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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

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/he

Review

A critical literature review on biohydrogen production by pure


cultures
Omneya Elsharnouby a, Hisham Hafez b,*, George Nakhla a,c, M. Hesham El Naggar a
a

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:

Global research is moving forward in developing hydrogen as a renewable energy source in

Received 19 November 2012

order to alleviate concerns related to carbon dioxide emissions and depleting fossil fuels

Received in revised form

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

Accepted 7 February 2013

mesophiles and thermophiles isolates and co-cultures of isolates have been investigated.

Available online 13 March 2013

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:

reporting on fermentative biohydrogen production by pure cultures of bacteria in different

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

for mono- and cultures are outlined.

Mesophilic

Copyright 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

Thermophilic

reserved.

Anaerobic

1.

Introduction

The challenges of environmental pollution and traditional


energy reserves depletion are focussing intensive research on
alternative energy production. Hydrogen is widely regarded as
one of the most promising energy carriers, because of its high
efficiency of conversion to usable power, non-polluting
oxidation products, and high gravimetric energy [1]. These
advantages render hydrogen as an attractive candidate to
reduce reliance on conventional fossil fuels.
Biological hydrogen production is suitable for a variety of
feedstocks including organic waste material, and is less

energy intensive compared to other hydrogen processes.


Biological methods include photosynthetic hydrogen production and dark fermentative hydrogen production.
Photosynthetic hydrogen production involves transforming
solar energy into hydrogen via photosynthetic bacteria.
However, its application is challenged by the low transfer
efficiency of light, complexity in reactors design, and low
hydrogen production rates [2,3]. On the other hand,
fermentative hydrogen production facilitates high hydrogen
production rate through a simple operation, making the
process an increasingly popular option for hydrogen
production.

* 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

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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

Table 1 e Operational and performance parameters of the reviewed 194 experiments.


Culture(s)

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]

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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

Chlorella vulgaris ESP6


(microalgal hydrolysate)
Molass
Glucose
Arabinose
Raffinose
Sucrose
Trehalose
Xylose
Cellobiose
Cellulose
Ribose
Galactose
Fractose
Mannose
Molasses
Glucose
Glucose

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

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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

(continued on next page)

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

ND: Not defined.


a Untreated potato steam peels, Molar yields were based on the amount of starch in untreated PSP assuming 100% starch consumption.
b The starch in the PSP was liquefied with alpha-amylase, and then the liquefied starch was further hydrolysed to glucose by amyloglucosidase.
c Mixture of glucose, xylose and sucrose.
d Sweet sorgham bagasse.
e Glucose: Xylose 7:3.
f Palm oil mill effluent.
g Mol H2/mol total sugar.
h Sugarcane bagasse hemicellulose hydrolysate.
i Up flow carrier free anaerobic system.
j Up flow anaerobic sludge blanket.
k g COD/L.
l g sugars/L.
m Controlled pH.
n 2 g/L peptone was added with the substrate.
o 8 g/L peptone was added with the substrate.
p 0.36 g/L, 1.4 g/L peptone and ammonium chloride respectively, were added with the substrate.

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]

ND: Not defined.


a Controlled pH.

reducing agent was inhibited by oxygen, and could not be


recovered at all even after 50 h, indicating complete damage
for the bacterial cells. In contrast, H2 production by the mixed
culture, which was initially inhibited by oxygen, was recovered immediately within 0.5 h. These results confirm that the
mixed culture can remove O2 in the reactor and recover H2
production immediately.
Similarly, Yokoi et al. [13] investigated the synergies between strict and facultative anaerobes. A sustained high biohydrogen yield of 2.7 mol H2/mol glucose was attained by a
mixed culture of C. butyricum and E. aerogenes HO-39. The
mixed bacteria utilized starch waste consisting of sweet potato starch residue as a carbon source, and corn steep liquor as
a nitrogen source. The experiment was conducted in a fed
batch culture, at a temperature of 37  C, and a controlled pH of
5.25. The results proved that a mixed culture of C. butyricum
and E. aerogenes could produce hydrogen from starch at a high
yield of more than 2 mol of hydrogen per 1 mol of glucose
without any reducing agents, since E. aerogenes, a facultative
anaerobe, removed oxygen and generated anaerobic conditions in the reactor.

