optimization of biohydrogen production from beer lees using anaerobic mixed bacteria

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 4 ( 2 0 0 9 ) 7 9 7 1 – 7 9 7 8

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Optimization of biohydrogen production from beer lees usinganaerobic mixed bacteria

Maojin Cui, Zhuliang Yuan, Xiaohua Zhi, 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 15 June 2009

Received in revised form

6 August 2009

Accepted 8 August 2009

Available online 27 August 2009

Keywords:

Beer lees

Anaerobic mixed bacteria

Biohydrogen production

Acidic pretreatment

Ferrous iron concentration

Initial pH value

* Corresponding author. Tel.: þ86 10 6262090E-mail address: [email protected] (J. Sh

0360-3199/$ – see front matter ª 2009 Profesdoi:10.1016/j.ijhydene.2009.08.022

a b s t r a c t

Beer lees are the main by-product of the brewing industry. Biohydrogen production from

beer lees using anaerobic mixed bacteria was investigated in this study, and the effects of

acidic pretreatment, initial pH value and ferrous iron concentration on hydrogen produc-

tion were studied at 35 �C in batch experiments. The hydrogen yield was significantly

enhanced by optimizing environmental factors such as hydrochloric acid (HCl) pretreat-

ment of substrate, initial pH value and ferrous iron concentration. The optimal environ-

mental factors of substrate pretreated with 2% HCl, pH¼ 7.0 and 113.67 mg/l Fe2þ were

observed. A maximum cumulative hydrogen yield of 53.03 ml/g-dry beer lees was achieved,

which was approximately 17-fold greater than that in raw beer lees. In addition, the

degradation efficiency of the total reducing sugar, and the contents of hemicellulose,

cellulose, lignin and metabolites are presented, which showed a strong dependence on the

environmental factors.

ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction hydrogen will be of great significance in solving environ-

In recent years, a large amount of cellulosic biomass such as

crop straws, leaves, weeds and beer lees have been burned or

discarded, which is not only a waste of resources, but also

pollutes the environment. Only in China, the annual yield of

waterish beer lees is about 10 million tons [1]. Most of them

were discarded as environmental pollutions, except that some

of them were utilized for feedstuff of livestock and fowl.

Processes which can convert cellulosic biomass into useful

products, e.g. methanol, ethanol, methane and hydrogen,

have attracted much attention. Compared with alcohols and

methane, hydrogen is superior because of its cleanness and

high energy-density (122 kJ/g) [2], which is the highest of all

known fuels. Therefore, converting cellulosic biomass into

3; fax: þ86 10 62559373.en).sor T. Nejat Veziroglu. Pu

mental pollution and energy shortage.

Recently, some studies on biohydrogen from cellulosic

biomass such as wheat straw, corn stalk, bulrush, and wheat

bran [3–8] have been carried out, however, the key issues

restricting the production of biohydrogen from cellulosic

biomass in these studies were selection of the optimal

pretreatment and the development of efficient hydrogen-

producing bacteria. Generally, the direct conversion of raw

cellulosic biomass into hydrogen by microorganisms is very

difficult due to the complicated polymer structure of hemi-

cellulose and cellulose, therefore, it is necessary to pretreat

the substrate in order to enhance hydrogen yield [9]. Nowa-

days, the main methods for pretreatment of cellulosic

biomass include steam explosion, mechanical, thermal,

blished by Elsevier Ltd. All rights reserved.

mailto:[email protected]

http://www.elsevier.com/locate/he


acidic, alkaline, ammonia, and oxidative pretreatments [3-8].

The hydrogen-producing bacteria using cellulosic biomass for

fermentation were mainly isolated from cow dung compost

[3,4], fistulated goat feed [5] and an anaerobic digester sludge

[6]. In addition, pure strains and mixed microflora were also

used as hydrogen-producing bacteria [7,8,10–12].

Bacterial metabolism during fermentation requires essen-

tial metals such as magnesium, zinc and iron. Among these

metals, iron is very important in the formation of hydrogenase

or other enzymes which are necessary for biohydrogen

production [13]. Our previous studies showed that SO42� had

no remarkable effect on hydrogen production [14,15], there-

fore, FeSO4$7H2O was used to study the influence of iron on

biohydrogen from beer lees.

