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2012
Hydrogen production and consumption of organic acids by a phototrophic microbial consortium INTERNATIONAL JOURNAL OF HYDROGEN ENERGY, OXFORD, v. 37, n. 16, supl. 1, Part 3, pp. 11691-11700, AUG, 2012 http://www.producao.usp.br/handle/BDPI/37914 Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo
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Hydrogen production and consumption of organic acids by a phototrophic microbial consortium Carolina Zampol Lazaro*, Daniele Vital Vich, Julia Sumiko Hirasawa, Maria Bernadete Amaˆncio Varesche Department of Hydraulics and Sanitation, School of Engineering of Sa˜o Carlos, University of Sa˜o Paulo, Av. Trabalhador Sa˜o-carlense, 400, 13566-590 Sa˜o Carlos, SP, Brazil
article info
abstract
Article history:
Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention
Received 1 February 2012
because its combustion releases only water, and it can be produced by microorganisms
Received in revised form
using organic acids as substrates. The aim of this study was to enrich a microbial
14 May 2012
consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge
Accepted 17 May 2012
Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other
Available online 19 June 2012
carbon sources were tested, such as acetate (30 mmol l1), butyrate (17 mmol l1), citrate
Keywords:
bated at 30 C under constant illumination by 3 fluorescent lamps (81 mmol m2 s1). The
Mixed culture
cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H2 l1 culture for
Rhodobacter
acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield
Sulfurospirillum
was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H2 mmol1 substrate for acetate, butyrate, citrate, lactate
Organic acids
and malate, respectively. The consumption of substrates was 43% for acetate, 37% for
Anaerobic batch reactors
butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the
(11 mmol l1), lactate (23 mmol l1) and malate (14.5 mmol l1). The reactors were incu-
clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum (29% of the total) were also found in the microbial consortium. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Rapid worldwide industrialization and urbanization as well as the increase in fossil fuel consumption have led to some concerns regarding environment pollution. Thus, attention has been given to alternative sources of energy that produce hydrogen as a clean energy carrier. Biological hydrogen production processes are known to be more environmental friendly and consume less energy compared to thermochemical and electrochemical processes because biological processes take place at ambient temperature and pressure [1].
Phototrophic bacteria are highlighted in the literature as promising microbial systems for the biological production of hydrogen because of their ability to consume organic substrates present in wastes, indicating the potential for combining wastewater treatment and energy generation. Simple organic molecules, such as organic acids generated from the anaerobic digestion of organic matter, can be used by phototrophic bacteria as substrates for H2 production. The maximum hydrogen yield from a particular substrate, assuming complete conversion of the substrate to H2 and CO2, can be calculated using Equation (1) [2,3].
* Corresponding author. Tel.: þ55 1633738357; fax: þ55 1633739550. E-mail addresses:
[email protected] (C.Z. Lazaro),
[email protected] (M.B.A. Varesche). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.05.088
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CaHbOc þ (2a c)H2O / (2a c þ 0.5b)H2 þ aCO2
(1)
The substrate conversion efficiency, which is a measure of how much of the substrate has been utilized for hydrogen production, is another useful parameter. The efficiency is determined by calculating the ratio of moles of hydrogen that have actually been produced to moles of hydrogen expected from stoichiometric conversion of the substrate [4]. Most studies of photobiological hydrogen production have been conducted using pure cultures, and very little information has been reported regarding photo-fermentative hydrogen production by mixed cultures of phototrophic bacteria [5,6]. The microorganisms most commonly used for the biological production of hydrogen are Rhodobacter sphaeroides [4,7,8], Rhodobacter capsulatus [9] and Rhodopseudomonas palustris [10e12]. Eroglu et al. [13] studied the photo-fermentative hydrogen production from olive mill wastewater (OMW) by R. sphaeroides O.U.001 under two different regimes of illumination, continuous and 12 h lighte12 h dark. Biomass submitted to continuous illumination reached a stationary phase of about 0.55 g dry cell weight l1 culture at 44 h after inoculation, with a maximum H2 productivity (based on biomass) of 2.37 ml H2 g1 h1. Initially, under 12 h lighte12 h dark cycles, the biomass showed a slow growth rate, which persisted for about 36 h. After this time, the culture showed an enhancement in the growth rate, reaching at stationary phase 0.5 g dry cell weight l1 culture at about 70 h after