The second type of co-cultures reported in the literature


was between cellulose degrading anaerobes and high
hydrogen producers via fermenting simple sugars. The most
common dark fermentation procedure employed to generate
hydrogen from cellulose materials involved expensive pretreatment processes, such as delignification, and hydrolysis
[14]. Since pre-treatment processes are expensive, fermentative hydrogen production from cellulosic materials is desirable. Therefore, many studies investigated employing cocultures of two bacterial strains: one with the capability of
hydrolysing cellulose, and the other is a high hydrogen producer utilizing simple sugars.
For example, Yan et al. [15] investigated biohydrogen production from cellulose using the thermophilic anaerobic
bacterium Clostridium thermocellum JN4, and the co-cultures of
the aforementioned bacterium with Thermoanaerobacterium
thermosaccharolyticum GD17. The C. thermocellum JN4 can
decompose cellulose but cannot completely utilize the cellobiose and glucose produced by the cellulose degradation,
while the T. thermosaccharolyticum GD17 can utilize monosugars. The experiment was conducted at pH of 4.4, and

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

temperature of 60  C in a batch reactor. The C. thermocellum JN4


resulted in hydrogen yield of about 0.8 mol H2/mol glucose,
with lactate as the main product. When C. thermocellum JN4
was co-cultured with T. thermosaccharolyticum GD17, hydrogen
production was doubled and H2 yield increased to a high level
of 1.8 mol H2/mol glucose. Butyrate was the most abundant
byproduct and lactate was not detected at the end of the cocultures process.
Aijie et al. [14] employed dark fermentation of microcrystalline cellulose to produce biohydrogen using mono and cocultures. Clostridium acetobutylicum ATCC 824(X), a high
hydrogen producer from microcrystalline cellulose was utilized to produce hydrogen at temperature of 37  C, and pH of
5.0. The mono-culture of X9 yielded hydrogen after a 5-h time
lag. The corresponding hydrogen yield, maximum hydrogen
production rate, and cellulose hydrolysis ratio reached
755 mL/L medium, 6.4 mmol H2/h/g dry cell, and 68.3%,
respectively. The co-cultures of C. acetobutylicum X9 and strain
Ethanoligenens harbinense B49, which can produce hydrogen
efficiently from both monosaccharides and microcrystalline
cellulose, yielded hydrogen immediately following initiation
of fermentation. The hydrogen yield, maximum hydrogen
production rate, and cellulose hydrolysis ratio of 1810 mL/L
medium, 55.4 mmol H2/h/g dry cell, and 77.6%, respectively,
were achieved. The strain B49 rapidly removed reduced sugars
produced by cellulose hydrolysis by X9, hence improving
cellulose hydrolysis and subsequent hydrogen production.
Another example of technical and economical efficiencies
attained by the synergies between co-cultures in fermentative
hydrogen production process is provided by Qian et al. [16].
The authors utilized a combination of cellulose-hydrolysing
bacteria and highly efficient hydrogen producing bacteria to
optimize hydrogen production from cellulosic waste. They
employed a mix of C. thermocellum and Clostridium thermosaccharolyticum utilizing corn stalk waste. C. thermocellum is a
cellulose-degrading bacterium, which has the potential for
direct hydrogen production from lignocellulosic waste, hence
eliminating the need for an extensive hydrolysing process, but
with low biohydrogen yield. The C. thermosaccharolyticum is a
non-cellulolytic high hydrogen-producing stain. The experiments were conducted in both batch and continuous-flow
modes at temperature of 55  C, and pH of 7.2. At the end of
the C. thermocellum mono-culture experiment, cellobiose,
glucose, and xylose contents were found in the fermentation
broth. However, cellobiose and xylose were not detected at the
end of the C. thermosaccharolyticum and C. thermocellum cocultures experiments because C. thermosaccharolyticum cultures produced hydrogen, organic acids (acetate), and other
components. The hydrogen yield in the co-culture batch
fermentation reached 68.2 mL/g-cornstalk, which was 94.1%
higher than that in the mono-culture, and the rate of
hydrogen production reached 14.1 mL H2/L/h. A hydrogen
yield of 74.9 mL/g cornstalk, as well as production rate of
18.5 mL H2/L/h were achieved using the optimized co-culture
method in the scaled-up reactor.
Alei et al. [17] investigated employing a cellulolytic
hydrogen-producing bacterium and a non-cellulolytic high
hydrogen-producing bacterium. In their study, C. thermocellum
DSM 1237, a cellulolytic hydrogen-producing bacterium, was
co-cultured with Clostridium thermopalmarium DSM 5974, a

