optimization of biohydrogen production from soybean straw using anaerobic mixed bacteria

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Optimization of biohydrogen production from soybean strawusing anaerobic mixed bacteria

Hongliang Han, Liling Wei, Biqian Liu, Haijun Yang*, Jianquan Shen*

Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of New Materials, Institute of Chemistry, Chinese Academy

of Sciences, Zhongguancun North First Street 2, Beijing 100190, PR China

a r t i c l e i n f o

Article history:

Received 29 December 2011

Received in revised form

7 March 2012

Accepted 11 March 2012

Available online 13 April 2012

Keywords:

Soybean straw

Pretreatment

Anaerobic mixed bacteria

Biohydrogen

Array testing strategy

* Corresponding authors. Tel.: þ86 10 626209E-mail address: [email protected] (J. Sh

0360-3199/$ e see front matter Copyright ªdoi:10.1016/j.ijhydene.2012.03.073

a b s t r a c t

In this study, batch experiments were carried out to produce biohydrogen from soybean

straw after pretreatment using anaerobic mixed bacteria at 35 �C. The effects of acid (HCl),

alkali (NaOH), hydrogen peroxide, acid peroxide and alkaline peroxide pretreatments on the

cellulosic biomass of soybean straw were studied. Furthermore, the effects of different

pretreatment on hydrogen production, together with their corresponding degradation effi-

ciencies for the total reducing sugar (TRS) and metabolites were investigated. The results

showed that 4%HClwas the best choice for the pretreatment of soybean straw. Array testing

strategy (L9(34)) was applied to design the experiments and analyze the effects of inoculum,

initial pH and ferrous iron and nickel concentrations on fermentative hydrogen production.

The results showed that hydrogen yield was significantly enhanced by optimizing envi-

ronmental factors. A maximum cumulative hydrogen yield of 60.2 mL/g-dry soybean straw

was achieved, which was approximately 11-fold greater than that in raw soybean straw.

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

reserved.

1. Introduction Recently, some studies on biohydrogen production from

Hydrogen is considered as a clean energy carrier in the future

because of its high energy-yield (122 kJ/g) and producing only

water upon combustion [1]. Conventional physicochemical

methods for hydrogen production are usually costly and

energy intensive [2,3]. Biohydrogen production has been given

considerable attention due to environmentally friendly and

energy saving process [4,5]. Extensive scientific attention has

been devoted to the conversion of sugar or soluble starch into

hydrogen gas by dark fermentation [6e8]. However, a low cost

of feedstock is a very important factor in establishing a cost-

effective hydrogen-producing technology. Renewable energy

sources, such as agricultural waste containing cellulosic

biomass, constitute an abundant, inexpensive and reliable

raw material for biohydrogen production and offer consider-

able advantages [9,10].

03; fax: þ86 10 62559373.en).2012, Hydrogen Energy P

agricultural waste such as wheat straws or corn stalks have

been carried out [11e13]. Generally, the direction conversion of

natural cellulosic biomass into hydrogen gas by hydrogen-

producing bacteria is very difficult due to the complicated

polymer structure of hemicellulose and cellulose [14,15]. Herein,

various pretreatment methods of cellulosic biomass have been

extensively described in order to enhance hydrogen yield. The

main pretreatments are based on mechanical, physical, biolog-

ical and chemical methods [16,17]. Among these pretreatment

technologies, chemical methods are considered to be efficient,

suchasacidic, alkalineandoxidativepretreatments. Inaddition,

several researches have shown that the operating condition

could improve the hydrogen yield of the fermentative biological

processes, such as initial pH and ferrous concentration [10,17].

Soybean straw is biodegradable cellulose resource, but it

has not been utilized for a long time. Most of the soybean

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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 3 2 0 0e1 3 2 0 8 13201

straw was directly combusted, resulting in resources waste

and air pollution [18]. In light of the above background, we

explored the feasibility of converting soybean straw into

hydrogen with acid, alkaline and hydrogen peroxide pretreat-

ments using anaerobic mixed bacteria. The objective of this

study was to investigate the feasibility of producing hydrogen

from soybean straw. For this purpose, the array testing

strategy (L9(34)) was applied to design the experiments and

analyze the effects of inoculum, initial pH and the concentra-

tion of ferrous iron and nickel on fermentative hydrogen

production in batch cultivation.

