review paper on biohydrogen

8/17/2019 Review Paper on Biohydrogen

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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, CanadabGreenField Ethanol Inc., Chatham, Ontario N7M 5J4, Canadac

Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Ontario N6A 5B9, Canada

a r t i c l e i n f o

Article history:

Received 19 November 2012

Received in revised form

21 January 2013

Accepted 7 February 2013

Available online 13 March 2013

Keywords:

Biohydrogen

Fermentation

Pure cultures

Mesophilic

Thermophilic

Anaerobic

a b s t r a c t

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

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

resources. Biohydrogen has the potential to replace current hydrogen production tech-

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

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

systems. Synergies between different types of bacteria, i.e. strict and facultative, and a

comparison between mono- and co-cultures, types of feedstocks, and preferred feedstocks

for mono- and cultures are outlined.

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

reserved.

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 pro-

duction 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.: þ1 519 784 6230; fax: þ1 519 352 9559.E-mail addresses: [email protected] (O. Elsharnouby), [email protected], [email protected] (H. Hafez),

[email protected] (G. Nakhla), [email protected] (M.H. El Naggar).

Available online at www.sciencedirect.com

j o u r n a l h o m e p a g e : w w w . e l s e v i er . c o m / l o c a t e / he

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

0360-3199/$ e see front matter Copyright ª 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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Several factors influence the fermentative hydrogen pro-

duction process, which have to be optimized for enhanced

performance. Chief among these factors are: inoculum, sub-

strate, reactor type, and temperature, which seem to impact

both hydrogen yield and hydrogen production rate, albeit with

varying importance [4]. Hydrogen yield is significantly influ-

enced by the inoculum type, as the fermentation end products

are influenced by the bacterial metabolism. The inoculumused 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 cul-

ture 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. Never-

theless, only a few review papers with limitedscope are found

in the literature addressing fermentative hydrogen produc-tion 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 dis-

cussed. 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 withrespect to operational and performance parameters. Sixteen

different types of pure cultures were employed in fermenta-

tive 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 ontechnical and economic efficiencies

Ten independent studies considered in this review havecompared the effectiveness of co-cultures of pure isolates

with their mono-cultures in fermentative hydrogen produc-

tion. Table 2 summarizes the operational and performance

parameters of the mono-cultures and co-cultures studied. In

allten studies, the co-cultures achievedbetterresults than the

mono-cultures with respect to different parameters. Exam-

ining 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, co-

cultures can improve the hydrolysis of complex sugars and

plant biomass, and can provide a wider range of pH for bac-

teria to fermenthydrogen.In moststudies, both economic and

technical reasons were important considerations and inter-

dependant. 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 facultativeanaerobes. 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 Clos-

tridium 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 mixedculture of Clostridium butyricum and Escherichia coli for stable H2production without any reducing agents. They utilized

glucose as substrate, at a temperature of 37 C, and an opti-

mum 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 greaterglucose conversion efficiency resulting in a higher overall

volumetric hydrogen production. These findings indicated

that employing co-cultures was more economical by elimi-

nating the expensive reducing agent, and technically more

effective with higher hydrogen production.

Haruhiko et al. [12] examined the O2 tolerance and H2-

producing stability of the mono- and mixed cultures of C.

butyricum and Enterobacter aerogenes in batch and continuous

flowstudies 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 caseof 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, H 2production by the mixed culture of C. butyricum and E. aero-

genes 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 bio-

hydrogen 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


http://dx.doi.org/10.1016/j.ijhydene.2013.02.032http://dx.doi.org/10.1016/j.ijhydene.2013.02.032http://dx.doi.org/10.1016/j.ijhydene.2013.02.032http://dx.doi.org/10.1016/j.ijhydene.2013.02.032


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

Culture(s) Reactortype

TC

Substratetype

Substrateconcentration

(g/L)

pH Hydrogen yie(mol H2 /mol glucose

equivalent)

