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Hydrogen production by anaerobic co-digestion of rice strawand sewage sludge

Mijung Kima, Yingnan Yang a,*, Marino S. Morikawa-Sakura a, Qinghong Wang a,Michael V. Lee b, Dong-Yeol Lee c, Chuanping Feng d, Yulin Zhou a, Zhenya Zhang a

aGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, JapanbWorld Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials

Science(NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, JapancEnvironmental Technology Team, GS Engineering & Construction, Namdaemun-Ro 5-Ga, Joong-Gu, Seoul 100-722, KoreadWater Resource and Environmental Engineering, China University of Geosciences, No.29 Xueyuan Road, Beijing 100083, China

a r t i c l e i n f o

Article history:

Received 22 June 2011

Received in revised form

19 October 2011

Accepted 22 October 2011

Available online 7 December 2011

Keywords:

Rice straw

Sewage sludge

Anaerobic co-digestion

Biohydrogen production

C/N ratio

* Corresponding author. Tel.: þ81 29 8538830E-mail address: yo.innan.fu@u.tsukuba.a

0360-3199/$ e see front matter Copyright ªdoi:10.1016/j.ijhydene.2011.10.116

a b s t r a c t

In this study, the rich carbon content of rice straw and the high nitrogen content of sewage

sludge make the straw a good potential substrate and the sludge a viable inoculum for

biohydrogen production. Two treatment conditions for the sewage sludge (raw and heat-

treated) were used in the present experiments. Batch test using a mixture of rice straw

and sewage sludge were carried out to investigate the optimum carbon to nitrogen (C/N)

ratio for effective biohydrogen production. The experimental results indicate that

untreated sludge could be used as the inoculum for efficient hydrogen production when

mixed with the appropriate proportion of rice straw. According to our results, biogas and

hydrogen production in all raw sludge cases ramped up more quickly and also exhibited

longer and higher hydrogen production in comparison with heat-treated cases. At the C/N

ratio of 25 in untreated sludge, hydrogen production was 33% higher than heat-treated one.

Additionally, under the same conditions, high and stable hydrogen content (58%) and the

maximal hydrogen yield (0.74 mmol H2/g-VS added straw) were obtained.

Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction grain. In particular, rice straw is one of the most abundant

Recently, biohydrogen has been attracting increasing attention

as a biofuel of the future because biohydrogen technology not

only constitutes a biofuel source, but also can be applied in the

disposal of various environmental pollutants, for instance,

sewage sludge, industrial or agriculture waste, and urban solid

waste [1,2]. Lignocellulosicmaterialshave ahighpolysaccharide

content (about 60%) containing three key structural compo-

nents: cellulose, hemicellulose, and lignin [3,4]. Also, lignocel-

lulosicmaterial used to produce biofuels does not competewith

; fax: þ81 29 8534922.c.jp (Y. Yang).2011, Hydrogen Energy P

agricultural wastes in Japan (9 million tons in 2006), with only

asmallquantityof rice strawbeingusedfor livestock feedstuffor

fertilizer. Themajority of the waste is plowed into the field, and

some is burned in open fields, causing air pollution [5].

Biofuels (ethanol and hydrogen) from lignocellulosic

materials, such as barley straw, wheat straw, rice straw, or

corn stalks, are an economically available renewable form of

energy. Previously, Komatsu et al. [6] investigated the feasi-

bility of anaerobic co-digestion of rice straw, pretreated with

water and enzymes, and sewage sludge for biogas production.

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

Table 1 e Characterization of raw materials used in theexperiments.

