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Review
Department of Civil and Environmental Engineering, The University of Western Ontario, London, Ontario N6A 5B9, Canada
GreenField Ethanol Inc., Chatham, Ontario N7M 5J4, Canada
c
Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Ontario N6A 5B9, Canada
b
article info
abstract
Article history:
order to alleviate concerns related to carbon dioxide emissions and depleting fossil fuels
resources. Biohydrogen has the potential to replace current hydrogen production tech-
21 January 2013
nologies relying heavily on fossil fuels. Batch and continuous systems employing pure
mesophiles and thermophiles isolates and co-cultures of isolates have been investigated.
The co-cultures of the isolates achieved better results than mono-cultures of the isolates
with respect to different parameters. This paper presents a critical review of the literature
Keywords:
Biohydrogen
systems. Synergies between different types of bacteria, i.e. strict and facultative, and a
Fermentation
comparison between mono- and co-cultures, types of feedstocks, and preferred feedstocks
Pure cultures
Mesophilic
Copyright 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Thermophilic
reserved.
Anaerobic
1.
Introduction
* Corresponding author. Tel.: 1 519 784 6230; fax: 1 519 352 9559.
E-mail addresses: oelsharn@uwo.ca (O. Elsharnouby), hishambahaa@gmail.com, h.hafez@greenfieldethanol.com (H. Hafez),
gnakhla@eng.uwo.ca (G. Nakhla), melnagga@uwo.ca (M.H. El Naggar).
0360-3199/$ e see front matter Copyright 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijhydene.2013.02.032
4946
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Several factors influence the fermentative hydrogen production process, which have to be optimized for enhanced
performance. Chief among these factors are: inoculum, substrate, reactor type, and temperature, which seem to impact
both hydrogen yield and hydrogen production rate, albeit with
varying importance [4]. Hydrogen yield is significantly influenced by the inoculum type, as the fermentation end products
are influenced by the bacterial metabolism. The inoculum
used for fermentative hydrogen production include: mixed
communities of anaerobic bacteria obtained from anaerobic
sludge digesters [5,6], compost piles [7], and pure cultures of
known species of hydrogen-producing bacteria. In pure culture systems, metabolic shifts are more easily detected due to
the reduced diversity of the biomass. Moreover, studies
employing pure cultures can reveal important information
regarding conditions that promote high hydrogen yield and
production rate [8].
Numerous pure bacterial cultures have been used in recent
studies to produce hydrogen from various substrates. Nevertheless, only a few review papers with limited scope are found
in the literature addressing fermentative hydrogen production by pure cultures [9,10]. For example, the review article [9]
merely presented data on the challenges and prospective of
biohydrogen production by pure cultures, without any critical
analysis, and provided minimal insight on the potential
applications.
In this paper, a critical review of 195 studies employing
pure cultures was conducted considering the most important
parameters [1e103]. The relative effectiveness of co-cultures
of pure isolates and mono-cultures of these isolates is discussed. In addition, comparative studies between employing
thermophilic and mesophilic cultures, batch and continuous
systems, and the different types of feedstocks, are evaluated.
Table 1 summarizes the data of considered studies with
respect to operational and performance parameters. Sixteen
different types of pure cultures were employed in fermentative hydrogen production processes solely or co-cultured in
the studies listed in Table 1. It should be noted that for ease of
comparison, the hydrogen production rate and hydrogen yield
from these studies were normalized to L H2/L/day and mol H2/
mol hexose equivalent, respectively.
2.
Effect of synergies between co-cultures on
technical and economic efficiencies
Ten independent studies considered in this review have
compared the effectiveness of co-cultures of pure isolates
with their mono-cultures in fermentative hydrogen production. Table 2 summarizes the operational and performance
parameters of the mono-cultures and co-cultures studied. In
all ten studies, the co-cultures achieved better results than the
mono-cultures with respect to different parameters. Examining the available literature, it is evident that the motivation
behind employing co-cultures, rather than mono-cultures,
was either economical or technical. From economy view
point, co-cultures can help maintain anaerobic conditions for
strict high hydrogen producers and eliminate the need for an
expensive reducing agent. From the technical view point, cocultures can improve the hydrolysis of complex sugars and
plant biomass, and can provide a wider range of pH for bacteria to ferment hydrogen. In most studies, both economic and
technical reasons were important considerations and interdependant. It was also found that there are primarily three
different types of co-culture of pure isolates. An explanation
of the synergistic effect in the co-culture processes for each
type is provided below.
The first type of co-cultures involves strict and facultative
anaerobes. Obligate anaerobes are extremely sensitive to O2,
and their H2-producing abilities are inhibited by a slight
amount of O2, which requires the addition of a reducing agent
such as L-cysteine to stabilize H2 production. In order to
eliminate the cost of the expensive reducing agent, facultative
anaerobes are used to consume O2 in a medium, so anaerobic
conditions are readily attained without the need for a
reducing agent. Therefore, a strict anaerobe such as Clostridium sp., and a facultative anaerobe such as Enterobacter sp.
are co-cultured in the same reactor under optimum culture
conditions for H2 production, to achieve stable and high-yield
H2 production without a reducing agent.
