Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 6528–6537

Anaerobic biohydrogen production from monosaccharidesby a mixed microbial community culture

Li Jianzheng a,*, Ren Nanqi a, Li Baikun b, Qin Zhi a, He Junguo a

a State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, 202 Haihe Road, Harbin 150090, Chinab Department of Civil and Environmental Engineering, University of Connecticut, Storrs, CT 06259, USA

Received 15 November 2005; received in revised form 12 November 2007; accepted 12 November 2007Available online 15 January 2008

Abstract

Monosaccharides (e.g. glucose and fructose) are produced from the hydrolyzation of macromolecules, such as starch, cellulose, hemi-cellulose and lignin, which are abundant in various industrial wastewaters. The elucidation of anaerobic activated sludge microbial com-munity utilizing monosaccharides will lay an important foundation for the industrialization of biohydrogen production. In this study, thehydrogen production by a mixed microbial culture on four monosaccharides (glucose, fructose, galactose and arabinose) was investi-gated in a batch cultures. The mixed microbial culture was obtained from anaerobic activated sludge in a continuous stirred-tank reactor(CSTR) after 29 days of acclimatization. The results indicated that glucose had the highest specific hydrogen production rate of 358 mL/g.g mixed liquid volatile suspended solid (MLVSS), while arabinose had the lowest hydrogen production rate of 28 mL/g.gMLVSS. Glu-cose also possessed the highest specific conversion rate to hydrogen of 82 mL/g glucose, while fructose had the highest specific conversionrate to liquid product of 443 mg/g fructose. Arabinose had the lowest conversion rates to both liquid products and hydrogen. Metabolicpathways and fermentation products were the major reasons for the difference in hydrogen production from these four monosaccharides.The complex fermentation pathways of arabinose reduced its hydrogen production efficiency and a long acclimation period (over 68 h)was required before the anaerobic activated sludge could effectively utilize arabinose in batch cultures.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Hydrogen; Fermentation; Mixed microbial community; Monosaccharide; Glycolysis

1. Introduction

Hydrogen, a renewable and clean energy, could lead tosocio-economic, environment-friendly and sustainabledevelopment. Currently, there are two types of biohydro-gen production: direct (convert solar energy into hydrogenthrough photosynthesis by blue algae) and indirect (anaer-obic fermentation of organic substrates) microbial meta-bolic pathways. Several breakthroughs have beenachieved in the fundamental understanding of biohydrogenproduction, including, the isolation of microbial strainswith high hydrogen production capability, identification

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.11.072

* Corresponding author. Tel.: +086 451 86283761; fax: +086 45186282103.

E-mail address: jianzhengli@hit.edu.cn (J. Li).

of high-efficiency and low-cost carbon sources, and optimi-zation of the microbial fermentation process (Ren et al.,1995,1997,2003; Taguchi et al., 1995; Rachman et al.,1997,1998; Li and Ren, 1998; Wang et al., 2003). In themean time, the optimization and industrial application ofbiohydrogen production have been extensively investigated(Benemann, 1996; Ueno et al., 1996; Tanisho et al., 1998;Lay et al., 1999; Lin and Chang, 1999; Li et al., 2002).

Biohydrogen production from wastewater through fer-mentation is carried out by anaerobic acidogenic bacteriawith highly diverse fermentation characteristics and hydro-gen production capabilities (Tanisho and Ishiwata, 1995;Ueno et al., 1996; Lay et al., 1999; Lin and Chang, 1999;Nielsen et al., 2001; Yu et al., 2002; Yokoi et al., 2002; Liet al., 2002, 2004a). Fermentation performance is dependenton a number of factors, such as temperature, pH, alkalinity,

