international journal of hydrogen energy 33 (2008) 43094317

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Fermentative hydrogen production by microbial consortium

Sandra I. Maintinguer*, Bruna S. Fernandes, Iolanda C.S. Duarte, Nora Katia Saavedra, M. Angela T. Adorno, M. Bernadete Varesche
Department of Hydraulics and Sanitation, School of Engineering of Sa Carlos, University of Sa Paulo, Av. Trabalhador Sa o o o-carlense, 400, 13566-590 Sa Carlos-SP, Brazil o

article info
Article history: Received 17 April 2008 Received in revised form 4 June 2008 Accepted 5 June 2008 Available online 8 August 2008 Keywords: Hydrogen production Sucrose Fermentation Clostridium sp

abstract
Heat pre-treatment of the inoculum associated to the pH control was applied to select hydrogen-producing bacteria and endospores-forming bacteria. The source of inoculum to the heat pre-treatment was from a UASB reactor used in the slaughterhouse waste treatment. The molecular biology analyses indicated that the microbial consortium presented microorganisms afliated with Enterobacter cloacae (97% and 98%), Clostridium sp. (98%) and Clostridium acetobutyricum (96%), recognized as H2 and volatile acids producers. The following assays were carried out in batch reactors in order to verify the efciencies of sucrose conversion to H2 by the microbial consortium: (1) 630.0 mg sucrose/L, (2) 1184.0 mg sucrose/L, (3) 1816.0 mg sucrose/L and (4) 4128.0 mg sucrose/L. The subsequent yields were obtained as follows: 15% (1.2 mol H2/mol sucrose), 20% (1.6 mol H2/mol sucrose), 15% (1.2 mol H2/mol sucrose) and 4% (0.3 mol H2/mol sucrose), respectively. The intermediary products were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Clean energy sources have been applied in order to satisfy the global energy demand. The hydrogen gas generated in the wastewater treatment by biological processes can be used as an alternative energy source. In this way, the knowledge about the hydrogen-producing microorganisms is fundamental to the development of alternative and cleaner sources of energy production. Hydrogen-producing fermentative anaerobic bacteria have potential to metabolize organic substrates as they produce cleaner and renewable energy in their process [1]. The fermentative production of hydrogen can be facilitated with methanogenesis inhibition as the methanogenic archaea use hydrogen in the anaerobic biological processes. The heat treatment of the seed sludge associated with the

pH control has been applied in the selection of hydrogenproducing bacteria as Clostridium sp. These bacteria, spore forming, are tolerant to high temperatures and adverse environmental conditions [2]. Fermentative processes of the production of hydrogen with acidogenic anaerobic bacteria, such as Clostridium, are being frequently applied by many authors [38]. The following pathways of the sucrose degradation can occur in fermentative processes of hydrogen gas production [5]: (1) consumption of sucrose and generation of acetic acid and (2) consumption of sucrose and generation of butyric acid, as described in Eqs. (1) and (2): C12 H22 O11 5H2 O / 4CH3 COOH 4CO2 8H2 (1)

C12 H22 O11 H2 O / 2CH3 CH2 CH2 COOH 4CO2 4H2

(2)

* Corresponding author. Tel.: 55 16 3373 8357; fax: 55 16 3373 9550. E-mail addresses: mainting@sc.usp.br (S.I. Maintinguer), varesche@sc.usp.br (M.B. Varesche). 0360-3199/$ see front matter 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2008.06.053

