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Biohydrogen production from apple pomace by anaerobicfermentation with river sludge

Xiaoqiong Fenga, Hui Wanga,*, Yu Wanga, Xiaofang Wanga, Jianxin Huangb

aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry (Ministry of Education), Department of Chemistry, Northwest

University, Xi’an 710069, PR ChinabCollege of Life Science, Northwest University, Xi’an, Shaanxi 710069, PR China

a r t i c l e i n f o

Article history:

Received 14 April 2009

Accepted 7 July 2009

Available online 26 July 2009

Keywords:

Biological H2 production

Apple pomace

Fermentation

Pretreatment

Natural mixed microorganisms

* Corresponding author. Tel.: þ86 029 883631E-mail address: huiwang@nwu.edu.cn (H

0360-3199/$ – see front matter ª 2009 Profesdoi:10.1016/j.ijhydene.2009.07.015

a b s t r a c t

The biological hydrogen (bio-H2) production from apple pomace (AP) by fermentation using

natural mixed microorganisms in batch process was studied under various experimental

conditions. The river sludge was used as a seed after being boiled for 15 min. The results

show that the optimal pretreatment for AP was to soak it in the ammonia liquor of 6% for

24 h at room temperature. An optimal fermentation condition for bio-H2 production was

proposed that the pretreated AP at 37 �C, the initial pH of 7.0 and the fermentation

concentration of 15 g/l could produce a maximum cumulative H2 yield (CHYm) of

101.08 ml/g total solid (TS) with an average H2 production rate (AHPR) of 8.08 ml/g TS/h.

During the conversion of AP into H2, acetic acid, ethanol, propionic acid and butyric acid

were main liquid end-products.

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

1. Introduction feasible for practical applications because it is rapid, simple

High dependence on fossil fuels has led to serious energy

crisis and environmental problems, so it is very urgent for the

exploration of clean and renewable substitutes. Among all the

alternative energy, hydrogen (H2) has attracted more and

more attention due to the characteristic of being clean, high

efficient and renewable. Currently, H2 can be generated by

many methods, including thermocatalytic reforming of H2-

rich organic compounds, electrolysis of water and biological

processes [1]. Among the methods, biological hydrogen (bio-

H2) production is found to be sustainable and environmental

friendly [2]. Moreover, organic wastes can be used as

fermentation substrates for bio-H2 production, which facili-

tates both waste treatment and energy recovery. For the two

biological routes, photosynthetic and fermentative H2

production, the fermentative H2 production seems more

15; fax: þ86 029 88303798. Wang).sor T. Nejat Veziroglu. Pu

and independent of weather condition [3].

The fermentative H2 production mainly depends on

temperature, pH and substrate concentration [4–7]. The

temperature and pH are two important factors in biological

fermentation process due to their effects on the substrate

utilization, H2 production and liquid end-product distribution

as well as bacterial growth. At present, some investigators

have reported the effect of temperature on the fermentative

H2 production using various natural mixed microorganisms

such as cow dung compost and anaerobic digester sludge [8,9].

However, the detailed information regarding the effect of

temperature on the fermentative H2 production with river

sludge as seed is still lacking. In addition, the reported optimal

initial pH values are different for different cellulosic materials,

varying from pH¼ 4.5 [9] for rice slurry, pH¼ 6.5 [10] for beer

lees to 7.0 or 8.0 for wheat straw [8].

.

blished by Elsevier Ltd. All rights reserved.

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 5 ( 2 0 1 0 ) 3 0 5 8 – 3 0 6 4 3059