2. Materials and methods

2.1. Materials

Soybean straw used in this study was collected in autumn

from a suburb of Cangzhou city Hebei province, China. The

straw dried in sunlight, and was then comminuted to more

than 20-mesh using a comminutor and dried again in a ther-

moelectrical oven for 3 h at 120 �C.

2.2. Inoculum and medium

The hydrogen-producing mixed cultures used here were

enriched from cracked cereals and were dominated by Clos-

tridium butyricum following identification of the bacteria in

culture [19]. The cultures were acclimated in a completely

stirred tank reactor in a chemostat for approximately one

month. The reactor was operated at 35 �C, an 8 h hydraulic

retention time, and stirred by gas circulation. The bacteria

were directly used as inoculum without any pretreatment.

One liter of culture medium used to ferment contained

NH4HCO3, 3770mg; K2HPO4, 125mg; Na2CO3, 2000mg; CuSO4 $

5H2O, 5mg; MgCl2 $ 6H2O, 100mg; MnSO4 $ 4H2O, 15mg; FeSO4

$ 7H2O, 25 mg; CoCl2 $ 6H2O, 0.125 mg.

2.3. Pretreatment

2.3.1. Acid and alkaline pretreatment1.0 g of soybean straw was mixed with 20 mL of dilute HCl (or

NaOH) aqueous solution with different concentrations, which

were 0.5%, 1%, 2%, 4%, and 8%, respectively, and boiled for

30 min in serum vials. The mixture was then neutralized to

pH 7.0 by addition of dilute NaOH (or HCl) aqueous solution

at different concentrations (0.5%, 1%, 2%, 4% and 8% (w/v),

respectively).

2.3.2. Hydrogen peroxide pretreatment1.0 g of soybean straw was mixed with 20 mL of dilute H2O2

aqueous solution with different concentrations, which were

0.5%, 1%, 2%, 4%, 8%, 16% and 30%, respectively, and boiled for

30min in serum vials. Themixturewas then neutralized to pH

7.0 by addition of dilute NaOH (or HCl) aqueous solution.

2.3.3. The co-pretreatment of acid (or alkaline) and 16% (w/v)hydrogen peroxide1.0 g of soybean straw was mixed with 20 mL of dilute HCl (or

NaOH) and hydrogen peroxide (16%w/v) aqueous solution and

boiled for 30 min in serum vials. The concentrations of HCl (or

NaOH) were 0.5%, 1%, 2%, 4% and 8% (w/v), respectively. The

mixture was then neutralized to pH 7.0 by addition of dilute

NaOH (or HCl) aqueous solution.

2.4. Experimental procedures

Batch experimentswere carried out in 120mL serumvialswith

a working volume of 80 mL. The fermentation broth compo-

sition consisted of 40 mL pretreated soybean straw solution,

20 mL inoculum, 10 mL distilled water, and 10 mL nutrient

solution. 1 L of the nutrient medium contained NH4HCO3,

30,160mg; K2HPO4, 1000mg; Na2CO3, 16,000mg; CuSO4 $ 5H2O,

40 mg; MgCl2 $ 6H2O, 800 mg; MnSO4 $ 4H2O, 120 mg; FeSO4 $

7H2O, 200mg; CoCl2 $ 6H2O, 1.0 mg. The air was removed from

the solution and the headspace by argon gas for 3 min before

the vials were cappedwith rubber septumstoppers and placed

in a reciprocal shaker (reciprocation: 5 cm � 120 strokes per

min). The batch experiments were performed at 35 �C and

initial pH 7.0 in the dark. Each experimental condition was

carried out in triplicate. The experimental results noted were

the averages (�standard deviation) of the values obtained in

independent experiments conducted in triplicate. All chem-

icals used in the experiments were of AR grade.

2.5. Analytical methods

Hydrogen content, ethanol and volatile fatty acids (VFAs, C2-

C6) analytical procedures were the same as our previous

work [4]. The concentration of the total reducing sugar was

determined by the phenol-sulfuric acid method using glucose

as a standard [20]. The contents of hemicellulose, cellulose

and lignin were determined according to Van Soest’s method

[21]. All gas production data reported were standardized to

standard temperature (0 �C) and pressure (760 mm Hg).