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The maximum theoretical conversion of sucrose to hydrogen is equal to 1 mol of sucrose that generates 8 mole of hydrogen gas in the pathway of acetate degradation. However, the generation of 4 mole of hydrogen per mole of consumed sucrose occurs if the sucrose is converted to butyrate. Then, the major theoretical production of hydrogen previews acetate as the nal product of the fermentation. On the other hand, in practice, the high hydrogen production is related to the mixture of fermentation products such as acetate and butyrate; and the low hydrogen production is associated to the formation of propionate and nal products that are lesser reduced, such as alcohols and lactic acid [9]. Batch reactors are used to investigate nutritional requirements and obtain hydrogen productivities from the organic substrates [7]. The hydrogen productions from complex carbohydrates are being evaluated in batch reactors because the particulate organic matter that is present in these compounds can difcult the operation in other congurations, such as starch. Moreover, higher velocities of H2 production are veried in the congurations of batch reactors in which the biomass remains retained in all of the operational periods when compared with continuous ow reactors. Hydrogen productions are being veried to higher sugar concentrations, even though these concentrations can be an inhibitory factor to the cellular growth. Although the sucrose can be fermented, it can resist to the microbial decomposition in high concentrations [10] and also be an inhibitory factor to the cellular growth and H2 generation. Wastewaters of beverage industries contain sucrose provided of bottles washing machines and syrup industries, washes of oor and machines in concentrations that can vary from 700 mg/L to 2000 mg/L. However, there are few studies of hydrogen production with reduced substratum concentrations, what it would be compatible with sugar concentrations of efuent industries for cooling, that they could be reused for the energy generation. The search for biotechnology to obtain hydrogen production from industrial efuents can be advantageous as an energy alternative. Therefore, the identication of the microorganisms that are responsible for this process has fundamental importance. In this way, researches are developed in order to obtain the biological processes stability to the hydrogen gas production. Different inoculums sources have been tested with efciencies conrmed in the biological hydrogen production as natural soil, anaerobic digestion sludge, aerobic compost, wastewater treating plants, lake sediment, and others. However these inoculums are coming from countries of tempered climate, in its majority. There are few studies of hydrogen production with inoculums from tropical countries as Brazil. In the present work the inoculum obtained from a granulated sludge of the Upow Anaerobic Sludge Blanket (UASB) reactor treating swine wastewaters was used (Jaboticabal Brazil). The batch reactors were used to determine the hydrogen production at control condition without nutritional limits (addition of vitamins and some nitrogen sources). The main goal of the study consists in the development commercial processes to obtain hydrogen production, applied for low organic matter wastewaters exploring the ability of tropical microorganisms by modern biotechnology practices.

This work applied classical anaerobic methodologies and also Molecular Biology was applied in order to purify, characterize and identify microorganisms that produce hydrogen gas to four different sucrose concentrations.

2.
2.1.

Experimental
Inoculum

The inoculum was obtained from a granulated sludge of the Upow Anaerobic Sludge Blanket (UASB) reactor treating swine wastewaters (Jaboticabal Brazil). The biomass was transferred to a mortar with pestle in order to undo the granular arrangement. This cellular suspension was preheated at 90  C for 15 min in order to inactivate the hydrogen consumers and harvest the spore-forming anaerobic bacteria such as Clostridium sp. [11].

2.2.

Biomass of heat-treated sludge

The pre-treated inoculum (20%) was transferred to the sterile culture medium [12] in asks of 100 ml under aseptic conditions. The increase of the biomass was done using the culture medium [12] formed by nutrient solutions A, B, C and D, sucrose (1800 mg/L), urea (40 mg/L), peptone (1000 mg/L) and 1 ml of vitamin solution [13] as described in Table 1. The vitamin solution was composed by p-aminobenzoic acid (40 mg/L) and biotine (10 mg/L). These anaerobic asks were kept with nal reaction volume of 70 ml and headspace of 30 ml. Afterwards, the asks were submitted to a He atmosphere (99.99%) for 5 min and incubated at 37  C.

2.3.

Operation of the batch reactors

The reactor preparation was performed as the following procedures; the reactivated biomass was submitted to washes in a refrigerated centrifuge at 8500 rpm and 3  C for 10 min. The pellet was resuspended in a new culture medium with the following composition: medium [12] with addition of urea, peptone, vitamin solution [13] as previously described and sucrose, respectively, to the four conditions (Table 1). The pH was adjusted to 5.5 with additions of chloridric acid or sodium hydroxide. The culture medium was ltered in a membrane of 0.22 mm in a ltration system that was previously sterilized in an autoclave (121  C, 1 atm, 20 min). The reactors were submitted to a He atmosphere (99.99%) for 20 min after the distribution of the solutions. After that, they were capped with butyl rubber stoppers, wrapped and kept at 37 1  C, with no agitation. In this study, anaerobic batch reactors of 2 L of volume were used in triplicate in four different conditions of sucrose concentrations: (1) 630.0 mg/L (1.9 mmol/L); (2) 1184.0 mg/L (3.5 mmol/L); (3) 1816.0 mg/L (5.3 mmol/L) and (4) 4128.0 mg/L (12.1 mmol/L), according to Table 1. The inoculum composed 20% of the total reaction volume that was composed by microbial consortium obtained from the heat-treated sludge.