According to the above background, the availability of

abundant beer lees coupled with anaerobic fermentation

resulting in hydrogen production is considered to be ideal as it

not only utilizes a renewable resource but also produces clean

hydrogen in a sustainable manner. The objective of this study

was to investigate the feasibility of producing hydrogen from

beer lees. For this purpose, the effects of HCl concentration,

initial pH value and ferrous iron concentration on hydrogen

production from beer lees were studied by anaerobic

fermentation in batch cultivation.

2. Materials and methods

2.1. Seed microorganisms

The hydrogen-producing mixed cultures used here were

enriched from cracked cereals and were dominated by Clos-

tridium pasteurianum following identification of the bacteria in

culture [16]. The cultures were acclimated in a completely

stirred tank reactor (CSTR) in a chemostat for approximately

one month. The CSTR was operated at 35 �C, with an 8 h

hydraulic retention time, and stirred by gas circulation [14]. 1 l

of culture medium used to ferment contained NH4HCO3,

3770 mg; K2HPO4, 125 mg; NaHCO3, 2000 mg; CuSO4$5H2O,

5 mg; MgCl2$6H2O, 100 mg; MnSO4$4H2O, 15 mg; FeSO4$7H2O,

25 mg; CoCl2$6H2O, 0.125 mg.

2.2. Acidic pretreatment of beer lees

The beer lees used in this study were obtained from the Five-

star Brewery, Beijing, China. 1.2 g of dry beer lees were mixed

with 20 ml of dilute HCl aqueous solution at different

concentrations (0.5%, 1%, 2%, 4%, w/v) separately (6% (w/v)

solids loading) and boiled for 30 min in serum vials. The

mixture was then neutralized to pH 7.0 by the addition of

dilute NaOH aqueous solution at different concentrations

(0.5%, 1%, 2%, 4%, w/v), respectively.

2.3. Experimental procedures

Batch experiments were carried out in 120 ml serum vials. The

total work volume was 80 ml (approximately 1.5% (w/v) solids

loading) in each case, which included approximately 40 ml of

mixture after pretreatment (20 ml of dilute HCl used to

pretreat substrate and approximately 20 ml of dilute NaOH

used to adjust pH), 20 ml inoculum, 10 ml nutrient solution (1 l

contained NH4HCO3, 30 160 mg; K2HPO4, 1000 mg; NaHCO3,

16 000 mg; CuSO4$5H2O, 40 mg; MgCl2$6H2O, 800 mg;

MnSO4$4H2O, 120 mg; FeSO4$7H2O, 200 mg; CoCl2$6H2O, 1 mg),

and approximately 10 ml distilled water. The air was removed

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

before the vials were capped with rubber septum stoppers and

placed in a reciprocal shaker (120 rpm). The batch experi-

ments were performed at 35 �C in the dark. Each experimental

condition was carried out in triplicate. All chemicals used in

the experiments were of AR grade.

2.4. Analytical methods

The hydrogen content was determined by a gas chromato-

graph (Techcomp. Co., China, 7890II) equipped with a thermal

conductivity detector (TCD) and a 2-m stainless steel column

packed with Porapak Q (80–100 mesh). The operating

temperatures of the injection port, the oven and the detector

were set at 70, 50 and 70 �C, respectively. Argon was used as

the carrier gas at a flow rate of 30 ml/min. At each time

interval, the total volume of biogas production was measured

by a plunger displacement method using appropriately sized

glass syringes, ranging from 10 to 100 ml [17]. The cumulative

hydrogen volume was calculated by equation (1) [18]:

VH;i ¼ VH;i�1 þ CH;i

�VG;i � VG;i�1

�þ VH;0

�CH;i � CH;i�1

�(1)

where VH, i and VH, i � 1 are cumulative hydrogen volumes at

the current (i) and previous (i� 1) time intervals, VG, i and VG,

i � 1 are the total biogas volumes in the current (i) and previous

(i� 1) time intervals, CH, i and CH, i � 1 are the fraction of

hydrogen in the headspace of the bottle at the current (i) and

previous (i� 1) intervals and VH,0 is the total volume of

headspace in the bottle. Detection of the alcohols and volatile

fatty acids (VFAs, C2–C5) were measured by a gas chromato-

graph using a flame ionization detector (FID) and a 2-m glass

column packed with Unisole F-200 (30–60 mesh). The

temperatures of the injection port, the oven and the detector

were set at 200, 165 and 200 �C, respectively. The carrier gas

was argon at a flow rate of 30 ml/min. The concentration of

the total reducing sugar was determined by the phenol–

sulfuric acid method using glucose as a standard [19]. The

contents of hemicellulose, cellulose and lignin were deter-

mined according to Van Soest’s method [20].

2.5. Model analysis

The cumulative hydrogen production in the batch experi-

ments followed the modified Gompertz equation [21]:

H ¼ P

�exp

�Rme

Pðl� tÞ þ 1

��(2)

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

hydrogen production potential (ml), Rm is the maximum

hydrogen production rate (ml/h), e¼ 2.71828, l is the lag-phase

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

values of P, Rm and l for each batch were estimated using

Origin 7.5, which is a scientific graphing and data analysis

software.

0 10 20 30 40 50 600

10

20

30

40

50

60

70

Cu

mu

la

tiv

e h

yd

ro

ge

n (m

l)

Time (hour)

UP

0.5%

1%

2%

4%

Fig. 2 – Cumulative hydrogen volumes from 1.2 g of dry

beer lees without pretreatment and pretreated by different

HCl concentrations (from 0.5% to 4%, w/v) versus

corresponding fermentation time. The operation was at

35 8C, 13.67 mg/l Fe2D and initial pH 7.0.

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 4 ( 2 0 0 9 ) 7 9 7 1 – 7 9 7 8 7973

3. Results and discussion

3.1. Effect of HCl pretreatment on hydrogen production

3.1.1. Effect of HCl concentration on the saccharification oflignocellulosic biomassIt is known that the main components of cellulosic biomass

are hemicellulose, cellulose and lignin. Previous studies have

shown that pretreatment had some influence on the contents

of hemicellulose, cellulose and lignin [4,22–24]. It was reported

that the major compositions of total sugars such as xylose,

glucose and arabinose were produced after the hydrolysis of

hemicellulose in acidic environments [25]. To investigate the

effect of HCl concentration on the cellulosic biomass, the

compositions and contents in raw beer lees and pretreated

samples were analyzed and are shown in Fig. 1. As shown in

Fig. 1, it can be seen that the contents of hemicellulose

apparently decreased (43.54–1.72%) after the pretreatment

process. The contents of hemicellulose after 2% and 4% HCl

pretreatment were 6.08% and 1.72%, respectively. It was

apparent that the contents of cellulose and lignin decreased

after HCl pretreatment. Therefore, it can be concluded that

the major reaction during acidic pretreatment was the

hydrolysis of hemicellulose, which was accompanied, to some

extent, by the hydrolysis of cellulose and lignin. This showed

that dilute HCl had an excellent effect on the degradation of

hemicellulose. The content of total reducing sugar was only

3.12% in raw beer lees. After HCl pretreatment, the total

reducing sugar significantly increased, and continued to

increase (30.80–37.39%) with increased HCl concentrations

(0.5–4%). The amount of total reducing sugar with acidic

treatment was approximately 10-fold to 12-fold compared

with that of the raw substrate, indicating that HCl pretreat-

ment increased the amount of total reducing sugar. The

soluble sugar from beer lees pretreated with 4% HCl was

highest (37.39%), so the cumulative hydrogen yield was

maximal (Fig. 2).

UP 0.5% 1% 2% 4%0

10

20

30

40

Co

nte

nt

(%

dry

we

ig

h)

HCl concentration (w/v)

Hemicellulose

Cellulose

Lignin

Total reducing sugar

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

total reducing sugar in beer lees without pretreatment and

pretreated by different HCl concentrations (from 0.5% to

4%, w/v) before and after pretreatment. UP means

unpretreatment.