Jenni et al. [11] investigated the applicability of mixed
culture of Clostridium butyricum and Escherichia coli for stable H2
production without any reducing agents. They utilized
glucose as substrate, at a temperature of 37 C, and an optimum pH of 6.5 in a batch reactor. The authors noted that the
gas production pattern of batch fermentations by E. coli and C.
butyricum differed, i.e. E. coli continued gas production long
after the exponential growth. Mono-cultures of E. coli and C.
butyricum achieved H2 yields of 1.45 mol-H2/mol-glucose
consumed, and 2.09 mol- H2/mol-glucose consumed, while
the co-cultures achieved 1.65 mol-H2/mol-glucose consumed.
It should be emphesized, however, that even though C.
butyricum achieved a higher molar hydrogen yield i.e. specific
hydrogen production, the co-culture facilitated a greater
glucose conversion efficiency resulting in a higher overall
volumetric hydrogen production. These findings indicated
that employing co-cultures was more economical by eliminating the expensive reducing agent, and technically more
effective with higher hydrogen production.
Haruhiko et al. [12] examined the O2 tolerance and H2producing stability of the mono- and mixed cultures of C.
butyricum and Enterobacter aerogenes in batch and continuous
flow studies using starch as a substrate at temperature of 37 C
and pHs of 6.5 and 5.5, respectively. In the batch study, E.
aerogenes hardly produced H2 from starch since it had no
ability to utilize starch. H2 production by C. butyricum without
a reducing agent occurred after a long lag time of 12 h. In case
of C. butyricum with 0.1% L-cysteine as reducing agent, H2 was
evolved after a short lag time of 5 h. On the other hand, H2
production by the mixed culture of C. butyricum and E. aerogenes occurred after even a shorter lag time of less than 2 h,
and the amount of H2 evolved was the largest at 175% of that
produced by C. butyricum with a reducing agent. This confirms
that the mixed culture could produce H2 without a reducing
agent, since E. aerogenes consumed O2 rapidly, and thus
maintaining the anaerobic conditions conducive for biohydrogen production. In the continuous-flow study, the case
of C. butyricum with a reducing agent, exhibited H2 production
after 15 h due to removal of O2 in the reactor by the reducing
agent. The hydrogen production by C. butyricum without the
T
C
Substrate
type
Batch
70
Batch
70
Batch
Batch
70
70
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
72
72
72
72
72
72
70
72
72
72
72
Batch
72
Batch
72
Pretreated wheat
straw
Pretreated barely
straw
Glucose
Carrot pulp
hydrolysate
Glucose
Glucose
PSPa
PSP-H2b
GXSc
SSBd
Sucrose
Glu/Xyle
Glu/Xyle
Glu/Xyle
Miscanthus
hydrolysate
Miscanthus
hydrolysate
Miscanthus
hydrolysate
CSABR
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
75
75
75
75
75
75
75
75
75
75
75
Batch
75
Batch
75
Reactor
type
Substrate
concentration
(g/L)
pH
Hydrogen yield
(mol H2/mol glucose, hexose
equivalent)
H2
production
rate
(L/L/d)
Ref. No.
1-Caldicellulosiruptor saccharolyticus
Xylose
Xylose
Glucose
Glucose
PSP-H2b
PSPa
Glucose
Glucose
Fructose
fructose
Glucose
Fructose
Glucose
Fructose
Carrot pulp
hydrolysate
7.2
3.8
ND
[54]
20l
ND
0.6
[55]
20
10
7
7
3.4
2.8
6.5
8.4
[26]
[26]
31
10
10
10
10
20l
10
10
14
28
10
7
7
7
7
6.8m
6.8m
7m
7
7
7
7
2.8
3.4
3.5
3.4
3.2
2.8
2.96
3.4
3.3
2.4
3.4
8.8
6.4
7
7.1
5
5.7
4.5
8.23
8.91
6.65
8.64
[56]
[56]
[56]
[56]
[50]
[50]
[28]
[51]
[51]
[51]
[51]
14
3.3
7.13
[51]
28
2.4
4.25
[51]
5
5
27
10
10
10
10
20
10
20
10
7
7.5
7
7
7
7
7
7
7
7
7
3.36
1.31
3
2.9
3.3
3.8
3.5
3.4
3.4
3.2
3.3
2.66
0.4
8.5
8.43
6.1
8.57
7.47
8.5
2.4
4.4
8
[30]
[30]
[30]
[30]
[30]
[30]
[26]
[26]
[26]
[26]
[26]
20
8.7
[26]
10
2.7
8.6
[26]
4947
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
2-Thermotoga neapolitana
DSM 4359
DSM 4359
10
Culture(s)
DSM 4359
Reactor
type
T
C
75
Fed
batchCSABR
Fed batchCSABR
Fed batchCSABR
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
80
Batch
80
BatchSerum
bottels
Substrate
concentration
(g/L)
pH
Hydrogen yield
(mol H2/mol glucose, hexose
equivalent)
H2
production
rate
(L/L/d)
Ref. No.