1-Caldicellulosiruptor saccharolyticus

Batch 70 Pretreated wheat

straw

10 7.2 3.8

Batch 70 Pretreated barelystraw 20l

7 ND

Batch 70 Glucose 20 7 3.4

Batch 70 Carrot pulp

hydrolysate

10 7 2.8



Batch 72 PSPa 10 7 3.5

Batch 72 PSP-H2b 10 7 3.4

Batch 72 GXSc 10 6.8m 3.2

Batch 72 SSBd 20l 6.8m 2.8

Batch 70 Sucrose 10 7m 2.96

Batch 72 Glu/Xyle 10 7 3.4



Batch 72 Miscanthushydrolysate

10 7 3.4

Batch 72 Miscanthus

hydrolysate

14 7 3.3

Batch 72 Miscanthus

hydrolysate

28 7 2.4

2-Thermotoga neapolitana

DSM 4359 CSABR 75 Xylose 5 7 3.36

DSM 4359 Batch 75 Xylose 5 7.5 1.31

Batch 75 Glucose 27 7 3


Batch 75 PSP-H2b 10 7 3.3

Batch 75 PSPa 10 7 3.8



Batch 75 Fructose 10 7 3.4 Batch 75 fructose 20 7 3.2

Batch 75 Glucose þ

Fructose

10 7 3.3

Batch 75 Glucose þ

Fructose

20 7 3


hydrolysate

10 7 2.7

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Table 1 e ( continued )


TC

Substratetype


(g/L)


equivalent)


hydrolysate

20 7 2.4

DSM 4359 Fed

batch-CSABR

75 Xylose 5 7.5m 2.66

Fed batch-

CSABR

75 Glucose 5 7.5m 3.2

Fed batch-

CSABR

75 Sucrose 5 7.5m 2.5

Batch 85 Glucose 2.5 7.5 3.75









Batch 80 Miscanthushydrolysate

10 7 2.9

Batch 80 Miscanthus

hydrolysate

14 7 3.2

Batch 80 Miscanthus

hydrolysate

28 7 2

Batch-

Serum

bottels

80 Glucose 5 7.5 3.85

3-Clostridium DMHC-10


4-Enterobacter Cloacae

IIT-BT08 Batch 36 Sucrose 10 6 3.014


Batch 36 Cellobiose 10 6 1.42

F.P01 Batch 36 Maltose 10 5 0.729 DM11 Batch 37 Glucose 10 6.5 3.31

Batch 37 Glucose 10 6.5 2.2


5-Clostridium beijerinckii

L9 Batch 35 Glucose 3 7.2 2.81

Fanp 3 Batch 35 Glucose 10 6.47e6.98 2.52

AM21B Batch 36 Glucose 10 6.5 2

AM21B Batch 36 Starch 10 6.5 1.8

RZF-1108 Batch 35 Glucose 9 7 1.97

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Batch 30 Glucose 2.34 6.3 ND

Batch 36 Starch 10 6.8 ND

Batch 41 Starch 10 6.8 ND

Batch 36 Starch 10 6m ND

Batch 36 Starch 10 7m ND

L9 Batch 35 Glucose 6 6.4 1.72

RZF-1108 Batch 35 Glucose 10 6.5 1.96

6-Clostridium butyricum

ATCC19398 Batch 36 Glucose 3 7.2 2.29

CGS5 Batch 37 Sucrose 17.8 5.5 1.39 W5 Batch 39 Glucose 10 6.5 0.81

EB6 Batch 37 POMEf ND 5.5 0.22

EB6 Batch 37 Glucose 10 6 0.6

Batch 37 SCB hemicellulose

hydrolysateh20k 5.5 1.73g

Batch 37 Glucose (and 200e

400 mg/l phenol))

5 6.5 1.46

CGS5 Batch 37 Xylan hydrolysate 14.2 7.5 0.84

CGS5 Batch 37 Pretreated straw

hydrolysate

9.2 7.5 0.91

CWBI1009 Batch 30 Glucose 5 5.2m 1.7

EB6 Batch 37 Glucose 15.7 5.6 2.2

TISTR 1032 Batch 37 Sugarcane juice 22.3

(sucrose)

6.5 1.33

TISTR 1032 Batch 37 Sucrose 22.3 6.5 1.34 TISTR 1032

(immobilized)

Repeated

batch

37 Sugarcane juice 22.3

(sucrose)

6.5 1.52

CGS5 Batch 37 Chlorella vulgaris ESP6

(microalgal hydrolysate)