J. Li et al. / Bioresource Technology 99 (2008) 6528–6537 6529

oxidation-reduction potential (Li and Ren, 1998). The vari-ations of these factors result in diverse microbial communi-ties, which finally lead to different fermentation types (Liet al., 2002; Wang et al., 2003; Qin et al., 2003; Li et al.,2004a). There are mainly four fermentation types in theanaerobic acidogenesis of organic matters (e.g. glucose),namely acetic acid fermentation, propionic acid type fer-mentation, butyric acid type fermentation and ethanol typefermentation (Zoetemeyer et al.,1982; van den Heuvel et al.,1988; Fox and Pohland, 1994; Ren et al., 1997; Himmi et al.,2000. Many microbial communities exhibit an acetic acidfermentation with acetic acid as the major product (Reac-tion (1)) (Datta, 1981; Chan and Holtzapple, 2003; Thanak-oses et al., 2003). The major products of propionic acid typefermentation are propionic and acetic acids (Reactions (1)and (2)), while the products of butyric acid type fermenta-tion include butyric and acetic acids (Reactions (1) and(3)). As for ethanol type fermentation, ethanol and aceticacid are the primary fermentation products (Reactions (1)and (4)) (Fox and Pohland, 1994; Ren et al., 1997).

C6H12O6 þ 4H2Oþ 2NADþ ! 2CH3COO� þ 2HCO�3 þ 2NADHþ 2H2 þ 6Hþ DG00 ¼ �215:67 kJ=mol ð1ÞC6H12O6 þ 2NADH! 2CH3CH2COO� þ 2H2Oþ 2NADþ DG00 ¼ �357:87 kJ=mol ð2ÞC6H12O6 þ 2H2O! CH3CH2CH2COO� þ 2HCO�3 þ 2H2 þ 3Hþ DG00 ¼ �261:46 kJ=mol ð3ÞC6H12O6 þ 2H2Oþ 2NADH! 2CH3CH2OHþ 2HCO�3 þ 2NADþ þ 2H2 DG00 ¼ �234:83 kJ=mol ð4Þ

Eff

luen

t

Feed tank

Waterlock

Biogas meter

Pump

Baffle

Biogas outlet

Water level

Thermostat sensor Motor

Blender

Fig. 1. Schematic diagram of the CSTR reactor for the acclimatization ofanaerobic activated sludge.

Reactions (1)–(4) show that hydrogen is generated fromacetic acid, butyric acid and ethanol fermentations, notfrom propionic acid fermentation. However, propionicacid fermentation supposedly occur much easier than otherfermentation types, due to its low Gibbs free-energy changeðDG00Þ (Reaction (2)) (Zoetemeyer et al., 1982; Ren et al.,1997). Propionic acid type fermentation is concurrent withother fermentation types capable of producing hydrogen(e.g. acetic acid-, butyric acid- and ethanol-type fermenta-tions) in a mixed microbial community. Therefore, hydro-gen can still be produced from anaerobic fermentation eventhough the production of propionic acid was high. Currentstudies have been focused on butyric acid and ethanol fer-mentation for hydrogen bioproduction (Ueno et al., 1996;Lay et al., 1999; Lin and Chang, 1999; Li et al., 2002,2004a; Wang et al., 2002; Ren et al., 2003), insufficientinformation is available on the biohydrogen productioncharacteristics of mixed acid type fermentation.

Besides fermentation types, hydrogen production capa-bility is also directly related with fermentation substrates.Each substrate has its own fermentation pathways andend-products, which leads to different hydrogen yields(Taguchi et al., 1995; Woodward et al., 1996; Aoyamaet al., 1997; Wang et al., 2002). Several monosaccharides,including hexoses and pentoses, have been often used asfermentation substrates. They are the hydrolysate of a widevariety of macromolecules, such as starch, cellulose and

hemicellulose, and are abundantly available in variousindustries. Therefore, a study of anaerobic activated sludgemicrobial community utilizing different monosaccharideswill lay an important foundation for the industrializationof biohydrogen production.

This research was focused on examining biohydrogenproduction by anaerobic activated sludge fermentationfrom a highly concentrated organic wastewater. The hydro-gen production from four monosaccharides (glucose, fruc-tose, galactose and arabinose) was investigated in batchtests and compared using anaerobic activated sludgemicrobial community.