Currently, substrates for the fermentative H2 production

mainly include simple sugars, starch and biomasses such as

crops, agricultural and industrial wastes and cellulosic

municipal solid wastes [11–14]. However, natural cellulosic

materials cannot be used to produce H2 directly because the

cellulose polymers in the cell wall are intricately associated

with lignin and hemicellulose. Therefore, cellulosic materials

must be pretreated so as to remove lignin and hemicellulose,

as well as to reduce cellulose crystallinity before utilizing

them. Although there were many studies on the fermentative

H2 production from cellulosic materials [15–17], few investi-

gations on H2 production by fermentation from apple pomace

(AP) were reported. In general, AP accounting for about 25% of

fresh fruit weight is mainly composed of pericarp, core and

pulp remains. In China, the yield of AP as a byproduct of juice

extraction is more than 1 million tons, but only a small amount

of AP is used for deep-processing, and the vast majority is not

effectively utilized yet. In addition, AP is highly biodegradable

and rich in sugars and fibres, its disposal as waste causes not

only a serious environmental problem but also a huge loss of

precious resources [18]. Therefore, it can be considered as

a very potential substrate to produce H2 by fermentation. The

purpose of this work was to determine the optimal condition

for the conversion of AP into H2 by the river sludge. A series of

batch experiments were conducted to investigate the effects of

substrate pretreatments, initial pH, temperature and substrate

concentration on the fermentative H2 production.

2. Materials and methods

2.1. Pretreatment of substrate

Apple pomace (AP) used in this work was obtained from

a juice-making factory of Xianyang city. Before analysis, AP

was dried at 65 �C for 24 h. Its main components analyzed are

as follows: reducing sugar 13.52, cellulose 16.91%, lignin

24.11%, and hemicelluloses 20.00%. There are still some other

components, including moisture, pectin, mineral, vitamin C,

crude protein, crude fat and so on [18]. The total solid (TS)

content was 97.13% in this study.

Before being degraded by microorganisms, AP was pre-

treated by the following two methods: Method 1, AP was

soaked in H2SO4 solution at room temperature for 12 h, or

treated in H2SO4 solution by ultrasonic with a frequency of

25 kHz at various time, and then adjusted pH to 7.0 with dilute

NaOH solution; Method 2, AP was first soaked in ammonia

liquor at room temperature at various time, and then the solid

was obtained by filtration and washed repeatedly with

distilled water until the wash water reached pH 7.0. The

above-obtained filtrate was concentrated at 50 �C to remove

ammonia from the solution, subsequently, the above solid

and concentrated filtrate were mixed to be as the fermenta-

tion substrate for the H2 production. The ratio of solid to liquid

was 0.1 g/ml for all pretreatments in this work.

2.2. Seed microflora

The seed used in this study was natural anaerobic activated

sludge, which was obtained from the Bahe river of Xi’an city.

Prior to use, the river sludge was boiled for 15 min to inhibit

the bioactivity of methane-forming bacteria and enrich H2-

producing bacteria.

2.3. Batch experiments

The batch experiments were conducted with 150 ml three-

necked flasks as reactors. In each run, the reactor was filled

with 100 ml mixture of heat-pretreated sludge, substrate and

nutrient stock solution. Then the reactor was sealed and

incubated with continuous stirring at 120 rpm to ensure

thorough mixing and facilitate the rapid diffusion of biogas.

One liter of culture medium contained: peptone, 4000 mg;

L-cysteine, 600 mg; NaCl, 2000 mg; KH2PO4, 2000 mg;

MgSO4$7H2O, 500 mg; FeSO4$7H2O, 6 mg. The volume and

composition of biogas were monitored once every time period.

Only carbon dioxide and H2 were detected from gas products.

The liquor samples were taken for the volatile fatty acids

(VFAs) and alcohols analysis after fermentation.

2.4. Analyses

The volume of biogas was measured by the water-displace-

ment method in a measuring cylinder. The composition and

proportion of biogas were analyzed by an on-line gas chro-

matograph (GC-SP-6890) with a thermal conductivity detector

(TCD) and 3 mm inside diameter stainless-steel column

packed with molecular sieve TDX-01. Argon was used as the

carrier gas at the flow rate of 40 ml/min. The operational

temperatures at the column oven, injection and detector were

kept at 90, 130 and 130 �C, respectively. The liquid end-prod-

ucts were determined by a second gas chromatography (SP-

6890) under the following conditions: column: 3 mm inside

diameter stainless-steel column packed with polymer beads

TDX-01, carrier gas: nitrogen (flow rate: 40 ml/min), detector:

flame ionization detector (FID), column temperature: 170 �C,

injection temperature: 200 �C, detector temperature: 200 �C.