20
2.4
6.2
[26]
75
Carrot pulp
hydrolysate
Xylose
7.5m
2.66
2.3
[70]
75
Glucose
7.5m
3.2
2.9
[70]
75
Sucrose
7.5m
2.5
1.3
[70]
85
77
70
65
60
80
80
80
80
80
2.5
2.5
2.5
2.5
2.5
7.5
10
14
28
10
7.5
7.5
7.5
7.5
7.5
7.5
7
7
7
7
3.75
3.85
3.18
3.09
2.04
1.84
3.3
3.2
2.5
2.9
0.54
0.56
0.32
0.19
0.033
6.05
9.9
8.16
4.18
9
[22]
[22]
[22]
[22]
[22]
[71]
[51]
[51]
[51]
[51]
14
3.2
8.4
[51]
28
3.7
[51]
80
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
Glu/Xyle
Glu/Xyle
Glu/Xyle
Miscanthus
hydrolysate
Miscanthus
hydrolysate
Miscanthus
hydrolysate
Glucose
7.5
3.85
1.2
[23]
Batch
37
Glucose
10
3.35
2.14
[19]
Batch
Batch
Batch
Batch
Batch
Batch
Batch
36
36
36
36
37
37
37
Sucrose
Glucose
Cellobiose
Maltose
Glucose
Glucose
Glucose
10
10
10
10
10
10
10
6
6
6
5
6.5
6.5
6.5
3.014
2.2
1.42
0.729
3.31
2.2
3.1
15.84
ND
15.6
1.92
ND
7.17
ND
[29]
[29]
[29]
[72]
[20]
[73]
[73]
Batch
Batch
Batch
Batch
Batch
35
35
36
36
35
Glucose
Glucose
Glucose
Starch
Glucose
3
10
10
10
9
7.2
6.47e6.98
6.5
6.5
7
2.81
2.52
2
1.8
1.97
1.9
9.36
15.84
9.84
0.5
[31]
[46]
[74]
[74]
[44]
3-Clostridium DMHC-10
4-Enterobacter Cloacae
IIT-BT08
F.P01
DM11
5-Clostridium beijerinckii
L9
Fanp 3
AM21B
AM21B
RZF-1108
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Batch
Substrate
type
4948
Table 1 e (continued )
30
36
41
36
36
35
35
Glucose
Starch
Starch
Starch
Starch
Glucose
Glucose
2.34
10
10
10
10
6
10
6.3
6.8
6.8
6m
7m
6.4
6.5
ND
ND
ND
ND
ND
1.72
1.96
1.7
8.64
9.48
8.97
9.36
ND
2.54
[75]
[76]
[76]
[76]
[76]
[77]
[78]
Batch
Batch
Batch
Batch
Batch
Batch
36
37
39
37
37
37
3
17.8
10
ND
10
20k
7.2
5.5
6.5
5.5
6
5.5
2.29
1.39
0.81
0.22
0.6
1.73g
1.52
3.9
5.3
24.8
4.128
1.611
[31]
[45]
[79]
[65]
[65]
[57]
Batch
37
6.5
1.46
1.02
[80]
CGS5
CGS5
Batch
Batch
37
37
14.2
9.2
7.5
7.5
0.84
0.91
1.5
0.64
[49]
[49]
CWBI1009
EB6
TISTR 1032
Batch
Batch
Batch
30
37
37
Glucose
Sucrose
Glucose
POMEf
Glucose
SCB hemicellulose
hydrolysateh
Glucose (and 200e
400 mg/l phenol))
Xylan hydrolysate
Pretreated straw
hydrolysate
Glucose
Glucose
Sugarcane juice
5.2m
5.6
6.5
1.7
2.2
1.33
3.02
ND
3
[81]
[82]
[58]
TISTR 1032
TISTR 1032
(immobilized)
CGS5
Batch
Repeated
batch
Batch
37
37
Sucrose
Sugarcane juice
6.5
6.5
1.34
1.52
3.11
3.5
[58]
[58]
37
5.5m
ND
5.8
[69]
W5
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
TM-9A
W5
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
37
37
37
37
37
37
37
37
37
37
37
37
37
39
37
37
5
15.7
22.3
(sucrose)
22.3
22.3
(sucrose)
9
100
10
10
10
10
10
10
10
10
10
10
10
10
100
3
3
7
8
8
8
8
8
8
8
8
8
8
8
8
6.5
6.5
6.5
1.63
3.1
0.06
2.7
1.49
1.61
0.59
0.94
0.06
0.84
0.86
0.84
0.67
1.85
2.09
1.65
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
11.9
0.41
0.52
[59]
[83]
[83]
[83]
[83]
[83]
[83]
[83]
[83]
[83]
[83]
[83]
[83]
[84]
[11]
[11]
L9
RZF-1108
6-Clostridium butyricum
ATCC19398
CGS5
W5
EB6
EB6
C. butyricum
and Escherichia
coli
4949
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Culture(s)
Reactor
type
T
C
Substrate
type
Substrate
concentration
(g/L)
pH
Hydrogen yield
(mol H2/mol glucose, hexose
equivalent)
H2
production
rate
(L/L/d)
Ref. No.