Parameter Rice straw Sewage sludge

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 ) 3 1 4 2e3 1 4 9 3143

Cheng et al. [7] reported the feasibility of hydrogen production

from lignocellulosic materials as a substrate using cultivated

mixed or pure hydrogen-producing bacteria. Nguyen et al. [4]

reported using a hyperthermophilic bacterium Thermotoga

neapolitana at 75 �C for five days to produce biohydrogen from

untreated rice straw and from rice straw pretreated with

ammonia and/or sulfuric acid. However, scaling up a biore-

actor for only pure hydrogen-producing bacteria is impractical

due to the sizable expenditure necessary for growth and

cultivation of the strain. Hence, practical biological hydrogen

production requires the elimination of the above pretreat-

ments for both substrate and inoculumwhile still maintaining

high yields. Until now, technologies useful for commercial-

izing biohydrogen production have not yet been clearly

defined. Therefore, basic research is critical in order to

advance the development of technologies useful for

commercializing biohydrogen production.

According to previous research, some hydrogen-producing

bacteria of the Clostridium species were reported to degrade

insoluble cellulose and cellulosic waste materials without any

chemical pretreatment [8,9]. In addition, Ueno et al. [10]

demonstrated a lack of detectable methane in the biogas

from cellulose by natural anaerobic microflora. We are inter-

ested in using rice straw directly with raw sewage sludge in

order to reduce the energy input and achieve cost-effective

hydrogen production.

In anaerobic biological processes, proper carbon to

nitrogen (C/N) ratio is important for efficient digestion.

Previously, Hills and Roberts [11] demonstrated that the

proper C/N ratio for anaerobic digestion was 25e30 from

lignocellulosic materials (C/N ratio of 35e118) mixed with

dairy manure (C/N ratio of around 12). Generally, organic

waste with high nitrogen content combined with lignocellu-

losicmaterial with high carbon content can provide a versatile

mixture for anaerobic processes that could be optimized for

each organic waste source to maximize the desired product.

As Kim et al. [12] reported biohydrogen production could be

enhanced by co-digestion of food waste and sewage sludge

due to the balanced C/N ratio. However, the desired C/N ratio

for efficient hydrogen formation by co-digestion of rice straw

and sewage sludge has not been reported in the literature.

Based on these requirements, we proposed a simple and

cost-effective hydrogen fermentation process without

pretreatment of substrate and inoculum, namely rice straw

and raw sewage sludge, as used in this study. The aim of this

study was to investigate the potential of biohydrogen

production from rice straw using sewage sludge in anaerobic

thermophilic digestion conditions, in order to (1) investigate

the possibility of biohydrogen production from raw rice straw

using sewage sludge directly, (2) compare the effects of raw

and heat-treated sewage sludge, and (3) estimate the

optimum C/N ratio in the anaerobic co-digestion process.

Total solids (%) 90.04 2.01

Volatile solids (%) 81.43 79.14

Carbon (%) 35.11 36.36

Nitrogen (%) 0.87 5.76

pH N.D. 6.35

N.D. - not determined. The percentages were calculated on the

basis of dry weight.

2. Materials and methods

2.1. Raw materials

Air-dried rice straw (species Koshihikari) was received from

a local rice farmer in Chiba-ken (Japan) in 2009. The rice straw

was chopped into 1e2 cm pieces and then milled and sieved

through a 2.0-mm screen and kept in a plastic bag at room

temperature until use. In this study, sewage sludge from the

wastewater treatment plant (Ibaraki, Japan) was used as the

inoculum, which was stored at 4 �C. The pH, alkalinity, and

volatile suspended solid (VSS) of the sewage sludge were 6.35,

4.8 g/l as CaCO3, and 7.4 g/l, respectively.

2.2. Heat treatment of inoculum

The heat treatment consisted of heating the sewage sludge at

100 �C for 15 min and then cooling to room temperature in

order to enrich the spore-forming bacteria of the Clostridium

species and inhibit hydrogen-consuming bacteria [13]. After

that, the activity of the microorganisms in the untreated and

heat-treated sludge was determined by measuring the ATP

concentration. Heat-treated sludge and untreated sludgewere

used as inocula in complementary experiments in order to

compare the effect on biohydrogen production with and

without heat treatment.