2. Methods

2.1. Acclimatization of anaerobic activated sludge microbecommunity in a continuous stirred-tank reactor

A 17 L continuous stirred-tank reactor (CSTR) with aneffective volume of 9.6 L was used to acclimatize activated

sludge to obtain a stable mixed fermentation communityfor batch culture study (Fig. 1). The temperature was main-tained at the level of 35 ± 1 �C. NaHCO3 was added to thefeeding solution to maintain pH of 6.5–7.5 in influent andto keep a pH level of 5.0 in the reactor. The feeding solu-tion containing diluted molasses, with a chemical oxygendemand (COD) concentration of 3000 mg/L, was pumpedinto the CSTR continuously. Hydraulic retention time(HRT) of the CSTR was 8 h. COD:N:P of the feeding solu-tion was maintained at an average ratio of 250:5:1 by add-ing synthetic fertilizer in order to supply microorganismswith adequate nitrogen and phosphorus. During theacclimatization period, the mixed liquor volatile suspendedsolid (MLVSS) in the reactor was approximately 13.5 g/L.

Rotary shaker water bath

Reactor

Liquidsampling port

Biogas canula

Gas measuringcollector

Gas sampling

Equilibriumbottle

Garden hose

Two-way

Fig. 2. Schematic diagram of batch culture tests for monosaccharides.

6530 J. Li et al. / Bioresource Technology 99 (2008) 6528–6537

When pH, biogas yield, hydrogen percentage and liquidfermentation products became stable in the CSTR reactor,a mixed microbial community of anaerobic activatedsludge was expected to be well developed (Li et al.,2004a,2004b). After the acclimatization, the mixed micro-bial community was inoculated into batch cultures forthe fermentation of monosaccharides.

2.2. Batch tests for the fermentation of monosaccharides

Batch cultures (Fig. 2) were used to compare the hydro-gen productivity of different substrates by anaerobic acti-vated sludge microbe community. The working volume ofthe Erlenmeyer flask was 500 mL, and the culture wasmaintained at a temperature 35 ± 1 �C in a rotary shakerwater bath. Initial substrate (monosaccharides) concentra-tion was set at 10 mg/L. Anaerobic activated sludge (mixedmicrobial community) was inoculated into the batch cul-ture at a concentration of 0.5 g MLVSS/L (gram mixedliquid volatile suspended solid (MLVSS) per liter). Severalminerals were added in the batch culture to meet therequirement for microbial growth (1.5 g KH2PO4, 2 g(NH4)2SO4, 0.1 g CaCl2 � 2H2O, 0.1 g MgCl2 6H2O, and3 mg FeSO4 7H2O in 1.0 L solution,). The total volumeof the batch culture was 500 mL, and pH was adjusted to6.0 using a 0.1 N NaOH solution. For every substratetested, the experiment was conducted with two parallel setsof duplicate batch cultures under identical experimentalconditions. In one of the duplicate cultures, the equilibriumbottle and gas measurement collector were filled with 20%NaOH solution to absorb CO2 in the biogas, so that thehydrogen content during the fermentation process could

Specific hydrogen production rate ¼ Total hydrogen produced in a batch testðmLÞReacted monosaccharideðgÞ � BiomassðMLVSSÞin a batch testðgÞ

be measured. In the other one of the duplicate cultures,the equilibrium bottle and gas measuring collector werefilled with water (pH 3.0) to measure the total volume ofthe biogas during the fermentation process. In order toensure anaerobic conditions in the batch culture, nitrogen

gas was spurged in the equilibrium bottle for 20 min beforethe batch culture operation started.

2.3. Analytical methods

COD and pH were measured daily in the CSTR. ThepH of batch cultures before and after batch tests were alsomeasured. Mixed liquid volatile suspended solid (MLVSS)analyses were carried out once a week. These analyseswere conducted according to standard methods (APHA,1995).