3.1.2. Effect of HCl pretreatment on hydrogen production andhydrolysis of lignin and carbohydratesIt was reported that biohydrogen production from cellulosic

materials could be greatly increased by pretreatment [3,4,7,9].

In our previous work, the anaerobic mixed bacteria used here

appeared to have a good capacity for producing hydrogen

using monosaccharides, disaccharides and polysaccharides

as substrates [14,26,27]. In the present study, hydrogen

production from 1.2 g of pretreated beer lees and 1.2 g of raw

beer lees were compared. The cumulative hydrogen volumes

are presented in Fig. 2. As shown in Fig. 2, the hydrogen

volume from raw beer lees was only 3.79 ml, however,

a significant enhancement of hydrogen volume was achieved

after HCl pretreatment. Maximal hydrogen volumes of 62.59–

63.64 ml were observed at HCl concentrations of 2–4%. The

increment of hydrogen, however, was only 1.68%, thus 2% HCl

was considered the best choice to pretreat beer lees in view of

hydrogen yield and production costs. It is worth noting that

the excess Cl� produced did not significantly inhibit hydrogen

production. These results were different from those of

previous studies [3,4,9], which showed that high Cl� concen-

trations greatly inhibited biohydrogen production. This

inconsistency may be attributed to the different hydrogen-

producing bacteria used.

To further investigate the effect of HCl concentrations on

hydrogen production, the data in Fig. 2 were simulated using

equation (2), and the hydrogen production characteristics are

shown in Table 1. According to the data in Table 1, all R2 were

more than 0.999, indicating that the fitted curves matched

well with the experimental points. The maximum specific

hydrogen rate of 6.68 ml/h was observed at 2% HCl, indicating

that 2% HCl was the best concentration to pretreat the

substrate. The shortest duration time of 6.03 h occurred with

the raw substrate, and the changes in duration time were

small with increased HCl concentrations. This may have

Table 1 – Hydrogen production characteristics at differentHCl concentrations (from 0 to 4%, w/v).

HCl (w/v) P (ml) Rm (ml/h) l (h) R2

0 3.79 3.79 6.03 0.9999

0.5% 26.04 3.40 7.42 0.9999

1% 40.82 3.64 7.48 0.9991

2% 62.59 6.68 7.47 0.9996

4% 63.64 5.37 7.51 0.9990

P is hydrogen production potential (ml).

Rm is the maximum hydrogen production rate (ml/h).

l is the lag-phase time (h).

R2 is correlation coefficient.


occurred because the microorganisms used were acclimated

to the raw beer lees.

To investigate the effect of anaerobic fermentation on the

cellulosic biomass, the compositions and contents in raw beer

lees and pretreated samples after fermentation were analyzed

and are shown in Fig. 3. The contents of hemicellulose,

cellulose and lignin in raw beer lees changed greatly before

and after fermentation (Figs. 1 and 3), indicating that hemi-

cellulose, cellulose and lignin were directly degraded by the

bacteria [28]. The cumulative hydrogen volume from raw beer

lees, however, was small (only 3.79 ml), thus, hemicellulose,

cellulose and lignin were not degraded into hydrogen but to

other products by the fermentation bacteria. As shown in

Fig. 3, only small amounts of hemicellulose, cellulose and

lignin in pretreated beer lees were degraded by bacteria,

which was different from the changes in raw beer lees. The

results might be explained by the process of acidic pretreat-

ment of beer lees which produced some biological inhibitors

like furfural, which inhibited some of the bacteria in the

anaerobic mixture to decompose hemicellulose, cellulose and

lignin [29], but had no remarkable inhibition on hydrogen-

producing bacteria. Significant reductions in sugar content

were found after the fermentation, which showed that almost

all the sugar in solution was consumed by the bacteria.