9 5.5m ND

W5 Batch 37 Molass 100 7 1.63

TM-9A Batch 37 Glucose 10 8 3.1

TM-9A Batch 37 Arabinose 10 8 0.06

TM-9A Batch 37 Raffinose 10 8 2.7

TM-9A Batch 37 Sucrose 10 8 1.49

TM-9A Batch 37 Trehalose 10 8 1.61

TM-9A Batch 37 Xylose 10 8 0.59

TM-9A Batch 37 Cellobiose 10 8 0.94

TM-9A Batch 37 Cellulose 10 8 0.06

TM-9A Batch 37 Ribose 10 8 0.84

TM-9A Batch 37 Galactose 10 8 0.86

TM-9A Batch 37 Fractose 10 8 0.84

TM-9A Batch 37 Mannose 10 8 0.67

W5 Batch 39 Molasses 100 6.5 1.85


C. butyricum

and Escherichia

coli


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TC

Substratetype


(g/L)


equivalent)

C. butyricum and

Enterobacter

aerogenes HO-39

Repeated

batch

37 Sw eet potato

starch residue

ND 5.25m 2.7

7-Thermoanaerobactermathranii A3N

A3N Batch 70 Sucrose 10 8 2.69

A3N Batch 70 Glucose 10 8 2.64

A3N Batch 70 Xylose 5 8 2.5

A3N Batch 70 Starch 5 8 ND

8-Thermoanaerobacterium thermosaccharolyticum

W16 Batch 60 Xylose 10 6.5 2.62

PSU-2 Batch 60 Sucrose 20 6.25 2.53

W16 Batch 60 Xylose þ

Glucose

10 6.5 2.45

W16 Batch 60 Glucose 10 6.5 2.42

W16 Batch 60 Hydrolysed

corn stover

ND 7 2.24g


W16 Batch 60 Glucose 10 7 ND W16 Batch 60 Xylose 10 7 ND

W16 Batch 60 Glucose þ

Xylose þ

Arabinose

7.5,2.2,0.3 7 ND

W16 Batch 60 Hydrolysed

corn stover

ND 7 ND

PSU-2 Cont. UAi 60 Sucrose 20 5.5 1.11

PSU-2 Cont.UASB j 60 Sucrose 20 5.5 1.77

W16 Batch 60 Xylose 12.24 6.8 2.84

Thermoanaerobacterm

thermosaccharolyticum

GD17 and C. thermocellum JN4

Batch 60 Cellulose 5 4.4 1.8

9-Ethanoligenens harbinese

YUAN-3 Batch 35 Glucose 10 5 1.91

YUAN-3 CSTR 35 Glucose 10 5 1.93 B49 Batch 37 Glucose 9 7 1.83

B49 Batch 37 Glucose 12 7 1.71




B49 Batch 35 Glucose 14.5 6 2.2


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Ethanoligenens

harbinese B49

and C. acetobutylicum X9

Batch 37 Microcrystalline

cellulose

10 5 1.32

10-Klebsiella pneumoniae

ECU-15 Batch 37 Glucose 10 6 2.07

DSM2026 Batch 37 Glycerol 20 6.5 0.53

11-Pantoea agglomerans



Batch 37 Glucose 20(saline

conditions)

7.2 3.3

12-Clostridium tyrobutyricum

JM1 Batch 37 Glucose 20 6.3 3.24

JM1 CSTR 37 Glucose 5 6.7 1.81

FYa102 Batch 35 Glucosen 3 7.2 1.47

FYa102 CSTR 35 Glucoseo 12 6 1.06

FYa102 CSTR 35 Glucosep 12 6 1.42

ATCC 25755 Fed batch 37 Glucose 50 5.7 2.33

13-Clostridium acetobutylicum

M121 Batch 37 Glucose 3 7.2 1.8

X9 Batch 37 Microcrystalline

cellulose

10 5 0.59

ATCC 824 Cont.

Trickling bed reactor

30 Glucose 10 6.2 0.9

ATCC 824 Batch 36 Cassava

wastewater

5k 7 2.41

14-Escherichia coli


S3 Batch 30 Glucose 5 6.8 0.84

S6 Batch 30 Glucose 5 6.8 0.49

WDHL Batch 37 Glucose 15 6 0.3

WDHL Batch 37 Galactose 15 6 1.12

WDHL Batch 37 Lactose 15 6 1.02

WDHL Batch 37 Glucose þ

Galactose

7.5, 7.5 6 1.02

DJT135 Batch 35 Arabinose 10 6.5 1.2

DJT135 Batch 35 Fractose 10 6.5 1.27

DJT135 Batch 35 Galactose 10 6.5 0.69

DJT135 Batch 35 Glucose 10 6.5 1.51

DJT135 Batch 35 Lactose 10 6.5 0.37

DJT135 Batch 35 Maltose 10 6.5 0.2

DJT135 Batch 35 Mannitol 10 6.5 0.88

DJT135 Batch 35 Sorbitol 10 6.5 1.36

DJT135 Batch 35 Sucrose 10 6.5 0.35

DJT135 Batch 35 Terhalose 10 6.5 0.52

DJT135 Batch 35 Xylose 10 6.5 0.68



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TC

Substratetype


(g/L)


equivalent)