4955

non-cellulolytic high hydrogen-producing bacterium. The


bacteria utilized cellulose as the sole substrate, at temperature of 55  C, and pH of 7.0. The co-culture produced nearly
double the amount of hydrogen produced in C. thermocellum
monocultures. Ethanol and acetate were the main metabolites
in C. thermocellum monocultures, whereas the co-cultures
produced butyrate as the main metabolite. These results
support the synergies between cellulolytic anaerobes and
high-yield biohydrogen producers. It is thought that using cocultures of various types of complex sugars degrading anaerobes in general and high hydrogen producers would give the
same results as using co-cultures of cellulose degrading anaerobes and high hydrogen producers.
The third type of co-cultures reported in the literature involves aciduric hydrogen producing microorganisms and high
hydrogen producers. Due to the inhibitory impact of organic
acids produced during fermentation on biohydrogen producers, near neutral or weak acidic conditions are mandatory
to attain high hydrogen yields. However, pH control is uneconomical due to the large quantity of chemicals needed.
Aciduric microorganisms can produce hydrogen at low pHs,
hence reducing or even eliminating buffering requirements.
Thus, using a hydrogen producing bacterium co-cultured with
aciduric microorganisms can achieve stable and highhydrogen production at low pHs [18]. In this study, a batch
experiment was carried out to measure the hydrogenproducing ability of a mixed culture of Candida maltosa HY35, an aciduric microorganism, which can produce hydrogen
at pH as low as 1.3, and a facultative anaerobe E. aerogenes W23, which has aciduric hydrogen producing properties (can
produce hydrogen at pH of 4.0). These mixed cultures at 35  C
attained a hydrogen yield of 1735 mL/L, representing 17.15%
and 119.90% higher yield than the monocultures E. aerogenes
W-23 and C. maltosa HY-35, respectively. Meanwhile, the
average hydrogen production rate of the mixed culture was
261.1 mL/h/L, which was 7.85% and 146.23% higher than those
of the monoculture of E. aerogenes W-23 and C. maltosa HY-35,
respectively. In this case, the co-cultures of hydrogen producing aciduric microorganism and high hydrogen producing
anaerobe allowed for dispensing or reducing amount of buffering agents by providing a wider range of pH for bacteria to
ferment within. In addition, Candida consumed the lactate,
succinic and citric acids produced by Enterobacter during
fermentation and slowed the shift in pH. These results
confirm the synergies between the mixed cultures.
The success of co-cultures of high hydrogen producing
bacteria and hydrogen producing aciduric bacteria emphasizes the potential for employing co-cultures of multi species
high-hydrogen producing-bacteria with different optimum pH
ranges to naturally realize hydrogen production over a wide
pH range. For instance, it was found that the optimum pH for
hydrogen production and growth rate for Clostridium DMHV-10
is 5.0 [19]. The bacterium fermented 10 g/L glucose with a yield
of 3.35 mol H2/mol glucose, at temperature of 37  C, and an
initial pH of 5.0. The bacterium is capable of growth and
hydrogen production within a pH range of 5.0e7.0. On the
other hand, it was reported that the optimum pH for hydrogen
production and growth rate for the hydrogen producer Enterobacter Cloacae was found to be 6.5 [20]. The bacterium yielded
3.31 mol H2/mol glucose from fermenting 10 g/L glucose at a

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

temperature of 37  C, and an initial pH of 6.5. This bacterium


was capable of growing and producing hydrogen within a pH
range of 4.5e8.0. Thus, these two strains could be co-cultured
at an initial pH of 6.5 eliminating the need for a buffer and a
reducing agent to maintain the anaerobic conditions for the
Clostridium bacteria.