3. Results and discussion

3.1. Effect of substrate pretreatment on H2 production

3.1.1. Effect of H2SO4 pretreatmentFig. 1 depicts the changes of cumulative H2 yield (CHY) verse

time at 37 �C, initial pH 6.0 and fermentation substrate

concentration 20 g/l. It is apparent from Fig. 1 that the CHY for

all the fermentation processes of AP increased rapidly with

time and finally reached a maximum value. As shown in

Fig. 1a, the CHY gradually increased with the increase of

H2SO4 concentration in the range of 0–0.5% under the condi-

tion of soaking for 12 h, and a maximum cumulative H2 yield

(CHYm) of 69.74 ml/g TS was obtained at the H2SO4 concen-

tration of 0.5%. While the CHY gradually decreased with the

increase of H2SO4 concentration in the range of 0.5–1.0%, was

only 48.39 ml/g TS at the H2SO4 concentration of 1.0%. As we

know, a relatively higher H2SO4 concentration is conducive to

the hydrolysis of substrate, however, when a much higher

SO42� anion was introduced into the culture medium, the

growth of H2-producing bacteria was also severely inhibited

20

40

60

0.0%

0.25%

0.5%

0.75%

1.0%

CH

Y(m

l/gT

S)

0

20

40

60

80

0.0%0.25%0.5%0.75%1.0%

CH

Y(m

l/gT

S)

0

20

40

60

0 5 10 15 20

15min30min45min60min75min

CH

Y(m

l/gT

S)

Time (h)

c

b

a

Fig. 1 – Changes of CHY vs. time at 37 8C, initial pH 6.0 and

substrate concentration 20 g/l for the various pretreatments

(a) soaking for 12 h at the H2SO4 concentrations of 0, 0.25,

0.5, 0.75 and 1.0%, (b) ultrasonic for 1 h at the H2SO4

concentrations of 0, 0.25, 0.5, 0.75 and 1.0%, and (c)

ultrasonic for 15, 30, 45, 60 and 75 min in 0.5% H2SO4.

0

1

2

3

4

5

6

7

0 0.25 0.5 0.75 1

Soaking for 12 hUltrasonic treatment for 1 h

Fig. 2 – Effects of two pretreatment methods (soaking for

12 h and ultrasonic for 1 h) on AHPR at the H2SO4

concentrations of 0, 0.25, 0.5, 0.75 and 1.0%.

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 5 ( 2 0 1 0 ) 3 0 5 8 – 3 0 6 43060

[19,20]. Similarly, from Fig. 1b, a CHYm of 76.68 ml/g TS also

occurred at the H2SO4 concentration of 0.5% under the

condition of ultrasonic for 1 h. It is concluded that an appro-

priate H2SO4 concentration for the substrate pretreatment

was 0.5%. In addition, it can be seen in Fig. 1c that ultrasonic

time also affected H2 yield for the acid pretreated substrate

and the optimal ultrasonic time was 1 h. As far as the average

H2 production rates (AHPRs) in Fig. 2 are concerned, compar-

isons of known show that the largest AHPRs occurred at the

H2SO4 concentration of 0.5% for soaking for 12 h and ultra-

sonic for 1 h, this also confirmed the results mentioned

previously. Accordingly, it is suggested that the optimal acid

pretreatment condition for AP was ultrasonic treatment in

0.5% H2SO4 for 1 h.

In order to study the effect of pretreatment on the

substrate, the composition of AP was analyzed. Compared

with the raw AP, the reducing sugar content in the AP treated

by ultrasonic in 0.5% H2SO4 for 1 h increased by 51.0%, while

the cellulose and hemicellulose contents decreased by 35.6%

and 26.9%, respectively. The increase of reducing sugar

content is just the reason why the CHYm of pretreated AP

(76.68 ml/g TS) was higher than that of raw AP (41.28 ml/g TS).