SH5 Fed-Batch 37 Soduim

formate

16.1 6.5 ND

15-Clostridium thermocellum

JN4 Batch 60 Cellulose 5 4.4 0.8

7072 Batch 55 Cellulose 5 7.4 1.2

7072 CSTR, 100 L Cron stalk 30 7.4 0.45

7072 CSTR, 10 L 55 Cron stalk 30 7.4 0.43

7072 Batch 55 Cron stalk 5 7.4 ND

ATCC 27405 CSTR 60 Cellulose 4 7 1.29



ATCC 27405 CSTR 60 Cellulose 1.5 7 0.98

ATCC27405 Batch 55 Delignefied

wood fibres

0.1 6.5 1.6

ATCC 27405 Batch 60 Cellulose 1 6.8 1.9

C. thermocellum

and C. thermosaccharolyticm

Batch 55 Corn stalk

waste

10 7.2 ND

C. thermocellum and

C.thermosaccharolyticm

CSTR 55 Corn stalk

waste

10 7.2 ND

C. thermocellum DSM1237

and C.thermopalmarium

DSM 5974

Batch 55 Cellulose 9 7 1.36

16- Enterobacter aerogenes

HO-39 Batch 38 Glucose 10 6.5 1

HO-39 Fed batch 37 Glucose 10 6.5 0.8

HO-39 Batch 38 Maltose 10 6.5 0.7

ATCC29007 Batch 38 Glucose 21.25 6.13 ND

Batch 38 Glucose 0.2 7 ND

Batch 37 Glycerol 20 7 0.2


E 82005 Batch 38 Glucose 10 5.8 1

IAM 1183 Batch 37 Xylose 5 6.3 2.64

IAM 1183 Batch 37 Galactose 10 6.3 2.82 IAM 1183 Batch 37 Mannose 25 6.3 0.96

IAM 1183 Batch 37 Rahamnose 5 6.3 0.48

IAM 1183 Batch 37 Arabinose 10 6.3 1.56

ATCC35029 Batch 37 Glycorel 21 ND 1.22

NCIMB 10102 Continuous

packed col.

40 Corm starch

hydrolysate

ND 5.5 2.55

NCIMB 10102 Batch 40 Starch

hydrolysate

20 6.5 1.09g


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Enterobacter aerogenes

W23 and Candida

maltosa HY-35



and C. butyricum

Batch 36 Starch ND 6.5 2


and C. butyricum

(immobiolized)

Repeated

batch

36 Starch ND 5.5 2.6

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 ¼ 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.


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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 recov-

ered immediately within 0.5 h. These results confirm that the

mixed culture can remove O2 in the reactor and recover H2production immediately.

Similarly, Yokoi et al. [13] investigated the synergies be-tween strict and facultative anaerobes. A sustained high bio-

hydrogen 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 po-

tato starch residue asa carbon source, and corn steepliquor as

a nitrogen source. The experiment was conducted in a fed

batchculture, 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 condi-

tions 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 pre-

treatment processes, such as delignification, and hydrolysis

[14]. Since pre-treatment processes are expensive, fermenta-

tive hydrogen production from cellulosic materials is desir-able. Therefore, many studies investigated employing co-

cultures of two bacterial strains: one with the capability of

hydrolysing cellulose, and the other is a high hydrogen pro-

ducer utilizing simple sugars.

For example, Yan et al. [15] investigated biohydrogen pro-

duction 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 cello-

biose and glucose produced by the cellulose degradation,

while the T. thermosaccharolyticum GD17 can utilize mono-

sugars. The experiment was conducted at pH of 4.4, and

Table 2 e Operational and performance parameters for studies employing mono and co-cultures.


TC

Substratetype


(g/L)

pH Hydrogenyield (mol H2 /mol/glucose,

hexose equivalent)

H2productionrate (L/L/d)

Ref. no.

