hidrogeno 2

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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 Sao Carlos, University of Sao Paulo, Av. Trabalhador Sao-carlense,

400, 13566-590 Sao Carlos-SP, Brazil

a r t i c l e i n f o

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

a b s t r a c t

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 treat-

ment. The molecular biology analyses indicated that the microbial consortium presented

microorganisms affiliated 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 efficiencies 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 molH2 /mol sucrose), 20% (1.6 mol H2/mol su-

crose), 15% (1.2 molH2 /mol sucrose) and 4% (0.3 molH2 /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 funda-

mental to the development of alternative and cleaner sourcesof 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 hydrogen-

producing bacteria as Clostridium sp. These bacteria, spore

forming, are tolerant to high temperatures and adverse envi-

ronmental 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 canoccur 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):

C12H22O11 5H2O/4CH3COOH 4CO2 8H2 (1)

C12H22O11 H2O/2CH3CH2CH2COOH 4CO2 4H2 (2)

* Corresponding author. Tel.: 55 16 3373 8357; fax: 55 16 3373 9550.E-mail addresses: [email protected] (S.I. Maintinguer), [email protected] (M.B. Varesche).

A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / h e

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

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 3 ( 2 0 0 8 ) 4 3 0 9 4 3 1 7
mailto:[email protected]:[email protected]://www.elsevier.com/locate/hehttp://www.elsevier.com/locate/hemailto:[email protected]:[email protected]


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Theconditions (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 condi-

tion (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.

2.4. Chemical and chromatographic analyses

Hydrogen content in biogas was determined by gas chroma-

tography (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 Chro-

matograph HP 6890/FID equipped with column of 30 m,

internal diameter of 0.25 mm and film thickness of 0.25 mm

[14].

The alcohols concentrations were measured by gas chro-

matography (GC 2010 Shimatzu), equipped with flame ioniza-

tion 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 film thickness).

The sucrose concentrations were determined by the color-

imetric method [15,16]. The volatile suspended solids concen-

trations (VSS) and pH values were accomplished in

accordance with Standard Methods for Examination of Water

and Wastewater [10].

2.5. H2 partial pressure verification

The H2 partial pressure in the headspace of the anaerobic

reactors was verified by Eq. (3)

PH2 nH2RT =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.

2.6. Cellular growth analysis

The cellular growth was based on optical density at 600 nm

(OD600), in accordance with Standard Methods for Examina-

tion 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 calcu-

lated by Eq. (4)

VSS 2:6691ABS600 0:0095 (4)

2.7. Microscopic analyses

Morphological characteristics of the microorganisms weremonitored by phase contrast microscopy using Olympus

BX60-FLA with software Image Pro-Plus. Gram staining was

carried out according to the methodology proposed by Scien-

tific Services of Cultures Collections of Germany [17].

2.8. Molecular analyses

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 3 0) in the 16S rRNA with GC

clamp (50- CGC CCG CCG GGG CGC GCC CCG GGC GGGGCG

GGG GCA CGG GGGG 3 0). 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 confirm the

amplification.

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 electro-

phoresed 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

Table 1 Composition of the synthetic substrate of the anaerobic batch reactors

Compound Anaerobic batch reactor

1 2 3 4

Sucrose (mg/L) 630.0 1184.0 1816.0 4128.0

Urea (mg/L) 10 20 40 80

Peptone (mg/L) 1000 1000 1000 1000

Vitamin solution 1 ml 1 ml 1 ml 1 ml

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)

0.5 ml 1.0 ml 2.0 ml 4.0 ml

B Nutrient: CaCl2.6H2O (2.06 g/L) 0.5 ml 1.0 ml 2.0 ml 4.0 ml

C Nutrient: SeO2 (0.144 g/L) 0.5 ml 1.0 ml 2.0 ml 4.0 ml

D Nutrient: KH2PO4 (5.36 g/L), K2HPO4 (1.30 g/L), Na2HPO4H2O (2.76 g/L) 0.5 ml 1.0 ml 2.0 ml 4.0 ml

Seed sludge (mL) (reactivated biomass) 300 300 300 200

Headspace He (ml) 500 500 500 1000

Initial pH 5.5 5.5 5.5 5.5

Ultra pure water (ml) 1500 1500 1500 1000

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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 polyacryl-

amide, immersed in 20 ml of ultra pure water for 24 h and

then PCR-amplified with the forward primer EUB968f without

GC clamp and reverse primer EUB968r. After PCR amplifica-

tion, PCR products were purified with Ultraclean PCR

Clean-up. All the strands of the purified PCR products weresequenced with primers EUB968F. Sequencing was performed

on an automated ABI 310 PRISM sequencer (Dye terminator

Cycle Sequencing Kit Applied Biosystems, USA) in accor-

dance 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 neighbor-

joining method with bootstrap values calculated from 500

replicate runs, using the routines included in MEGA software.