37
Sweet potato
starch residue
ND
5.25m
2.7
0.977
[13]
70
70
70
70
Sucrose
Glucose
Xylose
Starch
10
10
5
5
8
8
8
8
2.69
2.64
2.5
ND
2.4
2.4
1.8
1.3
[85]
[85]
[85]
[85]
60
60
60
10
20
10
6.5
6.25
6.5
2.62
2.53
2.45
5.71
6.5
5.97
[86]
[40]
[86]
W16
W16
Batch
Batch
60
60
10
ND
6.5
7
2.42
2.24g
6.88
ND
[40]
[60]
W16
W16
W16
W16
Batch
Batch
Batch
Batch
60
60
60
60
5
10
10
7.5,2.2,0.3
6.7
7
7
7
2.07
ND
ND
ND
ND
8.8
7.4
8.4
[87]
[52]
[52]
[52]
W16
Batch
60
ND
ND
7.7
[52]
PSU-2
PSU-2
W16
Thermoanaerobacterm
thermosaccharolyticum
GD17 and C. thermocellum JN4
9-Ethanoligenens harbinese
YUAN-3
YUAN-3
B49
B49
B49
B49
B49
B49
B49
Cont. UAi
Cont.UASBj
Batch
Batch
60
60
60
60
Xylose
Sucrose
Xylose
Glucose
Glucose
Hydrolysed
corn stover
Glucose
Glucose
Xylose
Glucose
Xylose
Arabinose
Hydrolysed
corn stover
Sucrose
Sucrose
Xylose
Cellulose
20
20
12.24
5
5.5
5.5
6.8
4.4
1.11
1.77
2.84
1.8
3.64
5.9
ND
0.33
[39]
[39]
[88]
[15]
Batch
CSTR
Batch
Batch
Batch
Batch
Batch
Batch
Batch
35
35
37
37
37
35
37
35
35
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
10
10
9
12
6
10
10
14.5
10
5
5
7
7
7
6
7
6
6
1.91
1.93
1.83
1.71
1.36
1.67
2.2
2.2
2.26
1.66
19.6
ND
ND
ND
ND
ND
ND
9.91
[38]
[38]
[89]
[89]
[89]
[90]
[91]
[92]
[48]
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
C. butyricum and
Repeated
Enterobacter
batch
aerogenes HO-39
7-Thermoanaerobacter
mathranii A3N
A3N
Batch
A3N
Batch
A3N
Batch
A3N
Batch
8-Thermoanaerobacterium thermosaccharolyticum
W16
Batch
PSU-2
Batch
W16
Batch
4950
Table 1 e (continued )
Ethanoligenens
harbinese B49
and C. acetobutylicum X9
10-Klebsiella pneumoniae
ECU-15
DSM2026
11-Pantoea agglomerans
Microcrystalline
cellulose
10
1.32
11.08
[14]
Batch
Batch
37
37
Glucose
Glycerol
10
20
6
6.5
2.07
0.53
10.08
12.2
[47]
[66]
Batch
Batch
Batch
37
37
37
Glucose
Glucose
Glucose
10
20
20
(saline
conditions)
7.2
7.2
7.2
3.8
4.2
3.3
1.82
0.78
0.61
[25]
[25]
[25]
Batch
CSTR
Batch
CSTR
CSTR
Fed batch
37
37
35
35
35
37
Glucose
Glucose
Glucosen
Glucoseo
Glucosep
Glucose
20
5
3
12
12
50
6.3
6.7
7.2
6
6
5.7
3.24
1.81
1.47
1.06
1.42
2.33
ND
7.21
1.6
10.3
3.1
16.7
[37]
[36]
[31]
[35]
[35]
[93]
Batch
Batch
37
37
3
10
7.2
5
1.8
0.59
1.42
21.33
[31]
[14]
Cont.
Trickling
bed reactor
Batch
30
Glucose
Microcrystalline
cellulose
Glucose
10
6.2
0.9
5.34
[94]
36
Cassava
wastewater
5k
2.41
1.32
[61]
S3
S6
WDHL
WDHL
WDHL
WDHL
Batch
Batch
Batch
Batch
Batch
Batch
Batch
37
30
30
37
37
37
37
3
5
5
15
15
15
7.5, 7.5
6.5
6.8
6.8
6
6
6
6
1.45
0.84
0.49
0.3
1.12
1.02
1.02
0.33
0.39
0.34
0.45
0.32
0.37
0.59
[11]
[95]
[95]
[96]
[96]
[96]
[96]
DJT135
DJT135
DJT135
DJT135
DJT135
DJT135
DJT135
DJT135
DJT135
DJT135
DJT135
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
35
35
35
35
35
35
35
35
35
35
35
Glucose
Glucose
Glucose
Glucose
Galactose
Lactose
Glucose
Galactose
Arabinose
Fractose
Galactose
Glucose
Lactose
Maltose
Mannitol
Sorbitol
Sucrose
Terhalose
Xylose
10
10
10
10
10
10
10
10
10
10
10
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
1.2
1.27
0.69
1.51
0.37
0.2
0.88
1.36
0.35
0.52
0.68
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
[27]
[27]
[27]
[27]
[27]
[27]
[27]
[27]
[27]
[27]
[27]
12-Clostridium tyrobutyricum
JM1
JM1
FYa102
FYa102
FYa102
ATCC 25755
13-Clostridium acetobutylicum
M121
X9
ATCC 824
ATCC 824
14-Escherichia coli
4951
37
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Batch
4952
Table 1 e (continued )
Culture(s)
Reactor
type
T
C
Substrate
type
Substrate
concentration
(g/L)
pH
Hydrogen yield
(mol H2/mol glucose, hexose
equivalent)
H2
production
rate
(L/L/d)
Ref. No.