inoculation, with maximum productivity of 4.4 ml H2 g1 h1. The calculated yield was the same for the two conditions, with 250 mmol H2 l1 OMW. Seifert et al. [14] also investigated the growth of R. sphaeroides O.U.001 on Biebl and Pfenning media using dairy wastewater instead of malic acid in its composition. They evaluated the influence of illumination, pH, dairy wastewater and inoculum concentrations. The highest hydrogen yield (3.2 l H2 l1) was reached with light intensities between 9 and 13 klux, 0.086 g dry weight l1, dairy wastewater at a concentration of 40% and pH controlled close to 7. The authors concluded that the amount of hydrogen produced from dairy wastewater in these conditions was 30% better than that observed with standard samples of malic acid (2.3 l H2 l1). Hydrogen production by different Rhodobacter sp. was also investigated by Kapdan et al. [15] using acid hydrolyzed wheat starch by photo-fermentation. The highest specific hydrogen production rate (46 ml H2 g1 biomass h1) and the yield (1.23 mol H2 mol1 glucose) were obtained with the R. sphaeroides RV culture. Avcioglu et al. [16] used the dark fermentation effluent of molasses as substrate for hydrogen production by two strains of R. capsulatus. The system used was an outdoor panel photobioreactor. The maximum hydrogen yield obtained was 78% (of the theoretical maximum). Hydrogen productivity of photosynthetic bacteria on fermented effluent of potato steam peels hydrolysate was searched by Afsar et al. [17] using strains of R. capsulatus, R. sphaeroides and R. palustris. In this study, the authors conclude that using defined cocultures might be advantageous for overcoming the difficulties of varying feedstock properties and may also be beneficial for obtaining a more stable operation for continuous cultures.
According to Li and Fang [6], the development of photofermentative hydrogen production is in its initial phase. There are some important aspects that need to be developed by further studies. One of these aspects is the use of mixed cultures from environmental samples for treating complex substrates. Thus, the aim of this study was to evaluate the hydrogen production from organic acids using an enriched phototrophic microbial consortium as the inoculum.
2.
Materials and methods
2.1.
Inoculating and enriching the microbial consortium
The Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) was obtained from anaerobic granular sludge from an Upflow Anaerobic Sludge Blanket reactor (UASB) used to treat pig slaughterhouse wastewater (Jaboticabal, Brazil). Firstly, the sludge was triturated to break down the granules. Then, 100 ml of the triturated sludge was transferred to a glass bottle (Duran) with 101 mm of diameter and total volume of 1 l containing 0.4 l of RCVB culture medium [18]. Helium gas (99%) was purged in the headspace to ensure the anaerobic conditions. The culture medium was supplemented with malate (30 mmol l1) and sodium glutamate (4 mmol l1). The initial pH was adjusted to 6.8e7.0 with NaOH (1 N). The bottle was incubated at 30 C in a growth chamber constantly illuminated by 3 fluorescent lamps (20 W). When a layer of purple biomass appeared, it was transferred to another glass bottle using a platinum loop and cultivated under the same conditions described above. This process of transferring the purple biomass to another glass bottle with fresh culture medium was performed about five times to primarily harvest the purple non-sulfur photosynthetic bacteria.
2.2.
Operation of the batch reactors
Microbial hydrogen production experiments were conducted in closed glass bottles (Duran) with 86 mm of diameter and total volume capacity of 0.5 ml containing 0.35 l culture medium [19]. Argon gas (99.99%) was purged to ensure the anaerobic conditions. The substrates were acetate (30 mmol l1), butyrate (17 mmol l1), citrate (11 mmol l1), lactate (23 mmol l1) or malate (14.5 mmol l1). Sodium glutamate (4 mmol l1) was used as the nitrogen source. The concentrations of carbon and nitrogen sources were set to maintain a balanced carbon and nitrogen ratio (C/N ratio equal to an average of 16). The bottles were kept in a controlled-temperature incubator at 30 C illuminated constantly by 3 fluorescent lamps (TLT 20W/75RS, extra day light, Philips). The light intensity at the surface of the bottle was approximately 81 mmol m2 s1. The photosynthetically active radiation measurement was performed with an LI-193 spherical quantum sensor coupled with datalogger LI-1400 (LI-COR). Before each assay, the biomass was centrifuged (10,000 rpm at 4 C for 5 min) and was washed with fresh medium. The culture medium [19] used for the hydrogen production experiments contained the following nutrients per liter of deionized water: 0.5 g KH2PO4; 0.4 g MgSO4 7H2O; 0.4 g
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NaCl; 50 mg CaCl2 2H2O; 3.9 mg Fe(III) citrate; 0.3 mg H3BO3; 0.03 mg Na2MoO4 2H2O; 0.1 mg ZnSO4 7H2O; 0.2 mg CoCl2 2H2O; 0.01 mg CuCl2 2H2O, 0.03 mg MnCl2 4H2O; 0.02 mg NiCl2 6H2O and 0.04 mg vitamin B12. The initial pH was adjusted to 6.8e7.0 with NaOH (1 N).
