th Proceedings of the 5 International Workshop on Hydrogen and Fuel Cells October 26 –29, 2010 Campinas, SP, Brazil ISSN 2179-5029
BIOLOGICAL HYDROGEN PRODUCTION FROM INDUSTRIAL WASTEWATERS
(1) GUILHERME PEIXOTO (1) JORGE LUIS RODRIGUES PANTOJA FILHO (1) MARCELO ZAIAT
(1) Department of Hydraulics and Sanitation, São Carlos School of Engineering (EESC), University of São Paulo (USP), São Carlos, São Paulo State, Brazil
ABSTRACT This research evaluates the potential for producing hydrogen in anaerobic reactors using industrial wastewaters (glycerol from biodiesel production, wastewater from the parboilization of rice, and vinasse from ethanol production). In a complementary experiment the soluble products formed during hydrogen production were evaluated for methane generation. The assays were performed in batch reactors with 2 liters volume, and sucrose was used as a control substrate. The acidogenic inoculum was taken from a packed-bed reactor used to produce hydrogen from a sucrose-based synthetic substrate. The methanogenic inoculum was taken from an upflow anaerobic sludge blanket reactor treating poultry slaughterhouse wastewater. Hydrogen was -1 -1 produced from rice parboilization wastewater (24.27 mL H 2 g COD) vinasse (22.75 mL H 2 g -1 COD) and sucrose (25.60 mL H 2 g COD), while glycerol only showed potential for methane generation.
KEY WORDS Hydrogen; biological process; wastewaters;
1. INTRODUCTION The bioproduction of hydrogen is an interesting option because organic waste can be used as feedstock for the process. The fermentative process could be another way of generating hydrogen besides electrolysis and methane steam reforming, which are the most common processes used for hydrogen production nowadays.
Hydrogen generation using the fermentation process is possible with various types of wastewater using either mixed or pure cultures [1]. The use of various effluents, mainly wastewater containing cellulose, pentose, and xylose [2] [3], glycerol, residues from biodiesel production [4], effluent from cheese processing [5], dairy wastewater [6], by-products of wheat flour processing [7], molasses [8], solid food wastes [9], effluent from paper production [10] and domestic sewage [11], among others, has been reported.
Industrial effluents are potential energy sources readily found in many communities. Thus, hydrogen generation from these wastewaters could be carried out using local feedstock, reducing the costs involved in transportation and storage.
It is worth mentioning that vinasse and glycerol are by-products of ethanol and biodiesel plants, respectively. Both are renewable fuels that would have theirs sustainability increased if hydrogen could be produced using by-products of these processes.
1 Correspondence should be addressed to Guilherme Peixoto: Phone: +55 (16) 3373-8360; fax: (16) 3373-9550; e-mail: [email protected]
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Glycerol and vinasse were chosen due to the Brazilian industrial expansion on the production of renewable fuels, such as biodiesel and ethanol. The rice parboilization wastewater was chosen due to the high content of carbohydrates in its composition. According to Hawkes et al. [12] they are the main precursors of hydrogen production.
Current glycerol generation in Brazil is about 17.6 million liters per year considering the study realized by Silva et al. [13], which estimates that biodiesel production generates 10% (weight/weight) of glycerol.
Vinasse generation is usually 14 times the volume of ethanol produced. Data from UNICA (Sugarcane Agroindustry Union of São Paulo State) show that it was produced 15 million cubic meters of ethanol in the 2004–2005 harvest, thus resulting in a 210 million cubic meter generation of vinasse.
According to researches realized in 2005 by EPAGRI (Rural Extension and Agropecuary Research Business), to parboil 1 kg of rice it is generated around 1 liter of effluent. Thus, it is estimated that in Brazil, where 24% of the rice is parboiled [14], it was generated around 2.8 million cubic meters of rice parboilization wastewater in 2006.
The production of methane can be easily carried out using the organic acids generated in the hydrogen production process. According to Mohan et al. [15] the utilization of effluents for the production of hydrogen followed by posterior methanization of the soluble products generated in the acidogenic systems offers a solution for the problem of the renewable fuels and reduces significantly the pollution impact of the wastewaters used in the process.
2. OBJECTIVE The objective of this paper is to evaluate the potential of industrial residues for hydrogen generation through simple assays in bench-scale batch reactors with sucrose as the control substrate.