Fed-Batch
37
Soduim
formate
16.1
6.5
ND
57.6
[97]
15-Clostridium thermocellum
JN4
7072
7072
7072
7072
ATCC 27405
ATCC 27405
ATCC 27405
ATCC 27405
ATCC27405
Batch
Batch
CSTR, 100 L
CSTR, 10 L
Batch
CSTR
CSTR
CSTR
CSTR
Batch
60
55
55
55
60
60
60
60
55
5
5
30
30
5
4
3
2
1.5
0.1
4.4
7.4
7.4
7.4
7.4
7
7
7
7
6.5
0.8
1.2
0.45
0.43
ND
1.29
1.53
1.65
0.98
1.6
0.01
ND
17.8
18.3
4.8
0.67
0.5
0.4
0.2
ND
[15]
[1]
[1]
[1]
[1]
[34]
[34]
[34]
[34]
[62]
Batch
Batch
60
55
1
10
6.8
7.2
1.9
ND
ND
0.34
[31]
[16]
CSTR
55
10
7.2
ND
0.44
[16]
Batch
55
Cellulose
Cellulose
Cron stalk
Cron stalk
Cron stalk
Cellulose
Cellulose
Cellulose
Cellulose
Delignefied
wood fibres
Cellulose
Corn stalk
waste
Corn stalk
waste
Cellulose
1.36
0.42
[17]
38
37
38
38
38
37
37
38
37
37
37
37
37
37
40
6.5
6.5
6.5
6.13
7
7
5.8
5.8
6.3
6.3
6.3
6.3
6.3
ND
5.5
1
0.8
0.7
ND
ND
0.2
0.89
1
2.64
2.82
0.96
0.48
1.56
1.22
2.55
7.2
2.88
8
1.31
4.8
3.56
1.3
ND
5.97
6.96
12.4
4.56
2.88
ND
6.3
[98]
[98]
[98]
[99]
[100]
[67]
[101]
[102]
[103]
[103]
[103]
[103]
[103]
[68]
[63]
40
20
6.5
1.09g
10.7
[64]
W23
Batch
35
Glucose
Glucose
Maltose
Glucose
Glucose
Glycerol
Glucose
Glucose
Xylose
Galactose
Mannose
Rahamnose
Arabinose
Glycorel
Corm starch
hydrolysate
Starch
hydrolysate
Glucose
10
10
10
21.25
0.2
20
10
10
5
10
25
5
10
21
ND
NCIMB 10102
Batch
Fed batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Continuous
packed col.
Batch
6.5
1.87
5.8
[18]
ATCC 27405
C. thermocellum
and C. thermosaccharolyticm
C. thermocellum and
C.thermosaccharolyticm
C. thermocellum DSM1237
and C.thermopalmarium
DSM 5974
16- Enterobacter aerogenes
HO-39
HO-39
HO-39
ATCC29007
E 82005
IAM 1183
IAM 1183
IAM 1183
IAM 1183
IAM 1183
ATCC35029
NCIMB 10102
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
SH5
Enterobacter aerogenes
W23 and Candida
maltosa HY-35
Enterobacter aerogenes
and C. butyricum
Enterobacter aerogenes
and C. butyricum
(immobiolized)
Batch
35
Glucose
6.5
2.19
6.27
[18]
Batch
36
Starch
ND
6.5
ND
[12]
Repeated
batch
36
Starch
ND
5.5
2.6
ND
[12]
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
4953
4954
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Table 2 e Operational and performance parameters for studies employing mono and co-cultures.
T
C
Substrate
type
Substrate
concentration
(g/L)
pH
Hydrogen
yield (mol H2/
mol/glucose,
hexose equivalent)
H2
production
rate (L/L/d)
Ref. no.
Culture(s)
Reactor
type
Clostridium butyricum
Escherichia coli
C. butyricum and
Escherichia coli
Enterobacter aerogenes
and C. butyricum
Enterobacter aerogenes
and C. butyricum
(immobiolized)
Enterobacter aerogenes
HO-39 and C. butyricum
Batch
Batch
Batch
37
37
37
Glucose
Glucose
Glucose
3
3
3
6.5
6.5
6.5
2.09
1.45
1.65
0.41
0.33
0.52
[11]
[11]
[11]
Batch
37
Starch
ND
6.5
ND
[12]
Repeated
batch
37
Starch
ND
5.5
2.6
ND
[12]
Repeated
batch
37
ND
5.25a
2.7
0.977
[13]
C. thermocellum JN4
Thermoanaerobacterium
thermosaccharolyticum
GD17 and C. thermocellum
JN4
C. acetobutylicum X9
Batch
Batch
60
60
Sweet
potato
starch
residue
Cellulose
Cellulose
5
5
4.4
4.4
0.8
1.8
0.01
0.33
[15]
[15]
Batch
37
10
0.59
21.33
[14]
C. acetobutylicum X9 and
Ethanoligenens harbinese
Clostridium thermocellum
and C. thermosaccharolyticum
Clostridium thermocellum
and C. thermosaccharolyticum
Clostridium thermocellum
DSM1237 and C.
thermopalmarium
DSM5974
Enterobacter aerogenes W23
Enterobacter aerogenes
W23 and Candida
maltosa HY-35
Batch
37
10
1.32
11.08
[14]
Batch
55
10
7.2
ND
0.34
[16]
CSTR
55
10
7.2
ND
0.44
[16]
Batch
55
Microcrystalline
cellulose
Microcrystalline
cellulose
Corn stalk
waste
Corn stalk
waste
Cellulose
1.36
0.42
[17]
Batch
Batch
35
35
Glucose
Glucose
5
5
6.5
6.5
1.87
2.19
5.8
6.27
[18]
[18]
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
4955
4956
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
3.