3.1.2. Effect of ammonia liquor pretreatmentFig. 3 describes the effect of ammonia liquor pretreatment on

the fermentative H2 production at 37 �C, initial pH 6.0 and

fermentation substrate concentration 20 g/l. As shown in

Fig. 3a, when ammonia liquor concentration rose in the range

of 2–6%, the CHY distinctly increased from 64.64 to 80.37 ml/g

TS. In contrast, the CHY dramatically declined from 80.37 to

52.16 ml/g TS when ammonia liquor concentration rose from

6 to 10%. The experiment phenomena might be explained as

follows: the further increase of ammonia liquor concentration

intensified the interaction of lignin and ammonia liquor, and

the dissolved lignin deposited on the surface of substrate with

the increase of degradation products, which prohibited the

substrate fermentation [16]. However, as can be seen from

Fig. 3b, the soaking time almost did not affect H2 yield for the

AP pretreated by ammonia liquor. The above results,

combined with Fig. 4, show that the optimal alkali pretreat-

ment condition for AP was to soak it in 6% ammonia liquor at

room temperature for 24 h, which resulted in an AHPR of

6.18 ml/g TS/h.

From the composition analysis of AP soaked by 6%

ammonia liquor for 24 h, it is found that the reducing sugar

content increased by 60.6% and the lignin content decreased

by 65.7% as compared with the raw AP. The results show that

ammonia liquor could delignify effectively. In fact, ammonia

liquor has high selectivity for the reaction with lignin rather

than carbohydrates, which cannot only reduce the

0

20

40

60

80

1002%4%6%8%10%12%

a

0

20

40

60

80

0 5 10 15

6h12h18h24h30h

Time (h)

b

Fig. 3 – Changes of CHY vs. time at 37 8C, initial pH 6.0 and

substrate concentration 20 g/l (a) for the substrate soaked

by ammonia liquor for 24 h at the concentrations of 2, 4, 6,

8, 10 and 12% and (b) for the substrate soaked by 6%

ammonia liquor for 6, 12, 18, 24 and 30 h.

b

0

1

2

3

4

5

6

7

8

6 12 18 24 30

AH

PR (

ml/g

TS/

h)

Soaking time (h)

0

1

2

3

4

5

6

7

2 4 6 8 10 12

AH

PR (

ml/g

TS/

h)

Ammonia liquor concentration (%)

a

Fig. 4 – Effect of ammonia liquor concentration on AHPR at

the fixed soaking time of 24 h (a); effect of soaking time on

AHPR at the fixed ammonia liquor concentration of 6% (b).

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 5 ( 2 0 1 0 ) 3 0 5 8 – 3 0 6 4 3061

crystallization of cellulosic materials, but also prevent from

some adverse reactions resulting in the decrease of reducing

sugar.

According to the results of fermentation experiments

under various pretreatments, an optimal pretreatment

condition for AP was suggested as follow: AP was soaked in

ammonia liquor of 6% for 24 h at room temperature.

3.2. Effect of initial pH on H2 production

Previously reported results showed that initial pH had influ-

ence on the fermentative H2 production [21]. Therefore, the

effect of initial pH on H2 production was investigated at 37 �C

and fermentation substrate concentration 20 g/l with the AP

soaked in ammonia liquor of 6% for 24 h as the fermentation

substrate. Fig. 5 illustrates the changes of CHY (a) and pH (b)

verse time at the initial pH values of 5.0, 6.0, 6.5, 7.0, 7.5, 8.0

and 9.0. It is obvious from Fig. 5a that the initial pH affected H2

yield, e.g., the CHY increased from 42.71 to 90.06 ml/g TS with

the increase of initial pH from 5.0 to 7.0, and the difference of

CHY between the initial pH 6.0 and 7.0 was slight. A CHYm of

90.06 ml/g TS occurred at the initial pH 7.0. But as the initial pH

was further increased, the CHY decreased gradually, and

a lowest CHY of 19.53 ml/g TS was observed at the initial pH 9.