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Table 1 Composition of the synthetic substrate of the anaerobic batch reactors Compound 1
Sucrose (mg/L) Urea (mg/L) Peptone (mg/L) Vitamin solution A Nutrient: NiSO4.6H2O (0.5 g/L), FeSO4.7H2O (2.5 g/L), FeCl3.6H2O (0.25 g/L), CoCl2.2H2O (0.04 g/L) B Nutrient: CaCl2.6H2O (2.06 g/L) C Nutrient: SeO2 (0.144 g/L) D Nutrient: KH2PO4 (5.36 g/L), K2HPO4 (1.30 g/L), Na2HPO4H2O (2.76 g/L) Seed sludge (mL) (reactivated biomass) Headspace He (ml) Initial pH Ultra pure water (ml) 630.0 10 1000 1 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 300 500 5.5 1500

Anaerobic batch reactor 2

1184.0 20 1000 1 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml 300 500 5.5 1500

3
1816.0 40 1000 1 ml 2.0 ml 2.0 ml 2.0 ml 2.0 ml 300 500 5.5 1500

4
4128.0 80 1000 1 ml 4.0 ml 4.0 ml 4.0 ml 4.0 ml 200 1000 5.5 1000

The conditions (1), (2) and (3) were prepared with 1500 ml of the reaction medium volume and 500 ml of headspace with He gas in order to guarantee the anaerobic conditions. The condition (4) was set up with 1000 ml of the reaction medium volume and 1000 ml of headspace in the same conditions of the previous assays. The condition (4) needed increase the headspace volume to avoid the increase of gas pressure in the reactor due the higher sucrose concentration than other conditions.

2.6.

Cellular growth analysis

The cellular growth was based on optical density at 600 nm (OD600), in accordance with Standard Methods for Examination of Water and Wastewater [10]. The cellular mass was expressed in volatile suspended solids (VSS, g/L) that was proportional to the optical density at 600 nm and was calculated by Eq. (4) VSS 2:6691 ABS600 0:0095 (4)

2.4.

Chemical and chromatographic analyses 2.7. Microscopic analyses

Hydrogen content in biogas was determined by gas chromatography (GC 2010 Shimatzu) using a thermal conductivity detector and argon as a carrier gas. The temperatures of the injector, detector and column were kept at 30  C, 200  C and 230  C, respectively. Volatile acid concentrations were assessed using Gas Chromatograph HP 6890/FID equipped with column of 30 m, internal diameter of 0.25 mm and lm thickness of 0.25 mm [14]. The alcohols concentrations were measured by gas chromatography (GC 2010 Shimatzu), equipped with ame ionization detector and sample introduction system to COMBI-PAL headspace (AOC 5000 model and HP-INNOWAX column of 30 m 0.25 mm 0.25 mm of lm thickness). The sucrose concentrations were determined by the colorimetric method [15,16]. The volatile suspended solids concentrations (VSS) and pH values were accomplished in accordance with Standard Methods for Examination of Water and Wastewater [10].

Morphological characteristics of the microorganisms were monitored by phase contrast microscopy using Olympus BX60-FLA with software Image Pro-Plus. Gram staining was carried out according to the methodology proposed by Scientic Services of Cultures Collections of Germany [17].

2.8.

Molecular analyses

2.5.

H2 partial pressure verication

The H2 partial pressure in the headspace of the anaerobic reactors was veried by Eq. (3) PH2 nH2 RT =V (3)

where T temperature, 310.15 K; R constant of gases (0.08206 L atm/mol K); n number of moles of H2; V headspace volume, ml.