Comparative study between thermophiles
and mesophiles
Temperature is one of the most important operational parameters in fermentative H2 production. Temperature affects
the growth rate metabolic pathways of microorganism, substrate hydrolysis rate and hydrogen production rate.
Fermentative reactions can be operated at mesophilic
(25e40 C), thermophilic (40e65 C) or hyperthermophilic
(>80 C) temperatures [21]. It has been demonstrated that
within a specific temperature range, increasing the temperature accelerates hydrogen production, with sharply dropping
activity of hydrogen producers outside the optimum temperature range [8]. The approximately 200 investigations
reviewed in this study can be classified into two groups: 116
studies were conducted within mesophilic range; and 78
studies were carried out within thermophilic range. Performing experiments employing mesophilic cultures is
generally less expensive. However, it was reported that thermophilic and hyperthermophilic cultures seem to exhibit superior performance in hydrogen production. The highest
reported hydrogen yields in the literature, which were close to
the theoretical maximum of 4.0 mol-H2/mol-glucose, were
achieved by using extreme thermophiles [22,23].
In general, thermophiles are thought to be robust microorganisms that produce stable enzymes. It is widely accepted
that more hydrogen can be produced under thermophilic
conditions than under mesophilic conditions [24]. However,
the data available in the literature does not always support
this hypothesis, and seem to be substrate dependant. This is
because some mesophilic bacteria have better bacterial kinetics than thermophilic ones utilizing the same substrate,
despite operating at much lower temperatures. For instance,
the hyperthermophilic bacterium, Thermotoga neapolitana in a
batch experiment at a temperature of 77 C, and a pH of 7.5,
was capable of producing 3.85 mol H2/mol glucose, from 2.5 g/
L glucose, with a hydrogen production rate of 0.56 L/L/d [22].
Giuliana et al. [23] reported that T. neapolitana achieved a
maximum hydrogen yield of 3.85 mol H2/mol and a maximum
hydrogen production rate of 1.2 L/L/d, utilizing 5 g/L of glucose
in serum bottles at a temperature of 80 C, pH of 7.5. On the
other hand, the maximum hydrogen yield of 3.8 mol H2/mol
glucose and hydrogen production rate of 1.82 L/L/d were
attained by the mesophilic bacterium Pantoea agglomerans
utilizing 10 g/L glucose as substrate, at a temperature of 37 C,
and a pH of 7.2 [25]. Although the latter bacterium was operating at mesophilic tempratures, it produced hydrogen at a
higher rate than the thermophilic one from glucose (monosaccharide), and with almost the same yield.
The maximum hydrogen yield reported in the literature by
a thermophile utilizing fructose (another type of monosaccharides) was 3.4 mol H2/mol hexose equivalent, with a
maximum hydrogen production rate of 2.4 L/L/d, by the bacteria T. neapolitana [26]. The bacteria utilized 10 g/L fructose at
a temperature of 75 C, and a pH of 7.0. Nevertheless, the
maximum hydrogen yield reported in the literature by a
mesophile utilizing 10 g/L fructose in a batch at a temperature
of 35 C, and a pH of 6.5 was 1.27 mol H2/mol hexose equivalent by the bacteria E. coli [27].
The maximum hydrogen yield attained by a thermophile
utilizing sucrose (i.e. di-saccharide) was 2.96 mol H2/mol
hexose equivalent with a hydrogen production rate of 4.5 L/L/
d, which was achieved using Caldicellulosiruptor saccharolyticus,
at a pH of 7, a temperature of 70 C, and an initial concentration of 10 g/L, in a batch reactor [28]. The maximum
hydrogen yield of a mesophile utilizing sucrose was reported
by Narendra et al. [29] for E. cloacae. They achieved a hydrogen
yield and a hydrogen production rate of 3.1 mol H2/mol hexose
equivalent and 15.84 L/L/d, respectively, at a temperature of
36 C, a pH of 6.0, and initial sucrose concentration of 10 g/L.
It has been recently reported [30] that mesophilic anaerobic
bacteria cannot utilize cellulose (i.e. complex sugars) effectively. The addition of exogenous cellulose enzymes is
necessary for hydrolysis of cellulose to generate H2 by mesophilic anaerobic bacteria. On the other hand, thermophilic
anaerobic bacteria can effectively utilize cellulose [31], and
therefore, they have a great potential for H2 production from
cellulose without the addition of exogenous cellulose [15]. In
addition, the high operating temperature of the thermophiles
enhances the hydrolysis rate. For example, Rumana et al. [32]
reported that the bacterium C. thermocellum utilized 1 g/L cellulose, and produced a hydrogen yield of 1.9 mol H2/mol
hexose equivalent. On the other hand, the maximum attained
hydrogen yield reported in the literature from mesophiles (C.
acetobutylicum and E. harbinense) utilizing cellulose at a concentration of 10 g/L was only 1.32 mol H2/mol hexose equivalent [14]. These results confirm that thermophiles can more
effectively utilize complex sugars for hydrogen production
than mesophiles.
