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Sequential generation of hydrogen and methane from xyloseby two-stage anaerobic fermentation

Jun Cheng a,*, Wenlu Song a,b, Ao Xia a, Huibo Su a, Junhu Zhou a, Kefa Cen a

a State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, ChinabCommittee of High Technology Development District, Jining 272000, China

a r t i c l e i n f o

Article history:

Received 9 April 2012

Received in revised form

8 June 2012

Accepted 15 June 2012

Available online 15 July 2012

Keywords:

Xylose

Hydrogen

Methane

Fermentation

Energy conversion efficiency

Methanogenesis

* Corresponding author. Tel.: þ86 571 879528E-mail address: juncheng@zju.edu.cn (J. C

0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2012.06.0

a b s t r a c t

The sequential generation of hydrogen and methane from xylose by two-stage anaerobic

fermentation was investigated for the first time in this study. The effects of substrate

concentration, bacteria domestication and nitrogen source on hydrogen yield were studied

in the first stage. The genetic characterization of the 16S rDNA was used to analyze the

flora of strains domesticated with xylose and glucose. The maximum hydrogen yield is

190.6 ml H2/g xylose when the xylose feedstock concentration is 1% (w/v), hydrogenogens

are domesticated with xylose and yeast extract is used as nitrogen source. The soluble

metabolite byproducts (SMB) from the hydrogen-producing stage were reutilized by

methanogens to produce methane in the second stage. Over 98 wt % of acetate and

butyrate in the SMB are reutilized to give a methane yield of 216.5 ml CH4/g xylose. The

sequential generation of hydrogen and methane from xylose markedly increases the

energy conversion efficiency to 67.5%.

Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction wastes and obtain a higher energy utilization rate, thus

On account of the fast depletion of fossil fuels and the serious

environmental pollution caused by them, it is necessary to

develop renewable energy resources. Hydrogen produced from

biomass is a promising energy alternative because it is clean,

renewable, having high-energy density, and is compatible with

both electrochemical and combustion processes [1,2].

Compared with other processes, bio-hydrogen production by

dark fermentation has increasingly attracted interest in recent

years because of its low cost, rapid bacteria growth, high

hydrogen-producing capacity, and abundant feedstocks [3e5].

The annual output of renewable straws in theworld reaches

4.5 billion tons [6], among which polysaccharides cellulose and

hemicellulose account for greater than 50% [7]. By using cheap

cellulosic biomass to produce clean gaseous fuels such as

hydrogen andmethane, it is possible to recycle the agricultural

89; fax: þ86 571 87951616heng).2012, Hydrogen Energy P49

promoting the hydrogen industry and the low-carbon

economy. However, the energy conversion efficiency is low by

yielding only hydrogen from fermentation of biomass. For

example, the theoretical energy conversion efficiency of only-

hydrogen production is just 19.1% by dark fermentation from

water hyacinth [8]. During hydrogen production, many soluble

metabolite byproducts (SMB) such as volatile fatty acids (VFAs)

are also produced. It is not only awaste of carbonaceous energy

resources, but also a secondary pollution to the environment.

Our previous study has reported that SMB in the residual of

hydrogen-producing solutions can be reutilized by the metha-

nogen community to further produce methane, thereby

dramatically increasing the energy conversion efficiency and

diminishing the pollution. For example, the theoretical energy

conversion efficiency is promoted to 63.1% by a combination of

hydrogen fermentation and methanogenesis from water

.

ublications, LLC. Published by Elsevier Ltd. All rights reserved.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 3 3 2 3e1 3 3 2 913324

hyacinth [8]. So, there is a broad industrial prospect to cogen-

erate hydrogen and methane from cellulosic biomass.

Direct fermentation of raw cellulosic feedstock is generally

inefficient because cellulose and hemicellulose are not

assimilable to most hydrogen-producing bacteria before

hydrolyzing to monomeric sugars [9]. Cellulose is a macro-

molecule made of glucose and some other hexose, while

hemicellulose is mostly composed of pentose (mainly xylose)

and some other monomeric sugars. In straws, the contents of

cellulose and hemicellulose are generally 30e40% and

20e30%, respectively [8,10,11]. In other words, about

333.3e444.4 g hexose and 227.2e340.8 g pentose can be

generated from 1 kg straws [8]. Fermentative hydrogen

production from glucose has beenwell-studied [12,13], andwe

have also reported both theoretical and experimental results

of cogenerating hydrogen and methane from glucose in our

former study [14]. According to the stoichiometric equation:

C6H12O6 þ 2H2O / 4CO2 þ 4H2 þ 2 CH4, 1 mol glucose can

cogenerate 4molH2 and 2mol CH4 theoretically, viz. 1 g glucose

(molecular weight ¼ 180 g/mol) can cogenerate 498 ml H2 and

249 ml CH4. The fermentative experiments show that the

maximum H2 and CH4 yields are 2.75 mol H2/mol glucose and

2.13 mol CH4/mol glucose. Some researchers have investigated

hydrogen production from xylose by fermentation. However,

the low energy conversion efficiencies through hydrogen

fermentation (�31%) [15e19]werenoteconomic,which resulted

in a bottleneck in industrial application. It is quite necessary to

enhance biogas production from xylose. A combination of

hydrogen fermentation and methanogenesis is an effective

methodto improveenergyconversionefficiency.Thesequential

generation of hydrogen andmethane from xylose by two-stage

anaerobic fermentationwas investigatedfor thefirst time inthis

paper. In the first stage, hydrogen was produced from xylose,

and the influences of substrate concentration, bacteria domes-

tication and nitrogen source (yeast extract) on hydrogen

yield were studied. In the second stage, the SMB from the

hydrogen-producing stage were reutilized by the methanogen

community to further produce methane, and consequently the

energy conversion efficiency was remarkably increased.

2. Materials and methods

2.1. Preparation and characterization of seed inocula

2.1.1. Enrichment of hydrogenogensThe anaerobic activated sludge was sampled from a methane

producing fermenter with swinemanure feedstock in Huzhou

Table 1 e Experimental design.

First stage: hydrogen production

No. Feedstock Hydrogenogenic bacteria Yeast

1 Xylose 3 g Domesticated with glucose, 10 ml e

2 Xylose 3 g Domesticated with xylose, 10 ml e

3 Xylose 3 g Domesticated with xylose, 10 ml 0

4 Xylose 6 g Domesticated with xylose, 10 ml e

5 Xylose 6 g Domesticated with xylose, 10 ml 0

marsh gas plant located in Zhejiang Province, China. The

method of pretreatment of the sludge was the same as we

used before [20], in which xylose was used as substrate to

domesticate the hydrogenogens. The preheated solid sludge

was inoculated with xylose feedstock (10 g/l and 20 g/l) to

begin the fermentation reaction. The preheated solid sludge,

which was domesticated with glucose instead of xylose [21]

was used as the control.

2.1.2. Phylogenetic characterization of hydrogenogen-enriched inoculumTotal DNAwas extracted from 0.1ml of preheated solid sludge

enriched in hydrogenogens, as described in the literature

[22]. Bacterial 16S rRNA genes were amplified with

27F (50-AGAGTTTGATCMTGGCTCAG-30) and 1390R (50-ACGGGCGGTGTGTACAA-30) primer set. Primer 519F (50-CAG-CAGCCGCGGTAATAC-30) was used for sequencing. Cloning

and sequencing of hydrogenogenic 16S rDNAwere carried out

as described previously [23].

The 16S rRNA gene sequences were compared with avail-

able database sequences via a BLAST search from GenBank to

determine their phylogenetic positions. The sequences were

grouped into operational taxonomic units (OTUs) based on

nucleic acid sequence similarity within each library. Phylo-

genetic trees were constructed by MEGA software version 3.0

with the neighbor-joining method based on nucleic acid

sequences [24].

The sequences determined in this study are available in

GenBank under HM238272 to HM238282 (16S rRNA gene

sequences of the bacteria).

2.1.3. Enrichment of methanogensThe original activated sludge was domesticated in a tradi-

tional growthmedia [21] to enrich themethanogens, and then

it was acclimated with SMB from the hydrogen-producing

stage to produce methane.