Clostridium butyricum Batch 37 Glucose 3 6.5 2.09 0.41 [11]

Escherichia coli Batch 37 Glucose 3 6.5 1.45 0.33 [11]C. butyricum and

Escherichia coli

Batch 37 Glucose 3 6.5 1.65 0.52 [11]


and C. butyricum

Batch 37 Starch ND 6.5 2 ND [12]


and C. butyricum

(immobiolized)

Repeated

batch

37 Starch ND 5.5 2.6 ND [12]


HO-39 and C. butyricum

Repeated

batch

37 Sweet

potato

starch

residue

ND 5.25a 2.7 0.977 [13]

C. thermocellum JN4 Batch 60 Cellulose 5 4.4 0.8 0.01 [15]

Thermoanaerobacterium

thermosaccharolyticum

GD17 and C. thermocellum JN4

Batch 60 Cellulose 5 4.4 1.8 0.33 [15]

C. acetobutylicum X9 Batch 37 Microcrystall ine

cellulose

10 5 0.59 21.33 [14]

C. acetobutylicum X9 and

Ethanoligenens harbinese

Batch 37 Microcrystall ine

cellulose

10 5 1.32 11.08 [14]

Clostridium thermocellum

and C. thermosaccharolyticum

Batch 55 Corn stalk

waste

10 7.2 ND 0.34 [16]


and C. thermosaccharolyticum

CSTR 55 Corn stalk

waste

10 7.2 ND 0.44 [16]


DSM1237 and C.

thermopalmarium

DSM5974

Batch 55 Cellulose 9 7 1.36 0.42 [17]

Enterobacter aerogenes W23 Batch 35 Glucose 5 6.5 1.87 5.8 [18]

Enterobacter aerogenesW23 and Candida

maltosa HY-35

Batch 35 Glucose 5 6.5 2.19 6.27 [18]

ND: Not defined.

a Controlled pH.


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11/22

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 co-

cultures process.Aijie et al. [14] employed dark fermentation of microcrys-

talline cellulose to produce biohydrogen using mono and co-

cultures. Clostridium acetobutylicum ATCC 824(X), a high

hydrogen producer from microcrystalline cellulose was uti-

lized 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 initiationof 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 reducedsugars

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 thermo-saccharolyticum 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 experi-

ments 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 co-

cultures experiments because C. thermosaccharolyticum cul-tures 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

non-cellulolytic high hydrogen-producing bacterium. The

bacteria utilized cellulose as the sole substrate, at tempera-

ture 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 andhigh-yield biohydrogen producers. It is thought that using co-

cultures of various types of complex sugars degrading anaer-

obes in general and high hydrogen producers would give the

same results as using co-cultures of cellulose degrading an-

aerobes and high hydrogen producers.

The third type of co-cultures reported in the literature in-

volves aciduric hydrogen producing microorganisms and high

hydrogen producers. Due to the inhibitory impact of organic

acids produced during fermentation on biohydrogen pro-

ducers, near neutral or weak acidic conditions are mandatory

to attain high hydrogen yields. However, pH control is un-

economical 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 high-

hydrogen production at low pHs [18]. In this study, a batch

experiment was carried out to measure the hydrogen-

producing ability of a mixed culture of Candida maltosa HY-

35, an aciduric microorganism, which can produce hydrogen

at pH as low as 1.3, and a facultative anaerobe E. aerogenes W-

23, 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, theaverage 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 pro-

ducing aciduric microorganism and high hydrogen producing

anaerobe allowed for dispensing or reducing amount of buff-

ering 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 empha-sizes 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]. Thebacterium 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 thatthe optimum pH for hydrogen

production and growth rate for the hydrogen producer Enter-

obacter 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

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



12/22

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 thermophilesand mesophiles

Temperature is one of the most important operational pa-

rameters in fermentative H2 production. Temperature affects

the growth rate metabolic pathways of microorganism, sub-

strate 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 tempera-

ture accelerates hydrogen production, with sharply dropping

activity of hydrogen producers outside the optimum temper-ature 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. Per-

forming experiments employing mesophilic cultures is

generally less expensive. However, it was reported that ther-

mophilic and hyperthermophilic cultures seem to exhibit su-

perior 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 micro-

organisms that produce stable enzymes. It is widely acceptedthat 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 ki-

netics 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 maximumhydrogen 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 oper-

ating at mesophilic tempratures, it produced hydrogen at a

higher rate than the thermophilic one from glucose (mono-

saccharide), and with almost the same yield.

