The objective of this study was to compare two start-up strategies for hydrogen production using granular sludge without any thermal pre-treatment.

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1 Comparison of two start-up strategies for hydrogen production with granular anaerobic sludge C. E. Hernández-Mendoza, Iván Moreno-Andrade and Germán Buitrón* Laboratory for Research on Advanced Processes for Water Treatment, Instituto de Ingeniería, Campus Juriquilla, Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, Querétaro, 76230, Mexico. Keywords: Hydrogen; anaerobic granules; sequencing batch reactor; hydraulic retention time Introduction Around 80% of the energy demands worldwide are satisfied by fossil fuels. However, they are non-renewable energy sources and also its combustion is detrimental to the environment. In this sense, it has become necessary to find new energy sources that also have to be environmentally friendly. Hydrogen gas has been recognized as an ideal alternative energy vector due to its high energy content (122 kj/g), which is 2.7 times greater than the energy content of the hydrocarbon fuels. Hydrogen can be produced by anaerobic fermentation using a wide range of substrates including wastes or rejected products. Carbohydrates, mainly glucose, are the preferred carbon sources in fermentation processes which mainly produce acetic and butyric acids together with the hydrogen. Up to now, the pre-treatment procedures, such as the heat shock, used at lab-scale can be prohibitive and not practical for use at large scale. So, the need to obtain a hydrogen producing inoculum employing more practical pre-treatments is becoming highly necessary. The target will always be to avoid the presence of hydrogen consuming microorganisms, such as propionate producing and methanogenic bacteria. An option to wash out those hydrogenophilic microorganisms is by the hydraulic retention time (HRT) which can be used as a microbial selection method allowing specific microbial consortiums remain into the reactor (Zhang et al., 2006). Also, the use of short HRT reduce methanogenic growth due to its growth rate is lower than the hydrogen producing bacteria (Jung et al., 2011). On the other hand, it has been stated that the biomass retention into the reactor is a promising alternative to improve the hydrogen production (Lee et al., 2009). A way to maintain the biomass into the reactor is by the use of granular sludge. The granulation is a complex process which involves many physicochemical and microbial interactions. Methanogenic granules can also be formed in anaerobic sequencing batch reactors (AnSBR) when selective conditions are used in order to maintain the heavier aggregates into the reactor and washing out the flocs with poor settling characteristics (Ong et al., 2002). The objective of this study was to compare two start-up strategies for hydrogen production using granular sludge without any thermal pre-treatment. Methods Seed sludge The sludge was collected from an UASB reactor treating the wastewater of a brewery Page 1 of 5

2 industry. The inoculum was selected in order to have an average granule diameter of 2.0 ± 0.5 mm. After that the sludge was put into the reactor. The inoculum had a density of kg/l with a settling velocity of 2.2 ± 0.4 cm/s. The sludge composition was evaluated using molecular biology methods using PCR and DGGE analysis. Substrate composition Glucose (5 g/l) was added as the sole carbon source. The substrate used contained nutrients as described by Mizuno et al. (2000). Experimental procedure Two start-up procedures were studied: discontinuous (strategy 1) and continuous (strategy 2) modes. In Strategy 1, an anaerobic sequencing batch reactor (AnSBR) was used with a useful volume of 1.0 L. The ph (5.5) was controlled by a ph sensor (Hanna BL ) which adjusted it by adding 2N NaOH according to the system requirements. Three HRT levels were tested (24, 12 and 6 h). For the Strategy 2, an UASB reactor was used with 2.0L of useful volume. The HRT was fixed at 6 h. ph was controlled at 4.5 ± 0.1. Since the beginning the reactor was operated at continuous mode. For both strategies the reactors were operated under mesophilic conditions (35 C) and mixed liquor volatile suspended solids (MLSS) concentration was 7.3 ± 0.4 gvss/l. Prior to each run the reactor was purged with nitrogen gas to ensure anaerobic conditions. Biogas production was measured by the acid brine displacement method. The inoculum was not previously acclimated to hydrogen production. Analytical methods The content of hydrogen in the biogas was determined by a gas chromatography (SRI 8610C) equipped with a thermal conductivity detector (TCD) and two packed columns (6' x 1/8" stainless steel silica gel packed column and 6' x 1/8" stainless steel molecular sieve 13X packed column). The temperatures of the injector and detector were 90 and 150 C, respectively. The column temperature events were as follows: the initial temperature was 40 C held for 4 minutes and then was gradually increased from 40 C to 110 C at a rate of 20 C/min the final temperature was held for 3 minutes. Nitrogen was used as a carrier gas at a flow rate of 20 ml/min. The analysis of the volatile fatty acids (VFAs) and alcohols in the effluent were done in a gas chromatography (Varian 3300) equipped with a flame ionization detector (FID) and a capillary column (Agilent mm ID, 15 m length). The temperatures of the injector and detector were 190 and 210 C, respectively. The column temperature events were the following: the initial temperature was 70 C and gradually increased from 70 to 130 C at a rate of 10 C/min, the final temperature was held for 7.5 minutes. Nitrogen was used as a carrier gas at a pressure of 483 kpa. Chemical oxygen demand (COD), total solids (TS), volatile solids (VS) and volatile suspended solids (VSS) were determined according to the standard methods (APHA, 1992). Glucose concentration was determined following the phenol-sulfuric acid method described by Dubois et al. (1956). Page 2 of 5