Biogas yield (m3/day) of the CSTR was measured dailyat room temperature by wet gas meter, and its constitu-ents were analyzed using a gas chromatography (SC-7,Shandong Lunan Instrument Factory, Qingdao, China).The gas chromatography was equipped with a thermalconductivity detector and a stainless steel column(2 m � 5 mm) filled with Porapak Q (50–80 meshes).Nitrogen was used as the carrier gas at a flow rate of40 mL/min. The dose of injected sample was 0.5 ml eachtime.

The analyses of volatile fatty acids (VFAs) and ethanolin the fermentation solution were performed using anothergas chromatography (GC112, Shanghai Anal. Inst. Co.,Shanghai, China) with a hydrogen flame ionization detec-tor and a stainless steel column (2 m � 5mm) packed withsupporter of GDX-103 (60–80 meshes). The operation ofthe stainless steel column was amenable to a temperatureprogramming process within 100–200 �C. N2 was used asthe carrier gas at a flow rate of 50 mL/min, H2 was thecombustion gas at 50 mL/min, and O2 was the combus-tion-supporting gas at 500 mL/min.

The concentration of saccharides was measured by a sul-furic acid-phenol method (He, 2004). Sulfuric acid-phenolreacted with saccharides (e.g. hexose, hentose) and gener-ated different colors. Pentose had the highest adsorptionat wavelength of 490 nm, while hexose and uronic acidhad the highest adsorption at 480 nm. The adsorption val-ues were in a linear relationship with saccharideconcentration.

2.4. Specific hydrogen production rate

Hydrogen productivity of each monosaccharide was cal-culated as specific hydrogen production rate (mL H2/gsubstrate.gMLVSS):

2.5. Specific conversion rate to liquid products/hydrogen

The conversion efficiency of each monosaccharide toliquid products and hydrogen was calculated as specificconversion rates:

Specific conversion rate to liquid productsðg=gÞ ¼ Liquid products concentrationðmg=LÞ � batch test volumeð500 mLÞReacted monosaccharideðgÞ

J. Li et al. / Bioresource Technology 99 (2008) 6528–6537 6531

In this study, ethanol, acetic acid, propionic acid, buty-ric acid and valeric acid were measured as liquid products.

Specific conversion rate to hydrogenðmL=gÞ ¼ Total volume o

R

0

2

4

6

8

10

0 10 15

Time (

Bio

gas

yiel

d (L

/d)

a

0

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Influent

a

b

0

500

1000

1500

2000

2500

VFA

s (m

g/L

)

Acetic acid Pr

Et

5

Valeric acid

Fig. 3. The operational status of the continuous stirred-tank reactor (CSTR)effluent pH (a), volatile fatty acids (VFAs) and ethanol (b), biogas yield and h

Reacted monosaccharide equals the difference between theinitial and final amounts of monosaccharide in batch tests.

f hydrogen produced in a batch testðmLÞeacted monosaccharideðgÞ

20 25 30

d)

0

10

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30

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50

Hyd

roge

n pe

rcen

tage

(%)

Biogas yield

Hydrogenpercentage

Effluent

opionic acid Butyric acid

hanol Total

during acclimatization process of anaerobic activated sludge. Influent andydrogen percentage (c).

6532 J. Li et al. / Bioresource Technology 99 (2008) 6528–6537

2.6. Statistic analysis

Liquid fermentation products (acetic acid, propionicacid, butyric acid, valveric acid, and ethanol) and hydrogenyield were analyzed after the lag stage in the monosaccha-

0

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Biogas

Hydrogen

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y = 0.8x

R2 = 0

0

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0

c

b

a

d

Cum

ulat

ive

biog

as a

nd h

ydro

gen

yiel

ds (

mL

)

0 5 10 2015

0 2015105

2015105

605040302010Time(ho

Fig. 4. The production of biogas from four monosaccharides by anaerobic aarabinose (d). The lag stage for glucose, fructose, galactose and arabinose waacceleration stage for each monosaccharide.