4.
Bioreactor configuration
Table 3 e Operational and performance parameters of the batch and their continuous counterparts reviewed studies.
Culture
Reactor
type
T C
Substrate
concentration
(g/L)
pH
Hydrogen yield
(mol H2/mol
glucose, hexose
equivalent)
H2 production
rate (L/L/d)
Ref. no.
55
55
55
Corn stalk
Corn stalk
Corn stalk
5
30
30
7.4
7.4
7.4
ND
0.43
0.45
4.8
18.3
17.8
[1]
[1]
[1]
35
35
35
37
37
Glucoseb
Glucosec
Glucosed
Glucose
Glucose
12, 8
12, 0.35, 1.4
3, 2
5
20
6
6
7.2
6.7
6.3
1.06
1.42
1.47
1.81
3.24
10.3
3.1
1.6
7.21
ND
[35]
[35]
[31]
[36]
[37]
35
35
Glucose
Glucose
10
10
5
5
1.91
1.93
1.66
19.6
[38]
[38]
75
75
Xylose
Xylose
5
5
7.5
7
1.31
3.36
0.4
2.66
[30]
[30]
60
60
Sucrose
Sucrose
20
20
5.5
6.25
1.77
2.53
5.9
6.5
[39]
[40]
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
1-Clostridium thermocellum
7072
Batch
7072
CSTR (10 L)
7072
CSTR (100 L)
2-Clostridium tyrobutyricum
FYa102
CSTR
FYa102
CSTR
FYa102
Batch
JM1
CSTR
JM1
Batch
3-Ethanoligenens harbinese
YUAN-3
Batch
YUAN-3
CSTR
4-Thermotoga neapolitana
DSM 4359
Batch
DSM 4359
CSTR
5-Thermoanaerobacterium thermosaccharolyticum
PSU-2
Cont. UASBa
PSU-2
Batch
Substrate
type
4957
4958
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Although the CSTRs were fed with a much higher concentration of glucose, the hydrogen yields were almost the same or a
bit less than those attained in the batch studies. This difference in hydrogen yields was attributed to the different
ambient pHs in the batches and the CSTRs experiments. It is
believed that if the CSTRs experiments were conducted at a
pH of 7.2, higher hydrogen yields would have been attained. It
must be noted, however, that in the CSTRs study [35], the
hydrogen production rate in the (GP) reactor was 3.5 times
higher than the (GA) due to a much higher organic loading
rate, but the yield of the (GP) was only 75% of that of the (GA)
due to a lower glucose fraction.
Ji et al. [36] immobilized the hydrogen producing anaerobe,
C. tyrobutyricum JM1 in a packed-bed reactor using polyurethane foam as support media. The hydraulic retention
time (HRT) condition for maximum hydrogen production rate
in this system was 2 h, where the main metabolite was
butyrate with low lactate concentration, and hydrogen yield of
1.81 mol H2/mol glucose was attained at a pH of 6.7, temperature of 37 C, and a feed glucose concentration of 5 g/L.
Therefore, the immobilized system was an effective and stable approach for continuous hydrogen production for efficient
utilization of carbon substrates with good hydrogenproducing performance. However, in a later study by the
same group [37], the effects of pH on hydrogen fermentation
of glucose by the same bacterium were investigated in batch
cultivations. The batches were conducted at different pHs (6.0,
6.3, 6.7), temperature of 37 C, and a glucose concentration of
20 g/L. The initial low glucose concentration (such as the 5 g/L
of glucose used in the previous study [36]) resulted in a low
fermentation rate, and consequently a low hydrogen yield. It
was proven that a pH of 6.3 was optimum for hydrogen production with a high concentration of butyrate, and a hydrogen
yield of 3.24 mol H2/mol glucose. The lower hydrogen yield
achieved in the continuous-flow system was attributed to the
un-optimized pH and substrate concentration.
Defeng et al. [38] studied hydrogen production of autoaggregative (self-flocculating granular) E. harbinense YUAN-3
in a batch reactor and a continuous stirred-tank reactor
(CSTR), with glucose as substrate under non-sterile conditions. In the batch reactor, the optimized operational conditions constituted a pH of 5.0, temperature of 35 C, and glucose
concentration of 10 g/L. The maximum hydrogen yield and
hydrogen production rate under the optimum operational
conditions were 1.91 mol H2/mol glucose and 1.66 L/L/d,
respectively. In the CSTR, hydrogen gas yield reached a
maximum of 1.93 mol H2/mol glucose, and H2 production rate
reached a maximum of 19.6 L/L/d. The strain YUAN-3 was well
retained in the reactor. The overflow rate of cells was less than
0.1% in the continuous flow reactor, at a dilution rate of 0.5/h.