The analysis of the microbial structure was performed at the end of the hydrogen process to all of the evaluated conditions. PCR/DGGE technique was applied with primers for Bacteria Domain: 968FGC and 1392R [18]. The region corresponds to positions 968F (50 - AACGCGAAGAACCTTAC 30 ) and 1392R (50 - AACG GGC GGT GTG TAC 30 ) in the 16S rRNA with GC clamp (50 - CGC CCG CCG GGG CGC GCC CCG GGC GGGGCG GGG GCA CGG GGGG 30 ). PCR products were electrophoresed on 1% (wt/vol) in agarose gel in 1 TAE, 50 V for 30 min. Then, the products were cored with etidium bromide to conrm the amplication. Denaturing gradient gel electrophoresis (DGGE) was carried out using the Dcode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA) in accordance with the manufacturer instructions. PCR products were electrophoresed on TAE buffer (1X) at 75 V for 16 h and 65  C on polyacrylamide gel (7.5%) containing linear gradient varying from 30% to 60% of denaturant. After electrophoresis, the

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polyacrylamide gel was cored with etidium bromide for 20 min and then visualized on UV transiluminator. Most of the bands were excised from DGGE polyacrylamide, immersed in 20 ml of ultra pure water for 24 h and then PCR-amplied with the forward primer EUB968f without GC clamp and reverse primer EUB968r. After PCR amplication, PCR products were puried with Ultraclean PCR Clean-up. All the strands of the puried PCR products were sequenced with primers EUB968F. Sequencing was performed on an automated ABI 310 PRISM sequencer (Dye terminator Cycle Sequencing Kit Applied Biosystems, USA) in accordance with the manufacturer instructions. The search of the GenBank database was conducted using the BLAST program. Phylogenetic analyses of the sequences were performed using the Molecular Evolutionary Genetic Analysis 3.1 (MEGA 3.1) software [19]. Evolutionary distances were based on the Kimura [20] model and tree reconstruction on the neighborjoining method with bootstrap values calculated from 500 replicate runs, using the routines included in MEGA software.

10000

8000

mol H2/VSS (g)

6000

4000

2000

0 0 20 40 60 80 100 120 140 160 180 200 220 240

Time (h)
Fig. 1 Hydrogen production in the anaerobic reactors to conditions: (-) 630.0 mg sucrose/L; (C) 1184.0 mg sucrose/ L; (,) 1816.0 mg sucrose/L; and (:) 4128.0 mg sucrose/L.

2.9.

Experimental data tting

and 222 h of the operation to the conditions (1), (2), (3) and (4), respectively. Lag phase was not observed in the H2 production to the conditions (1) and (2), indicating that these sucrose concentrations were not inhibitory. The H2 generation was equal to 614.4 mmol H2/g VSS with 2 h of operation and 344.9 mmol H2/ g VSS with 8 h, respectively, to the conditions (1) and (2). The maximum H2 generations were 2147.3 mmol H2/g VSS in 14 h and 3201.6 mmol H2/g VSS in 48 h. In this case, the imposed conditions favoured the H2 generation in the rst hours of the operation. On the other hand, the conditions (3) and (4) had four behaviours: Lag phase, preliminary period of H2 production, uninterrupted period of H2 production and decay period. To the conditions (3) and (4), H2 generation of 1297.1 mmol H2/g VSS with 24 h of operation and 284.2 mmol H2/g VSS with 17.5 h was veried, respectively. The maximum H2 generations were 10,059.5 mmol H2/g VSS in 178 h and 2663.1 mmol H2/g VSS in 219.5 h. Table 2 summarizes the results of the four studied conditions. This fact was correlated to the higher substrate concentrations that inhibited the bacterial growth and consequently the H2 generation in the reactors to the conditions (3) and (4). However, the maximum H2 specic production rate (10059.5 mmol H2/g VSS h) occurred in the reactors of condition (3) after adaptation to the nutritional conditions (Table 2). The microbial growth to the four studied conditions is illustrated in Fig. 2a and b. To the conditions (1) and (2), the microbial growth with consequent consumption of sucrose and generation of intermediary products, such as volatile fatty acids and alcohols, occurred in the period of 28 h and 96 h, respectively. The Lag phase of microbial growth did not occur in those cases, and consequently the biomass did not suffer inhibition with the imposed substrate concentration and its adaptation was not necessary to this condition. Initial microbial growth of 0.07 g VSS/L and 0.08 g VSS/L was observed in the conditions (1) and (2), respectively, and maximum with 9.5 h (0.26 g VSS/L) and 20 h (0.3 g VSS/L) of operation. Cellular

The experimental data were tted to mean values obtained from triplicates of the reactors using Microcal Origin 5.0 software. The maximum specic activities of the hydrogen gas production were obtained by non-linear sigmoidal adjustment of the Boltzman function. The maximum sucrose consumption velocity was veried by the major angular coefcient generated from the screened lines between the points representing the experimental sucrose concentrations in relation to the time.