ride batch cultures. Hydrogen production rate in the accel-eration stage was calculated by linear regression inMicrosoft EXCEL. It was considered to be equal the gra-dient of the equation (Y = aX � b) obtained from the lin-ear regression.

y = 15.4x - 200

R2 = 0.98

y = 20.4x - 273

R2 = 0.98

y = 6.9x - 91

R2 = 0.99

- 57

.98

4540353025

45403530 25

4540353025

130120110100908070urs)

ctivated sludge in batch tests. Glucose (a), fructose (b), galactose (c) ands 13, 13, 17, and 68 h, respectively. A linear regression was made for the

Table 1Hydrogen production by anaerobic activated sludge fermentation fromfour monosaccharides in batch test (the culturing period for glucose,fructose and galactose was 45 h, and the culturing period for arabinosewas 121 h)

Parameters Glucose Fructose Galactose Arabinose

Inoculated biomass(gMLVSS/L)

0.46 0.69 0.46 0.69

Lag stage (hours) 13 13 17 68Reacted monosaccharide

(g)2.1 2.3 2.8 3.5

Accumulative biogas yield(mL)

256 177 192 59

Accumulative hydrogenyield (mL)

173 139 133 34

Hydrogen percentage inbiogas (%)a

68 ± 5 78 ± 3 69 ± 3 58 ± 5

Conversion rate tohydrogen (mL/gsubstrate)

82.4 60.4 47.5 9.7

Hydrogen production rate(mL/h)b

20.4 15.4 6.9 0.8

Specific hydrogenproduction rate (mL/gsubtrategMLVSS)

358 175 206 28

a The hydrogen percentage in biogas was obtained after the lag stage.b The hydrogen production rate was calculated through the linear

regression of the acceleration stage data (Fig. 4).

J. Li et al. / Bioresource Technology 99 (2008) 6528–6537 6533

3. Results and discussion

3.1. Acclimatization of propionic acid type fermentation

microbial consortium in the CSTR

In order to accelerate the acclimatization of the anaero-bic activated sludge, pH of the feeding solution was keptaround 5.5 � 6.0 and pH in the CSRT reactor was main-tained at 5.0 (Fig. 3a) based on the previous results (Renet al., 2002; Qin et al., 2003). When operated at tempera-ture of 35 ± 1 �C and organic loading rate (OLR) of9 kg/m3 d (HRT 8 h, influent COD: 3000 mg/L), the CSTRtook 24 days to achieve its stable status. Propionic acidaccounted for approximately 25% among liquid fermenta-tion products from the 7th to the 15th days (Fig. 3b). Thisshort stable period ended with the drastic changes of otherliquid fermentation products, especially butyric acid, fromthe 7th to the 24th day. The concentration changes of thesefermentation products indicated a shifting phase for differ-ent acidogenic microbial communities in the CSTR.During the stabilized stage (24th–29th days), the totalamount of liquid fermentation products was 1627 ± 107mg/L, containing ethanol (83 ± 21 mg/L), acetic acid(465 ± 64 mg/L), propionic acid (1018 ± 59 mg/L), butyricacid (38 ± 20 mg/L) and valeric acid (18 ± 6 mg/L) withthe percentages of 5 ± 1%, 28 ± 3%, 62 ± 2%, 2 ± 1%and 1 ± 0.4%, respectively.

Biogas production and hydrogen percentage in biogassteadily increased in the CSTR reactor during the first14 days and reached 3.26 L/d and 4.6% on the 14th day(Fig. 3c), and then dropped rapidly. During the stabilizedstage (24th–29th days), biogas yield was 1.13 ± 0.57 L/dand hydrogen was only 6.7 ± 1.2% in biogas. This resultcorresponded with the fermentation theory that propionicacid fermentation exhibited a low hydrogen productioncapability (Reaction (2)). Because other fermentationpathways (e.g. ethanol, butyric, and acetic acid type fer-mentations) carried out by the mixed microbial consor-tium could produce hydrogen (Reaction 1, 3, and 4), itwas expected that this mixed microbial consortium fromthe CSTR should still produce hydrogen in batch culturetests.