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

Fermentative hydrogen production was applied in both batch


reactors and continuous systems. Batch-mode reactors are
easily and flexibly operated. This has resulted in the wide
utilization of batch reactors for determining the biohydrogen
potential of organic substrates. In industrial context, however,
continuous bioprocesses are recommended for practical
considerations such as waste stock management, economic
feasibility, and practical engineering design [33].
Seven cases of batch studies are reported in the literature
to be successfully scaled up or applied to continuous-flow
systems. The performance of scaled up continuous systems
varied from one study to another. In some cases, the same or
even better performance was achieved compared to their
batch counterparts. In other cases, however, the continuous
systems displayed lesser but stable performance. Table 3 illustrates the differences in performance and operational parameters between the batches and continuous-flow systems
employing the same bacteria, and utilizing the same substrates. Scrutinizing the results in Table 3, it may be concluded

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]

ND: Not defined.


a UASB up flow anaerobic sludge blanket.
b 2 g/L peptone was added with the substrate.
c 8 g/L peptone was added with the substrate.
d 0.36 g/L, 1.4 g/L peptone and ammonium chloride respectively, were added with the substrate.

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

that optimizing the operational parameters is the key for


achieving a successful continuous-flow system.
Continuous-flow system performance was superior to the
batch in thermophilic fermentation of cornstalk by C. thermocellum 7072 [1]. In the batch test involving a substrate of 5 g/
L cornstalk at a temperature of 55  C and a pH of 7.4, hydrogen
yield and hydrogen production rate of 38.8 mL/g and 201.4 mL/
L/h were achieved. The continuous stirred tank reactor (CSTR)
was fed with 30 g/L of cornstalk, and operated at the same
temperature and pH as the batch test. The maximum
hydrogen production rate in the 10 L CSTR, and the 100 L CSTR
was 767.5, and 739.9 mL/L/h, respectively. The authors
attributed the higher hydrogen production rates and shorter
lag-phase period observed in the two CSTRs to the improved
mixing conditions in the two reactors, compared to the
anaerobic bottles. The hydrogen yield in the 10 and 100 L
CSTRs reached 58.3 and 61.4 mL/g of cornstalk. Acetate and
ethanol were the major end products of fermentation by C.
thermocellum for cornstalk, and the ratio of ethanol/acetate
was lower in both CSTRs than in the 125 mL anaerobic bottles.
The substrate concentration was the only different operational parameter between the CSTRs and batch tests. Thus, it
was presumed that it also contributed to the higher production rate and yield, as higher substrate concentration increases fermentation rate. The shift from the ethanol to the
acetate pathway in the CSTRs explained the higher attained
hydrogen yields in the CSTRs.
Cheng and Liu [1] used C. thermocellum and achieved a
hydrogen yield of 1.2 mol H2/mol hexose equivalent, at a pH of
7.0, and a temperature of 60  C. These results are consistent
with the 0.98e1.65 mol H2/mol hexose equivalent observed by
Lauren et al. [34] employing a CSTR at neutral pH, and influent
of cellulose concentration in the range of 1.5e4 g/L.
In another study, Liang-Ming et al. [35] investigated the
fermentative biohydrogen production in CSTRs using Clostridium tyrobutyricum FYa102. Two CSTRs were employed in
this study: one, denoted (GP), was fed with 12 g/L of glucose
and 8 g/L of peptone, while the other, denoted (GA), was fed
with 12 g/L of glucose, 1.4 g/L of ammonium chloride, and
0.360 g/L of peptone. The experiments were carried out at a
temperature of 35  C, and a pH of 6.0. The hydrogen yield and
hydrogen production rate achieved for the (GA) and (GP) reactors were 1.42 mol H2/mol glucose, and 3.1 L/L/d, and
1.06 mol H2/mol glucose, and 10.3 L/L/d, respectively. On the
other hand, Pei-Ying et al. [31], conducted biochemical
hydrogen potential (BHP) tests to investigate the metabolism
of glucose fermentation and hydrogen production performance of C. tyrobutyricum FYa102 in batches. Glucose and
peptone were used in the fermentation medium at initial
concentrations of 3 g/L, and 2 g/L, respectively. The experiment was conducted at a temperature of 35  C and a pH of 7.2.
The attained hydrogen yield and hydrogen production rates
were 1.47 mol H2/mol glucose, and 1.6 L/L/d, respectively. In
glucose re-feeding experiments, the C. tyrobutyricum FYa102
fermented additional glucose during re-feeding tests, producing a substantial quantity of hydrogen. The higher
hydrogen production rates attained in the CSTRs were
attributed to the higher rate of fermentation resulting from
the higher concentrations of glucose and peptone in the medium, and the better mixing conditions in the CSTRs.