2.6. Model analysis

Themodeling of hydrogen production in the batch experiments

is based on the following modified Gompertz equation [22]:

H ¼ Pexp

�� exp

�RmeP

ðl� tÞ þ 1

��

where H is the cumulative hydrogen production (mL), P is

hydrogen production potential (mL), Rm is the maximum

hydrogen production rate (mL/h), e is 2.71828, l is the lag-

phase time (h), and t is the incubation time (h). The kinetic

parameters (P, Rm and l) were estimated via Origin 7.5.

3. Results and discussion

3.1. Effect of HCl pretreatment on hydrogen production

Lignocellulosic biomass is mainly composed of hemicellulose,

cellulose and lignin. Previous researches have shown that

pretreatment could affect the contents of hemicellulose,

cellulose and lignin [12,23,24]. The content decrease mainly

came from the degradation of components and other soluble

extractives. Acid catalysed hydrolysis was used to cleave the



0 10 20 30 40 50 60 70

0

10

20

30

40

50

Cu

mu

la

tiv

e h

yd

ro

ge

n (m

L)

Time (hour)

UP

0.5%

1%

2%

4%

8%

Fig. 2 e Cumulative hydrogen volumes from 1.0 g of dry

soybean straw pretreated by different HCl concentrations

versus corresponding fermentation time. The operation

was at initial pH 7.0 and 35 �C. UP means unpretreatment.


intrachain linkages in hemicellulose and cellulose chains

contained in biomass to produce soluble sugars and signifi-

cant quantities of by-products, such as acetate, phenol, and

furan compounds [25]. The effects of HCl concentrations on

the cellulosic biomass, the compositions and contents in raw

soybean straw and pretreated samples were analyzed. As

shown in Fig. 1, it can be seen the content of hemicellulose

(14.45e2.80%) and lignin (23.31e13.31%) apparently decreased

with the increase of HCl concentration. But the content of

cellulose had small changes (39.93e33.49%). Therefore, it can

be concluded that the major reaction during acidic pretreat-

ment was the hydrolysis of hemicellulose and lignin, which

was accompanied, to some extent, by the hydrolysis of

cellulose. This showed that diluted HCl had an excellent effect

on the degradation of hemicellulose and lignin. The content of

total reducing sugar (TRS) was only 3.72% in raw soybean

straw. After HCl pretreatment, the TRS significantly increased,

and continued to increase (14.95e46.83%) with the increase of

HCl concentration (0.5e8%), indicating that HCl pretreatment

increased the amount of TRS.

The effect of HCl concentration on the cumulative

hydrogen volume is presented in Fig. 2. 1.0 g of dry soybean

straw pretreated with HCl pretreatment which was used as

the substrate to produce hydrogen at an initial pH of 7.0. It can

be seen from Fig. 2 that the cumulative hydrogen volume from

raw soybean straw was only 5.46 mL but that the cumulative

hydrogen volumes continued to increase (17.26e47.65 mL)

when HCl concentration increased (0.5e4%). The maximum

hydrogen volume of 47.65 mL was observed at 4% HCl, which

was 8.73-fold higher than that from the raw substrate. When

HCl concentration increased to 8%, the hydrogen volume

dramatically decreased to 40.77 mL. It is possible because HCl

pretreatment produced some inhibitors such as furfural that

inhibited the activity of hydrogen-producing bacteria [26].

Gas product analyses indicated that only H2 and CO2 were

present in the biogas, without any detectable CH4 during the

course of hydrogen production, suggesting that there were no

UP 0.5%HCl 1%HCl 2%HCl 4%HCl 8%HCl

0

5

10

15

20

25

30

35

40

45

50

Co

nten

t (%

d

ry

we

ig

h)

HCl concentration (w/v)

Hemicellulose

Cellulose

Lignin

Total reducing sugar

Fig. 1 e Contents of hemicellulose, cellulose, lignin and

total reducing sugar in soybean straw without

pretreatment and pretreated by different HCl

concentrations (from 0.5% to 8%, w/v) before and after

pretreatment. UP means unpretreatment.