3.2. Hydrogen production from four monosaccharides in

batch tests

Batch test results showed that the production of biogasand hydrogen in the mixed microbial community fermenta-tion was different for four substrates tested. The biogasproduction capability on glucose was the highest amongthe substrates, with the accumulative hydrogen yield of173 mL (Fig. 4) and the specific hydrogen production rateof 358 mL/g.gMLVSS (Table 1). The production capabilityfrom both fructose and galactose were observably lowerthan glucose, with the specific hydrogen production ratesof 175 and 206 mL/g.gMLVSS (Table 1), respectively. Spe-cific conversion rates from glucose, fructose and galactose

to hydrogen were 82.4, 60.4 and 47.5 mL/g substrate,respectively. During the acceleration stage, the hydrogenproduction rates of glucose, fructose and galactose were20.4, 15.4, and 6.9 mL/h, respectively (Fig. 4a–c).

Arabinose fermentation by the anaerobic activatedsludge exhibited a lag stage of 68 h (Fig. 4d), which wasobservably longer than the other three substrates (glucose,fructose and galactose, Table 1). In addition, the biogasproduction capability of arabinose fermentation was low.After the fermentation ended at 121 h, the accumulativehydrogen yield was 34 mL and specific hydrogen produc-tion rate was 28 mL/g.gMLVSS (Table 1). Specific conver-sion rate of arabinose to hydrogen was 9.7 mL/g substrate,the hydrogen production rate was 0.8 mL/h (Fig. 4d), andhydrogen was only 58% in the biogas (Table 1). The varia-tion of the hydrogen production capabilities of monosac-charides might be attributed to different fermentationpathways.

3.3. Liquid fermentation products from monosaccharides in

batch tests

Each substrate not only possessed different hydrogenproductivities, but also generated different amounts ofliquid products. When the mixed microbial communitywas cultured for 45 h, the total amount of the liquid prod-ucts from fructose fermentation was 2038 mg/L (Fig. 5b)with propionic acid and acetic acid as major liquid products(Table 2), highest among the four substrates. Total liquidproducts from galactose fermentation were 2023 mg/L(Fig. 5c). Although glucose had the highest hydrogen

6534 J. Li et al. / Bioresource Technology 99 (2008) 6528–6537

production, its liquid products ranked the third as 1577 mg/L (Fig. 5a). The total amount of arabinose liquid productswas 1487 mg/L (Fig. 5d), the lowest in the four substrates.Although the amounts of liquid fermentation products var-

0

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0 10 15 20

Acetic acid

Propionic acid

Butyric acid

Valeric acid

Ethanol

Total

Liq

uid

ferm

enta

tion

prod

ucts

(m

g/L

)

c

b

a

d

5

5

5

Fig. 5. Liquid fermentation products from four monosaccharides by anaerobicarabinose (d).

ied with monosaccharide in the batch tests, propionic acidand acetic acid were the major liquid product species (Table2), which sustained the stability of the inoculated anaerobicactivated sludge from the CSTR reactor.

25 30 35 40 45

25 30 35 40 45

60 75 105 120

(hours)

25 30 35 40 45

90

activated sludge in batch tests. Glucose (a), fructose (b), galactose (c) and

J. Li et al. / Bioresource Technology 99 (2008) 6528–6537 6535

3.4. The comparison of specific conversion rates of

monosaccharides to liquid products and hydrogen

Several factors, such as metabolic pathways, fermenta-tion products, and enzymes, could lead to the different con-version rates from monosaccharides to hydrogen. Becausethe mixed microbial community used for batch tests wascollected from the same source (the CSTR reactor), itwas assumed that the initiative enzyme levels were the samein the cultures of these four monosaccharides. Therefore,the main reasons for the different specific substrate conver-sion rates to hydrogen are more than likely the metabolicpathways.