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

above results that optimizing the operational parameters for


the auto-aggregative bacteria achieved continuous stable
hydrogen production, despite the occurrence of microbial
shift.
Tien et al. [30] investigated biohydrogen production from
xylose by T. neapolitana in batch culture using serum bottles
and a continuously stirred anaerobic bioreactor (CSABR). A
maximum hydrogen production rate of 0.44 L H2/L/d and a
maximum hydrogen yield of 1.31 mol H2/mol hexose equivalent were obtained in the serum bottles test, at an initial
xylose concentration of 5.0 g/L, and a pH of 7.5. The CSABR
was run at uncontrolled and controlled pH conditions. In the
uncontrolled pH experiments, the fermentation process
ceased before the complete consumption of substrate due to
the drastic decrease in pH. In pH-controlled cultures, much
higher H2 production and xylose utilization rates were achieved as evident from the levels of acetic acid varying from 2.5
to 3.5 g/L compared to 2.5 g/L in the uncontrolled batches. In
contrast to acetic acid production, lactic acid production was
the lowest under pH-controlled conditions. Subsequently, the
H2 production rate increased exponentially reaching the
maximum level. The maximum H2 yield, and hydrogen production rate of 2.8 mol H2/mol xylose consumed, and 2.66 L H2/
L/d were measured while the pH was maintained at 7.0. It
appears that controlling pH at neutral limit instead of an
initial pH of 7.5 was the key for a stable continuous system.
Another example of the effect of un-optimized operational
parameters on the performance of continuous-flow system
was provided by Sompong et al. [39]. They investigated the
fermentation of 20 g/L sucrose by the bacterium T. thermosaccharolyticum strain PSU-2 in an UASB bioreactor. The system
was stable, and the hydrogen yield and production rate of
1.77 mol H2/mol hexose and 5.9 L H2/L/d were achieved at a
temperature of 60  C and a pH of 5.5. However, the same authors investigated in another study [40] the fermentation of
the same concentration of sucrose under the same temperature, and a wide range of pH (4.0e9.0) by the same bacterium
and observed maximum hydrogen yield and hydrogen production rate of 2.53 mol H2/mol hexose equivalent, and 6.5 L/L/
d at a pH of 6.25. Therefore, it is presumed that if the continuous system was operated at the optimum pH, a higher
hydrogen yield and production rate would have been realized.

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%

Fig. 1 e Percentage of the usage of pure and real waste


substrates in the reviewed literature.

feedstocks are discussed in terms of their applicability and


operational challenges, as well as the motivation for their use
in fermentative hydrogen production.

5.1.

Pure carbohydrates (synthetic waste)

Pure carbohydrate sources are expensive raw materials for


real scale hydrogen production (which can only be viable
when based on renewable and low cost sources). Nevertheless, the majority of the reviewed studies utilized pure carbohydrates as substrate, including: monosaccharides
(glucose, xylose, fructose, arabinose, mannose and ribose);
disaccharides (sucrose, cellubiose, maltose, and lactose); or
polysaccharides (starch, cellulose, and xylan).
Simple sugars such as glucose, sucrose, and lactose are the
most commonly used pure substrates due to their ease of
biodegradability, relatively simple structures, and presence in
real industrial effluents [42,43]. Unlike starch and cellulose,
they require short fermentation times (i.e. process HRT),
which makes these substrates preferred model substrates for
hydrogen production studies. Model substrates are employed
in fermentative hydrogen production processes to study bacterial kinetics, assess adequate nutrients, and identify the
optimized operational parameters for the process [19,44e48].
However, real life applications involve complex sugars, and
thus it is indispensable to employ these substrates in the dark
fermentation process in order to provide relevant insight into
system performance [1,49].
Hydrolysis of real waste comprising different sugars was
modelled by co-digestion of various pure substrates. The
control experiments assessed the fermentation preferences of
the bacteria among the different types of sugars. Panagiotopoulos et al. [50] conducted four experiments to evaluate the hydrogen production and the main organic acids by