2.3.
Analytical methods
The hydrogen content in the biogas was determined by analyzing a gas samples (250 ml) from each reactor using a gas chromatograph (GC 2010, Shimadzu, Tokyo, Japan) equipped with a Carboxen 1010 PLOT column (30 m, 0.53 mm), a thermal conductivity detector (TCD) and argon as a carrier gas. The temperatures of the injector and the detector were 130 C and 230 C, respectively. The column temperature program was as follows: 40 C for 2 min, heated to 60 C at the rate of 5 C min1, followed by an increase to 95 C at the rate of 25 C min1, and then increasing at 5 C min1 to a final temperature of 200 C. Organic acids concentrations were measured using a high performance liquid chromatograph (HPLC) (Shimadzu) equipped with an Aminex HPX-87H column (300 mm, 7.8 mm; Bio-Rad) with column oven (CTO-20A) at 64 C, UV-diode array detector (SPD-M10A VP), system controller (SCL-10A VP) and pump (LC-10AD VP). The mobile phase was H2SO4 solution (0.01 N) at 0.8 ml min1. The cellular growth was determined spectrophotometrically based on optical density at 660 nm (OD660nm) (DR/4000 Spectrophotometer-HACH) and the biomass dry weight was measured as volatile suspended solids (VSS) [20]. Standard-growth curve experiment was carried out in order to establish the ratio between cell density and biomass dry weight. In this experiment, there were collected samples for the optical cell density and VSS analysis. After that the data were plotted and it was found that an optical cell density of 1.0 corresponded to 0.92 g dry weight l1 culture (Equation (2)). VSS (g l1) ¼ 0.9204 OD660 nm þ 0.0019
2.4.
Molecular biology
2.4.1.
Nucleic acid extraction
(2)
The analysis of the microbial diversity was performed using sample collected at the end of the enrichment phase. DNA extraction was performed according to the procedure described by Griffiths et al. [21], modified by the use of glass beads and a phenol:chloroform:buffer mixture (1:1:1 v/v). A segment of the 16S rRNA gene was amplified by PCR using the eubacterial primers 27F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and 1100R (50 -AGG GTT GCG CTC GTT G-30 ) [22].
2.4.2.
Cloning and determination of the 16S rRNA sequence
The PCR products were cloned using the pGEM Easy Vector System (Promega) according to the manufacturer’s specifications. For this purpose, 2 ml of PCR product was used in the ligation with pGEM vector and subsequent transformation in competent cells. 100 ml of the transformated culture was plated onto LB/Ampicilin/IPTG/X-Gal plates and incubated overnight at 37 C. After this period, 50 clones were randomly
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selected, incubated in 5 ml of liquid Luria Bertani media (LB) (37 C, overnight) and submitted to PCR using the primers M13F (50 -CGC CAG GGT TTT CCC AGT CAC GAC-30 ) and M13R (50 -TTT CAC ACA GGA AAC AGC TAT GAC-30 ) [23]. The PCR products were purified with Illustra GFX (GE Healthcare) kit. Nucleotide sequencing was performed on an automated ABI Prism 310 sequencer (Dye terminator cycle sequencing kit, Applied Biosystems) according to the manufacturer’s instructions, using the M13F primer. The nucleotide sequences analysis was processed according to the procedure described by Oliveira et al. [24]. The phylogenetic tree was constructed using PHYLIP 3.6 and was calculated using the neighbor-joining algorithm [25]. Bootstrap re-sampling analysis for 100 replicates was performed to estimate the confidence of tree topologies. The nucleotide sequences determined in this study and included in the phylogenetic tree have been deposited in GenBank under the accession numbers JQ437239eJQ437255.