Monosaccharide fermentation was accomplished by gly-colysis. Three hexoses: glucose, fructose, and galactose,have a similar fermentation pathway (Hames and Hooper,2000). They were initially converted to fructose 6-phos-phate through one reaction or multiple reactions. Fructose6-phsphate was then converted to 3-phosphoglyceralde-hyde by the catalysis of fructose diphosphate aldolase.Although the rest metabolic pathways were similar forthese three hexoses, their hydrogen yields were different.From the data illustrated in Table 3, it can be derived thata high conversion to liquid products led to a low conver-sion to biogas and hydrogen. This could explain thatalthough fructose had a higher hydrogen percentage in bio-

Table 3Specific liquid product conversion rate (g/g) and hydrogen conversion rate (m(The culturing period for glucose, fructose and galactose was 45 h, and the cu

Parameters

pH Initial valueFinal value

Monosaccharide concentration (g/L) Initial concentrationFinal concentrationReacted value

Fermentation liquid products (mg)The percentage of propionic acid and valveric acid in liquid products (%)Specific conversion rate to liquid product (mg/g)Accumulative biogas yield (mL)Accumulative hydrogen yield (mL)Hydrogen percentage in biogas (%)Specific conversion rate to biogas (mL/g)Specific conversion rate to hydrogen (mL/g)

Table 2Liquid products from the fermentation of four monosaccharides in batch testsculturing period for arabinose was 121 h)

Parameters Glucose

Lag stage (hours) 13Total fermentation liquid products (mg/L)a 1577Acetic acid (mg/L)a/percentage (%)b 489/30 ± 2Propionic acid (mg/L)a/percentage (%)b 571/24 ± 5Butyric acid (mg/L)a/percentage (%)b 428/34 ± 4Valeric acid (mg/L)b/percentage (%)b 25/1 ± 0.8Ethanol (mg/L)a/percentage (%)b 64/5 ± 1

a Total fermentation liquid products were the accumulative values throughob The hydrogen percentage in biogas was obtained after the lag stage.

gas than glucose and galactose, its higher conversion rateto liquid products resulted in the lower conversion rate tohydrogen.

The constituent of liquid fermentation products wasanother reason for the different substrate conversion rates.The production of propionic acid did not generate hydro-gen (Reaction (2)), and the production of valeric acidwas not through glycolysis pathway and did not generatehydrogen either. A high level of these two liquid productsindicates a fermentation pathway insufficient at hydrogenproduction. The percentages of propionic acid and valericacid together were 25% for glucose, 33% for fructose,36% for galactose and 38% for arabinose (Table 3). Thisexplained the lower conversion rates of fructose and galact-ose to hydrogen than that of glucose.

Arabinose had the lowest hydrogen conversion rate of10 mL/g. There might be two possible reasons. First, themetabolic pathway of arabinose (a pentose) was differentfrom hexoses (Rosenberg, 1980; Hames and Hooper,2000). The fermentation of arabinose required variousenzymes, and hence its biochemical reactions were rela-tively complex. Arabinose was converted to xylulose 5-phosphate and ribose 5-phosphate by the isomerase,kinase, and epimerase. Xylulose 5-phosphate and ribose5-phosphate were subsequently converted to glyceraldehy-des 3-phosphate and fructose 6-phosphate by transaldolase

L/g) from the fermentation of four monosaccharides after the lag stagelturing period for arabinose was 121 h)

Glucose Fructose Galactose Arabinose

6.0 6.0 6.0 6.03.4 3.4 3.6 4.510.1 10.7 10.5 9.95.9 6.1 4.9 2.94.2 4.6 5.6 7.0788 1019 1011 74425 33 36 38375 443 361 212256 177 192 59173 139 133 3468 ± 5 78 ± 3 69 ± 3 58 ± 5122 77 68 1782 60 47 10

(The culturing period for glucose, fructose and galactose was 45 h, and the

Fructose Galactose Arabinose

13 17 682038 2023 1488710/36 ± 1 738/36 ± 2 644/44 ± 1814/32 ± 6 667/31 ± 1 489/33 ± 1357/21 ± 6 308/15 ± 3 142/9 ± 0.228/1 ± 0.5 119/5 ± 2 73/4 ± 0.6129/9 ± 3 191/14 ± 4 140/8 ± 2

ut the culturing period.