2.3. Batch experiments of anaerobic co-digestion of ricestraw and sewage sludge

Biohydrogen production from rice straw by bacteria naturally

present in sewage sludge was studied under thermophilic

anaerobic conditions. The sewage sludge was sieved through

a 2.0-mm screen in order to filter out impurities. With

different masses of rice straw mixed with sewage sludge, five

with different C/N ratios were prepared in the batch experi-

ments (Table 2). 150-ml of untreated sludge or heat-treated

sludge was put into 500-ml bottles (SIBATA) with 5, 14, 27, or

41 g of rice straw. Fermentation of sewage sludge without

added straw was used as the control. The contents of each

bottle were mixed and the initial pH was adjusted to 7.0 with

2 N sodium hydroxide. The bottles were sealed with silica gel

stoppers, and the air was purged with N2 to produce absolute

anaerobic conditions. The batch experiments were operated

without agitation at 55 �C (thermophilic conditions) for ten

days. The effect of the amount of rice straw added was eval-

uated based on the hydrogen content of the gas produced. The

hydrogen production was calculated as follows:

H ¼ ðPV1 � PV2Þ=A (1)

where H (ml H2/g-added straw) is the hydrogen production;

PV1 (ml) is the volume of potential daily hydrogen production

Table 2 e Details of batch experiments design withdifferent amounts of rice straw added to identical 150-mlvolumes of untreated sludge and heat-treated sludge.

Experiment Number Rice straw (g) TSa (%) C/Nb ratio

R1 0 2 6

R2 5 5 15

R3 14 10 20

R4 27 17 25

R5 41 23 30

H1 0 2 6

H2 5 5 15

H3 14 10 20

H4 27 17 25

H5 41 23 30

a TS, Total solid.

b C/N, carbon/nitrogen. Experiments for: R1, untreated sludge

(control); R2-R5, untreated sludge plus 5 g, 14 g, 27 g, and 41 g,

respectively of added rice straw; H1, heat-treated sludge (control);

H2-H5, 5 g, 14 g, 27 g, and 41 g of rice straw respectively added to

heat-treated sludge.

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 ) 3 1 4 2e3 1 4 93144

from adding the rice straw; PV2 (ml) is the volume of potential

daily hydrogen production from control; A (g) is the added rice

straw. Volatile fatty acid (VFA) content and pH, as well as the

biogas concentration and composition, were measured every

day as performance indicators. All of the results were reported

as averages from triplicate experiments.

2.4. Analytical methods

TS, volatile solid (VS), VSS and alkalinity were determined

according to standard methods [14]. The composition of the

biogas, including hydrogen, methane, and carbon dioxide,

was determined by gas chromatography (GC-8A, SHIMAZU,

Japan) using a thermal conductivity detector (TCD, 80 �C) anda Porapak Q column (60 �C) with N2 as the carrier gas. The

concentrations of metabolites such as acetate, propionate,

and butyrate were analyzed by HPLC (Jasco Co., Japan)

equipped with a UV/VIS, RI detectors, and a COSMOGEL 5C18-

AR-II Packed Column (4.6 � 250 mm) at 40 �C using 20 mM

phosphate buffer (pH 2.5) as the mobile phase. The sample

was centrifuged at 10,000 rpm for 10 min, after which the

supernatant was filtered (0.45 mm membrane) and the filtered

solution was immediately analyzed with a flow rate of 1.0 ml/

min. The pH value was measured by a pH meter (TES-1380).

The C and N contents were analyzed on a PerkineElmer 2400

CHN Elemental Analyzer. The ATP concentration was

measured by a Bac Titer-Glo� Microbial Cell Viability Assay

(Promega, USA).