2.2. Fermentation reactor and experiments

A 350-ml glass bottle with a fermentation volume of 300 ml

was used as the fermentation reactor. Five groups of exper-

iments on cogenerating hydrogen and methane from xylose

feedstock were carried out at 37 �C, as shown in Table 1. The

pure xylose chemical (Sinopharm Chemical Reagent Co.Ltd,

China) as the feedstock was inoculated with the preheated

solid sludge enriched in hydrogenogens in the fermenter,

which was purged with N2 gas for 20 min to create the

anaerobic environment. The pH value was adjusted to 6 � 0.1

Second stage: methane production

extract No. Feedstock Methanogenicbacteria

10 Residual of 1 Domesticated with

nitrilotriacetic acid, 10 ml20 Residual of 2

.5 g 30 Residual of 3

40 Residual of 4

.5 g 50 Residual of 5

XY04 (3/58)

XY08 (7/58)

XY24 (2/58)

Clostridium leptum (AJ305238)

Clostridium sporosphaeroides (GQ243738)

XY02 (3/58)

GL05 (5/57)

Clostridium tepidiprofundum (EF197795)

Clostridium neonatale (EU869234)

XY07 (5/58)

Clostridium butyricum (AJ002592)

GL11 (29/57)

Clostridium cochlearium (M59093)

GL37 (2/57)

GL12 (2/57)

XY49 (2/58)

Clostridium ultunense (NR_026531)

XY39 (3/58)

Sphingomonas echinoides (EU730918)

XY35 (2/58)

Bacillus subtilis (EU304958)

XY57 (6/58)

GL50 (4/57)

100

100

100

77

99

95

100

100

100

100

100

86

100

94

86

72

99

99

44

33

0.02

Fig. 1 e Phylogenetic tree of hydrogenogenic 16S rRNA gene sequences (XY stands for strains domesticated with xylose, GL

stands for strains cultured with glucose).

100

150

200

12345

gen

yiel

d (m

l/g)

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 en e r g y 3 7 ( 2 0 1 2 ) 1 3 3 2 3e1 3 3 2 9 13325

in the hydrogen-producing stage [20]. The influences on

hydrogen production of substrate concentration, bacteria

domestication, and nitrogen source (yeast extract) were

investigated. Then, the terminal fermentation byproducts in

the residual hydrogen-producing solution were inoculated

with the original activated sludge enriched in methanogens

to further produce methane at the pH of 8 � 0.1. The exper-

iments were performed in triplicate for all the conditions to

give the average data. 2.3. Analysis of biogas and liquid

byproducts.

The compositions of biogas and liquid byproducts were

determined on GC as we used in our former research [14,20].

0 8 16 24 32 40 48 56 64 72 80 88 96 1040

50

Hyd

ro

Time (h)

Fig. 2 e Hydrogen yield from xylose feedstock by anaerobic

fermentation (1: xylose feedstock [ 1%, inoculum

domesticated with glucose, no nitrogen source; 2: xylose

feedstock [ 1%, inoculum domesticated with xylose, no

nitrogen source; 3: xylose feedstock [ 1%, inoculum

domesticated with xylose, 0.5 g yeast extract; 4: xylose

feedstock [ 2%, inoculum domesticated with xylose, no

nitrogen source; 5: xylose feedstock [ 2%, inoculum

domesticated with xylose, 0.5 g yeast extract.).

3. Results and discussion

3.1. Strains of hydrogenogens before and after thedomestication

After the boiling pretreatment, spore-forming hydrogenogens

harvested from anaerobic activated sludge are usually

cultured with media containing glucose as carbon source [21].

In order to promote the hydrogen yield from xylose in this

study, the hydrogenogens were acclimated with xylose for

many times to enrich the strains, which are strong hydrogen

producers with xylose. 16S rRNA profiles of gene sequences

before and after domestication with xylose is displayed in

Fig. 1, in which XY and GL stand for strains domesticated with

0 8 16 24 32 40 48 56 64 72 80 88 96 1040

20

40

60

80

100

120

140

Hyd

roge

n pr

oduc

tion

rate

(ml /

(L·h

))

Time (h)

12345

Fig. 3 e Hydrogen production rate from xylose feedstock by

anaerobic fermentation (1: xylose feedstock [ 1%,

inoculum domesticated with glucose, no nitrogen source;

2: xylose feedstock [ 1%, inoculum domesticated with

xylose, no nitrogen source; 3: xylose feedstock [ 1%,

inoculum domesticated with xylose, 0.5 g yeast extract; 4:

xylose feedstock [ 2%, inoculum domesticated with

xylose, no nitrogen source; 5: xylose feedstock [ 2%,

inoculum domesticated with xylose, 0.5 g yeast extract.).