6536 J. Li et al. / Bioresource Technology 99 (2008) 6528–6537

and transketolase. Thereafter, glyceraldehydes 3-phosphateand fructose 6-phosphate entered the glycolysis pathwayand continued their degradation. The complexity of meta-bolic pathways and the involvement of various enzymes ledto the long lag stage (68 h, Fig. 4d) and the low liquid prod-uct yields (212 mg/g, Table 3) and biogas yield (34 mL,Table 3). Second, the percentage of propionic acid andvaleric acid produced from arabinose was 38% in the liquidproducts, which was more than that of glucose (25%), fruc-tose (33%) and galactose (35%) (Table 3). However, theconversion of arabinose to propionic acid and valeric aciddid not produce hydrogen, which led to the low hydrogenpercentage (58%) in the biogas of arabinose (Table 3).

The integrated analysis of the lag stage, specific hydro-gen production rate, and specific conversion rate to liquidproducts and hydrogen revealed that glucose had the high-est production rate and conversion rate for hydrogen, andfructose had the highest production rate and conversionrate for liquid, while arabinose exhibited the lowest pro-duction rate and conversion rate and the longest lag stage.Fermentation pathways and product constituents were themajor reason for the difference in hydrogen productionfrom these four monosaccharides. The hydrogen produc-tion capabilities on the four monosaccharides were in theorder as follows: glucose > fructose > galactose > arabi-nose. Therefore, this study recommended that glucose,fructose and galactose can be used for substrates for biohy-drogen production by anaerobic activated sludgefermentation.

4. Conclusion

An extensive biohydrogen production study was con-ducted on anaerobic activated sludge fermentation fromfour monosaccharides (glucose, fructose, galactose andarabinose). Several major conclusions can be drawn fromthis study. First, although propionic acid fermentationcould not produce hydrogen theoretically, it could occurin an anaerobic fermentation system and could still pro-duce hydrogen when concurrent with other metabolic path-ways by a mixed microbial community. Second, batchculture tests indicated that hydrogen production capabili-ties varied among these four monosaccharides, with glu-cose having highest hydrogen production rate of 358 mL/ggMLVSS, and arabinose having lowest production rateof 28 mL/ggMLVSS. Specific conversation rate to hydro-gen from glucose was more than thirteen times as arabi-nose. Fermentation pathways and product constituentswere the major reasons for the difference in hydrogen pro-duction from these four monosaccharides. Complex fer-mentation pathways of arabinose reduced its hydrogenproduction efficiency. Third, the conversion abilities ofanaerobic activated sludge from monosaccharides tohydrogen were drastically different. The lag stages of hex-oses fermentation (glucose, fructose, and galactose) in thebatch cultures were less than 17 h, while pentose (arabi-nose) fermentation exhibited a lag stage as long as 68 h.

The hydrogen production capabilities were in the orderas follows: glucose > fructose > galactose > arabinose.Fourth, a monosaccharide cannot have both high liquidproduction rate and high hydrogen production rate, dueto the mass balance of liquid products and gas productsfrom the fermentation reactions. Fructose had the highestliquid production rate, while glucose had the highesthydrogen production rate. Monosaccharides (glucose, fruc-tose, galactose and arabinose) tested in this study can beproduced from the hydrolyzation of a wide variety of mac-romolecules that are abundantly available in various indus-trial wastewaters. Therefore, the results of the study will beimportant to a certain extent for the industrialization ofbiohydrogen production.

Acknowledgements

The authors would like to thank the National NaturalScience Foundation of China (Contract No. 50378025),the National High Technology Research and Develop-ment Program of China (863 Program, Grant No.2006AA05Z109) and Provincial Science Foundation ofHeilongjiang (Grant No. E0305) for their supports for thisstudy.

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