Universidade de São Paulo Biblioteca Digital da Produção Intelectual - BDPI Departamento de Hidráulica e Saneamento - EESC/SHS Artigos e Materiais de Revistas Científicas - EESC/SHS 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 international journal of hydrogen energy 37 (2012) 11691e11700 Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/he 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 À À Available online 19 June 2012 carbon sources were tested, such as acetate (30 mmol l 1), butyrate (17 mmol l 1), citrate À À À (11 mmol l 1), lactate (23 mmol l 1) and malate (14.5 mmol l 1). The reactors were incu- À À Keywords: bated at 30 C under constant illumination by 3 fluorescent lamps (81 mmol m 2 s 1). The À1 Mixed culture cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H2 l culture for Rhodobacter acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield À1 Sulfurospirillum was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H2 mmol 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 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 Sul- furospirillum (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 Phototrophic bacteria are highlighted in the literature as promising microbial systems for the biological production of Rapid worldwide industrialization and urbanization as well as hydrogen because of their ability to consume organic the increase in fossil fuel consumption have led to some substrates present in wastes, indicating the potential for concerns regarding environment pollution. Thus, attention combining wastewater treatment and energy generation. has been given to alternative sources of energy that produce Simple organic molecules, such as organic acids generated hydrogen as a clean energy carrier. Biological hydrogen from the anaerobic digestion of organic matter, can be used by production processes are known to be more environmental phototrophic bacteria as substrates for H2 production. The friendly and consume less energy compared to thermochem- maximum hydrogen yield from a particular substrate, ical and electrochemical processes because biological assuming complete conversion of the substrate to H2 and CO2, processes take place at ambient temperature and pressure [1]. 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 11692 international journal of hydrogen energy 37 (2012) 11691e11700 þ À / À þ þ CaHbOc (2a c)H2O (2a c 0.5b)H2 aCO2 (1) According to Li and Fang [6], the development of photo- fermentative hydrogen production is in its initial phase. The substrate conversion efficiency, which is a measure of There are some important aspects that need to be developed how much of the substrate has been utilized for hydrogen by further studies. One of these aspects is the use of mixed production, is another useful parameter. The efficiency is cultures from environmental samples for treating complex determined by calculating the ratio of moles of hydrogen that substrates. have actually been produced to moles of hydrogen expected Thus, the aim of this study was to evaluate the hydrogen from stoichiometric conversion of the substrate [4]. production from organic acids using an enriched phototrophic Most studies of photobiological hydrogen production have microbial consortium as the inoculum. been conducted using pure cultures, and very little informa- tion has been reported regarding photo-fermentative hydrogen production by mixed cultures of phototrophic 2. Materials and methods bacteria [5,6]. The microorganisms most commonly used for the biological production of hydrogen are Rhodobacter sphaer- 2.1. Inoculating and enriching the microbial consortium oides [4,7,8], Rhodobacter capsulatus [9] and Rhodopseudomonas palustris [10e12]. The Phototrophic Hydrogen-Producing Bacterial Consortium Eroglu et al. [13] studied the photo-fermentative hydrogen (PHPBC) was obtained from anaerobic granular sludge from an production from olive mill wastewater (OMW) by R. sphaeroides Upflow Anaerobic Sludge Blanket reactor (UASB) used to treat O.U.001 under two different regimes of illumination, contin- pig slaughterhouse wastewater (Jaboticabal, Brazil). Firstly, uous and 12 h lighte12 h dark. Biomass submitted to contin- the sludge was triturated to break down the granules. Then, uous illumination reached a stationary phase of about 0.55 g 100 ml of the triturated sludge was transferred to a glass bottle À Ò dry cell weight l 1 culture at 44 h after inoculation, with (Duran ) with 101 mm of diameter and total volume of 1 l a maximum H2 productivity (based on biomass) of 2.37 ml containing 0.4 l of RCVB culture medium [18]. Helium gas (99%) À1 À1 e H2 g h . Initially, under 12 h light 12 h dark cycles, the was purged in the headspace to ensure the anaerobic condi- biomass showed a slow growth rate, which persisted for about tions. The culture medium was supplemented with malate À À 36 h. After this time, the culture showed an enhancement in (30 mmol l 1) and sodium glutamate (4 mmol l 1). The initial the growth rate, reaching at stationary phase 0.5 g dry cell pH was adjusted to 6.8e7.0 with NaOH (1 N). The bottle was À weight l 1 culture at about 70 h after inoculation, with incubated at 30 C in a growth chamber constantly illumi- À1 À1 maximum productivity of 4.4 ml H2 g h . The calculated nated by 3 fluorescent lamps (20 W). When a layer of purple yield was the same for the two conditions, with 250 mmol biomass appeared, it was transferred to another glass bottle À1 H2 l OMW. Seifert et al. [14] also investigated the growth of R. using a platinum loop and cultivated under the same condi- sphaeroides O.U.001 on Biebl and Pfenning media using dairy tions described above. This process of transferring the purple wastewater instead of malic acid in its composition. They biomass to another glass bottle with fresh culture medium evaluated the influence of illumination, pH, dairy wastewater was performed about five times to primarily harvest the and inoculum concentrations. The highest hydrogen yield purple non-sulfur photosynthetic bacteria. À1 (3.2 l H2 l ) was reached with light intensities between 9 and À 13 klux, 0.086 g dry weight l 1, dairy wastewater at a concen- 2.2. Operation of the batch reactors tration of 40% and pH controlled close to 7. The authors concluded that the amount of hydrogen produced from dairy Microbial hydrogen production experiments were conducted Ò wastewater in these conditions was 30% better than that in closed glass bottles (Duran ) with 86 mm of diameter and À1 observed with standard samples of malic acid (2.3 l H2 l ). total volume capacity of 0.5 ml containing 0.35 l culture Hydrogen production by different Rhodobacter sp. was also medium [19]. Argon gas (99.99%) was purged to ensure the investigated by Kapdan et al. [15] using acid hydrolyzed wheat anaerobic conditions. The substrates were acetate À À À starch by photo-fermentation. The highest specific hydrogen (30 mmol l 1), butyrate (17 mmol l 1), citrate (11 mmol l 1), À1 À1 À1 À1 production rate (46 ml H2 g biomass h ) and the yield lactate (23 mmol l ) or malate (14.5 mmol l ). Sodium À1 À1 (1.23 mol H2 mol glucose) were obtained with the R. sphaer- glutamate (4 mmol l ) was used as the nitrogen source. The oides RV culture. concentrations of carbon and nitrogen sources were set to Avcioglu et al. [16] used the dark fermentation effluent of maintain a balanced carbon and nitrogen ratio (C/N ratio molasses as substrate for hydrogen production by two strains equal to an average of 16). The bottles were kept in of R. capsulatus. The system used was an outdoor panel a controlled-temperature incubator at 30 C illuminated photobioreactor. The maximum hydrogen yield obtained was constantly by 3 fluorescent lamps (TLT 20W/75RS, extra day 78% (of the theoretical maximum).