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 3 3 2 3e1 3 3 2 913326

xylose and glucose, respectively. There are 58 operative

sequences of XY and the coverage degree is

(3 þ 7þ2 þ 3þ5 þ 2þ3 þ 2þ6)/58 ¼ 57%, while there are 57

operative sequences of GL and the coverage degree is

(5 þ 29þ2 þ 2þ4)/57 ¼ 74%. So the flora is more complex after

domestication with xylose. It can be seen from the phyloge-

netic tree that XY have nine dominant floras, while GL have

five. It is reported that in the spore-forming hydrogenogens

harvested from boiled anaerobic activated sludge, Clostridium

is the dominant flora [25e27]. This study proves that in GL,

Clostridium butyricum is the distinct dominant flora with

a proportion of about 50%. In XY, however, there are only

about 10% of C. butyricum and dominant floras of several other

strains, which aremost related to Clostridium leptum (AJ305238,

similarity of 92%), Clostridium sporosphaeroides (GQ243738,

similarity of 92%), Clostridium ultunense (NR026531, similarity

of 94%) [27], Sphingomonas echinoids (EU730918, similarity of

100%), and Bacillus subtilis (EU304958, similarity of 95%) [28].

Several Clostridium species have been shown to metabolize

Table 2 e Soluble metabolite byproducts from the H2 and CH4-

No.a Ethanol/% (V) Acetate/% (V) Propio

H2 stage CH4 stage H2 stage CH4 stage H2 stage

1/10 0.008 0.0001 0.581 0.005 0.049

2/20 0.011 0.0006 0.626 0.01 0.012

3/30 0.014 0.0002 0.928 0.009 0.027

4/40 0.018 0.0004 1.021 0.012 0.014

5/50 0.019 0.0004 1.254 0.013 0.031

a Conditions of group 1/10e5/50 are the same as Table 1.

xylose by early researches [29e31]. C. butyricum is a strong

hydrogen producer with glucose, but maybe inefficient with

xylose [32]. By enrichment with xylose, strains which

decompose xylose well become dominant in the sludge, and

are expected to give higher hydrogen production rate and

hydrogen yield.

3.2. Hydrogen production from xylose feedstock

The yields (ml/g) and production rates (ml/(L$h)) of hydrogen

fermentation from xylose are shown in Fig. 2 and Fig. 3,

respectively. It is found that when the concentration of xylose

is 1% (w/v), fermentation of strains domesticated with glucose

give a longer lag-phase with a hydrogen yield of

114.1 � 17.2 ml/g (group 1). When strains domesticated with

xylose are used as hydrogenogens and no nitrogen source is

added, the maximum hydrogen production rate is promoted

from 35.8 � 5.5 ml/(L$h) to 48.4 � 1.4 ml/(L$h) and peak time is

advanced from 64 h to 40 h, while the hydrogen yield is

increased from 114.1 � 17.2 ml/g to 165.5 � 7.8 ml/g (group 2).

Themain reason is that the process of domestication changed

the dominant strains in the sludge. Before domestication with

xylose, some genuses of bacteria in the hydrogenogens are

unable to grow with xylose as a sole carbon source due to the

lack of a xylose uptake system [33], resulting in a lower yield of

hydrogen. After the domestication, more hydrogen-producing

bacteria, which could contribute to the degradation of xylose,

reproduce themselves to change the microflora. The results

indicate that the type of feeding carbon substrate is important

in determining the evolution of characteristic hydrogen-

producing communities. The domestication enhances the

microflora’s ability to degrade organics.

The hydrogen production profile of hydrogenogens was

found maximum in the exponential phase of the growth [34],

and most xylose utilizers require yeast extract for growth on

pentose media [35]. In order to get a maximum hydrogen yield

with the bacteria domesticated with xylose, 0.5 g yeast extract

was added into the initial fermentation medium (lack of

nitrogen source) as nitrogen source. It is shown by experiments

that when yeast extract is added as nitrogen source with

bacteria domesticated with xylose, the maximum hydrogen

production rate is promoted from 48.4 � 1.4 ml/(L$h) to

59.6� 6.6 ml/(L$h) and peak time is advanced from 40 h to 24 h,

while the hydrogen yield is further increased to 190.6 � 1.4 ml/

g, which is equivalent to 1.28 � 0.01 mol/mol-xylose (group 3).

The main reason is that yeast extract provides sufficient

producing stages.

nate/% (V) Butyrate/% (V) Valerate/% (V)

CH4 stage H2 stage CH4 stage H2 stage CH4 stage

0.005 0.391 0.001 0.028 0.002

0.004 0.417 0.001 0.014 0.001

0.006 0.411 0.001 0.017 0.001

0.004 0.535 0.001 0.006 0

0.005 0.764 0.002 0.015 0.002

Table 3 e Energy conversion efficiencies in hydrogen and methane sequential generation from xylose.