Meanwhile, the initial pH also had influence on the lag time

(Fig. 5b), e.g., the lag time was only 5.5 h at the initial pH 7.0. At

the initial pH 9.0, the lag time prolonged to 13 h and H2

production stopped when pH dropped to around 6.0. In addi-

tion, the initial pH had effects on AHPR and the maximum H2

percentage of mixing gas. As can be seen from Fig. 6,

a maximum AHPR of 6.39 ml/g TS/h and H2 percentage of 53%

both were obtained at the initial pH 7.0. The results show that

too low or high initial pH did not favor the fermentative H2

production from AP by the river sludge. Because the envi-

ronmental pH has great influence on the vital activities of H2-

producing bacteria, it not only affects the absorption of

nutrient through changing the cell membrane charge, but also

influences enzyme activity in metabolic process [22].

3.3. Effect of temperature on H2 production

The AP soaked in ammonia liquor of 6% for 24 h was used as

the fermentation substrate to investigate the effect of

temperature on the fermentative H2 production at initial pH

7.0 and fermentation substrate concentration 20 g/l. Fig. 7

shows the effects of temperature on the CHYm and AHPR (a),

and the changes of H2 percentage of mixing gas with time at

various temperatures (b). As can be seen from Fig. 7a, the

temperature seriously affected the fermentative H2 produc-

tion from the AP. As the temperature increased from 33 to

5

6

7

8

0 5 10 15 20 25 30 35

pH=5.0pH=6.0pH=6.5pH=7.0pH=7.5pH=8.0pH=9.0pH

Time (h)

0

20

40

60

80

100pH=5.0pH=6.0pH=6.5pH=7.0pH=7.5pH=8.0pH=9.0

CH

Y (

ml/g

TS)

a

b

Fig. 5 – Changes of (a) CHY and (b) pH vs. time at 37 8C and

substrate concentration 20 g/l for the initial pH values of

5.0, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0.

0

1

2

3

4

5

6

7

8

0

10

20

30

40

50

60

4 5 6 7 8 9 10

AHPR

Maximum H2 percentage

AH

PR (

ml/g

TS/

h)

Maxim

umH

2percentage

ofm

ixinggas

(%)

Initial pH

Fig. 6 – Effects of initial pH values on AHPR and the

maximum H2 percentage of mixing gas at 37 8C and

substrate concentration 20 g/l.

b

0

10

20

30

40

50

60

Hyd

roge

n pe

rcen

tage

(%

)

a

0

20

40

60

80

100

0

1

2

3

4

5

6

7

8

33 35 37 39 41

CHYm

AHPR

CH

Ym

(ml/g

TS)

AH

PR

(ml/g

TS

/h)

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 5 ( 2 0 1 0 ) 3 0 5 8 – 3 0 6 43062

37 �C, the CHYm and AHPR gradually rose from 40.80 to

90.06 ml/g TS and from 3.57 to 6.00 ml/g TS/h, respectively.

However, a sharp decrease in both the CHYm and AHPR

occurred with further increasing the temperature from 37 to

41 �C, and H2 production was almost inhibited at 41 �C only

with a CHYm of 5.41 ml/g TS and an AHPR of 0.42 ml/g TS/h. It

is worth noting that the CHYm at 35 �C (86.08 ml/g TS) was

approximate to that at 37 �C (90.06 ml/g TS), but the AHPR at

35 �C (3.82 ml/g TS/h) was far less than that at 37 �C (6.00 ml/g

TS/h). This is because, in low-temperature condition, the

metabolic activity of H2-producing bacteria is relatively slow

resulting in a longer fermentation time. Furthermore, it can be

seen from Fig. 7b that the temperature also affected H2

percentage of mixing gas. The maximum H2 percentage at

each temperature was different, e.g., they were 50%, 53% and

22% at 33, 37 and 41 �C, respectively. This is because the

change of temperature affects the physiological activities of

H2-producing bacteria and further affects H2 production

capability due to the change in the metabolic pathway of H2-

producing bacteria. The results show that the optimal

temperature for the conversion of AP to H2 by fermentation

was 37 �C at the initial pH 7.0.

0 5 10 15 20 25

Time (h)

Fig. 7 – Effects of temperature on CHYm and AHPR at initial

pH 7.0 and substrate concentration 20 g/l (a); changes of H2

percentage of mixing gas vs. time at various temperatures

(33, 35, 37, 39 and 41 8C) (b).