methanogens in the anaerobic mixed bacteria used here. The

effects of HCl concentration on the sugar degradation effi-

ciency, hydrogen yields, and liquid metabolites are shown in

Table 1. The sugar degradation efficiencies exceeded 93% and

the changes were small with increasing HCl concentration,

indicating that most of the sugar was consumed by the

bacteria. The hydrogen yield in biogas from raw soybean

straw was only 5.46 mL/g-substrate. The increase of hydrogen

yield was found after HCl pretreatment, and a maximum

hydrogen yield of 47.65 mL/g-substrate was achieved at 4%

HCl. The liquid product analyses showed that the metabolites

found after fermentation were ethanol, acetic acid, propionic

acid, n-butyric acid, iso-butytic acid, iso-valeric acid and n-

valeric acid. It should be noted that high yields of ethanol and

propionic acid were obtained from the raw soybean straw,

however, low yields of acetic acid, n-butyric acid, iso-butytic

acid, iso-valeric acid and n-valeric acid were also found. It

corresponded to the lower hydrogen yield for the raw soybean

straw. The reason for this may be due to the varied metabo-

lism, in that some hemicellulose, cellulose and lignin may

have been consumed in the production of ethanol and VFAs,

however, only sugar was metabolized to produce hydrogen by

anaerobic mixed bacteria [17]. When the HCl concentration

increased from 0.5% to 8%, the yield of ethanol decreased

gradually (38.45e20.71 mg/g-substrate), then increased to

35.05 mg/g-substrate. But the yields of acetic acid, propionic

acid, n-butyric acid, iso-butytic acid, iso-valeric acid and n-

valeric acid showed an irregular variation. The mechanism of

hydrogen production from glucose and xylose by bacterial

fermentation has been reported previously [27,28]. The

production of acetic and n-butyric acid favors the production

of hydrogen, and in contrast, the production of propionic acid

results in less hydrogen. As shown in Table 1, the maximum

yield of acetic and n-butyric acid appeared at 4% HCl, which

was in agreement with the data in Fig. 2 and Table 1. This also

showed that 4% HCl was the best choice for the pretreatment

of soybean straw.



Table 1 e Effects of HCl, NaOH and H2O2 concentrations on the degradation efficiency of total reducing sugar, hydrogenyield and yields of ethanol and volatile fatty acids.

w/v DESb HYc Ethanol(mg/L)

AceticAcid (mg/L)

PropionicAcid (mg/L)

iso-ButyricAcid (mg/L)

n-ButyricAcid (mg/L)

iso-ValericAcid (mg/L)

n-ValericAcid (mg/L)

UPa 93.01% 5.46 82.49 364.30 617.03 417.93 665.27 432.58 648.76

HCl 0.5% 97.75% 17.26 38.45 403.56 545.21 578.16 719.96 550.68 711.26

1% 98.33% 25.01 32.94 460.14 616.09 519.79 816.29 577.36 804.86

2% 98.71% 33.77 24.36 396.47 534.13 584.85 748.78 551.43 735.69

4% 98.87% 47.65 20.71 475.93 590.91 615.27 852.29 594.16 737.78

8% 98.10% 40.77 35.05 415.48 590.71 611.27 842.89 575.36 651.05

NaOH 0.5% 89.12% 10.47 83.57 548.49 588.94 365.96 738.83 382.53 695.69

1% 90.07% 9.98 89.67 522.09 598.65 505.14 694.14 491.04 731.67

2% 90.11% 7.63 31.37 460.29 551.71 487.75 684.3 468.14 685.41

4% 92.14% 6.60 32.38 418.95 501.01 517.81 646.67 475.80 631.41

8% 86.85% 3.14 158.24 483.77 594.25 442.68 734.87 457.37 722.22

H2O2 0.5% 65.83% 1.76 112.36 152.14 173.68 131.04 269.72 150.34 213.92

1% 64.80% 2.85 61.43 203.32 225.85 181.48 309.32 194.57 280.59

2% 60.32% 4.48 25.9 220.18 273.68 252.37 379.67 242.48 268.11

4% 55.56% 6.20 23.13 251.40 291.64 240.94 387.07 256.00 356.45

8% 54.17% 13.99 21.92 275.90 314.51 235.32 416.45 257.25 388.87

16% 47.15% 23.00 25.30 282.12 355.17 363.14 476.05 328.69 383.00

30% 45.66% 18.04 23.49 214.75 268.92 293.49 367.32 266.98 274.93

a UP means unpretreatment.

b DES means degradation efficiency of total reducing sugar.

c HY means hydrogen yield (mL H2/g-Substrate).