No.a Hydrogen yield(ml/g)

Methane yield(ml/g)

Efficiency inhydrogen-onlyproduction (%)

Efficiency in hydrogenand methane production (%)

1/10 114.1 � 17.2 173.5 � 3.8 8.5 � 1.3 51.2 � 2.2

2/20 165.5 � 7.8 188.9 � 3.2 12.3 � 0.6 58.8 � 1.4

3/30 190.6 � 1.4 216.5 � 5.7 14.2 � 0.1 67.5 � 1.5

4/40 124.8 � 0.2 172.6 � 9.9 9.3 � 0.01 51.8 � 2.5

5/50 177.8 � 2.2 186.7 � 15.7 13.2 � 0.2 59.2 � 4.0

a Conditions of group 1/10e5/50 are the same as Table 1.

0 1 2 3 4 5 6 7 8 9 10 11 120

50

100

150

200

250

Met

hane

yie

ld (m

l/g)

Time (d)

1' 2' 3' 4' 5'

Fig. 4 e Methane yield from residual hydrogen-producing

solutions of xylose (10: residual of condition 1, xylose

feedstock [ 1%, inoculum domesticated with glucose, no

nitrogen source; 20: residual of condition 2, xylose

feedstock [ 1%, inoculum domesticated with xylose, no

nitrogen source; 30: residual of condition 3, xylose

feedstock [ 1%, inoculum domesticated with xylose, 0.5 g

yeast extract; 40: residual of condition 4, xylose

feedstock [ 2%, inoculum domesticated with xylose, no

nitrogen source; 50: residual of condition 5, xylose

feedstock [ 2%, inoculum domesticated with xylose, 0.5 g

yeast extract.).

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 en e r g y 3 7 ( 2 0 1 2 ) 1 3 3 2 3e1 3 3 2 9 13327

nutrition for the growth of the hydrogenogens during

fermentation, resulting in the improvement of hydrogen

production. On one hand, hydrogenogens keep growing with

yeast extract and maintain high metabolic activities; on the

other hand, the growth of hydrogenogens ensures high quan-

tity of hydrogenogenic cells, which is favorable for hydrogen

production.

In this research, the influence of xylose concentration on

hydrogen production was also investigated. When the xylose

concentration (w/v) is increased from 1% to 2%, the maximum

hydrogenproduction rate ispromoted from48.4�1.4ml/(L$h) to

60.0 � 7.0 ml/(L$h), but the hydrogen yield is decreased from

165.5� 7.8 ml/g to 124.8� 0.2 ml/g without yeast extract (group

4); whilewith yeast extract themaximumhydrogen production

rate is greatly promoted from 59.6 � 6.6 ml/(L$h) to 135.3 � 5.5

ml/(L$h),but thehydrogenyield isdecreasedfrom190.6�1.4ml/

g to 177.8 � 2.2 ml/g (group 5). This result may be due to the

excessive xylose concentration resulting in an accumulation of

byproductssuchasVFAsandanincrease inthepartialhydrogen

pressure, in which case the microflora would inhibit hydrogen

production [36]. Thus, a xylose concentration of 1% (w/v) gives

the maximum hydrogen yield, but a higher concentration may

bemore promising to give a higher hydrogen rate.

3.3. Methane production from residual hydrogen-producing solutions

The SMB of hydrogen production from xylose (such as

ethanol, acetate, propionate, butyrate, and valerate) can be

reutilized by the methanogen community to further produce

methane [20]. As shown in Table 2, SMB in residual solutions

after the first hydrogen-producing stage mainly include

acetate and butyrate (more than 90% of the total SMB), and

their concentrations are proportional to the cumulative

hydrogen production (ml) in the first stage. The results imply

that the hydrogen production is through acetate- and

butyrate-type fermentation. In the second stage, metha-

nogens can directly convert acetate to methane through ace-

toclastic pathway. Methanogens split acetate into a methyl

group and an enzyme-bound CO, with the CO subsequently

oxidized to provide electrons for the reduction of the methyl

group to methane [37]. Butyrate and other byproducts can be

converted into acetate by the mixed bacteria, and then

utilized by the methanogens to produce methane. The results

show that after the second stage, over 98 wt % of acetate and

butyrate in residual SMB of the first stage are reutilized by the

methanogen community to produce methane.