UP 0.5% HCl 1% HCl 2% HCl 4% HCl0

2

4

6

8

10

12

Co

nte

nt (%

d

ry

w

eig

h)

HCl concentration (w/v)

Hemicellulose

Cellulose

Lignin

Total reducing sugar

Fig. 3 – Contents of hemicellulose, cellulose, lignin and

total reducing sugar in beer lees without pretreatment and

pretreated by different HCl concentrations (from 0.5% to

4%, w/v) after fermentation.

To sum up, the hydrogen-producing bacteria used here

could not directly convert hemicellulose, cellulose and lignin

into hydrogen, but only converted soluble sugar into hydrogen.

3.1.3. Effect of HCl pretreatment on the distribution ofmetabolitesGas product analyses showed 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

methanogens in the anaerobic mixed bacteria used here

which benefited hydrogen production. The effect of HCl

pretreatment on H2 contents, H2 yields and liquid metabolites

are shown in Table 2. The H2 content in biogas from raw beer

lees was only 14.78%. The increase in H2 content in the biogas

was found after HCl pretreatment, and a maximum H2

content of 39.44% was achieved at an HCl concentration of 2%.

The H2 yield in biogas from raw beer lees was only 3.16 ml/g-S.

The increase in H2 yield in the biogas was found after HCl

pretreatment, and a maximum H2 yield of 53.03 ml/g-S was

achieved at an HCl concentration of 4%.

The liquid product analysis showed that the metabolites

found after fermentation were ethanol, acetic acid, propionic

acid, n-butyric acid, iso-valeric acid and n-valeric acid. It

should be noted that high yields of ethanol and VFAs were

obtained from the raw beer lees, however, a low cumulative

hydrogen yield (3.16 ml/g-S) was produced. The reason for this

may be due to the varied metabolism, in that some hemi-

cellulose, 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. When the HCl concentration increased from 0.5% to

4%, the yield of ethanol increased gradually (5.29–21.73 mg/g-

S), along with an initial increase in acetic acid and n-butyric

acid which then decreased. Propionic acid and n-valeric acid

decreased gradually (100.00–25.60 mg/g-S and 9.33–0 mg/g-S,

respectively) and an irregular variation in iso-valeric yield was

found. The mechanism of hydrogen production from glucose

and xylose by bacterial fermentation has been reported

previously [30,31].

Theoretically the metabolic pathways of the three main

products, acetic acid, propionic acid and n-butyric acid from

xylose are as follows:

C5H10O5 þ 1:67H2O/1:67CH3COOHþ 3:33H2 þ 1:67CO2

DG� ¼ �195:5 KJ=mol ð3Þ

C5H10O5 þ 1:67H2/1:67CH3CH2COOHþ 1:67H2O

DG� ¼ �315:1 KJ=mol ð4Þ

C5H10O5/0:83CH3ðCH2Þ2COOHþ 1:67H2 þ 1:67CO2

DG� ¼ �233:9 KJ=mol ð5Þ

Theoretically the metabolic pathways of the three main

products, acetic acid, propionic acid and n-butyric acid from

glucose are as follows:

C6H12O6 þ 2H2O/2CH3COOHþ 4H2 þ 2CO2

DG� ¼ �215:7 KJ=mol ð6Þ

C6H12O6 þ 2H2/2CH3CH2COOHþ 2H2O

DG� ¼ �357:9 KJ=mol ð7Þ

Table 2 – Effect of pretreatment with different HCl concentrations (from 0 to 4%, w/v) on the H2 contents, H2 yields and yieldsof ethanol and VFAs after fermentation.

HCl(w/v)

H2

contenta

(%)

H2 yield(ml/g-S)

Ethanol(mg/g-S)

Aceticacid

(mg/g-S)c

Propionic acid(mg/g-S)

n-Butyric acid(mg/g-S)

iso-Valeric acid(mg/g-S)

n-Valeric acid(mg/g-S)

0 14.78 3.16 11.13 82.67 65.87 42.60 10.60 9.93

0.5% 28.81 21.70 5.29 88.67 100.00 83.33 27.20 9.33

1% 35.37 34.01 8.40 138.67 50.73 122.00 29.87 3.69

2% 39.44 52.16 9.33 179.33 26.80 135.33 23.20 0.69

4% 38.69 53.03 21.73 130.00 25.60 106.00 26.47 NDb

a H2 content means percentage of the total cumulative hydrogen produced to total biogas.

b ND means no detect.

c (mg/g-S) means (mg/g-dry beer lees).