3.2. Effect of NaOH pretreatment on hydrogenproduction

NaOH is a strong agent for extracting both hemicelluloses and

lignin, and the pretreatment significantly cleaved the a-ether

bonds between lignin and hemicelluloses from the cell walls

[29]. The effects of NaOH concentrations on components of

cellulosic biomass are shown in Fig. 3. It is seen that the

contents of hemicellulose, cellulose and lignin had different

rate reductions, and the content of TRS clearly increased with

increasing NaOH concentrations. But, in comparison, the

content of TRS pretreated with the same concentration of HCl

and NaOH, the content of TRS with HCl pretreatment was

UP 0.5% NaOH 1% NaOH 2% NaOH 4% NaOH 8% NaOH

0

5

10

15

20

25

30

35

40

45

Co

nte

nt (%

d

ry

w

eig

h)

NaOH concentration (w/v)

Hemicellulose Cellulose

Lignin Total reducing sugar


total reducing sugar in soybean straw pretreated by

different NaOH concentrations. UP means unpretreatment.

always higher than that with NaOH pretreatment. These

results are consistent with previous studies [12,17,30], indi-

cating that HCl pretreatment was superior to NaOH pretreat-

ment. The effect of NaOH concentration on the cumulative

hydrogen volume is presented in Fig. 4. The cumulative

hydrogen volume gradually decreased as NaOH concentration

increases, in addition, the cumulative hydrogen volume of

3.14 mL for 8% NaOH pretreatment was lower than that from

the raw soybean straw (5.46 mL). The reasons might be that

the process of pretreatment produced some biological inhib-

itors, which inhibited the activities of hydrogen-producing

0 10 20 30 40 50

0

2

4

6

8

10

Cu

mu

la

tiv

e h

yd

ro

ge

n (m

L)

Time (hour)

UP 0.5% 1% 2% 4% 8%


soybean straw pretreated by different NaOH

concentrations versus corresponding fermentation time.

The operation was at initial pH 7.0 and 35 �C. UP means

unpretreatment.



0 5 10 15 20 25 30 35 40 45

0

3

6

9

12

15

18

21

24

Cu

mu

la

tiv

e h

yd

ro

ge

n (m

L)

Time (hour)

0.5%

1%

2%

4%

8%

16%

30%


soybean straw pretreated by different H2O2 concentrations

versus corresponding fermentation time. The operation

was at initial pH 7.0 and 35 �C.


bacteria. Gossett reported that the solubilized lignin compo-

nents often have an inhibitory effect for microbes [31]. High

concentrations of alkaline result in higher yields of extraction,

indicating a disruption of stronger linkages, such as ferulic

acid bridges between hemicelluloses and lignin [32]. The

maximum cumulative hydrogen volume of 10.47 mL was

achieved at 0.5% NaOH and was much smaller than HCl

pretreatment. Table 1 shows that the sugar degradation effi-

ciency decreased to about 90% and the hydrogen yield slowly

decreased with the increase of NaOH concentration. The

distribution of metabolites had no distinguished regulation,

but maximum acetic and n-butyric acid appeared at a NaOH

concentration of 0.5%, which was in agreement with the

maximum hydrogen yield.

3.3. Effect of H2O2 pretreatment on hydrogen production

Hydrogen peroxide has been identified as promising chem-

icals for delignification of agricultural residues [33]. The

decomposition of hydrogen peroxide generates active radi-

cals, such as hydroxyl radicals (HO$) and superoxide anion

radicals (O2$), participating in the degradation reaction of

lignin and hemicelluloses [34,35].

The effect of H2O2 concentration on components of cellu-

losic biomass is shown in Fig. 5. It is seen that the contents of

hemicellulose, cellulose and lignin had different rate reduc-

tions, and the content of TRS increased but the contents of

TRS had low levels. It suggested that H2O2 could degrade-

lignocellulose efficiently, but could not convert into sugar in

full. Fig. 6 shows that the cumulative hydrogen volumes

gradually increased as H2O2 concentration rose from 0.5% to

16%, and then decreased to 18.04 mL for 30% H2O2. A

maximum cumulative hydrogen volume of 23.00 mL was

achieved at 16% H2O2. Dissolution and oxidation of lignin by

peroxide was used to produce low molecular weight aromatic

products [33], which inhibited hydrogen production of

hydrogen-producing bacteria. As shown in Table 1, the

content of VFAs gradually increased with the growing H2O2

0.5% 1% 2% 4% 8% 16% 30%

0

5

10

15

20

25

30

35

40

45

Co

nte

nt (

% d

ry

w

eig

h)

H2O

2concentration (w/v)





different H2O2 concentrations.

concentration, but it was lower than HCl and NaOH pretreat-

ment. The maximum acetic and n-butyric acid appeared at

16% H2O2, which was in agreement with the maximum of

hydrogen yield.