2.9. Experimental data fitting

The experimental data were fitted to mean values obtained

from triplicates of the reactors using Microcal Origin 5.0 soft-

ware. The maximum specific activities of the hydrogen gas

production were obtained by non-linear sigmoidal adjustment

of the Boltzman function. The maximum sucrose consump-

tion velocity was verified by the major angular coefficient

generated from the screened lines between the points repre-

senting 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 tothe 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 identified 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 produc-

tion. Fig.1 presents the behaviour of the hydrogen gas produc-

tion 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

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 concentra-

tions were not inhibitory. The H2 generation was equal to

614.4 mmolH2/g VSS with 2 h of operation and 344.9 mmolH2/

g VSS with 8 h, respectively, to the conditions (1) and (2). The

maximum H2 generations were 2147.3 mmolH2/g VSS in 14 h

and 3201.6 mmolH2 /gVSS in 48 h. In this case, the imposed

conditions favoured the H2 generation in the first hours of

the operation.

On the other hand, the conditions (3) and (4) had fourbehaviours: 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 284.2mmolH2/g VSS

with 17.5 h was verified, respectively. The maximum H2generations were 10,059.5 mmolH2 /g V SS in 178 h and

2663.1 mmolH2 /g VSS in 219.5 h. Table 2 summarizes the

results of the four studied conditions.

This fact was correlated to the higher substrate concentra-

tions that inhibited the bacterial growth and consequently the

H2 generation in the reactors to the conditions (3) and (4).

However, the maximum H2 specific production rate

(10059.5 mmolH2/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 intermediaryproducts, 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 micro-

bial 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

0 20 40 60 80 100 120 140 160 180 200 220 2400

2000

4000

6000

8000

10000

molH2/VSS(g)

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.



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growth (0.45 g VSS/L) was verified 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 concentra-

tions 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 bio-

logical hydrogen production with reduction in the applied

organic load. The inoculum (10 g) was coming from agricul-

tural ground sample head-treated (100 C for 2 h) to select

Table 2 Results of the studied conditions (1); (2); (3) and (4)

Studied parameters (1) 630.0 mg/L (2) 1184.0 mg/L (3) 1816.0 mg/L (4) 4128.0 mg/L

Sucrose decomposition (%) 80.4 87.2 83.0 70.3

VSS (g/L) 0.26 0.30 0.45 0.34

Period (h) 9.5 20 96 201.5

pH (experiment end) 4.1 4.0 4.6 3.8Operation time (h) 28 96 202 222

max. conc. (mg/L)

Acetic Acid 131.5 146.6 537.0 204.7

Butyric Acid 1.2 3.3 252.5 281.4

Propionic Acid 0 0 3.3 0.7

isobutyric Acid 0 0 11.2 0.6

isovaleric Acid 0 0 23.2 5.6

Methanol 20.9 29.1 22.0 21.0

Ethanol 42.5 46.2 38.1 23.0

H2 Partial Pressure headspace (atm) 0.068 0.116 0.488 0.062

Maximum specific sucrose consumption (mmol sucrose/L h) 0.26 0.16 0.11 0.11

Period (h) 3.55.0 812 7292 162186

Maximum H2 specific production rate (mmol H2 /g VSS h) 2147.3 3201.6 10059.5 2663.1

Hydrogen yield (mol H2 /mol sucrose) 15% (1.2) 19.8% (1.6) 15% (1.2) 3.8% (0.3)

0 10 15 20 25 30

0,05

0,10

0,15

0,20

0,25

0,30

VSS(g/L)

Time (h)

0 20 40 60 80 100

0,05

0,10

0,15

0,20

0,25

0,30

0,35

VSS(g/L)

Time (h)

0 50 100 150 200

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45

0,50

VSS(g/L)

0 50 100 150 200 2500,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

VSS(g/L)

Time (h)

Time (h)

5

a b

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 glucoseand 2.2 molH2/mol glucose. The ratio of gas hydrogen in head-

space 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 fermen-

tative 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 condi-

tions (1) and (2) (Fig. 3a). To the conditions (3) and (4), produc-

tion of isobutyric and isovaleric acids was also observed in

reduced concentrations (Fig. 3b). In this experiment, the twodegradation pathways occurred but the sucrose conversion

to acetic acid was favoured in the first operational hours.

High hydrogen gas productions were also verified 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 presentmicrobial

consortium did not consume this intermediary product that

was in the anaerobic reactors. The ethanol production wasgradual and increased in relation to the operational period

of time. The maximum generations of ethanol were verified

in the final samplings of all the studied conditions ( Table 2).