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

C. saccharolyticus from the hydrolysed sweet sugar bagasse


(SSB) and from a mixture of pure sugars (glucose, sucrose and
xylose). They employed SSB in 2 experiments at sugar concentrations of 10 g/L and 20 g/L, and conducted two control
experiments employing mixtures of pure sugars of glucose,
xylose, and sucrose, at concentrations of 10 g/L and 20 g/L. At a
sugar concentration of 10 g/L, consumption of pure sugars and
sugars of SSB hydrolysate was complete within similar
fermentation time. At the 20 g/L of pure sugars, consumption
was still incomplete at 72 h, while sugars of SSB hydrolysate
were completely consumed at 70 h. The consumption pattern
of 20 g/L sugars of SSB hydrolysate sugars differed markedly
from that of pure sugars. Lactate production only occurred in
fermentations on SSB hydrolysate. Therefore, hydrogen production and hydrogen yields were higher in fermentations on
pure sugars than of SSB hydrolysate. The high rate of glucose
consumption in the fermentation of 20 g/L of SSB hydrolysate
sugars coincided with the high rate of lactate production in
this fermentation. Basically, at sugar concentration of 10 g/L,
the batch fermentations under controlled conditions
confirmed the results of the fermentability tests, but at higher
substrate concentrations, lactate production increased
dramatically at the expense of hydrogen production.
Trus de Vrije et al. [51] investigated thermophilic hydrogen
production using C. saccharolyticus and T. neapolitana on hydrolysate of the lignocellulosic feedstock Miscanthus (obtained
from enzymatic hydrolysis) in batch tests. Control experiments were also conducted at different mixing ratios of xylose
and glucose to assess the utilization preference of the bacteria
for a sugar type over the other and the optimum substrate
concentration. The authors observed that T. neapolitana
showed a preference for glucose over xylose, which were the
main sugars in the hydrolysate, while C. saccarolyticus
consumed both at a similar rate. Lactate production by C.
saccarolyticus was very low in fermentations on pure sugars, as
well as on hydrolysate. T. neapolitana produced more lactate
on the hydrolysate than on pure sugars. The optimum total
sugars concentration was 17 g/L, and C. saccharolyticus offered
the advantage of nearly 10% higher hydrogen yield during
growth on Miscanthus hydrolysates as compared to T. neapolitana, but the rates of substrate consumption and hydrogen
production by T. neapolitana were 5e18% higher.
Trus de Vrije et al. [26] investigated hydrogen production
from carrot pulp hydrolysate (obtained from enzymatic hydrolysis) by the same thermophilic bacteria C. saccharolyticus
and T. neapolitana. The main sugars in the hydrolysate were
glucose, fructose, and sucrose. Therefore, they initially
investigated hydrogen production from different concentrations of glucose, fructose, and mixtures of glucose and fructose as control experiments. in order to assess the adequate
degree of hydrolysation and optimized substrate concentration by determining the preferred sugar type for bacteria. They
observed that in fermentations of 10 g/L glucose and 10 g/L
fructose, C. saccharolyticus could virtually completely consume
all substrates with almost identical rates of consumptions. In
contrast, T. neapolitana consumption trend was different for
glucose than fructose, suggesting a preference for glucose. In
fermentations of 20 g/L of substrate, the consumption of
substrate was incomplete for both cultures even after 2 days.
Also, they found that the cultures productivities were