3.4. Effect of mixed acid or alkaline with hydrogenperoxide co-pretreatment on hydrogen production

Gould stated that the delignification reaction is strongly

dependent on pH [36,37]. Under acidic conditions, protonation

of hydrogen peroxide forms a hydroxonium ion, which is

a strong electrophilic agent and attacks the lignin’s aromatic

nuclei.

H2O2 þHþ4HOþ þH2O

In the alkaline peroxide treatment process, hydroperoxide

ion (HOO�) forms in alkaline media, which is the principal

active species in hydrogen peroxide delignificating systems.

Fig. 7 shows the effect of acid and alkaline peroxide treatment

on the components of cellulosic biomass. It is seen that the

contents of hemicellulose, cellulose and lignin had different

rate reduction, and the content of TRS clearly increased. But

the content of TRS for acidic peroxide pretreatment was

higher than alkaline peroxide pretreatment.

As can be seen in Fig. 8, acid and alkaline peroxide treat-

ment for soybean straw considerably affected the hydrogen

productivity. Under acidic peroxide, the cumulative hydrogen

volume initially increased and then decreasedwith increasing

HCl concentration. The maximum cumulative hydrogen

volume of 11.37 mL was observed at 2% HCl. Under alkaline

peroxide, the cumulative hydrogen volume dramatically

decreased with the increase of NaOH concentration, from

41.00 (0.5% NaOH) to 17.36 mL (1% NaOH). With NaOH

concentration up almost 1%, the cumulative hydrogen volume

remained 1.0 mL. The results indicated that the higher alka-

line concentration was not beneficial to hydrogen production.



0.5%HCl 1%HCl 2%HCl 4% HCl 8%HCl

0

5

10

15

20

25

30

35

40

45Hemicellulose

Cellulose

Lignin

Total reducing sugar

Co

nte

nt (

% d

ry

w

eig

h)

HCl concentration (w/v)

a

0.5%NaOH 1%NaOH 2%NaOH 4% NaOH 8%NaOH

0

5

10

15

20

25

30

35

40

Co

nte

nt (%

dry

we

ig

h)

NaOH concentration (w/v)



b



different concentrations of acid (a) and alkaline (b) peroxide

pretreatment (16% H2O2).

0 10 20 30 40

0

2

4

6

8

10

12

Cu

mu

la

tiv

e h

yd

ro

gen

(m

L)

Time (hour)

0.5%

1%

2%

4%

8%

a

0 10 20 30 40

0

10

20

30

40

Cu

mu

lativ

e h

yd

ro

ge

n (m

L)

Time (hour)

0.5%

1%

2%

4%

8%

b


soybean straw pretreated by different concentrations of

acid (a) and alkaline (b) peroxide pretreatment (16% H2O2).

The operation was at initial pH 7.0 and 35 �C.


Table 2 depicted the liquid product analyses, showing higher

hydrogen yield corresponded with the higher contents of

acetic acid and n-butyric acid.

Comparing the cellulosic biomass of acid or alkaline

peroxide pretreatment with sole acid, alkaline and hydrogen

peroxide pretreatment, we found that the contents of

hemicellulose, cellulose and lignin obviously decreased, but

the content of TRS did not increase even decrease. It indi-

cated that the degradation of hemicellulose, cellulose and

lignin could not convert into TRS in full. The degradation of

hemicellulose, cellulose and lignin could produce the

inhibitor of hydrogen-producing bacteria [26,33]. In addition,

only sugar was metabolized to produce hydrogen by anaer-

obic mixed bacteria [17]. And this caused the decrease of

hydrogen production. Table 3 shows that the modified

Gompertz model fits experimental data for the optimum of

different pretreatment methods well with the R2 values,

which are all greater than 0.99. It indicated that the

maximum cumulative hydrogen, the minimum lag-phase

time and the maximum hydrogen production rate were

produced at 4% HCl, 16% H2O2 and alkaline peroxide with

a composition of 0.5% NaOH and 16% H2O2. In summary, 4%

HCl was the best choice for pretreatment of soybean straw

in view of hydrogen yield.