3. METHODOLOGY
3.1. BATCH REACTORS The reactors were composed of 2 liters glass flasks, consisting of 1 liter of liquid volume and 1 liter of headspace .
3.2. WASTEWATERS Three effluents were tested (vinasse; glycerol; rice parboilization wastewater), and sucrose was used as a control substrate.
Vinasse was obtained from an ethanol production plant (Usina Nova Era, Ibaté, SP, Brazil), and glycerol was obtained as a by-product after the esterification reaction of vegetable oil in a biodiesel production plant (Granol, Anápolis, GO, Brazil). The rice parboilization wastewater was obtained as a residue of the parboiled rice production process by a food production plant (Nelson Wendt Alimentos, Pelotas, RS, Brazil). The sucrose-based synthetic wastewater was prepared with organic demerara sugar (Native Natural Products, São Francisco S/A, Sertãozinho, SP, Brazil).
Basic characterizations of the wastewaters are presented in Table 1.
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Table 1 . Characterization of the wastewaters. Parameters Vinasse Glycerin Rice WW Sucrose COD (mg L -1) 20731 1108230* 5354 364 pH 3.8 ND 4.6 5.5 -1 Alkalnity (mg L CaCO 3) 0 ND 0 0 TS (mg L -1) 18170 ND 657 0 VSS (mg L -1) 14440 ND 537.5 0 FS (mg L -1) 3270 ND 119.6 0 Total N (mg L -1 N) 187.5 0 104.6 0 -1 3- Total P (mg L PO 4 ) 133 0 124.5 0 *For aqueous solution of 1g L -1 glicerol; COD – Chemical Oxygen Demand; TS – Total Solids; VSS – Volatile Suspended Solids; FS – Fixed Solids; N – Nitrogen; P – Phosphorous; ND – Not Determined.
3.3. Nutritional medium The reactors were filled with filtered wastewater (1.2 µm membrane) diluted with tap water to reach a chemical oxygen demand (COD) of approximately 300 mg L -1 in each flask. A -1 nutritional medium containing (in mg L ): CH 4N2O (6), NiSO 4.6H 2O (0.15), FeSO 4.7H 2O (0.75), FeCl 3.6H 2O (0.075), CoCl 2.2H 2O (0.012), CaCl 2.6H 2O (0.618), SeO 2 (0.0108), KH 2PO 4 (1.608), KHPO 4 (0.39) and Na 2HPO 4.2H 2O (0.828) was added to all reactors. Sodium bicarbonate (500 mg L -1) and chloridric acid (10 mol L -1) were added to each reactor to maintain the pH level of approximately 5.5.
3.4. Inoculum The acidogenic inoculum with around 30 g L -1 VSS was obtained from fixed-bed anaerobic reactors used for hydrogen production from sucrose-based synthetic wastewater [16].These reactors were filled with low density polyethylene as a support material for the attachment of biomass and kept at 25 ºC for 0.5 h of hydraulic detention time.
The metanogenic inoculum used in the complementary experiment had around 37 g L -1 VSS and was obtained from a UASB (Upflow Anaerobic Sludge Blanket Reactor) treating poultry slaughterhouse wastewater [17]. The granular sludge was taken directly from the middle of the reactor blanket zone.
3.5. Experimental procedure The duration of the experiment was 36 h. The complementary evaluation took about 156 h. The tests were performed in 4 batch reactors (in duplicate). Argon was fluxed for 20 min to guarantee an anaerobic environment in each reactor before closing them. The reactors were placed in a thermostatic chamber at 25.0±0.9ºC without agitation. Gas and liquid samples were taken periodically from the reactors for analysis. Internal pressure of the flask was measured using a pressure gauge with detection range of 0 to 500 mbar. Less than 10% of the overall bulk liquid volume was taken throughout the experiment.
In the acidogenic step (0 to 36 h) the liquid phase (1 liter) consisted of the diluted effluent, the nutritional medium utilized by Fernandes et al. [18] and around 400 mg L -1 VSS of inoculum taken from a packed-bed reactor used to produce hydrogen from a sucrose-based synthetic substrate [16].
In the complementary experiment (48 to 224 h), the reactors from the first step were opened and their products were filtered with a 0.45 µm membrane to remove the acidogenic biomass. After this procedure they were complemented with the same nutritional medium of the acidogenic step, inoculated with around 500 mg L -1 VSS of the methanogenic inoculum taken from an UASB [17], adjusted for pH 7.0 and fluxed with Argon.