The maximum hydrogen yield reported in the literature by

a thermophile utilizing fructose (another type of mono-

saccharides) was 3.4 mol H2 /mol hexose equivalent, with a

maximum hydrogen production rate of 2.4 L/L/d, by the bac-

teria 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 H 2 /mol hexose equiva-

lent by the bacteria E. coli [27].

The maximum hydrogen yield attained by a thermophileutilizing 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 concen-

tration 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 beenrecently reported [30] that mesophilic anaerobic

bacteria cannot utilize cellulose (i.e. complex sugars) effec-

tively. The addition of exogenous cellulose enzymes isnecessary for hydrolysis of cellulose to generate H2 by meso-

philic 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 cel-

lulose, 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 con-

centration of 10 g/L was only 1.32 mol H2 /mol hexose equiv-alent [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 practicalconsiderations 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 il-

lustrates the differences in performance and operational pa-

rameters between the batches and continuous-flow systems

employing the same bacteria, and utilizing the same sub-

strates. Scrutinizing the results in Table 3, it may be concluded




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Table 3 e Operational and performance parameters of the batch and their continuous counterparts reviewed studies.

Culture Reactortype

TC Substratetype


(g/L)

pH Hydrogen yield(mol H2 /mol

glucose, hexoseequivalent)

1-Clostridium thermocellum

7072 Batch 55 Corn stalk 5 7.4 ND

7072 CSTR (10 L) 55 Corn stalk 30 7.4 0.43

7072 CSTR (100 L) 55 Corn stalk 30 7.4 0.45 2-Clostridium tyrobutyricum

FYa102 CSTR 35 Glucoseb 12, 8 6 1.06

FYa102 CSTR 35 Glucosec 12, 0.35, 1.4 6 1.42

FYa102 Batch 35 Glucosed 3, 2 7.2 1.47

JM1 CSTR 37 Glucose 5 6.7 1.81

JM1 Batch 37 Glucose 20 6.3 3.24

3-Ethanoligenens harbinese

YUAN-3 Batch 35 Glucose 10 5 1.91

YUAN-3 CSTR 35 Glucose 10 5 1.93

4-Thermotoga neapolitana

DSM 4359 Batch 75 Xylose 5 7.5 1.31

DSM 4359 CSTR 75 Xylose 5 7 3.36

5-Thermoanaerobacterium thermosaccharolyticum

PSU-2 Cont. UASBa 60 Sucrose 20 5.5 1.77

PSU-2 Batch 60 Sucrose 20 6.25 2.53

ND: Not defined.

a UASB ¼ 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.



14/22

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. ther-

mocellum 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 201.4mL/

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 opera-tional parameter between the CSTRs and batch tests. Thus, it

was presumed that it also contributed to the higher produc-

tion rate and yield, as higher substrate concentration in-

creases 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 thefermentative biohydrogen production in CSTRs using Clos-

tridium 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) re-

actors 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 perfor-mance 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 experi-

ment 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, pro-

ducing 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 me-

dium, and the better mixing conditions in the CSTRs.

Although the CSTRs were fed with a much higher concentra-

tion of glucose, the hydrogen yields were almost the same or a

bit less than those attained in the batch studies. This differ-

ence 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], thehydrogen 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 poly-

urethane 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, andhydrogen yield of

1.81 mol H2 /mol glucose was attained at a pH of 6.7, temper-

ature of 37 C, and a feed glucose concentration of 5 g/L.

Therefore, the immobilized system was an effective and sta-ble approach for continuous hydrogen production for efficient

utilization of carbon substrates with good hydrogen-

producing 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 batcheswere 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 pro-

duction with a high concentration of butyrate, and a hydrogen

yield of 3.24 mol H2 /mol glucose. The lower hydrogen yieldachieved in the continuous-flow system was attributed to the

un-optimized pH and substrate concentration.