3.5. Effects of inoculum, pH, ferrous and nickel onhydrogen production

The previous works showed that inoculum volume [38], initial

pH [15,23], ferrous [8,17,23,39] and nickel [40] could affect

hydrogen production. The 9 orthogonal experimental condi-

tions (L9(34)) were designed to analyze the effect on hydrogen

production of four factors (inoculum volume, initial pH, FeSO4

and NiCl2) at three levels, as shown in Table 4 and Table 5.

The previous studies reported SO42- and Cl� had no remark-

able effect on hydrogen production [8,23], therefore, FeSO4

and NiCl2 was used to study the influence of iron and nickel

on biohydrogen from soybean straw. Soybean straw pre-

treated with 4% HCl was then used for biohydrogen produc-

tion according with orthogonal experimental conditions. The



Table 2 e Effects of different HCl or NaOH concentrations in 16%(w/v) H2O2 on the degradation efficiency of total reducingsugar, hydrogen yield and yields of ethanol and volatile fatty acids.

w/v DESa HYb Ethanol(mg/L)

AceticAcid (mg/L)

PropionicAcid (mg/L)

iso-ButyricAcid (mg/L)

n-ButyricAcid (mg/L)

iso-ValericAcid (mg/L)

n-ValericAcid (mg/L)

HCl 0.5% 50.71% 7.75 23.72 261.53 299.00 230.77 387.77 223.01 301.60

1% 45.85% 9.69 24.62 253.76 314.48 307.29 425.36 280.93 335.22

2% 51.82% 11.37 26.60 384.52 336.33 200.05 421.55 178.26 326.72

4% 28.92% 6.88 28.65 354.75 334.48 169.29 412.38 155.40 292.82

8% 6.29% 0.72 43.48 301.18 333.91 181.64 411.67 169.76 301.86

NaOH 0.5% 92.43% 41.00 21.19 414.92 296.53 147.29 545.48 129.35 238.12

1% 32.01% 17.36 29.41 379.33 293.62 277.49 389.97 260.10 287.93

2% 13.54% 1.02 41.10 323.38 360.30 376.80 380.68 337.40 395.45

4% 10.35% 0.95 43.98 340.76 346.20 239.43 327.40 229.71 372.87

8% 8.53% 1.25 41.51 335.36 463.50 419.08 305.92 290.14 396.85

a DES means degradation efficiency of total reducing sugar.

b HY means hydrogen yield (mL H2/g-Substrate).


parameter K was the statistical average of hydrogen volume

at one level (for one factor). The parameter R was the statis-

tical range of K1eK3 for one factor. The different values of R

revealed the effects of the four factors on hydrogen produc-

tion, while the different values of K showed the effects of the

three levels on hydrogen production [41]. It was found that

FeSO4 (R ¼ 6.83) asserted the most important influence on

hydrogen production, while inoculum (R ¼ 0.3) the least.

Because K2 ¼ 56.00 was the maximum value in the column C

(FeSO4), the level two of factor C (marked as “C2”) was selected

as a component of the theoretical optimal medium. The A3, B2and D2 were similarly selected as the other four components

Table 3 e Kinetic parameters for hydrogen productionusing various pretreatment conditions.

Pretreatment conditions P (mL) Rm (mL/h) l (h) R2

Unpretreatment 5.46 0.26 16.64 0.996

4% HCl 47.65 2.26 13.28 0.997

0.5% NaOH 10.47 0.86 16.29 0.993

16% H2O2 23.00 3.55 1.00 0.997

2% HCl, 16% H2O2 11.37 2.57 1.07 0.997

0.5% NaOH, 16% H2O2 41.00 11.17 1.14 0.999

P means hydrogen production potential.

Rm means the maximum hydrogen production rate.

l means the lag-phase time.

R2 means correlation coefficient.

Table 4 e Factors and levels in orthogonal experimentaldesign.