The biological hydrogen production can be affected by the

H2 partial pressure [23]. This fact was verified in the experi-

ment. 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 justified 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 inhibi-

tion 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

0%

20%

40%

60%

80%

100%

3,5 9,5 28

0%

20%

40%

60%

80%

100%

0 12 16 20 25 30 34 40

VolatileFattyAcids

(%)

VolatileFattyAcids(%

)

Time (h)

Time (h)

0%

20%

40%

60%

80%

100%

0 24 48 72 96 106 120 168

Time (h)

0%

20%

40%

60%

80%

100%

0 12 16 20 25 30 34 40

Time (h)

VolatileFattyAcids(%

)

VolatileFattyAcids

(%)

0 5 8 11 13 15

4 8 4 8

a b

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

4128.0 mg sucrose/L.



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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 unfavoura-

ble thermodynamically.Many methods to diminish the partialpressure of H2 can cause benefic 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 produc-

tion 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 (condi-

tion 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 path-

ways 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 efficiency of the conversion of sucrose to

acetate [9]. Kim et al. [11] operated a continuous flow reactorwith 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

identified as Clostridium ssp. and Bacillus racemilacticus.

Fig. 4 presents the profile 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 identified 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 thepre-treated inoculum that was responsible for the H2 produc-

tion 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 Clos-

tridium [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 H2with glucose (2.2 mol H2 /mol glucose) and cellobiose

(5.4 mol H2/mol cellobiose).

The band 2 presented 98% of phylogenetic similarity withBurkholderia cepacia. The band 2 that is present to condition

(1) was also observed in the conditions (2) and (4). This bacte-

rium 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, pesti-

cides 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-fixing 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 thepresence of sucrose and peptone.

A D

1

2

3

4

5

6

B C

Fig. 4 DGGE-profiles of 16S rRNA gene fragment of the

eubacterial populations at each experimental condition: (A)

630.0 mgsucrose/L, (B) 1184.0 mg sucrose/L; (C)

1816.0 mg sucrose/L and (D) 4128.0 mg sucrose/L.

Table 3 Affiliation of DGGE fragments determined by 16S rRNA sequence

Band Microorganisms Accessnumber (Genbank)

Similarity % References

1 Enterobacter cloacae EF120473.1 98 Feng, R.H. (2006). Not published

2 Burkholderia cepacia DQ387437.1 98 [19]

3 Enterobacter cloacae EF059865.1 97 Iversen, C. et al. (2007). Not published

4 Clostridium sp. DQ196619.1 90 [31]

5 Clostridium sp. AY862512.1 98 Zhang, T. (2004). Not published

6 Clostridium acetobutylicum U17030.1 96 [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 studiedconditions. 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 carbo-hydrates or peptones [3]. Some species fix 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 ofClostridium

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 specific

nutritional medium and pH control to 5.5 enriched these pop-

ulations [4]. Pure culture ofC. acetobutyricum was used in the

H2 production in reactors fed with glucose (10.5 g/L). H2conversion efficiency of 22%, specific production of glucose

of 1270 mlH2 /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 fermen-

tative products in the effluent 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 verified 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 1and 3 were related phylogenetically to E. cloacae. The

sequences referred to the band 2 were related phylogeneti-

cally 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 produc-

tion 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 efficiency 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 devel-

opment of microbial hydrogen-producing consortia.The conditions (1) and (2) presented H2 generation in the

first operation hours indicating that the substrate concentra-

tions 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 purified

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, pro-

pionic, isobutyric and isovaleric acids to the conditions (3) and

(4) was verified.

The best efficiency of the sucrose conversion to H2 was

obtained in the condition (2). The maximum specific activityof H2 production was observed in the condition (3).

The analyses of Molecular Biology revealed that the biolog-

ical production of H2 was due to the presence of the species E.

Band 1

Band 3

Enterobacter cloacae (EF120473.1)

Enterobacter cloacae (EF059865.1)

Band 2

Burkholderia cepacia (DQ387437.1)

Clostridium acetobutyricum (U17030.1)Band 6

Band 5

Clostridium sp.(AY862512.1)

Clostridium acetobutyricum (X81021)

Clostridium butyricum (AY458857)

Band 4

Clostridium sp.(DQ196619.1)

100

97

99

82

77

44

53

78

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.



9/9

cloacae, Clostridium sp. and C. acetobutyricum; recognized as H2and 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 identified as

Clostridium and E. cloacae.

Acknowledgements

The authors gratefully acknowledge the financial support of

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo

(FAPESP) and Conselho Nacional de Pesquisa e Desenvolvi-

mento (CNPq).

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