3.

Results and discussion

It was possible to obtain a microbial consortium with the predominance of Gram positive and Gram negative rods and absence of methanogenic archaea with predominance of methanogenic inoculum. The imposed conditions of pre-treatment and pH equal to 5.5 for the inoculum favoured the maintenance of the tolerant species and endospores forming in the microbial consortium. The association of these two factors caused the inhibition of bacteria and hydrogen-consuming methanogenic archaea. The methanogenic archaea grow satisfactorily in pH varying from 6.0 to 8.0 [21]. Therefore, they were unfavoured due to the experimental conditions related mainly to pH and heat treatment. On the other hand, these conditions supported Clostridium species (positive endospores) that were selected from different environments by means of heat treatment. Fang et al. [22] observed rods identied phylogenetically as Clostridium sp. in pH equal to 4.5. The anaerobic reactors fed with sucrose in the four studied conditions had different behaviours with hydrogen production. Fig. 1 presents the behaviour of the hydrogen gas production in the anaerobic reactors. The consumption of sucrose was not complete in the four studied conditions (Table 2). It was noted 80.4%, 87.2%, 83.0% and 70.3% of the sucrose consumption with 28 h; 96 h; 202 h

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Table 2 Results of the studied conditions (1); (2); (3) and (4) Studied parameters
Sucrose decomposition (%) VSS (g/L) Period (h) pH (experiment end) Operation time (h) max. conc. (mg/L) Acetic Acid Butyric Acid Propionic Acid isobutyric Acid isovaleric Acid Methanol Ethanol H2 Partial Pressure headspace (atm) Maximum specic sucrose consumption (mmol sucrose/L h) Period (h) Maximum H2 specic production rate (mmol H2/g VSS h) Hydrogen yield (mol H2/mol sucrose)

(1) 630.0 mg/L

80.4 0.26 9.5 4.1 28

(2) 1184.0 mg/L

87.2 0.30 20 4.0 96

(3) 1816.0 mg/L

83.0 0.45 96 4.6 202

(4) 4128.0 mg/L

70.3 0.34 201.5 3.8 222

131.5 1.2 0 0 0 20.9 42.5 0.068 0.26 3.55.0 2147.3 15% (1.2)

146.6 3.3 0 0 0 29.1 46.2 0.116 0.16 812 3201.6 19.8% (1.6)

537.0 252.5 3.3 11.2 23.2 22.0 38.1 0.488 0.11 7292 10059.5 15% (1.2)

204.7 281.4 0.7 0.6 5.6 21.0 23.0 0.062 0.11 162186 2663.1 3.8% (0.3)

growth (0.45 g VSS/L) was veried until 96 h of operation to the condition (3). The microbial growth was very slow in the condition (4) probably due to the higher sucrose concentrations than those studied previously, that became toxic. The maximum microbial growth of 0.34 g VSS/L was achieved in 201.5 h of operation to the condition (4).

Van Ginkel and Logan [24] had proved the increase in applied organic loads was inversely proportional to the hydrogen production. The authors had studied the high biological hydrogen production with reduction in the applied organic load. The inoculum (10 g) was coming from agricultural ground sample head-treated (100  C for 2 h) to select

0,30 0,25

b
VSS (g/L)

0,50 0,45 0,40 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00

VSS (g/L)

0,20 0,15 0,10 0,05 0 5 10 15 20 25 30

50

100

150

200

Time (h)
0,35 0,30 0,40 0,35 0,30

Time (h)

VSS (g/L)

0,25 0,20 0,15 0,10 0,05 0 20 40 60 80 100

VSS (g/L)

0,25 0,20 0,15 0,10 0,05 0,00 0 50 100 150 200 250

Time (h)

Time (h)

Fig. 2 (a) Cellular growth at each experimental condition: (1) 630.0 mg sucrose/L and (2) 1184.0 mg sucrose/L. (b) Cellular growth at each experimental condition: (3) 1816.0 mg sucrose/L and (4) 4128.0 mg sucrose/L.