3. Results and discussion

3.1. Characterization of raw materials

The initial physical and chemical characteristics of rice straw

and sewage sludge are summarized in Table 1. The rice straw

is a high-solids substrate, with an average total solid content

of 90.04%, while the sewage sludge contains little organics at

only 2.01%. The majority of the total solids present in rice

straw and sewage sludge were volatile solids, which account

for 81.43% and 79.14% of the total solid, respectively. Themain

elemental composition of the rice straw and sewage sludge

were 35.11% and 36.36% of carbon, and 0.87% and 5.76% of

nitrogen, respectively. The average C/N ratio of the rice straw

was 40, and that of the sewage sludge was 6. These values

indicate that rice straw has high carbon content while sewage

sludge has high nitrogen content, it should thus be easy to

adjust the optimal digestion C/N ratio to improve the bio-

hydrogen production.

3.2. Effects of heat treatment and added rice straw

Rice straw was used to produce hydrogen with untreated

sludge and heat-treated sludge. Fig. 1 shows the cumulative

biogas production from each of the experiments: R1 and H1

(control), R2 and H2 (5 g/150 ml), R3 and H3 (14 g/150 ml), R4

and H4 (27 g/150 ml), and R5 and H5 (41 g/150 ml) at initial pH

7.0 and constant temperature of 55 �C. Compared with the

control, high biogas production was obtained with the

addition of rice straw, indicating that the rice straw was

degraded through the fermentation process and converted to

biogas. In the untreated sludge cases (Fig. 1a), the cumulative

biogas production on day ten for the R1, R2, R3, R4, and R5

was 389, 794, 299, 673, and 813 ml, respectively. On the other

hand, in the heat-treated sludge cases (Fig. 1b), the cumu-

lative biogas production on day ten for the H1, H2, H3, H4,

and H5 was 24, 109, 262, 480, and 659 ml, respectively. It

showed that there is more biogas production in untreated

sludge case than in heat-treated case. In the R3 case, the

hydrogen content decreased significantly from the first day

(58% of the biogas) to day five (2%), and the cumulative

biogas production of 239 ml was completed on day five (Figs.

1a and 2a). After that, only minimal biogas was produced,

but the methane content increased slightly from 20% to 51%

by the end of the batch test (data not shown). In comparison,

the R2 case produced much more biogas (over about 450 ml)

was produced from the day six and the cumulative biogas

increased gradually which the main component of the biogas

was methane (about 65%). In contrast, both the R4 and R5

cases produced more biogas with a steady hydrogen content

until the end of the experiment. This means that the small

amount of rice straw during anaerobic co-digestion process

produce methane more easily than hydrogen. Therefore, in

co-digestion of the rice straw and sewage sludge, the

optimum proportion of both raw materials is very important

for biohydrogen production.

The cumulative hydrogen production and hydrogen

content are given in Fig. 2. In the untreated sludge cases

(Fig. 2a), when 27 g of rice straw (R4) was added, themaximum

hydrogen content accounted for around 65% of the biogas on

the first day, and maintained an average hydrogen content of

about 58% until the end of the batch test. Moreover, the

maximal cumulative hydrogen production was observed with

R4 to be 18 ml H2/g-added straw. In the cases of the R1, R2 and

R3, the biohydrogen production was low, and methane began

to appear from day two.

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 C

um

ula

tive

bio

gas p

rod

ucti

on (m

l)

Time (d)

b

H1 H2 H3 H4 H5

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Cu

mu

lati

ve b

ioga

s pro

duc

tion

(ml)

Time (d)

a

R1 R2 R3 R4 R5

Fig. 1 e Variation of cumulative biogas production with 5 g, 14 g, 27 g and 41 g of rice straw added to identical 150-ml

volumes of untreated sludge (a) or heat-treated sludge (b). Experiments for: R1 and H1 (control) representing untreated

sludge and heat-treated sludge, respectively, with no rice straw added; R2 and H2, 5 g/150ml; R3 and H3, 14 g/150ml; R4 and

H4, 27 g/150 ml; R5 and H5, 41 g/150 ml.