Factor Factors and symbols

A Inoculum(mL)

B pH C FeSO4

(mg/L)D NiCl2(mg/L)

Level 1 10 6.0 50 12.5

Level 2 20 7.0 100 25

Level 3 30 8.0 200 50

of the theoretical optimal medium. Accordingly, the theo-

retical optimal medium (A3B2C2D2) compositions were: inoc-

ulum 30 mL, pH 7.0, FeSO4 100 mg/L and NiCl2 25 mg/L. The

experimental optimal medium A1B2C2D2 (condition No. 2),

which produced the experimental maximum hydrogen

volume of 58.8 mL, was directly selected from the 9 practical

experimental conditions (Table 5). The experimental optimal

medium (A1B2C2D2) compositions were inoculum 10 mL, pH

7.0, FeSO4 100 mg/L and NiCl2 25 mg/L.

A new experiment with the theoretical optimal medium

(A3B2C2D2) was carried out to verify the practical hydrogen

production, the result of which was compared with that of the

experimental optimal medium (A1B2C2D2) in Fig. 9. The theo-

retical optimal medium of A3B2C2D2 derived from the orthog-

onal analysis yielded a hydrogen volume of 60.2 mL, which

Table 5 e Hydrogen production in dark fermentationunder orthogonal experimental conditions.

Experimentalcondititions

A B C D H2

productiona

(mL)Inoculum pH FeSO4 NiCl2

1 1b 1 1 1 51.5 � 0.3

2 1 2b 2 2 58.8 � 0.7

3 1 3 3b 3 46.5 � 0.4

4 2 1 2 3 54.2 � 0.5

5 2 2 3 1 50.6 � 0.3

6 2 3 1 2 52.2 � 0.7

7 3 1 3 2 50.4 � 0.2

8 3 2 1 3 52.3 � 0.5

9 3 3 2 1 55.0 � 0.4

K1c 52.27 52.03 52.00 52.37

K2 52.33 53.90 56.00 53.80

K3 52.57 51.23 49.17 51.00

Rd 0.3 2.67 6.83 2.80

a Data are the average of the values obtained in independent

experiments conducted in triplicate (n ¼ 3).

b Designed levels 1e3 for different factors (shown in Table 4).

c K: the average of hydrogen volume of three experiments at one

level (for one factor).

d R: the range of K1eK3 for one factor.



0 20 40 60 80

0

10

20

30

40

50

60

70

Cu

mu

la

tiv

e h

yd

ro

ge

n (m

L)

Time (hour)

A3B

2C

2D

2

A1B

2C

2D

2

Fig. 9 e Hydrogen production from soybean straw with the

theoretical optimal medium and experimental optimal

medium. - theoretical optimal medium: inoculum 30 mL,

pH 7.0, FeSO4 100 mg/L and NiCl2 25 mg/L. D experimental

optimal medium: inoculum 10 mL, pH 7.0, FeSO4 100 mg/L

and NiCl2 25 mg/L. Data are the averages of triplicate

independent experiments.


was slightly higher (by 2.4%) than 58.8 mL produced by the

experimental optimal medium A1B2C2D2 (condition No. 2)

selected from the 9 experimental conditions. In addition, the

cumulative hydrogen yield of 60.2 mL/g-dry soybean straw

was achieved, which was approximately 11-fold greater than

that in raw soybean straw (5.46 mL/g-dry soybean straw). The

modified Gompertz equation was used to fit the experimental

data. The results showed that the maximum hydrogen

production rate of 2.29 ml H2/h produced by the theoretical

optimal medium was also higher than the 2.21 ml H2/h

produced by the experimental optimal medium. In addition,

the lag-phase times decreased to 10.56 h for the theoretical

optimal medium, which was much shorter than the 12.35 h of

the experimental optimal medium. Therefore, the perfor-

mance of the theoretical optimalmediumwas superior to that

of the experimental optimal medium. The complete optimal

medium was finally determined as inoculum 30 mL, pH 7.0,

FeSO4 100 mg/L and NiCl2 25 mg/L.

4. Conclusions

In this work, different methods, acid (HCl), alkaline (NaOH),

hydrogen peroxide, acid peroxide and alkaline peroxide

treatment, were used to pretreat soybean straw. The results

showed that acid pretreatment is an effective method for

enhancing the hydrogen yield from soybean straw. The

maximumhydrogen yield of 60.2mL/g-dry soybean strawwas

achieved by orthogonal testing strategy optimizing hydrogen-

producing conditions, which was approximately 11-fold

greater than that in raw soybean straw. Therefore, hydrogen

production from soybean straw through dark fermentation is

feasible.

Acknowledgments

The authors would like to thank the Chinese Academy of

Sciences for financial support (Item No. KJCX2-YW-H21).

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