2.4.3. Modeling hydrogen production using the Gompertz equation The cumulative hydrogen production (H ) data were fitted to a modified Gompertz equation [26], which is a suitable model for describing the progressive accumulation of hydrogen in a batch experiment (Equation (3)): Rm $e ðl tÞ þ 1 H ¼ P$exp exp P
(3)
where P is the hydrogen production potential (mmol H2 l1 culture), Rm is the maximum H2 production rate (mmol H2 l1 culture d1), l is lag-phase time (d) and e is 2.718281828. This curve-fitting was accomplished using Statistica 8.0 software. The experiments were carried out in duplicates.
3.
Results and discussion
Many studies have been done using pure cultures of phototrophic purple bacteria to the photo-fermentative hydrogen production. However, the hydrogen production performed by microbial consortia must be studied as a way to evaluate the economic viability of the process. Hence, this study evaluated the photo-fermentative hydrogen production by microbial consortium from organic acids.
3.1. Enrichment of Phototrophic Hydrogen-Producing Bacterial Consortium The Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) obtained was composed mainly of Gram-negative rods, but Gram-positive rods were found as well. Approximately 33% of the clones were similar to bacteria belonging to Alphaproteobacteria, including Rhodobacter, Rhodospirillum and Rhodopseudomonas [27]. These bacterial genera have been widely reported in studies focusing on hydrogen production by phototrophic purple non-sulfur bacteria [6]. Clones similar to the genus Sulfurospirillum were also found in the microbial consortium. Sulfurospirillum are able to consume acetate, butyrate, citrate, lactate and malate, but they cannot produce hydrogen gas. Moreover, these microorganisms can use
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hydrogen gas as an electron donor in the presence of acetate [28]. The phylogenetic tree was constructed using 42 sequences obtained by phylogenetic analysis of 16S rRNA gene fragments (Fig. 1). These 42 sequences were grouped into 17 operational taxonomic units (OTUs), with 97% similarity (Fig. 2). OTU_1 is comprised of a clone similar to Acholeplasma sp. (EU517562 e 92%), a microorganism of the class Mollicutes (phylum Firmicutes), which contains bacteria that have no cell wall. They are not related to the production of hydrogen and are described in the literature as pathogens of plants and animals [29]. The source of the inoculum used in this study may explain the presence of this microorganism in the microbial consortium. Clones similar to uncultured anaerobic bacteria belonging to the class Clostridia (phylum Firmicutes) were grouped into two different OTUs. One clone was placed in OTU_2 (AY953192 e 100%), and other 5 clones were grouped in OTU_16 (EU864476 e 100%). Clostridium species are Gram-positive spore-forming bacteria that can produce hydrogen by the fermentative metabolism of carbohydrates. They are frequently reported in the literature as hydrogen-producing bacteria that use organic molecules (like sucrose and glucose) as carbon sources and produce H2, CO2, organic acids and alcohols as byproducts [30e32]. Zhang et al. [5], performed a DNA cloning analysis of a phototrophic sludge producing H2 from acidified wastewater and reported that approximately
81% of species were closely related to R. capsulatus (99% similarity), while two unidentified species were related to the Bacillus/Clostridium. These microorganisms most likely propagated in the microbial consortium due to their metabolic versatility and their ability to sporulate, although the environmental conditions imposed in this study were not suitable for the enrichment of these microorganisms. OTU_7 and OTU_8 contained 2 clones each, which were similar to uncultured bacteria (AY945866 e 94%). OTU_9 contained 3 clones similar to Propionicimonas paludica (EF515420 e 89.5%), which is capable of fermenting glucose to produce acetic, lactic, succinic and propionic acids and CO2. Akasaka et al. [33] isolated two strains under anaerobic conditions that were able to utilize several carbon sources (arabionose, xylose, fructose, glucose and sucrose) and also had the ability to grow well on pyruvate and lactate and less well in malate, fumarate and succinate. OTU_13 contained 3 clones similar to uncultured Magnetospirillum sp. (EF176784 e 86%), which is a genus belonging to the class Alphaproteobacteria. They are unable to grow photosynthetically but can use organic acids as a nutrient source [34], which explains their presence in the phototrophic microbial consortium; however, these microorganisms have not been described in the literature as hydrogen producers. OTU_14 contained 3 clones similar to Propionivibrio (AY928207 e 90%). Tanaka et al. [35] isolated the strain CreMal1, which they described as a member of Propionivibrio