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 ) 3 1 4 2e3 1 4 9 3145

In the heat-treated cases (Fig. 2b), the maximum cumula-

tive hydrogen production was observed with H4 and was

measured to be 12 ml H2/g-added straw, which is 33% lower

than in untreated case. In the heat-treated case of H4, the

hydrogen content was around 60% for only four days, and

then sharply decreased. Additionally, in experiment H1 and

R1, almost no hydrogen production was observed. It means

that adding rice straw as a substrate is an important factor in

successful biohydrogen production in this study.

In the case of R4, the hydrogen production was about 33%

higher than when heat-treated sludge was used (H4). In

addition, R4 showed dramatically enhanced biohydrogen

production in comparisonwith H4, and also exhibited a longer

period of hydrogen production.Moreover, the volatile solids in

the reactors of the R4 and H4 experiments on day ten were

reduced by 19% and 16%, respectively, relative to the starting

value. In all experiments, during fermentative hydrogen

production from rice straw with sewage sludge, the reactor of

R4 obtained not only the maximal hydrogen production

potential and hydrogen yield, but also the highest VS reduc-

tion efficiency. Besides that, from the experiment of R4, the

highest hydrogen production of 18 ml/g-TS (21 ml/g-VS) from

the rice straw was obtained using raw sewage sludge to

provide themicrobes. This is higher than the prior studies that

use no pretreatment of lignocellulosic materials. Zhang et al.

[15] and Li and Chen [16] presented hydrogen production of 3

ml/g-VS and 9 ml/g-VS, respectively from corn stalks using

cultivated mixed or pure hydrogen-producing bacteria.

Nasirian et al. [17] reported hydrogen production of 6 ml/g-VS

from wheat straw using acclimated mixed hydrogen-

producing bacteria. These results indicated that raw sewage

sludge could be to provide hydrogen producing-bacteria for

efficient hydrogen production without any growth and culti-

vation of a specific strain when mixed with the appropriate

proportion of rice straw.

In all experiments using untreated sludge with rice straw,

biogas and hydrogen production were significantly higher in

comparison with the heat-treated sludge cases. Heat treat-

ment is believed to be advantageous in repressing some

hydrogen-consuming bacteria like methanogens that do not

form spores, but alternatively, a decrease in microbial diver-

sity could be undesirable for decomposition of rice straw for

biohydrogen production, as was observed in the present

study. The raw sewage sludge should contain a variety of

microorganismswhich apply various enzymes to promote the

solubilization of the substrate. Hence, greater microbial

diversity may more rapidly break a substance down into its

components [18].

3.3. Optimal C/N-ratio in the co-digestion

Our study is the first report on biohydrogen production from

raw rice straw using sewage sludge directly from a waste-

water treatment plant that still contains the original pop-

ulation of anaerobic microbial strains. Table 3 lists the

hydrogen yields and the C/N ratio for various amounts of rice

0

4

8

12

16

20

0 2 4 6 8 10

Cu

mu

lati

ve H

2p

rod

uct

ion

(ml/g

-ad

ded

stra

w)

Time (d)

a

R1 R2 R3 R4 R5

0

20

40

60

80

100

0 2 4 6 8 10

H2co

nte

nt (

%)

Time (d)

0

4

8

12

16

20

0 2 4 6 8 10

Cu

mu

lati

ve H

2p

rod

uct

ion

(m

l/g-a

dd

ed st

raw

)

Time (d)

b

H1 H2 H3 H4 H5

0

20

40

60

80

100

0 2 4 6 8 10

H2co

nte

nt (

%)

Time (d)

Fig. 2 e Cumulative H2 production and H2 content for differentmasses of added rice straw in the untreated sludge (a) or heat-

treated sludge (b). Experiments for: R1 and H1 (control), representing untreated sludge and heat-treated sludge, respectively,

with no rice straw added; R2 and H2, 5 g/150 ml; R3 and H3, 14 g/150 ml; R4 and H4, 27 g/150 ml; R5 and H5, 41 g/150 ml.