28 th Proceedings of the 5 International Workshop on Hydrogen and Fuel Cells October 26 –29, 2010 Campinas, SP, Brazil ISSN 2179-5029
3.6. Analytical methods The composition of the biogas (hydrogen, carbon dioxide, and methane) was analyzed via gas chromatography (Shimadzu GC/TCD) [18]. Organic acids were measured using gas chromatography (HP GC 6890/FID), following the procedures described in [19]. Ethanol concentration was determined using gas chromatography (Shimadzu GC/FID) with an automatic injector (COMBI-PAL, AOC 5000, headspace mode) equipped with an HP-INNOWAX column.
Chemical oxygen demand (COD), pH, alkalinity, total solids (TS), volatile suspended solids (VSS), fixed solids (FS), nitrogen and phosphorous were analyzed according to the Standard Methods [20]. Sucrose was measured using the method described by Dubois et al. [21].
3.7. Data analysis The temporal profiles of hydrogen and methane concentration obtained for each reactor were subjected to non-linear regression analysis given by the Levenberg–Marquardt method [22], ® using the Microcal Origin 7.0 software. The maximum hydrogen production (P max ) was obtained by dividing the highest hydrogen concentration reached in each experiment by the biomass concentration, which was measured as volatile suspended solids (VSS).
A Boltzman function (sigmoid) was fitted to each temporal profile of hydrogen concentration (C), and the hydrogen production rates (v) were obtained by deriving this function according to the hydrogen balance in the batch reactor, as
dC v = (1) dt
The maximum specific hydrogen production rates (v max ) were calculated for each curve, as
dv v = = 0 (2) max dt
Aside from the maximum specific hydrogen production rates, the hydrogen yield (Y) was used to analyze the results and was calculated as the relationship between the hydrogen concentration (Y), or volume (Y v), and the organic matter concentration, measured as COD.
The data for COD and carbohydrates were fitted by exponential decay whereas first-order reaction, as described in kinetic Equation 3.
dC − A = k .C (3) dt 1 A
Where C A is the concentration of reagent; t is time; k1 is the first-order reaction constant.
4. RESULTS The results obtained following the proposed methodology are presented in Figures 1, Figure 2 and Table 2.
29 th Proceedings of the 5 International Workshop on Hydrogen and Fuel Cells October 26 –29, 2010 Campinas, SP, Brazil ISSN 2179-5029
Figure 1 . (A) Carbohydrates profile; (B) Volumetric H 2 profile; (C) Specific H 2 production; (D) COD profile; (E) Glycerol Soluble Metabolites Production; (F) Rice Wastewater Soluble Metabolites Production; (G) Vinasse Soluble Metabolites Production; (H) Sucrose Soluble Metabolites Production; ( . . . ) Exponential decay fit; (- - -) Boltzman sigmoidal fit; (HCi) Citric acid; (HMa) Malic acid; (HSu) Succinic acid; (HLa) Latic acid; (HFo) Formic acid; (HAc) Acetic acid; (HPr) Propionic acid; (HBu) Butiric acid; (HCa) Caproic acid; (EtOh) Ethanol.
30 th Proceedings of the 5 International Workshop on Hydrogen and Fuel Cells October 26 –29, 2010 Campinas, SP, Brazil ISSN 2179-5029
Figure 2 . (A) Volumetric CH 4 profile; (B) Specific CH 4 production.
Table 2 . Biomass concentration (C x), maximum specific hydrogen and methane production rates (v max ), hydrogen and methane yields (Y) and maximum hydrogen and methane production (P max ). Effluent Hydrogen Methane ■ □ ◊ ○ ■ □ ◊ ○ Cx vmax Ymax Pmax Cx vmax Ymax Pmax Glycerol 422.5 - - - 585.0 0.57 118.60 71.70 Rice WW 465,0 0.92 24.27 15.38 667.5 2.11 104.90 44.48 Vinasse 285.0 5.02 22.75 23.83 477.5 2.80 113.50 66.77 Sucrose 426.2 1.35 25.60 21.04 441.2 0.42 36.90 33.51 ■ □ ◊ ○ mg L -1 VSS, mL g -1 VSS h -1 , mL g -1 COD, mL g -1 VSS.
