas concentrations
Despite the repeated demonstrations that H2
yields are higher when dissolved gas removal
techniques are employed, the specific mechanisms of this improvement have yet to be
elucidated. Several possible hypotheses are: (1)
decreased negative feedback regulation by H2 on
the NFOR enzyme, (2) decreased substrate
availability for acetogens, and (3) decreased
inhibition by CO2. Each hypothesis will be
evaluated in turn.
Higher H2 yields have most frequently been
attributed to decreased inhibition by H2 on the
NADH:Fd oxidoreductase enzyme thereby allowing H2 yields >2 mol/mol glucose (refer to Fig. 2
and discussion above). However, this has always
been an assumption because the dissolved H2
concentrations were never measured. Recently,
Kraemer and Bagley (2006) measured dissolved
H2 and CO2 concentrations with and without N2
sparging. They demonstrated that although dissolved H2 and CO2 concentrations were significantly lower during sparging, H2 was still 1000fold higher than the regulatory level for the
NFOR enzyme. It is possible for N2 sparging to
alter the relative amounts of acetate and butyrate
in pure-cultures of clostridia, but such a change
was reported for sparging rates 10 times higher
than those of Table 2 (Crabbendam et al. 1985).
Thus, altered H2 thermodynamic regulation is
unlikely to have been the cause of higher H2
yield.

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691

Decreasing the dissolved concentrations of H2

and CO2 has been proposed for limiting H2
consumption by acetogens thereby increasing the
H2 yield (Hussy et al. 2003; Kim et al. 2006a;
Kraemer and Bagley 2006; Park et al. 2005).
Acetogenesis was observed in batch culture by
Park et al. (2005) from a heat-treated inoculum.
Kraemer and Bagley (2006) observed decreases
of 65% and 90% for dissolved H2 and CO2, so the
effective reduction in substrate concentrations
(since both gases are needed) would have been
96.5%. However, the authors did not measure
VFAs or microbial community so changes in
acetogens cannot be substantiated. Additionally,
the extent to which acetogenesis actually occurs
in continuous cultures is not yet known.
High concentrations of CO2 are known to be
inhibitory to bacteria and this fact has led to the
use of CO2 in food packaging to prevent spoilage
(Dixon and Kell 1989). Partial CO2 pressures >0.5
atm have been observed to decrease microbial
yield in Clostridium sporogenes (Dixon and Kell
1989) and pure CO2 sparging drastically decreased microbial diversity in a continuous mixed
culture (Kim et al. 2006a). Without sparging,
Kraemer and Bagley (2006) measured the dis-

solved CO2 concentration to be in the inhibitory

region (~0.56 atm) and sparging at 160 ml/min
significantly reduced it (~0.06 atm). Thus, it is
possible that relatively low rates of sparging could
remove CO2 sufficiently to remove its inhibitory
effect.

Varying the organic loading rate

Changing the organic loading rate (OLR = feed
concentration/hydraulic retention time) can increase the H2 yield. Table 3 summarizes studies
that used mixed cultures and assessed multiple
substrate concentrations at a constant hydraulic
retention time (HRT). There is disagreement in
the literature as to whether higher H2 yields are
achieved with lower or higher OLRs, and this
division is shown in Table 3. In some cases higher
OLRs decreased the H2 yield whereas in others
higher OLRs increased the H2 yield. In the latter
case, as OLRs increased the H2 yield usually
became constant or eventually began to decrease
thereby providing an optimal OLR (maximum H2
yield) (Kim et al. 2006b; Wu et al. 2006; Yang
et al. 2006).

Table 3 Comparison of studies that varied the organic loading rate (OLR) by changing the substrate concentration
Study

Substrate

Lower OLR improves H2 production

Kyazze et al. (2006)
sucrose
Oh et al. (2004b)
glucose
Van Ginkel and Logan (2005a) glucose
Van Ginkel and Logan (2005b) glucose
glucose
Wu et al. (2006)
sucrose
sucrose
Yu et al. (2002)
rice winery WW
Higher OLR improves H2 production
Kim et al. (2006b)
sucrose
Lin et al. (2006)
sucrose
sucrose
Yang et al. (2006)
citric acid WW
Zhang et al. (2004)
glucose

S0 (g COD l1)

Low

high

11.2
7.3
10.7
2.5
2.5
10
10
14

56.1
29.2
32
10
10
40
40
36

10
5
5
varied
5

60
40
40
varied
15

HRT (h) OLR (g COD l1 d1) H2 Yield [mol H2

(mol hexose
converted)1]
low

high

low
OLR

high
OLR

12
4
10
10
2.5
6
2
2

22.4
43.9
25.6
6
24
40
120
168

112.2
175.4
76.8
24
96
160
480
432

1.65
1.30
2.20
2.80
2.40
1.84
2.10
1.89

0.81
1.05
2.00
2.20
1.90
1.36
1.96
1.79

12
8.9
6
varied
4.5

20
13.5
20
10
26.7

120
107.9
160
40
80

0.25
1.69
1.34
0.50
0.72

1.00
2.49
2.17
0.85
1.04

Abbreviations: S0 = influent substrate concentration, HRT = hydraulic retention time, WW = wastewater