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 ) 3 1 4 2e3 1 4 93146

straw used in conjunction with untreated and heat-treated

sludge. Out of all the experiments, the maximal hydrogen

yield was produced with 0.74 mmol H2/g-VS straw at the C/N

ratio of 25 (R4). Although the hydrogen yield in the heat-

Table 3 e Hydrogen yield from the experiments with differentuntreated sludge and heat-treated sludge.

Experiment number R1 R2 R3

Hydrogen yield (mmol H2/geVS added straw) 0.00 0.20 0.4

treated series was lower than in the raw sludge series, C/N

ratio of 25 still showed the highest hydrogen yield. According

to this result, the C/N ratio of 25 provides the most efficient

biohydrogen production. This agrees with the result from

amounts of rice straw added to identical 150-ml volumes of

R4 R5 H1 H2 H3 H4 H5

4 0.74 0.57 0.00 0.18 0.52 0.54 0.43

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 ) 3 1 4 2e3 1 4 9 3147

Hills and Roberts [11], who reported the optimum C/N ratio of

25 for biogas production from manure with rice straw in

anaerobic digestion. As a result, this optimal C/N ratio is

important and necessary to increase the process stability and

hydrogen yield.

3.4. pH change during the co-digestion period

Fig. 3 shows the change of pH during the fermentation process

of R1, R4, H1 and H4. In R1 and H1 experiments, the pH range

remained around 7.0 during the fermentation period, this pH

value is not a suitable for biohydrogen production, but rather

it is representative of the best conditions for methanogens

survival [19]. Actually, almost no hydrogen production was

detected in R1 and H1. In the R4 experiment, the hydrogen

production coincided with a pH range between 4.5 and 5.5

during fermentation period; in the H4 experiment this range

was between 5.5 and 6.0. As shown in Fig. 2, R4 displayed the

highest hydrogen production, which was higher and more

stable in comparison with the H4 experiments at the higher

pH. This indicates that the optimum pH of hydrogen produc-

tion is in the range of 4.5e5.5 for the co-digestion of rice straw

with sewage sludge.

This result is consistent with the pH range of 4.6e5.4 re-

ported by Zhang et al. [15] for biohydrogen production from

cornstalk wastes, also at initial pH 7.0 in batch experiments.

Additionally, Antonopoulou et al. [20] reported an optimum

pH range of 4.7e5.3 for biohydrogen production from sweet

sorghumextract in a continuous stirred tank bioreactor. Other

prior studies from batch experiments reported that the

optimum pH for hydrogen production was dependent on the

substrates, e.g., pH of 4.9 for sucrose [21], and pH of 4.5 for rice

slurry [22]. These studies suggested that biohydrogen

4.0

5.0

6.0

7.0

8.0

9.0

10.0

0 2 4 6 8 10

pH

Time (d)

R1 R4

H1 H4

Fig. 3 e Daily variations in pH with 27 g of rice straw added

to identical volumes of untreated sludge (R4) or heat-

treated sludge (H4). R1 and H1 (control experiments with

no rice straw added) use 150-ml of the untreated sludge

and heat-treated sludge, respectively.

production was possible at even lower controlled pH without

deterioration. Fermentative hydrogen production by anaer-

obic co-digestion of rice straw and sewage sludge at a pH

range of 4.5e5.5 is probably advantageous in terms of the

inhibition of hydrogen-consuming bacteria, based on our

investigation.

3.5. VFA change during the co-digestion period

Fig. 4 shows the VFA concentration during the fermentation

process of R4 and H4. During the co-digestion period, the

main VFA consists of acetate, butyrate, and propionate; and

the average concentration of acetate and butyrate accoun-

ted for approximately 90% of the total VFA. By summing the

acetate, butyrate, and propionate concentrations in the R4

and H4, the total VFA reached maximum yields on the

seventh day of 8301 and 6944 mg/l, respectively. In addition,

in the case of R4 (Fig. 4a), the concentration of butyrate was

about two times higher during the co-digestion period than

was observed in H4 (Fig. 4b). This correlates a higher yield

for hydrogen production with a higher butyrate content.