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bacteria endospores forming, as Clostridium ssp. Four reactors of complete mixture had been operated at 30  C for COD glucose concentrations (g/L): 10; 7.5; 5.0 and 2.5. The reactors had been kept with 10 h HDT. The best yield of hydrogen production had been achieved with reduced glucose at the following concentrations: 2.5 g COD/L, 5.0 g COD/L, 7.5 g COD/ L and 10 g COD/L were gotten, respectively, for 2.8 mol H2/ mol glucose; 2.5 mol H2/mol glucose; 2.4 mol H2/mol glucose and 2.2 mol H2/mol glucose. The ratio of gas hydrogen in headspace had varied from 60 to 72% for all the studied conditions. The glucose removals had been 90% in all experiments and 97% for 2.5 g COD glucose/L. Two pathways of sucrose degradation can occur in fermentative processes of hydrogen gas production as follows: (1) consumption of sucrose generating acetic acid (Eq. (1)) and consumption of sucrose generating butyric acid (Eq. (2)), as previously described. The intermediary generated products were acetic acid, butyric acid and hydrogen gas to the conditions (1) and (2) (Fig. 3a). To the conditions (3) and (4), production of isobutyric and isovaleric acids was also observed in reduced concentrations (Fig. 3b). In this experiment, the two degradation pathways occurred but the sucrose conversion to acetic acid was favoured in the rst operational hours. High hydrogen gas productions were also veried when butyric acid was detected in the reactors. Generations of acetic acid were noted in the maximum proportions of 98.8%, 97.7%, 69.3% and 42.1%, respectively, and also butyric acid of 0.2%, 2.6%, 30.7% and 57.9% in the four studied conditions (Fig. 3a and b). Khanal et al. [5]

observed the generation of acetic, butyric and propionic acids to pH 5.0 in reactors fed with sucrose (1500 mg/L), with production of 240 mg H2/g COD. The generations of methanol remained constant in all of the operational periods of the anaerobic reactors to the studied conditions (Table 2). In this case, the present microbial consortium did not consume this intermediary product that was in the anaerobic reactors. The ethanol production was gradual and increased in relation to the operational period of time. The maximum generations of ethanol were veried in the nal samplings of all the studied conditions (Table 2). The biological hydrogen production can be affected by the H2 partial pressure [23]. This fact was veried in the experiment. The anaerobic reactors of the condition (3) fed with 1816.0 mg/L of sucrose presented higher H2 partial pressure (0.488 atm). Consequently, the H2 that was produced in the liquid phase remained in this phase or the effect of the partial pressure on the headspace carried the H2 from the gaseous to the liquid medium. This fact also justied the change in the headspace volume from 500 ml in the conditions (1), (2) and (3) to 1000 ml in the condition (4). However, to the condition (4) with 4128.0 mg/L of sucrose, there were no higher H2 partial pressures as the high sucrose concentrations caused inhibition in the microbial culture growth with reduced productions of H2 in the headspace. Van Ginkel and Logan [24] obtained high H2 productions with mechanisms that reduced the partial pressures in the headspace. According to Kim et al. [11], the partial pressure of H2 is one of the key factors that affect the fermentative

a
Volatile Fatty Acids (%)

Volatile Fatty Acids (%)

0 3,5 5 8 9,5 11 13 15 28

100% 80% 60% 40% 20% 0%

100% 80% 60% 40% 20% 0% 0 24 48 72 96 106 120 168

Time (h)
100% 100%

Time (h)

Volatile Fatty Acids (%)

Volatile Fatty Acids (%)

0 4 8 12 16 20 25 30 34 40

80% 60% 40% 20% 0%

80% 60% 40% 20% 0% 0 4 8 12 16 20 25 30 34 40

Time (h)

Time (h)

Fig. 3 (a) Proportions of the generated volatile fatty acids (- acetic; , butyric; isovaleric and F isobutyric) at each experimental conditions: (1) 630.0 mg sucrose/L and (2) 1184.0 mg sucrose/L. (b) Proportions of the generated volatile fatty acids (- acetic; , butyric; isovaleric and F isobutyric) at each experimental conditions: (3) 1816.0 mg sucrose/L and (4) 4128.0 mg sucrose/L.