3.4. Effect of fermentation substrate concentration on H2

production

To obtain the optimal fermentation substrate concentration,

the fermentative H2 production from the AP soaked in

ammonia liquor of 6% for 24 h with various substrate

concentrations was investigated at 37 �C and initial pH 7.0,

and the results were indicated in Figs. 8 and 9. It is apparent

from Fig. 8 that the CHYm increased with the increase of

substrate concentration, e.g., while the substrate concentra-

tion rose from 5 to 15 g/l, the CHYm rapidly increased from

46.31 to 101.08 ml/g TS. However, the CHYm declined to

0

20

40

60

80

100

120

0 5 10 15 20

5g/l

10g/l

15g/l

20g/l

CH

Y (

ml/g

TS)

Time (h)

Fig. 8 – Changes of CHY vs. time at 37 8C and initial pH 7.0

for the substrate concentrations of 5, 10, 15 and 20 g/l.

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 5 ( 2 0 1 0 ) 3 0 5 8 – 3 0 6 4 3063

90.06 ml/g TS as the substrate concentration further increased

to 20 g/l. From Fig. 9, a maximum AHPR of 8.08 ml/g TS/h was

obtained at the substrate concentration of 15 g/l. These results

show that the substrate concentration has a significant effect

on the fermentative H2 production. A relatively higher

concentration was favorable for H2 production, while a certain

concentration threshold was exceeded, it would result in the

increase of H2 partial pressure, and thus H2-producing

bacteria might shift their metabolism from H2 and VFAs to

alcohol and lactate production [23]. In addition, too high

substrate concentration would also cause the accumulation of

VFAs and a fall of pH, thus inhibiting the growth of H2-

producing bacteria [24]. So, the optimal substrate concentra-

tion for the fermentative H2 production from AP was 15 g/l

with a CHYm of 101.08 ml/g TS and an AHPR of 8.08 ml/g TS/h

(Fig. 9).

3.5. Biodegradation characteristics of substrate underthe optimal condition

Each species of fermentation bacteria has special fermenta-

tion types, such as butyric acid type, propionic acid type,

0

3

6

9

0 5 10 15 20 25

AH

PR (

ml/g

TS/

h)

Substrate concentration (g/l)

Fig. 9 – Effect of substrate concentration on AHPR at 37 8C

and initial pH 7.0.

ethanol fermentation, etc. Different fermentation types

produce different liquid end-products (VFAs and alcohols)

that can reflect the metabolic pathways of H2-producing

bacteria. When the available substrate was consumed up, H2

production stopped, while VFAs and alcohols remained in the

reactor. Under the optimal fermentation condition of this

work, the liquid end-products mainly consisted of acetic acid

(2055.65 mg/l), butyric acid (739.74 mg/l), ethanol (222.40 mg/l)

and propionic acid (121.41 mg/l), in which acetic acid

accounted for 65% of total liquid end-products and the

content of propionic acid was very low. So the acetic acid type

fermentation was dominant fermentation type during the

conversion of AP to H2 under the optimal fermentation

condition of this work.

4. Conclusions

The potential of AP as carbon source for the fermentative H2

production was investigated at various experiment condi-

tions. An optimal fermentation condition in this work was

suggested that the AP soaked in 6% ammonia liquor at room

temperature for 24 h was fermented at 37 �C, the initial pH 7.0,

and the fermentation substrate concentration 15 g/l, it would

produce a CHYm of 101.08 ml/g TS with an AHPR of 8.08 ml/g

TS/h. During the conversion of AP into H2, acetic acid, ethanol

and butyric acid were main liquid end-products. Future work

will focus on the fermentative H2 production from the enzy-

matic hydrolysates of pretreated AP.

Acknowledgments

The authors acknowledge the financial supports of the

National Hi-Tech. Research and development program (863) of

China (No. 2007AA05Z116), the National Natural Science

Foundation of China (No. 20873099, 20673082), the scientific

research foundation for ROCS, SEM. (No.2006331), the key

project of science and technology of Shaanxi Province

(No.2005k07-G2), and the natural science foundation of

Shaanxi education Committee (No.06JK167).

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