These results could indicate that VFA is generally related to

pH during the fermentation process. By comparing the

result of Fig. 4a and b, the high content of acetate (about

48%) and butyrate (about 44%) was also observed when the

pH was in the range of 4.5e5.5 in the R4 experiment during

fermentation period, while high content of acetate (about

60%) and low content of butyrate (about 30%) were observed

in pH range of 5.5e6.0 in the H4 experiment. This result was

agrees closely with a previous work [15], that also showed

the acetate and butyrate were at nearly the same level when

the pH of the mixture in the reactor was in the range of

4.6e5.4 for higher hydrogen production from cornstalk

wastes.

The propionate concentration decreased during the

fermentation process in R4, but it increased in H4. The

propionate may have been consumed by the activity of the

diverse microorganisms in the untreated sludge bioreactor as

part of the process to improve the hydrogen production. Ren

et al. [23] reported that the increase of propionate could be

a result of inhibiting hydrogen-producing bacteria, making it

undesirable for anaerobic fermentation processes. So this VFA

change during the co-digestion period is also an important

indicator for efficiency hydrogen production. After hydrogen

generation, the digestion residue (mainly acetate and butyrate

byproducts) could be readily converted into methane by

methanogens and there are many such studies [1,24,25].

Therefore, the wastes from the biohydrogen process can be

utilized further by methanogens for methane production in

the two-phase anaerobic process of continuous hydrogen and

methane generation.

3.6. Microorganism activity

According to above results, untreated sludge mixed with the

appropriate proportion of rice straw obtained higher and

stable hydrogen content during biogas production in

comparison with heat-treated sludge. In our previous work,

the microbial activity was indicated by the concentration of

ATP value, which is an indicator of metabolically active cells

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

1 2 3 4 5 6 7 8 9 10

VF

A c

once

ntr

atio

n (m

g/l)

Time (d)

a

Acetate Propionate Butyrate

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

1 2 3 4 5 6 7 8 9 10

VF

A c

once

ntr

atio

n (m

g/l)

Time (d)

bH4R4

Fig. 4 e VFA concentration during the co-digestion period in R4 (a) and H4 (b).

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 ) 3 1 4 2e3 1 4 93148

and an index of microbial density [26]. For this reason, ATP

concentration was measured on untreated sludge and heat-

treated sludge.

Fig. 5 shows the ATP concentration on raw sludge and

heat-treated sludge. The microbial activity of the raw sludge

was much higher than the heat-treated sludge. After heat

treatment, the ATP value was sharply decreased. Wang and

Wan [27] reported that a longer heat pretreatment may

repress the activity of the some hydrogen-producing bacteria.

Therefore, during biohydrogen production with different

masses of rice straw added to constant volumes of untreated

or heat-treated sludge for co-digestion, not only the diversity,

but also the activity of microorganisms could be a very

important factor.

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

raw sludge heat-treated sludge

AT

P (µ

mol

/l)

Fig. 5 e ATP values for the raw sludge and heat-treated

sludge.

4. Conclusion

In this research, a simple and effective biohydrogen produc-

tion process through co-digestion of rice straw and sewage

sludge was proposed. Based on the results, adding the

optimum proportion of rice straw as a substrate and main-

taining the diversity and activity of microorganisms are

important factors in successful biohydrogen production. It

can be concluded from the results that (1) the C/N ratio of 25 is

optimal for H2 production; (2) hydrogen production was

enhanced in the optimum pH range of 4.5e5.5; and (3) raw

sludge co-digestion process showed longer and higher H2

production when compared with heat-treated sludge.

Acknowledgments

This work was supported in part by Grant-in-Aid for Research

Activity Start-up 22880007 from Japan Society for the Promo-

tion of Science (JSPS). The author wishes to express gratitude

to Prof. Norio Sugiura and Prof. Motoo Utsumi for their kind

and excellent help with the experiments conducted in this

study.

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