Sulfurospirillum deleyanum (AB368775) OTU_17 Epsilonproteobacteria Sulfurospirillum cavolei (AB246781) Sulfurospirillum sp. (AY780560) OTU_15 OTU_14 Betaproteobacteria Propionivibrio sp. (AY928207) OTU_6 OTU_5 Rhodospirillum photometricum (D30777) Rhodopseudomonas palustris (AB017261) OTU_4 Rhodoplanes cryptolactis (AB087718) OTU_3 Magnetospirillum sp. (EF176784) Alphaproteobacteria OTU_13 Rhodobacter sp. (AB017799) OTU_12 Rhodobacter blasticus (DQ342322) OTU_10 Rhodobacter blasticus (AM690347) Rhodobacter capsulatus (D16427) OTU_11 OTU_16 Unidentified Clostridiales (EU864476) Clostridia Unidentified Clostridia (AY953192) OTU_2 Unidentified Clostridia (EU234230) OTU_9 Actinobacteria Propionicimonas sp. (EF515420) Propionicimonas paludicola (AB078859) OTU_1 Mollicutes Acholeplasma sp. (EU517562) OTU_8 Unidentified Bacteria (AY945866) OTU_7 E. coli 0.1
Fig. 1 e Phylogenetic tree of the OTUs identified from the Phototrophic Hydrogen-Producing Bacterial Consortium and their closest relatives, based on partial 16S rRNA gene sequences. The phylogenetic tree was constructed using neighbor-joining algorithm with 100 bootstraps. Escherichia coli was selected as the outgroup species. The scale bar represents 0.1 substitutions per nucleotide position (C, OTUs).
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Number of clones
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10 9 8 7 6 5 4 3 2 1 0 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 OTU
Fig. 2 e Number of clones grouped within each OTU.
Fig. 3 e Percentage of clones and their phylogenetic affiliations.
dicarboxylicus gen. nov., sp. nov. a mesophilic Gram-negative strict anaerobe that was able to grow on maleate, fumarate and malate, producing propionate and acetate as end products. The metabolic features of the members of this genus could explain their presence in the microbial consortium. OTU_15 contained 3 clones similar to Sulfurospirillum sp. (AY780560 e 92%), and OTU_17 contained 9 clones similar to Sulfurospirillum cavolei (AB246781 e 100%), which are Gramnegative, slightly curved rods that can grow fermentatively on fumarate, pyruvate and malate. The optimum growth of these microorganisms occurs at 30 C and pH 7 [28]. The conditions used in this study, including temperature, carbon source and pH, were favorable to their growth; however, these microorganisms may not be responsible for the H2 production. Although they are not responsible for the hydrogen production, they are able to use acetic acid, lactic acid and hydrogen
as electron donors for sulfate reduction [28]. In this study, the presence of acetic acid and H2, which are byproducts of phototrophic metabolism, combined with the presence of sulfate, may have enabled the growth of these microorganisms. According to Scoma et al. [36] side-process as acetate consumption by sulfate-reducing bacteria can occur when a microbial consortium is used. The species of interest for this study showed a wide morphological and metabolic diversity and were affiliated to the phototrophic purple non-sulfur bacteria group. These bacteria belong to Proteobacteria, the largest phylum within the Bacteria. The OTUs containing these microorganisms will be discussed below. OTU_3 was phylogenetically similar to Rhodoplanes cryptolactis (AB087718 e 96%), which is a species that preferentially grows by anaerobic photoorganotrophy metabolism, consuming pyruvate, acetate, malate, succinate and butyrate as carbon sources [37]. OTU_4 was similar to R. palustris (AB017261 e 99%), which is commonly described in the literature as hydrogen producer from various organic acids [38]. OTU_5 and OTU_6 contained clones that were similar to Rhodospirillum photometricum (D30777 e 85%). Another species that belongs to the same genus, Rhodospirillum rubrum, is a purple non-sulfur photosynthetic bacterium that can produce hydrogen from various organic acids [39], and also by a water-gas shift reaction [40]. OTU_10 contained 2 clones and OTU_11 contained 3 clones similar to R. capsulatus (D16427 e 98%). OTU_12 contained 3 clones similar to Rhodobacter sp. (AB017799 e 93%). These microorganisms are able to use various organic acids as substrates for hydrogen production and thus could have been responsible for generating hydrogen in this study. Fig. 3 shows the microbial diversity related to the percentage of clones belonging to different class: Alphaproteobacteria, Betaproteobacteria, Epsilonproteobacteria and Clostridia. Thirty-three percent of the clones were classified as part of the class Alphaproteobacteria, which includes phototrophic purple non-sulfur bacteria. Seven percent of the clones were related to Betaproteobacteria, which also contains some species of phototrophic bacteria. Clones similar to Epsilonproteobacteria represented 29% of the total and were similar to the genus Sulfurospirillum. Fourteen percent of the clones were phylogenetically related to
Table 1 e Summary of the results of hydrogen production experiments from five different carbon sources.