equivalent or higher than the productivities achieved with the


corresponding pure sugars (mixtures of glucose and fructose)
at 10 g/L sugars. Doubling the hydrolysate concentration had
adverse effect on hydrogen production, with a severe decrease
in yield in C. saccharolyticus cultures and a decrease in productivity with T. neapolitana.
Nan-Qi Ren et al. [52] investigated the utilization of an agrowaste, corn stover, as a renewable lignocellulosic feedstock for
the fermentative H2 production by the moderate thermophile
T. thermosaccharolyticum W16. The corn stover was hydrolysed
by cellulase with supplementation of xylanase after delignification with 2% NaOH, producing glucose, xylose, and arabinose. To determine the fermentative behaviour of the
bacterium, a set of control experiments supplemented with
glucose, xylose, and a mixture of glucose, xylose, and arabinose at a fixed total sugar quantity of 10 g/Lwere undertaken.
The concentrations of glucose, xylose, and arabinose in the
mixture were at the same levels as found in the corn stover
hydrolysate. It was observed that the bacterium showed preference for glucose over the other types of sugars. The bacterium grew well on the hydrolysate and reached a similar
optical density and maximum hydrogen production rate as on
simulated medium, although hydrogen yield was slightly
higher on hydrolysate. Although the molar carbon balances in
the control experiments closed at 100%, carbon balances did
not close in the hydrolysate, most probably due to the interference of unidentified components in the hydrolysate.

5.2.

Sustainable feedstocks (real waste)

For sustainable biohydrogen production, the feedstock has to


be cheap and would have to meet the following criteria: carbohydrate produced from sustainable resources; sufficient
concentration that fermentative conversion and energy recovery is energetically favourable; and minimum pretreatment [53].
Biomass is a viable renewable resource. It includes agricultural residues, energy crops, and industrial wastes, which
can be used for the production of power, heat and biofuels
[50]. Producing hydrogen from biomass greatly enhances the
security of supply [26]. Therefore, most recent studies
employed biomass for biological conversion via fermentation
processes. As shown in Table 4, sugar-containing crops
(e.g.sweet sorghum and sugar beet), starch-based crops (e.g.
corn and wheat), ligno-cellulosics (e.g. fodder grass and miscanthus), and food industry by-products are all biomass types
used as substrates in the literature [1,13,16,26,49e52,54e64].
In view of the increasingly negative public reaction to the
use of food for biofuel production, employing energy crops as
feedstocks for biofuels generation (e.g. wheat straw, barely
straw, corn stalk, miscanthus, and cassava) is widely accepted
in the scientific community [1,51,54,55,61]. They are
commonly referred to as second generation cellulosic
biomass. Additionally, utilizing industrial and agricultural
waste residues (e.g. delignified wood fibres, and corn stalk
waste) [16,35,49,52,60], or food industry waste (e.g. carrot pulp,
potato steam peels, sugarcane waste, sweet sorghum syrup,
corn starch, and sweet potato starch) [13,26,50,56e59,63,64]
addresses the concerns of skyrocketing food and energy
prices.

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]

(continued on next page)

4961

C. butyricum TISTR 1032


( immobilized)
C. butyricum W5
C. butyricum and Enterobacter
aerogenes HO-39
Thermoanaerobacterium
thermosaccharolyticum W16
Thermoanaerobacterium
thermosaccharolyticum W16
Clostridium acetobutylicum
ATCC 824
Clostridium thermocellum 7072
Clostridium thermocellum 7072
Clostridium thermocellum 7072
Clostridium thermocellum
ATCC27405
Clostridium thermocellum and
C. thermosaccharolyticum
Clostridium thermocellum and
C. thermosaccharolyticum

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

1-Current sustainable feedstocks:


Pretreated wheat straw
Caldicellulosiruptor saccharolyticus
Pretreated barely straw
Caldicellulosiruptor saccharolyticus
Carrot pulp hydrolysate
Caldicellulosiruptor saccharolyticus
Caldicellulosiruptor saccharolyticus
PSPa
Caldicellulosiruptor saccharolyticus
PSP-H2b
Caldicellulosiruptor saccharolyticus
SSBc
Miscanthus hydrolysate
Caldicellulosiruptor saccharolyticus
Miscanthus hydrolysate
Caldicellulosiruptor saccharolyticus
Miscanthus hydrolysate
Caldicellulosiruptor saccharolyticus
Thermotoga neapolitana
PSP-H2b
Thermotoga neapolitana
PSPa
Carrot pulp hydrolysate
Thermotoga neapolitana
Carrot pulp hydrolysate
Thermotoga neapolitana
Miscanthus hydrolysate
Thermotoga neapolitana
Miscanthus hydrolysate
Thermotoga neapolitana
Miscanthus hydrolysate
Thermotoga neapolitana
C. butyricum
SCB hemicellulose
hydrolysatef
Pretreated straw
C. butyricum CGS5
hydrolysate
Sugarcane juice
C. butyricum TISTR 1032