C6H12O6/CH3ðCH2Þ2COOHþ 2H2 þ 2CO2�

DG ¼ �261:5 KJ=mol ð8Þ

According to equations (3)–(8), 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 is shown in Table 2, maximum acetic and n-

butyric acid appeared at an HCl concentration of 2%, which

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

showed that 2% HCl was the best choice for the pretreatment

of beer lees.

3.2. Effect of initial pH value on hydrogen production

To investigate the effect of initial pH value on hydrogen

production, 1.2 g of beer lees pretreated with 2% HCl were then

used for biohydrogen production at different initial pH values

from 4.0 to 10.0. The obtained results are summarized in Fig. 4.

It was found that the cumulative hydrogen volumes were

highest at initial pH values of 6–8, and the maximum

hydrogen volume of 62.59 ml was achieved at an initial pH of

0 20 40 60 80 100 120 1400

10

20

30

40

50

60

70

Cu

mu

la

tiv

e h

yd

ro

ge

n (m

l)

Time (hour)

pH=4.0

pH=5.0

pH=6.0

pH=7.0

pH=8.0

pH=9.0

pH=10.0


beer lees pretreated by 2% (w/v) HCl versus corresponding

fermentation time at 35 8C, 13.67 mg/l Fe2D and different

initial pH values (from 4.0 to 10.0).

7.0. The results revealed that initial pH value had a significant

influence on the activity of hydrogen-producing bacteria,

which was in agreement with previous studies [4,9,32].

To further investigate the effect of initial pH values on

hydrogen production, the data in Fig. 4 were simulated using

equation (2), and the hydrogen production characteristics are

shown in Table 3 (when the initial pH values were 4.0 and 10.0,

the fitted curves did not match the experimental points well,

so these are not listed). According to the data in Table 3, all R2

were more than 0.990, indicating that the fitted curves

matched well with the experimental points. The maximum

specific hydrogen rate of 6.68 ml/h was observed at an initial

pH of 7.0, and the shortest duration time of 7.47 h was also

observed at an initial pH of 7.0, indicating that the optimal

initial pH value was 7.0.

The content of total reducing sugar from 1.2 g of dry beer

lees pretreated by 2% HCl was 36.55%, and the efficiency of

sugar degradation under various initial pH values is depicted

in Fig. 5, which describes the variation in sugar content before

and after fermentation. Low amounts of sugar degradation

(0.70% and 8.11%) were found at initial pH values of 4.0 and 5.0,

suggesting that it was difficult for the bacteria to consume the

sugar for hydrogen production at this level and the bacterial

activity was low. When the initial pH values changed from 6.0

to 10.0, all sugar degradation efficiencies were more than 92%,

and the diversification was small, indicating that almost all

soluble sugar was consumed by the bacteria at this pH level.

Table 3 – Hydrogen production characteristics at differentinitial pH values (from 5.0 to 9.0).

Initial pH P (ml) Rm (ml/h) l (h) R2

5.0 24.58 0.70 45.00 0.9990

6.0 53.75 2.37 10.71 0.9959

7.0 62.59 6.68 7.47 0.9996

8.0 55.34 6.65 7.50 0.9988

9.0 33.53 2.61 16.67 0.9982

P is hydrogen production potential (ml).

Rm is the maximum hydrogen production rate (ml/h).

l is the lag-phase time (h).

R2 is correlation coefficient.

4 5 6 7 8 9 10

0

20

40

60

80

100

Deg

rad

atio

n efficien

cy o

f to

tal red

ucin

g

su

gar (%

)

Initial pH

Fig. 5 – Degradation efficiency of total reducing sugar in

beer lees pretreated by 2% (w/v) HCl at different initial pH

values (from 4.0 to 10.0) after fermentation.