Defeng et al. [38] studied hydrogen production of auto-

aggregative (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 condi-

tions. In the batch reactor, the optimized operational condi-

tions constituteda 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 ratereached a maximum of 19.6 L/L/d.The strain YUAN-3 was well

retained in thereactor. 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 opera-

tion of the reactor. These results indicate that a successful

continuous operation was achieved. It is evident from the




15/22

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 amaximum hydrogen yield of 1.31 mol H2 /mol hexose equiva-

lent 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 ach-

ieved 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 themaximum level. The maximum H2 yield, and hydrogen pro-

ductionrateof 2.8 molH2 /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. thermo-

saccharolyticum 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 au-thors investigated in another study [40] the fermentation of

the same concentration of sucrose under the same tempera-

ture, and a wide range of pH (4.0e9.0) by the same bacterium

and observed maximum hydrogen yield and hydrogen pro-

duction 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 contin-

uous 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 carbo-hydrates. Nevertheless, 80% of the studies reported in the

surveyed literature have investigated hydrogen production by

dark fermentation from pure sugars, such as glucose, or su-

crose as substrate. Only a few studies have focussed on sus-

tainable substrate conversion (Fig. 1). However, for real value

to the society and environment, biohydrogen should be pro-

duced from renewable feedstocks (real waste) [41]. The po-

tential feedstocks include: biomass, agricultural waste bi-

products, lignocellulosic products (wood and wood waste),

waste from food processing, aquatic plants, algae, agricul-

tural, and livestock effluents. If used under appropriate con-

trol, these resources would become the major source of

energy in the future. In this study, different types of

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 viablewhen based on renewable and low cost sources). Neverthe-

less, the majority of the reviewed studies utilized pure car-

bohydrates 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 employedin fermentative hydrogen production processes to study bac-

terial 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. Pan-

agiotopoulos et al. [50] conducted four experiments to eval-

uate the hydrogen production and the main organic acids by

Pure monosaccharides

59%

Pure polysaccharides

11%

Sustainable

feedstocks

(current+future)

20%

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

substrates in the reviewed literature.




16/22

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 con-

centrations 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 similarfermentation 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 pro-

duction 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 highersubstrate 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 hy-

drolysate of the lignocellulosic feedstock Miscanthus (obtained

from enzymatic hydrolysis) in batch tests. Control experi-

ments 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. neapo-

litana, 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 hy-

drolysis) by the same thermophilic bacteria C. saccharolyticus

and T. neapolitana. The main sugars in the hydrolysate were

glucose, fructose, and sucrose. Therefore, they initiallyinvestigated hydrogen production from different concentra-

tions of glucose, fructose, and mixtures of glucose and fruc-

tose as control experiments. in order to assess the adequate

degree of hydrolysation and optimized substrate concentra-

tion 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

adverseeffect on hydrogen production, with a severe decrease

in yield in C. saccharolyticus cultures and a decrease in pro-

ductivity with T. neapolitana.

Nan-Qi Ren et al. [52] investigated the utilization of an agro-

waste, corn stover, as a renewable lignocellulosic feedstockforthe fermentative H2 production by the moderate thermophile

T. thermosaccharolyticum W16. The corn stover was hydrolysed

by cellulase with supplementation of xylanase after delignifi-

cation with 2% NaOH, producing glucose, xylose, and arabi-

nose. To determine the fermentative behaviour of the

bacterium, a set of control experiments supplemented with

glucose, xylose, and a mixture of glucose, xylose, and arabi-

nose 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 pref-

erence for glucose over the other types of sugars. The bacte-

rium grew well on the hydrolysate and reached a similaroptical 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 inter-

ference 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: car-

bohydrate produced from sustainable resources; sufficient

concentration that fermentative conversion and energy re-covery is energetically favourable; and minimum pretreat-

ment [53].

Biomass is a viable renewable resource. It includes agri-

cultural 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 mis-

canthus), and food industry by-products are all biomass types

used as substrates in the literature [1,13,16,26,49e

52,54e

64].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.




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Table 4 e Operational and performance parameters of the studies utilizing sustainable feedstocks.

Substrate types Culture(s) Reactor type TC Substrateconcentration

(g/L)

pH Hydrogen yi(mol H2 /mol glu

hexose equiva

1-Current sustainable feedstocks:

Pretreated wheat straw Caldicellulosiruptor saccharolyticus Batch 70 10 7.2 3.8

Pretreated barely straw Caldicellulosiruptor saccharolyticus Batch 70 20h 7 ND

Carrot pulp hydrolysate Caldicellulosiruptor saccharolyticus Batch 70 10 7 2.8

PSPa Caldicellulosiruptor saccharolyticus Batch 72 10 7 3.5 PSP-H2b Caldicellulosiruptor saccharolyticus Batch 72 10 7 3.4