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5 3 6 1

4 2

Fig. 4 DGGE-proles of 16S rRNA gene fragment of the eubacterial populations at each experimental condition: (A) 630.0 mg sucrose/L, (B) 1184.0 mg sucrose/L; (C) 1816.0 mg sucrose/L and (D) 4128.0 mg sucrose/L.

production of H2. In accordance with the authors, the partial pressures of H2 have negative effects in the H2 production that is responsible for the decay in the hydrogenase activity. This activity makes the reaction of H2 production unfavourable thermodynamically. Many methods to diminish the partial pressure of H2 can cause benec effect, such as gas spraying that enhances the mixture, mainly in batch reactors. There is an increase in the hydrogenase activity with the decay of the H2 partial pressure that makes the reaction of H2 production favourable thermodynamically. However, it was chosen to maintain the system with no changes of the headspace in the present work. Maximum conversions for sucrose of 1.2 mol H2/mol was noted to keep the reactors fed with 630.0 mg sucrose/L (condition 1) and 1816.0 mg sucrose/L (condition 3) and sucrose of 1.6 mol H2/mol to the reactors with 1184.0 mg sucrose/L (condition 2). Kawagoshi et al. [2] obtained maximum hydrogen generation conversions of glucose of 1.4 mol H2/ mol, with inoculum from anaerobic digestion. The inoculum

was submitted to heat treatment in batch reactors fed with glucose (6000 mg/L) and kept at 35  C. The degradation pathways were acetic and butyric acids. The maximum conversions of sucrose to H2 of 20% (1.6 mol H2/mol sucrose) occurred to the reactors of the condition (2) that were fed with 1184.0 mg sucrose/L when compared to the theoretical efciency of the conversion of sucrose to acetate [9]. Kim et al. [11] operated a continuous ow reactor with variations of 30006000 mg COD/L of sucrose, pH of 5.4 and 35  C. The authors obtained a maximum conversion of 13.5% (1.09 mole of H2/mol of sucrose) and the bacteria were identied as Clostridium ssp. and Bacillus racemilacticus. Fig. 4 presents the prole of the DGGE bands to the four studied conditions. The predominance of some bands in the samples was noted. This fact corroborated that the inoculum heat treatment and the pH control selected some bacteria that prevailed in the imposed conditions of the experiment. The enumerated bands were excised and identied as described in Table 3. The bands 1 and 3 were considered as the same population. This bacterium was one of the microorganisms present in the pre-treated inoculum that was responsible for the H2 production in the imposed conditions. The populations represented by bands 1 and 3 in the condition (1) showed phylogenetic similarity with Enterobacter cloacae (98% and 97%, respectively). The most known fermentative bacteria in the hydrogen production include species of Enterobacter, Bacillus and Clostridium [2]. Kumar and Das [25] inoculated anaerobic batch reactors with E. cloacae, Gram negative bacteria and facultative anaerobic, and obtained 6 mole H2/mol of sucrose of H2 yield with pH of 6.0 and 36  C. This bacterium also produced H2 with glucose (2.2 mol H2/mol glucose) and cellobiose (5.4 mol H2/mol cellobiose). The band 2 presented 98% of phylogenetic similarity with Burkholderia cepacia. The band 2 that is present to condition (1) was also observed in the conditions (2) and (4). This bacterium is commonly found in the soil, water, roots of the plants and associated to the fungi mycelium [26]. These bacteria, Gram negative rods, are utilized in bioremediation processes. Besides toxic compounds, such as organophosforates, pesticides were degraded by this species [27], benzene, toluene, xylene, phenol and chlorophenols [28]. There are no reports about this bacterium associated to H2 production. However, it is a nitrogen-xing bacterium when associated to roots of plants and can use substrates as sucrose, glucose, fructose and maltose [27]. Therefore, in this work, the B. cepacia was present in the anaerobic reactors, probably favoured by the presence of sucrose and peptone.

Table 3 Afliation of DGGE fragments determined by 16S rRNA sequence Band

1 2 3 4 5 6

Microorganisms
Enterobacter cloacae Burkholderia cepacia Enterobacter cloacae Clostridium sp. Clostridium sp. Clostridium acetobutylicum

Access number (Genbank)

EF120473.1 DQ387437.1 EF059865.1 DQ196619.1 AY862512.1 U17030.1

Similarity %
98 98 97 90 98 96

References
Feng, R.H. (2006). Not published [19] Iversen, C. et al. (2007). Not published [31] Zhang, T. (2004). Not published [32]