0 10 20 30 400

10

20

30

40

50

60

70

Cu

mu

la

tiv

e h

yd

ro

ge

n (m

l)

Time (hour)

13.67 mg/l

113.67 mg/l

213.67 mg/l

313.67 mg/l

413.67 mg/l

513.67 mg/l


beer lees pretreated by 2% (w/v) HCl versus corresponding

fermentation time at different Fe2D concentration (from

13.67 mg/l to 513.67 mg/l). The operation was at 35 8C and

initial pH 7.0.


The changes in initial pH values not only affected the

physical conditions of microorganisms and the dynamic

balance of NADH/NADþ in the cells, but also the types and

quantity of dominant bacteria in the mixed microbial culture

medium. The final pH, H2 contents, H2 yields and yields of

metabolites after fermentation are shown in Table 4. The

maximal hydrogen content was found to be 42.67% at pH 8.0,

and the maximal hydrogen yield was 52.16 ml/g-S at pH 7.0.

The metabolites after fermentation were ethanol, acetic acid,

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

acid. As shown in Fig. 5 and Table 4, when the initial pH values

were 4.0 and 5.0, the degradation of total reducing sugar from

beer lees pretreated with 2% HCl was small, and the yields of

all the metabolites were low, thus, the activity of mixed

bacteria was low at this pH level; when the initial pH value

changed from 6.0 to 8.0, the yield of n-butyric acid was highest

(86.67–135.33 mg/g-S), indicating that n-butyric acid was the

main fermentative product at this pH level; when the initial

pH values were 9.0 and 10.0, ethanol and acetic acid were the

main fermentative products. This might be because the

activity of homoacetogens increased at high initial pH values

(9.0 and 10.0). It was reported that the concentration of acetic

acid was maximum at pH 11.0 using homoacetogens [33].

Table 4 – Effect of initial pH values (from 4.0 to 10.0) on the finaafter fermentation.

InitialpH

FinalpH

H2

content(%)

H2 yield(ml/g-S)

Ethanol(mg/g-S)

Acetic acid(mg/g-S)

Pro

4.0 3.51 1.47 0.34 8.87 17.00

5.0 4.55 23.01 20.48 6.32 55.67

6.0 4.80 37.66 44.79 8.20 64.20

7.0 6.56 39.44 52.16 9.33 179.33

8.0 7.15 42.67 46.12 8.47 250.00

9.0 7.21 32.55 27.94 12.20 331.33

10.0 7.70 41.35 8.51 11.40 456.67

In this study, hydrogen can be consumed by HCO3� to produce

acetic acid at high pH values (9.0 and 10.0) according to

equation (9), thus, the hydrogen yields decreased [34]. In

addition, with the increased yield of acetic acid, it could react

with hydrogen to produce ethanol according to equation (10),

thus decreasing the hydrogen yields [34].

4H2 þ 2HCO�3 þHþ/CH3COO� þ 2H2O DG� ¼ �70:3 KJ=mol

(9)

CH3COOHþH2/C2H5OHþ 4H2O DG� ¼ �49:51 KJ=mol

(10)

3.3. Effect of Fe2þ concentration on hydrogen production

In our previous work, the addition of Fe2þ showed some effect

on hydrogen production [15]. In this study, various concen-

trations of Fe2þ were added to 1.2 g of dry beer lees substrate,

which was pretreated by 2% HCl. The cumulative hydrogen

volumes are depicted in Fig. 6. The maximum hydrogen

volume was found to be 63.38 ml with the addition of

l pH, H2 contents, H2 yields and yields of ethanol and VFAs

pionic acid(mg/g-S)




3.84 1.87 3.35 1.07

4.14 1.59 2.29 0.46

8.53 108.67 2.73 1.26

26.80 135.33 23.20 0.69

35.20 86.67 25.73 1.48

49.73 42.47 30.33 5.93

30.00 21.33 25.00 0.63

Table 5 – Effect of Fe2D concentrations (from 13.67 mg/l to 513.67 mg/l) on the degradation efficiency of total reducing sugar,H2 contents, H2 yields and yields of ethanol and VFAs after fermentation.