1
Initial substrate concentration (mmol l ) Substrate utilization (%) Byproducts-acetic acid (mmol) H2 yield (mmol H2 mmol1 substrate) Substrate efficiency conversion (%) Cumulative hydrogen production (mmol H2 l1 culture) a Hydrogen production potential (mmol H2 l1 culture) a Lag-phase (days) a Maximum rate of hydrogen production (ml H2 l1 culture h1) a 2 R
Acetate
Butyrate
Citrate
Lactate
Malate
30 43 e 0.6 14.5 7.8 8.8
17 37 1.4 1.4 13.8 9.0 8.7
11 100 3.5 0.7 8.2 7.9 8.1
23 49 1.2 0.5 8.2 5.6 5.9
14.5 100 0.8 0.9 15.6 13.9 13.2
2.8 0.4 0.991
2.9 0.9 0.993
0.5 0.6 0.989
e 0.3 0.984
2.2 1.0 0.993
a Values obtained by adjusting the data according to the modified Gompertz equation.
12
7
5
8
4
6
3
Acetate (mmol l-1)
10
6
4
2 2
A
1 0 0
5
10
15
20
25
0
30
Time (d)
20
12
18 10
14 12 10
6
8 4
6 4
2
B
0 0 12
7
18
Butyrate (mmol l-1)
16
8
5
10
15 Time (d)
20
25
2 0
30 14
C
12
10
10
8
8 6 6 4
4
2
Citrate (mmol l-1)
Hydrogen production (mmol l-1 culture)
2 0
0 0
5
10
15
20
25
30
Time (d)
25
6
4
15
3
10
2
Lactate (mmol l-1)
20
5
5
1
D
0 0
5
10
15
20
25
0
30
Time (d)
E
16 14
16 14 12
12
10
10
8
8
6
6 4
4
2
2
Malate (mmol l-1)
The PHPBC was capable of metabolizing all of the five different substrates tested in order to produce hydrogen. However, the percentage of the consumption varied among the substrates. Citrate and malate were totally consumed, while acetate (43%), butyrate (37%) and lactate (49%) were not (Table 1). For the substrates acetate, butyrate and malate, the production of hydrogen gas began after two days of incubation, for citrate and lactate the production started earlier. The hydrogen production stabilized after approximately 25 days of incubation for all the substrates tested. Interestingly, hydrogen production continued even after citrate and malate were completely consumed (Fig. 4). It is possible that the acetic acid produced (data not shown) in these reactors may have been used as a substrate to maintain hydrogen production. The PHPBC consumed 43% of acetate and produced 7.8 mmol H2 l1 culture. For butyrate, 37% of the substrate was consumed and hydrogen production was 9 mmol H2 l1 culture. In this experiment, acetic acid concentrations increased during the incubation period, reaching a maximum value of 1.4 mmol of acetic acid after 28 days of incubation. The citrate was totally consumed in 4 days of incubation, and the cumulative hydrogen production was 7.9 mmol H2 l1 culture. Acetic acid was generated simultaneously with citrate consumption. At the peak of production (the fourth day), the acetic acid content reached a concentration of 6.0 mmol, which dropped to 3.5 mmol by the twenty-fifth day of incubation. Probably, the acetic acid generated was most likely also used as a substrate. A total of 49% of lactate was consumed after 28 days of incubation, and the acetic acid concentration reached 1.2 mmol by the end of the experiment. The cumulative hydrogen production for this substrate was 5.6 mmol H2 l1 culture. The malate was consumed in its entirety in 9 days of incubation. In this case, acetic acid concentrations reached a maximum value of 2.5 mmol on the fourth day of incubation. The concentration of acetic acid at the end of the experiments was 0.8 mmol. The highest generation of hydrogen observed was 13.9 mmol H2 l1 culture in the reactors fed with malate (Table 1). By means of a modified Gompertz equation, the hydrogen production potential was determined and the results were statistically significant, as indicated by the R2 values shown in
14
8
Hydrogen production (mmol l-1 culture)
3.2. Hydrogen production from different individual carbon sources
9
Hydrogen production (mmol l-1 culture)
unidentified Clostridia. Other bacteria were found in smaller proportions: 2% of the clones belong to class Mollicutes, 5% were similar to Actinobacteria, and 10% were related to unidentified bacteria. According to these results we concluded that the set of experimental conditions (pH, temperature, source of light, carbon source and substrate concentration) were not feasible for the enrichment of microbial consortium predominantly composed by phototrophic microorganisms. Solely one third of clones were related to this group of bacteria. Another third of clones were related to Sulfurospirillum, which are not desirable microorganisms for the photo-fermentative hydrogen production. The high percentage of these microorganisms (29%) may be related with the low efficiency of the process.