In the second stage, the cumulative methane production

(ml) is proportional to the content of SMB produced in the first

stage. The methane yields are shown in Fig. 4. The maximum

methane yield of 216.5 � 5.7 ml/g is obtained when the xylose

feedstock concentration is 1% (w/v), hydrogenogens are

domesticated with xylose, and yeast extract is used as

nitrogen source in the first stage.

3.4. Energy conversion efficiency

The energy conversion efficiency of cogenerating hydrogen and

methane from xylose is calculated based on the heating values

of hydrogen, methane, and raw xylose. The heating value of

xylose is 14.525 kJ/g, which is measured by combustion calo-

rimetry. The heating values of hydrogen and methane are

242 kJ/mol and 801 kJ/mol, respectively. The maximum energy

conversion efficiency of 67.5% (hydrogen yield: 190.6 ml/g;

methane yield: 216.5 ml/g) was obtained (Table 3). A

Table 4 e A comparison of energy conversion efficiencies from xylose in the present study and those reported in relevantstudies.

Microorganism Fermentation type Fermentationtemperature (�C)

Energy conversionefficiency (%)

References

Granulated sludge UASB, hydrogen fermentation 37 8.9 [15]

sewage sludge CSTR, hydrogen fermentation 50 15.5 [16]

Clostridium sp. strain No. 2 Batch, hydrogen fermentation 35 22.9 [17]

Sewage sludge Batch, hydrogen fermentation 35 25.0 [18]

Thermotoga neapolitana

strain DSM 4359

Batch, hydrogen fermentation 75 31.1 [19]

Activated sludge Batch, hydrogen fermentation

and methanogenesis

37 67.5 In the present

study

CSTR, continuously stirred tank reactor.

UASB, upflow anaerobic sludge blanket.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 3 3 2 3e1 3 3 2 913328

comparison of energy conversion efficiencies fromxylose in the

present study and those reported in relevant studies is shown

in Table 4. Energy conversion efficiencies ranged from 8.9% to

31.1% through hydrogen fermentation in the literature [15e19].

In the present study, the energy conversion efficiency of 67.5%

through sequential generation of hydrogen and methane was

much higher than those obtained in previous literature.

Up to now, no research about cogenerating hydrogen and

methane from xylose has been reported. Xie et al. obtained

a hydrogen yield of 342.2ml/g and amethane yield of 265.1ml/

g from glucose (energy conversion efficiency: 82.0%) [14].

Cheng et al. obtained a hydrogen yield of 51.7 ml/g and

a methane yield of 143.4 ml/g TVS from water hyacinth

(energy conversion efficiency: 33.2%) [8]. Cheng et al. obtained

a hydrogen yield of 58.0ml/g and amethane yield of 200.9ml/g

TVS from corn straw (energy conversion efficiency: 67.1%) [38].

The energy conversion efficiency of xylose through hydrogen

andmethane cogeneration was lower than that of glucose but

higher than that of cellulosic biomass. This result may be

attributed to two reasons: (1) hexose is more favorable for

bacterial utilization than pentose; (2) some hydrolysis

byproducts from cellulosic biomass inhibit the hydrogen and

methane fermentation.

4. Conclusions

This study demonstrates the feasibility of cogenerating

hydrogenandmethane fromxylose (pentose),which isa typical

hydrolysis product of hemicellulose in biomass, by two-stage

anerobic fermentation. In the first hydrogen production stage,

the maximum hydrogen yield is 190.6 ml H2/g xylose. The SMB

from the hydrogen-producing stage were reutilized by

methanogens to produce methane in the second stage. The

maximum methane yield is 216.5 ml CH4/g xylose. The

sequential generation of hydrogen and methane from xylose

markedly increases the energy conversion efficiency to 67.5%.

Acknowledgments

This study is supportedbyNationalNatural Science Foundation

of China (51176163), International Sci. & Tech. Cooperation

Program of China (2010DFA72730, 2012DFG61770), National

High Technology R&D Program of China (2012AA050101),

National Key Technology R&D Program of China

(2011BAD14B02), Specialized Research Fund for the Doctoral

Program of Higher Education (20110101110021), Program for

New Century Excellent Talents in University (NCET-11-0446),

Key Natural Science Foundation of Zhejiang Province

(Z1090532), Major Sci. & Tech. Special Project of Zhejiang Prov-

ince (2008C13023-3), Fundamental Research Funds for the

Central Universities (2011XZZX007), and Program of Intro-

ducing Talents of Discipline to University (B08026).

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