Fe2þ

(mg/l)DESa

(%)H2

content(%)

H2 yield(ml/g-S)

Ethanol(mg/g-S)

Acetic acid(mg/g-S)

Propionic acid(mg/g-S)




13.67 97.47 39.44 52.16 9.33 179.33 26.80 135.33 23.20 0.69

113.67 97.43 41.86 52.82 21.07 166.00 22.53 106.00 24.40 7.13

213.67 97.59 39.43 48.43 20.00 168.00 21.53 120.00 22.33 5.55

313.67 97.70 46.87 44.37 16.33 195.33 23.20 140.00 20.33 2.97

413.67 97.76 45.89 42.49 11.53 154.67 17.33 108.67 17.40 2.53

513.67 97.67 42.61 38.56 11.07 199.33 21.00 133.33 14.73 0.47

a DES means degradation efficiency of total reducing sugar.


113.67 mg/l Fe2þ. However, concentrations exceeding this

level reduced the cumulative hydrogen volume, suggesting

that excessive Fe2þ was unfavorable for enhancing hydrogen

volume. These results were consistent with our previous

findings where we added Fe2þ to a soluble starch substrate for

hydrogen production [15].

The sugar degradation efficiency, H2 contents, H2 yields

and metabolites after fermentation are summarized in Table

5. It was found that the sugar degradation efficiency reached

more than 97% at different concentrations of Fe2þ, indicating

that almost all the sugar was consumed by the bacteria.

Hydrogen content in the biogas varied from 39.43% to 46.87%.

The maximal hydrogen yield was 52.82 ml/g-S with the addi-

tion of 113.67 mg/l Fe2þ. The metabolites in solution were

ethanol, acetic acid, propionic acid, n-butyric acid, iso-valeric

acid and n-valeric acid. The metabolic pathways of the three

main products, acetic acid, propionic acid and n-butyric acid

followed equations (3)–(8). As shown in Table 5, increased Fe2þ

concentrations thus affected the production of ethanol, iso-

valeric acid and n-valeric acid, which initially increased then

decreased, and the production of acetic acid, propionic acid

and n-butyric acid, which were found to vary irregularly. This

variation might be because the bacterial activity was influ-

enced by the Fe2þ concentration in the substrate. On the one

hand, Fe2þ which was the main component of ferredoxin or

iron–sulfur protein in the hydrogenase system directly

affected the microbial bio-oxidation and dehydrogenation

process; on the other hand, the strong reduction capacity of

Fe2þ played an important role in reducing the ORP value of the

fermentation environment and improved the metabolic

activity of hydrogen-producing bacteria [35]. Excessive Fe2þ

concentration, however, inhibited the activity of hydrogen-

producing bacteria, thereby resulting in a decline in hydrogen-

producing capability.

4. Conclusions

In this work, hydrogen production from beer lees using

anaerobic fermentation was demonstrated in batch experi-

ments. It was found that acidic pretreatment of substrate and

initial pH value was important in hydrogen production.

Maximum hydrogen yield (53.03 ml/g-dry beer lees) was ach-

ieved under 4% HCl pretreatment conditions, and was

enhanced by approximately 17-fold compared with that from

raw beer lees. Fe2þ iron was also added to the substrate to

investigate its effect on hydrogen production. The results

showed that moderate addition of Fe2þ at 113.67 mg/l

enhanced the hydrogen yield and excessive Fe2þ inhibited

hydrogen production. In conclusion, from the analysis of the

contents of hemicellulose, cellulose, lignin and total reducing

sugar before and after fermentation, we found that acidic

pretreatment may convert hemicellulose into soluble sugar

which can be consumed by bacteria for hydrogen production.

In addition, some hemicellulose, cellulose and lignin could be

directly utilized by the bacteria to produce alcohols, VFAs and

other products. Therefore, cumulative hydrogen production,

amounts of metabolites and the bacterial metabolic pathway

were strongly dependent on environmental factors.

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