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Effects of OLR
The reason for such diverse H2 yield observations
for lower or higher OLRs is unknown. For those
studies where higher OLRs decreased H2 production some possible reasons are: (1) increased
inhibition by VFAs at higher OLR, (2) lower
dissolved H2 concentrations at lower OLRs
decrease thermodynamic regulation, (3) OLR
affects acetogenic activity, and (4) lower dissolved
CO2 concentrations decrease CO2 inhibition.
These hypotheses will each be discussed.
VFA inhibition at higher OLRs appears to be a
valid explanation. The addition of external VFAs
has been demonstrated to decrease or inhibit H2
production in mixed-culture continuous-flow systems, although there is consensus that butyrate
causes greater inhibition than acetate. Kyazze
et al. (2006) observed inhibition of H2 production
when 4 g butyrate/l was added to the reactor
(18.9 g butyrate/l in total) whereas no inhibition
was observed when only 2 g butyrate/l was added
(12.2 g butyrate/l in total). Van Ginkel and Logan
(2005a) attributed the inhibition to the undissociated form of the VFAs because changing the pH
from 5.5 to 5.0 (increasing the un-ionized fraction
of the VFAs) at the same total acid concentration
decreased H2 production. However, this hypothesis does not support their results when the OLR
was changed. The H2 yield was constant at 2 mol
H2/mol glucose for 1030 g glucose/l (producing
42116 mM total VFAs) and decreased to 1.6 mol
H2/mol glucose at 40 g glucose/l (producing
75 mM total VFAs). Thus, the total VFAs (and
un-ionized VFAs) were lower at 40 g/l than at
30 g/l, so undissociated VFAs may not be the only
contributing factor to the inhibition of H2 production at higher OLRs.
Van Ginkel and Logan (2005b) suggested
lower OLRs may decrease the dissolved H2
concentration thereby removing the thermodynamic inhibition on hydrogenase. This is the same
hypothesis as discussed above for dissolved gas
removal. Although no one has measured dissolved gas concentrations while varying the OLR,
it is unlikely that changes in the OLR can
decrease the dissolved H2 concentration sufficiently to alter NADH:Fd oxidoreductase thermodynamics. The OLR used by Kraemer and

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Biotechnol Lett (2007) 29:685695

Bagley (2006) was 33 g COD l1 d1 for which the

dissolved H2 concentration (without sparging)
was 1.385 lM. The lowest OLR used to date has
been 6 g COD l1 d1 (Table 3) (Van Ginkel and
Logan 2005b), approximately 6-fold lower than
Kraemer and Bagley (2006), but a 1000-fold
decrease in dissolved H2 to <0.50.8 lM is needed
to alter NFOR thermodynamic regulation (Angenent et al. 2004; Hallenbeck 2005). Thus, decreased dissolved H2 thermodynamic regulation is
unlikely to be the explanation for improved H2
yields during OLR variations.
Another possible cause is a decrease in H2consuming acetogenesis. Intuitively, lower OLRs
would be expected to decrease acetogenesis since
this should decrease the dissolved H2 and CO2
substrate concentrations. However, Kim et al.
(2006b) observed Clostridium scatologenes (an
acetogen) at 10 g sucrose/l but not at 30 or 60 g/l.
Thus, C. scatologenes was not present when a
higher amount of substrate was available. More
research is needed to determine if OLR variations affect acetogenesis.
As discussed above for gas sparging, CO2 can
be inhibitory at sufficiently high partial pressures
(>0.5 atm). Thus, lower OLRs may decrease CO2
inhibition. This could explain why Kyazze et al.
(2006) could only achieve stable H2 production at
an OLR of 112 g COD l1 d1 when N2 sparging
was employed.

Practical implications and future research

Heat treatment dramatically changes the microbial community. However, such a microbial community will not necessarily maximize H2
production because spore-forming H2-consuming
bacteria will remain (Kim et al. 2006b; Park et al.
2005). Non-spore-forming facultative aerobes,
such as Enterobacter spp., may be beneficial for
consuming oxygen that enters the reactor with the
feedstock. In addition, the decrease in microbial
diversity caused by heat treatment could be
undesirable when complex substrates are to be
degraded, such as primary and secondary wastewater sludges, because greater microbial diversity
would provide a higher number of metabolic
degradation pathways.

Biotechnol Lett (2007) 29:685695

At present, there is not enough evidence to

support the use of inoculum heat treatment. Heat
treatment does not maximize H2 yield compared
to other pre-treatments (BES, iodopropane, perchloric acid) and at full-scale would be energetically costly. Moreover, non-sterile feedstocks
would negate heat treatment because incoming
organisms could consume H2 or decrease its
production. Methanogen control through heat
treatment should be replaced by operational controls using low pH and short solids retention time.
Further research is necessary to determine the
specific mechanisms by which sparging and other
dissolved gas removal techniques cause the H2
yield to increase. For example, to what extent
does H2-consumption (e.g. acetogenesis) exist in
continuous-flow H2-producing systems? Can this
H2-consumption be decreased by changes in
dissolved gas concentrations, organic loading rate
or other means?
From a practical perspective, sparging is
energy intensive and therefore minimizing the
use of sparging will improve the overall energy
balance of a fermentative H2 system. Lower
sparging rates than previously reported in the
literature should be investigated because our
discussion above indicated that H2 yields may
still be improved to the same extent as for higher
sparging rates. This would be an important
finding because the energy required for sparging
would be reduced and the product gas would be
less dilute. Nevertheless, full-scale implementations would require appropriate assessments to
determine whether the increased H2 yield from
sparging would be worth the added energy and
economic cost of using sparging at all.
The disagreement in the literature about the
effect of OLR on H2 yields does not allow any
meaningful conclusions to be drawn at present.
Further research is needed to determine the
specific mechanisms by which changes in organic
loading rate affect the H2 yield. Practically, the
highest allowable OLR would be preferred so as
to minimize the size of the reactor. That is, there
may be tradeoffs between H2 yield and reactor
size. Even if lower OLRs increase the H2 yield, a
minimum OLR will exist for practical H2 recovery so that it is not all lost in the dissolved phase.

693
Acknowledgements The authors would like to thank the
William H. Doherty Ontario Graduate Scholarship in
Science & Technology and the Natural Sciences and
Engineering Research Council (NSERC) of Canada for
funding.

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