However, after 7 days of continuous operation some other
hydrogen-producing bacterial species appeared and formed a
stable community with YUAN-3. The hydrogen yield
decreased from 0.93 mol H2/mol glucose to 1.5 mol H2/mol
glucose and stabilized thereafter. The dominant populations
in the continuous-flow reactor were affiliated with M. hominis,
and M. sueciensis, and the majority of dominant populations
belonged to E. harbinense, which were enriched during operation of the reactor. These results indicate that a successful
continuous operation was achieved. It is evident from the
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
5.
Feedstocks
Hydrogen can be produced from a wide spectrum of carbohydrates. Nevertheless, 80% of the studies reported in the
surveyed literature have investigated hydrogen production by
dark fermentation from pure sugars, such as glucose, or sucrose as substrate. Only a few studies have focussed on sustainable substrate conversion (Fig. 1). However, for real value
to the society and environment, biohydrogen should be produced from renewable feedstocks (real waste) [41]. The potential feedstocks include: biomass, agricultural waste biproducts, lignocellulosic products (wood and wood waste),
waste from food processing, aquatic plants, algae, agricultural, and livestock effluents. If used under appropriate control, these resources would become the major source of
energy in the future. In this study, different types of
4959
Sustainable
feedstocks
(current+future)
20%
Pure polysaccharides
11%
Pure monosaccharides
59%
5.1.
4960
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
5.2.
Table 4 e Operational and performance parameters of the studies utilizing sustainable feedstocks.
Substrate types
Culture(s)
Sugarcane juice
Molass
Sweet potato starch
residiue
Hydrolysed corn stover
Hydrolyzed corn stover
Cassava wastewater
Cron stalk
Cron stalk
Cron stalk
Delignefied wood fibres
Corn stalk waste
Corn stalk waste
Substrate
concentration
(g/L)
pH
Hydrogen yield
(mol H2/mol glucose,
hexose equivalent)
H2 production
rate (L/L/d)
Ref. no.
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
70
70
70
72
72
72
72
72
72
75
75
75
75
80
80
80
37
10
20h
10
10
10
20h
10
14
28
10
10
10
20
10
14
28
20g
7.2
7
7
7
7
6.8i
7
7
7
7
7
7
7
7
7
7
5.5
3.8
ND
2.8
3.5
3.4
2.8
3.4
3.3
2.4
3.3
3.8
2.7
2.4
2.9
3.2
2
1.73e
ND
0.6
8.4
7
7.1
5.7
8.64
7.13
4.25
6.1
8.57
8.6
6.2
9
8.4
3.7
1.611
[54]
[55]
[26]
[56]
[56]
[50]
[51]
[51]
[51]
[56]
[56]
[26]
[26]
[51]
[51]
[51]
[57]
Batch
37
9.2
7.5
0.91
0.64
[49]
Batch
37
6.5
1.33
[58]
Repeated
batch
Batch
Repeated
batch
Batch
37
6.5
1.52
3.5
[58]
37
37
22.3
(sucrose)
22.3
(sucrose)
100
ND
7
5.25i
1.63
2.7
ND
0.977
[59]
[13]
60
ND
2.24e
ND
[60]
Batch
60
ND
ND
7.7
[52]
Batch
36
5g
2.41
1.32
[61]
CSTR, 100 L
CSTR, 10 L
Batch
Batch
55
55
55
55
30
30
5
0.1
7.4
7.4
7.4
6.5
0.45
0.43
ND
1.6
17.8
18.3
4.8
ND
[1]
[1]
[1]
[62]
Batch
55
10
7.2
ND
0.34
[16]
CSTR
55
10
7.2
ND
0.44
[16]
4961
T C
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Reactor type
Reactor type
T C
40
ND
5.5
2.55
6.3
[63]
Starch hydrolysate
Enterobacter aerogenes
NCIMB 10102
Continuous
packed col.
Batch
40
20
6.5
1.09e
10.7
[64]
Batch
Batch
Batch
Batch
37
37
37
37
ND
20
20
21
5.5
6.5
7
ND
0.22
0.53
0.2
1.22
24.8
12.2
3.56
ND
[65]
[66]
[67]
[68]
Batch
37
5.5i
ND
5.8
[69]
2-Future sustainable
feedstocks:
POMEd
Glycerol
Glycerol
Glycorel
Chlorella vulgaris ESP6
(microalgal hydrolysate)
C. butyricum EB6
Klebsiella pneumoniae DSM2026
Enterobacter aerogenes
Enterobacter aerogenes
ATCC35029
C. butyricum CGS5
Substrate
concentration
(g/L)
pH
Hydrogen yield
(mol H2/mol glucose,
hexose equivalent)
H2 production
rate (L/L/d)
Ref. no.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
Culture(s)
Substrate types
4962
Table 4 e (continued )
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 4 9 4 5 e4 9 6 6
The reported hydrogen yields from biomass used as substrates varied greatly from approximately 20% to more than
90% of the theoretical 4 moles of H2 per mol of hexose. The
diversity of the applied feedstocks and pretreatment methods
hardly allow a comparison of hydrogen production efficiency.
5.3.
Future feedstocks
6.
Concluding remarks
4963
Acknowledgement
The authors acknowledge NSERC, GreenField Ethanol, Union
Gas, and Admira Energy for their financial support of the
project, as well as the Ontario Trillium Ph.D. Scholarship
Program awarded to Ms. Omneya Elsharnouby.
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4964
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