Hydrogen production (mmol l-1 culture)
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Hydrogen production (mmol l-1 culture)
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0
0 0
5
10
15
20
25
30
Time (d)
Fig. 4 e (C) Cumulative hydrogen production (mmol H2 lL1 culture) and (-) substrate concentration (mM) for (A) Acetate; (B) Butyrate; (C) Citrate; (D) Lactate; (E) Malate.
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 7 ( 2 0 1 2 ) 1 1 6 9 1 e1 1 7 0 0
Table 1. Thus, this model accurately predicted the cumulative phototrophic production of hydrogen. Table 2 presents the results obtained in the present work (substrate conversion efficiency and maximum hydrogen production rate) and also data from other researches to enable an easier comparison among the studies. A wide range of values of substrate conversion efficiency was reported by different authors. It could be due to the various experimental conditions used in each study such as microbial strain, carbon source and substrate concentration [41]. Working with a pure culture of R. palustris and using acetate (22 mmol l1), butyrate (27 mmol l1), lactate (50 mmol l1) and malate (15 mmol l1) as substrates, Barbosa
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et al. [38] found substrate conversion efficiencies of 14.8%, 0%, 12.6% and 36%, respectively. The authors tested another inoculum, which they called the Microbiology strain, with the same substrates, and observed substrate conversion efficiencies of 35.3%, 0.3%, 14.4% and 36.7%. In the present study, the values found for acetate (30 mmol l1), butyrate (17 mmol l1), lactate (23 mmol l1) and malate (14 mmol l1) were 14.5%, 13.8%, 8.2% and 15.6%, respectively. These values are close to those found by Barbosa et al. [38] for R. palustris, except for the substrates malate and butyrate. Barbosa did not observe any hydrogen production by R. palustris using butyrate as a substrate, and for malate, the substrate efficiency conversion was higher than that in the present study. For the
Table 2 e Comparison of hydrogen production by different microorganisms. Microorganisms
Rhodobacter sphaeroides O.U.001
Rhodopseudomonas palustris
Microbiology strain
ZX-5strain
Rhodobacter sphaeroides O.U.001 Rhodobacter sphaeroides O.U.001 hup-mutant Rhodobacter sphaeroides O.U.001 Rhodobacter sphaeroides ZK1 Rhodobacter sphaeroides SCJ Rhodobacter sphaeroides SCJ Rhodopseudomonas palustris WP3-5 Rhodopseudomonas sp. R. palustris W004 Rubrivivax sp. Rhodobacter sphaeroides KD131 Rhodobacter sphaeroides P1 PHPBC
Carbon source
Initial concentration (mmol l1)
Maximum rate of H2 production (ml l1 h1)
Substrate conversion efficiency (%)
Reference
Acetate Butyrate Lactate Propionate Malate Acetate Butyrate Lactate Malate Acetate Butyrate Lactate Malate Acetate Butyrate Succinate Malate Malate
30 15 20 20 15 22 27 50 15 22 27 50 15 35 50 50 30 15
20 5 20 22 24 2.2 e 9.1 5.8 5.3 0.2 7.9 6.0 90 110 94 92 6.9
33 14 31 31 50 14.8 e 12.6 36 35.3 0.3 14.4 36.7 69 74.6 81.4 78.9 70.5
[4]
Malate
15
9.2
85.2
Acetate Malate Acetate Malate Acetate
30 15 30 15 25
e e e e 26.9
4.6 58 5.5 71.5 19.5
Butyrate
25
31.8
8
Acetate
31.4
32.4
73.3
[47]
Butyrate, acetate, propionate, ethanol
24.5, 18.3, 2.8, 12.1 60 30 60 30 30 17 11 23 14
56.5 51.6 7.7 17.5 24.7 36.25 45.9 14.5 13.8 8.2 8.2 15.6
[44]
Acetate Butyrate Acetate Butyrate Acetate Butyrate Citrate Lactate Malate
26.5 10.3 16.4 e e e e 0.4a 0.9a 0.6a 0.3a 1.0a
a Maximum rate of H2 production (mmol H2 l1 culture d1).