Reactor type

Reactor type

T C

Corn starch hydrolysate

Enterobacter aerogenes NCIMB 10102

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)

ND: Not defined.


a Untreated potato steam peels, Molar yields were based on the amount of starch in untreated PSP assuming 100% starch consumption.
b The starch in the PSP was liquefied with alpha-amylase, and then the liquefied starch was further hydrolyzed to glucose by amyloglucosidase.
c Sweet sorgham bagasse.
d Palm oil mill effluent.
e mol H2/mol total sugar.
f Sugarcane bagasse hemicellulose hydrolysate.
g g COD/L.
h g sugars/L.
i Controlled pH.

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

Based on the reviewed literature, It is evident that the current


focus is primarily on food, agriculture, and industry-related
substances to provide sustainable feedstocks. The biohydrogen technology would have a rather limited scope if the
feedstock range and sources cannot be expanded intensively
[41]. Furthermore, some wastewaters have promising potential for biohydrogen production process (Table 4), including:
oil industry wastewaters [65]; and biodiesel wastes containing
glycerol [66e68]. In addition, microalgal biomass, which is
produced by CO2 fixation through photosynthesis of microalgae, was proven to be a good sustainable feedstock [69].
Although these industrial by-products are considered promising approaches for sustainable biohydrogen production, the
yields reported from utilizing these wastewaters were still low
compared to the traditional sustainable feed stocks. Further
research in utilizing these substrates via dark fermentation,
and the adequate pretreatments methods is required.

6.

Concluding remarks

Based on the findings of this literature review, the following


remarks can be drawn:
 Attaining technical and economic efficiencies is the main
drive behind employing co-cultures of pure bacteria in
fermentative hydrogen production.
 There are three types of co-cultures of pure isolates
a) Co-cultures of strict high hydrogen producers and
facultative anaerobes, which is used to attain anaerobic conditions without the need to add expensive
reducing agents. These co-cultures yield better performance parameters, especially for complex sugars
substrates.
b) Co-cultures of cellulose-degrading anaerobes and high
hydrogen producers capable of producing hydrogen
from simpler forms of sugars. These co-cultures offer
economical and technical advantages over cellulose
degrading anaerobe solely or enzymatically hydrolysed cellulose. They produce hydrogen in two steps;
cellulose degrading anaerobe via the initial degradation followed by high hydrogen-producing anaerobe
from the degraded sugars.
c) Co-cultures of aciduric microorganisms and hydrogen
producers which reduces alkali consumption, hence
reducing or eliminating the need for a buffer to
maintain a neutral or weak acidic pH.
 The perceived advantages of thermophiles over mesophiles
appear to be substrate-dependant:
a) For simple sugars (monosaccharides and disaccharides), either mesophiles or thermophiles could
produce more hydrogen from fermenting simple

4963

sugars depending on bacterial kinetics, and the substrate type.


b) For complex sugars, thermophiles outperform mesophiles in terms of hydrogen production due to their
ability to degrade complex substrates, in addition to
the increased hydrolysis and fermentation rates
associated with the high operating temperature.
 It is essential to first determine optimal operational conditions batch studies. Continuous systems can then be operated under these optimal operational conditions to achieve
a sustainable system with same or better performance parameters than its batch counterpart.
 Biodiesel wastes, oil industry wastewaters, and microalgal
biomass have significant potential as sustainable feed
stocks in the near future.
 Certain aspects of biohydrogen production merit further
research, including:
a) Employing co-cultures of high hydrogen producers of
facultative and strict anaerobes with different optimum pH ranges.
b) Use of co-cultures of complex sugars degrading anaerobes and high hydrogen producers.
c) Enhancement of the hydrogen yields and hydrogen
production rates from dark fermentation of emerging
sustainable feedstocks.

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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