SSBc Caldicellulosiruptor saccharolyticus Batch 72 20h 6.8i 2.8

Miscanthus hydrolysate Caldicellulosiruptor saccharolyticus Batch 72 10 7 3.4



PSP-H2b Thermotoga neapolitana Batch 75 10 7 3.3

PSPa Thermotoga neapolitana Batch 75 10 7 3.8

Carrot pulp hydrolysate Thermotoga neapolitana Batch 75 10 7 2.7

Carrot pulp hydrolysate Thermotoga neapolitana Batch 75 20 7 2.4

Miscanthus hydrolysate Thermotoga neapolitana Batch 80 10 7 2.9

Miscanthus hydrolysate Thermotoga neapolitana Batch 80 14 7 3.2

Miscanthus hydrolysate Thermotoga neapolitana Batch 80 28 7 2

SCB hemicellulose

hydrolysatef C. butyricum Batch 37 20g 5.5 1.73e

Pretreated straw

hydrolysate

C. butyricum CGS5 Batch 37 9.2 7.5 0.91

Sugarcane juice C. butyricum TISTR 1032 Batch 37 22.3

(sucrose)

6.5 1.33

Sugarcane juice C. butyricum TISTR 1032

( immobilized)

Repeated

batch

37 22.3

(sucrose)

6.5 1.52

Molass C. butyricum W5 Batch 37 100 7 1.63

Sweet potato starch

residiue

C. butyricum and Enterobacter

aerogenes HO-39

Repeated

batch

37 ND 5.25i 2.7

Hydrolysed corn stover Thermoanaerobacterium

thermosaccharolyticum W16

Batch 60 ND 7 2.24e

Hydrolyzed corn stover Thermoanaerobacterium

thermosaccharolyticum W16

Batch 60 ND 7 ND

Cassava wastewater Clostridium acetobutylicum

ATCC 824

Batch 36 5g 7 2.41

Cron stalk Clostridium thermocellum 7072 CSTR, 100 L 55 30 7.4 0.45

Cron stalk Clostridium thermocellum 7072 CSTR, 10 L 55 30 7.4 0.43

Cron stalk Clostridium thermocellum 7072 Batch 55 5 7.4 ND

Delignefied wood fibres Clostridium thermocellum

ATCC27405

Batch 55 0.1 6.5 1.6

Corn stalk waste Clostridium thermocellum and

C. thermosaccharolyticum

Batch 55 10 7.2 ND

Corn stalk waste Clostridium thermocellum and

C. thermosaccharolyticum

CSTR 55 10 7.2 ND

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Substrate types Culture(s) Reactor type TC Substrateconcentration

(g/L)

pH Hydrogen yi(mol H2 /mol glu

hexose equiva

Corn starch hydrolysate Enterobacter aerogenes NCIMB 10102 Continuous

packed col.

40 ND 5.5 2.55

Starch hydrolysate Enterobacter aerogenes

NCIMB 10102

Batch 40 20 6.5 1.09e

2-Future sustainablefeedstocks:

POMEd C. butyricum EB6 Batch 37 ND 5.5 0.22

Glycerol Klebsiella pneumoniae DSM2026 Batch 37 20 6.5 0.53

Glycerol Enterobacter aerogenes Batch 37 20 7 0.2

Glycorel Enterobacter aerogenes

ATCC35029

Batch 37 21 ND 1.22

Chlorella vulgaris ESP6

(microalgal hydrolysate)

C. butyricum CGS5 Batch 37 9 5.5i ND

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.

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19/22

The reported hydrogen yields from biomass used as sub-

strates 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 bio-

hydrogen technology would have a rather limited scope if the

feedstock range and sources cannot be expanded intensively

[41]. Furthermore, some wastewaters have promising poten-

tial 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 micro-

algae, was proven to be a good sustainable feedstock [69].

Although these industrial by-products are considered prom-

ising approaches for sustainable biohydrogen production, theyields 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 infermentative 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 anaer-

obic conditions without the need to add expensive

reducing agents. These co-cultures yield better per-

formance 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 hydro-lysed cellulose. They produce hydrogen in two steps;

cellulose degrading anaerobe via the initial degrada-

tion 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 di-

saccharides), either mesophiles or thermophiles could

produce more hydrogen from fermenting simple

sugars depending on bacterial kinetics, and the sub-

strate type.

b) For complex sugars, thermophiles outperform meso-

philes 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 condi-tions batch studies. Continuous systems can then be oper-

ated under these optimal operational conditions to achieve

a sustainable system with same or better performance pa-

rameters 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 opti-

mum pH ranges.

b) Use of co-cultures of complex sugars degrading an-aerobes 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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