5. DISCUSSION The fermentative hydrogen production from carbohydrates occurs simultaneously with the production of organic acids, and can also be redirected to the generation of alcohols, as described by the elementary reactions 4 to 7 [23].
+ → + + ↑ Acetic acid production: C6 H12 O6 2H 2O 2CH 3COOH 2CO 2 4H 2 (4)
→ + + ↑ Butyric acid production: C6 H12 O6 CH 3CH 2CH 2COOH 2CO 2 2H 2 (5)
+ → + Propionic acid production: C6 H12 O6 2H 2 2CH 3CH 2COOH 2H 2O (6)
→ + Ethanol production: C6 H12 O6 2CH 3CH 2OH 2CO 2 (7)
According to Figure 1A, the highest carbohydrates concentration was found in the flasks containing sucrose solution. Hawkes et al. [12] describe the carbohydrates as the main immediate compounds for hydrogen production. As observed in the same figure, glycerol showed no carbohydrates in its composition, which may have caused the inobservance of hydrogen from this effluent (Figure 1B). Carbohydrates are easier to degrade compared to the long chain compounds present in glycerol, thus the experiment length could not have been large enough to permit the degradation of glycerol and the production of hydrogen.
Despite this fact, propionic acid (48.43 mg L -1) was produced using glycerol, indicating the ability of the selected microorganisms to degrade the organic matter.
In Figure 1D, the COD of each wastewater showed reduction of 43%, 26% and 11% for vinasse, parboiled rice wastewater and sucrose, respectively. These differences on
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carbonaceous organic matter degradation are probably due to the different metabolic pathways that occurred in the conversion of the substrates into CO 2, H 2, acids and solvents. Generally, low organic matter removal efficiency is also observed in other studies, such as that of van Ginkel et al. [11], which was performed in batch reactors and found removal efficiencies ranging from 5% to 11.1% in terms of COD. The lowest COD reduction found for sucrose seems to be related to the production of the metabolites depicted in Figure 1H. Among them, ethanol, which achieved 72.48 mg L -1 (t=24h). It is worth mentioning that generation of more reduced endproducts such as ethanol, buthanol, and lactate reduces the amount of hydrogen generated because these compounds contain additional unreleased hydrogen atoms [24].
In the flasks containing parboiled rice wastewater, there was mainly the generation of acetate and lactate (Figure 1F). It is known that simultaneously with the production of acetate occurs the hydrogen release, according to the stoichiometry showed in the study of Khanal et al. [25], while the production of lactate as endproduct does not releases hydrogen [26], which can be one of the reasons for the relative low performance on hydrogen production showed by the parboiled rice wastewater (Figure 1B).
The flasks containing vinasse achieved the highest COD reduction. Probably due to the large conversion of the organic matter into CO 2. In this essay (Figure 1G) it was possible to observe that the major metabolites produced were acetic (28.12 mg L -1) and butiric acid (42.48 mg L -1), just the same observed in the hydrogen production from sucrose (Figure 1H).
The worst results in hydrogen production were observed for glycerol (Figure 1E). Even so, these results are positive, since this wastewater is generated from the production process of a renewable fuel and additional energy can be obtained from the process by the methanization of the organic acids generated in the acidogenic step, contributing to the attenuation of the negative environmental impacts associated with the discharge of this wastewater, which can compromise the sustainability of the production of this biofuel.
The temporal variation of hydrogen concentration for each wastewater is presented in Figure 1B. All systems showed an acclimation phase for hydrogen production, which lasted approximately 6, 9 and 12 h for parboiled rice wastewater, sucrose and vinasse, respectively. Logan et al. [27] observed different timing for the acclimation phase using different substrates in batch reactors (glucose, sucrose, molasses, lactate, cellulose, and potato extract - COD normalized at 1 g L -1), with shortest acclimation time found in molasses and glucose.
Maximum hydrogen production values (P max ) and maximum specific hydrogen production rates (v max ), were obtained from the temporal profiles of hydrogen concentration (Figure 1B) and specific hydrogen production (Figure 1C).