[38]
[43]
[45]
[46]
[41]
[48]
Present study
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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 7 ( 2 0 1 2 ) 1 1 6 9 1 e1 1 7 0 0
Microbiology strain, the substrate conversion efficiency was higher than that observed for R. palustris and also higher than the conversion efficiencies documented in the present study, except for butyrate. He et al. [42] reported 52.7% and 68.2% substrate conversion efficiency using lactate (30 mmol l1) by R. capsulatus JP91 and by R. capsulatus IR3 at 30 C, respectively. Tao et al. [43], using RCVB medium at 30 C, isolated a purple non-sulfur bacterium (ZX-5) similar to R. sphaeroides (99.7% similarity) that showed substrate conversion efficiencies of 69% for acetate (35 mmol l1), 74.6% for butyrate (50 mmol l1), 78.9% for malate (30 mmol l1) and 81.4% for succinate (50 mmol l1). Uyar et al. [4] showed that R. sphaeroides O.U.001 growing in Biebl and Pfenning medium and using acetate (30 mmol l1), butyrate (15 mmol l1), lactate (20 mmol l1) or malate (15 mmol l1) as a carbon source and sodium glutamate (10 mmol l1) as nitrogen source exhibited substrate conversion efficiencies of 33%, 14%, 31% and 50%, respectively. The values are higher than those obtained in the present study. Wu et al. [44] studied the hydrogen production by Rhodopseudomonas sp., R. palustris W004 and Rubrivivax sp. by individual and mixed carbon source. The authors concluded that butyrate was more desirable substrate to produce hydrogen than acetate. Furthermore, Rhodopseudomonas sp. produced more hydrogen (2192 ml l1 culture) than R. palustris W004 (1086 ml l1 culture). Moreover, using the mixture of acetate, butyrate, propionate and ethanol, Rhodopseudomonas sp. showed the higher substrate conversion efficiency (56.5%) in comparison to R. palustris W004 (51.6%) and Rubrivivax sp. (7.7%). Values of substrate conversion efficiency obtained by [4,43,45e48] are higher than those obtained in the present study, which is most likely due to the use of pure cultures instead of a bacterial consortium. According to ValdezVazquez and Poggi-Varaldo [49], in most anaerobic environments, H2 is consumed very quickly by different microbial groups. Moreover, microorganisms present in the microbial consortium could compete with the phototrophic purple nonsulfur bacteria by the substrates, causing the decrease in the yield of the H2 production process.
4.
Conclusions
The hydrogen production by the enriched microbial consortium was not feasible considering the low efficiency of the processes in terms of hydrogen yield. Although the use of microbial consortium is a crucial point for scale up the biological hydrogen production, it can create a number of problems due to the concomitant presence of H2 producers and consumers. Physiological parameters such as pH, temperature, and substrate, as well as, process parameters as hydraulic retention time and organic loading rate, could be adjusted in reactors operating in a continuous mode to favor some microorganisms in spite of others. Based on molecular biology analysis, the consortium was composed of phototrophic purple non-sulfur bacteria, which may be responsible for the photobiological production of H2. However, other microorganisms, including Sulfurospirillum, were also found, which are not related to H2 production, and furthermore
might reduce the substrate conversion efficiency by consuming the substrate or even the H2 produced. Thus, more studies are necessary to evaluate the feasibility of using Phototrophic Hydrogen-Producing Bacterial Consortium instead of pure cultures to produce hydrogen gas.
Acknowledgments The authors wish to thank Sa˜o Paulo Research Foundation (FAPESP) for the financial support of this study and National Council for Scientific and Technological Development (CNPqProcess number 131296/2007-8) for the postgraduate scholarship.
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