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The bands 4 and 5 presented phylogenetic similarity (90% and 98%) with Clostridium sp. The sequence referring to the band 6 was 96% similar to Clostridium acetobutyricum. These bands were repeated in other studied conditions. The majority of the studies describe Clostridium species, Gram positive rods, endospores forming, as responsible for anaerobic hydrogen production. These species survive in pH similar to 4.0 [22], produce hydrogen gas, organic acids and alcohol from carbohydrates or peptones [3]. Some species x atmospheric nitrogen, while others carry out fermentative metabolism. This evidence was expected as the imposed conditions to the experiment propitiated the maintenance of Gram positive rods, endospores forming, that is a characteristic of some hydrogen-gas-producing bacteria [29]. Many factors contributed to the maintenance of Clostridium species in the anaerobic reactors as the following description. The increase of peptone to the nutritional medium [3]; the heat treatment of the inoculum associated to the specic nutritional medium and pH control to 5.5 enriched these populations [4]. Pure culture of C. acetobutyricum was used in the H2 production in reactors fed with glucose (10.5 g/L). H2 conversion efciency of 22%, specic production of glucose of 1270 ml H2/L, velocity of H2 molar production of 8.9 0.8 mmol/L h and velocity of volumetric production of H2 of 220 8 ml/L h of the reactor were obtained. The fermentative products in the efuent were acetate and butyrate [30]. The anaerobic reactors operated with 1184.0 mg sucrose/L (condition 2) obtained better H2 yield (1.6 mol H2/mol sucrose). This yield was veried with 48 h of operation. Fig. 5 presents the consensus phylogenetic tree obtained with primers for Bacteria Domain from the information of sequences obtained in the four studied conditions. The sequence of the bands 4, 5 and 6 were associated to Clostridium species. The sequences referred to the bands 1 and 3 were related phylogenetically to E. cloacae. The sequences referred to the band 2 were related phylogenetically to B. cepacia.

The experimental results of generation of acetic and butyric acids to the four studied conditions demonstrated that the microorganisms from the inoculum had metabolic function similar to Clostridium species. This fact revealed that the heat-treated inoculum had high capacity of degrading sucrose and generating hydrogen gas. The biological production of hydrogen gas was due to the microorganisms consortia that were present in the studied conditions.

4.

Conclusions

Although H2 generation was observed, production of methane gas in the four operational conditions was not detected. This corroborated the heat treatment efciency and the pH control in order to inhibit the bacteria and H2 methanogenic archaea consumers. The additions of peptone and vitamin solution contributed to the H2 generation in lower interval of time and to the development of microbial hydrogen-producing consortia. The conditions (1) and (2) presented H2 generation in the rst operation hours indicating that the substrate concentrations were not inhibitory. The conditions (3) and (4) showed Lag phase of H2 generation. The condition (4) presented reduced rates of H2 production. The imposed conditions of high substrate concentrations became toxic to the puried consortia causing slowness in the process of H2 generation. The generated volatile fatty acids were acetic and butyric to the conditions (1) and (2). Production of acetic, butyric, propionic, isobutyric and isovaleric acids to the conditions (3) and (4) was veried. The best efciency of the sucrose conversion to H2 was obtained in the condition (2). The maximum specic activity of H2 production was observed in the condition (3). The analyses of Molecular Biology revealed that the biological production of H2 was due to the presence of the species E.

Band 1
100

Band 3 Enterobacter cloacae (EF120473.1)

99 44

Enterobacter cloacae (EF059865.1) Band 2

97 Burkholderia cepacia (DQ387437.1)

53

Clostridium acetobutyricum (U17030.1) Band 6

82

Band 5 Clostridium sp.(AY862512.1)

Clostridium acetobutyricum (X81021)

78 Clostridium butyricum (AY458857)

Band 4
77

Clostridium sp.(DQ196619.1)

0.02

Fig. 5 Consensus phylogenetic tree based on the DGGE excised sequences of bands with primers for Bacteria Domain obtained from the anaerobic reactors fed with sucrose. The bootstrap values indicate the repetition percentages (500 replicate runs). GenBank accession numbers are listed after species names.

international journal of hydrogen energy 33 (2008) 43094317

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cloacae, Clostridium sp. and C. acetobutyricum; recognized as H2 and volatile acid producers. The microscopic analyses showed the presence of rods, endospores and rods with endospores, proving the occurrence of the H2 gas-producing species that are mainly identied as Clostridium and E. cloacae.

Acknowledgements
The authors gratefully acknowledge the nancial support of Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq).

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