Figure 1B shows that sucrose, vinasse and parboiled rice effluent obtained 9.0 mL H 2, 6.8 mL H2 and 6.3 mL H 2, respectively. Sucrose achieved the highest hydrogen production. This behavior was expected since sucrose had the highest concentration of carbohydrates in its composition, and the acidogenic inoculum was already adapted for the hydrogen production from sucrose. In fact, Kawagoshi et al. [28] verified that the inoculum is one of the determining factors that influence the production of hydrogen. In their work, hydrogen production ranged -1 from 0.15 mol H 2 mol glucose when the inoculum from an activated sludge reactor was used, -1 to 1.35 mol H 2 mol glucose when a digested anaerobic sludge was utilized.
In Figure 1C the hydrogen production was normalized for 1 g L -1 VSS. -1 According to the calculations, vinasse and sucrose presented 23.83 mL H 2 g VSS and 21.04 -1 mL H 2 g VSS, respectively. It is also possible to observe in Figure 1C that vinasse was the wastewater that presented the highest rate in specific hydrogen production (t=12h).
-1 The highest specific hydrogen production (23.83 mL H 2 g VSS) and rate of specific hydrogen -1 -1 production (5.02 mL H 2 g VSS h ) achieved by vinasse are possibly related to the
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macronutrients presented in its own composition, as shown in Table 1. These macronutrients may have enhanced the growth of the microorganisms involved on hydrogen production.
5.1. Potential for methane production In the methanogenic step it was possible to verify the decreasing of carbonaceous organic matter for all effluents tested. In flasks containing sucrose and parboiled rice effluent, the removal percentage was 74.7% and 54.0%, respectively. COD removal for glycerol and vinasse was 31.7% and 35.5%, respectively. With respect to carbohydrates, the exception of sucrose which showed an initial concentration of 87 mg L -1, carbohydrate availability for all other flasks was insignificant. This behavior was expected, since carbohydrates are rapidly consumed and lipids and proteins are hydrolyzed to acids and aminoacids at the beginning of the anaerobic digestion process, still in the acidogenic step [29].
Regarding the methane production, we noticed that all effluents evaluated showed potential for its production, as shown in Figures 2A and 2B. The methane production was normalized to permit the comparison between different effluents. According to the data, glycerol and vinasse -1 presented the highest potential, with 71.7 and 66.8 mL CH 4 g VSS, respectively. These results for glycerol show that even with no hydrogen production detected during the acidogenic step, this previous stage was important to prepare this effluent by the hand of substrate conversion into soluble metabolites available for methanization.
5.2. Potential of the assayed wastewaters Comparing the parameter values of Table 2, we found that among the wastewaters studied vinasse and parboiled rice wastewater showed immediate potential for hydrogen production.
Altough the Y max (Table 2) was very similar between the mentioned effluents, the result of v max (maximum specific hydrogen production rate) was quite different. Similar behavior was observed in the work of Fernandes et. al. [18], where vinasse also presented much higher specific hydrogen production rate. Possibly the high concentration of macro and micronutrients of vinasse (Table 1), may have favored the microorganisms involved in hydrogen production.
For methane production, the effluent which showed highest potential was glycerol with 71.70 -1 -1 mL CH 4 g VSS and 118.60 mL CH 4 g COD, although its v max was quite lower compared to that obtained by parboiled rice wastewater and vinasse.
Considering the results obtained (Table 2) and the volumetric flow of effluent (1500 m 3 d -1) from the ethanol production plant where vinasse was colected, it is possible to estimate that hydrogen and methane production from vinasse can achieve around 535680 L d -1 and 1521694 L d -1, respectively, assuming 1 atm and 25ºC for calculations.
6. CONCLUSIONS -1 -1 Considering the highest performance obtained in SHPR (5.02 mL H 2 g VSS h ), SMPR (2.80 -1 -1 -1 mL CH 4 g VSS h ) and P max (23.83 mL H 2 g VSS), vinasse was the wastewater which showed better potential for generation of hydrogen and methane.
Vinasse's high potential for hydrogen production is extremely important in solving the severe environmental problem caused by the large amounts of vinasse generated per unit of produced ethanol, since the fermentative process used for hydrogen production is capable to reduce significantly the organic matter concentration of this industrial effluent.
The methane produced from the metabolites generated during hydrogen production can be used as reagent for extra hydrogen production by methane steam reforming process.
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7. ACKNOWLEDGEMENTS This work was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The authors also acknowledge the grants received from FAPESP and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.
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