3 Results For the discontinuous operation, the biogas produced was composed by hydrogen, carbon dioxide and methane. Hydrogen production was significantly when the 12 and 6h HRT (55 to 60% in average) were used while at 24h-HRT hydrogen production was quite low (5% in average). Methane was produced in all the cases, but the minimum value was observed for the lowest HRT (15% in average). For the case of the strategy 2, methane was not detected after 14 days of continuous operations. The maximum methane concentration (68%) was detected during the first day and was attributed to the high ph into the system but it decreased after 9 days. The hydrogen content in the biogas was in the range of 80% ± 5% after only 14 days of operation and it was maintained during a month. For the Strategy 1, the average methane concentration was 32% with high variations, despite the acidic conditions prevailing in the reactor (ph= 5.5). The hydrogen yield reached a maximum of 1.3 mol/mol substrate at a maximum volumetric production rate of 34 ml/l reactor /h. When the HRT decreased to 12 h, the hydrogen content in the biogas increased and it varied from 36% to 77%, while the methane concentration in the biogas varied from 7% to 27%, decreasing in relationship with the percentage observed for the previous condition. The volumetric hydrogen production varied from 0.2 L to 1.2 L with a maximum yield of 2.0 mol/mol substrate at a maximum volumetric production rate of ml/l reactor /h when the discontinuous mode was applied (Figure 1A). At the 24 th cycle the HRT was decreased to 6 h. The methane content in the biogas varied from 10% to 16% which was lower than the obtained at a HRT of 12 h and was attributed to a shift composition in the microbial community. The hydrogen content in the biogas was in the range from 51% to 71%. For the case of the continuous mode, the maximal hydrogen production was 4.2 L. The volumetric hydrogen production rate (VHPR) and yield followed a parallel pattern. From day 7 to 15 the hydrogen yield was 2.6 ± 0.8 mol H 2 /mol glucose while the VHPR was 65 ml/l/h. From day 16 to 37 the hydrogen yield and VHPR were 2.6 ± 0.2 mol H 2 /mol glucose and 75 ml/l/h (Figure 1B). Independently from the HRT employed a mixed fermentation type was done in Strategy 1. At 24h-HRT the principal soluble product was propionate. When the HRT was shortened to 12 h the acetate and butyrate concentration increased and the propionate concentration was quite lower than the acetate concentration. This suggests the development of a mixed acetate-butyratepropionate fermentation type. At 6h-HRT the principal soluble metabolites were butyrate and acetate while the propionate concentration decreased markedly; so, a butyrate-acetate fermentation type was carried out in the system. It was found that as the HRT was reduced, the volumetric hydrogen production rate was increased and the HPr/HBu and the HPr/HAc, HAc/HBu ratios decreased. (A) Page 3 of 5

4 VHPR (ml/l/h) (B) HRT = 24 h HRT = 12 h HRT = 6 h Cycle Discussion and Conclusions It was possible to generate hydrogen producing granules using the pressure selection of HRT and ph. In general, the continuous mode of operation was more efficient in the selection of the hydrogenproducing bacteria and in the inhibition of the methanogenic ones than the discontinuous mode. For the discontinuous mode it was observed that as the hydraulic retention time was reduced the methanogenic activity as well as the HPr/HBu and HPr/HAc ratios decreased. Fig. 1. Volumetric hydrogen production rate in: (A) discontinuous and (B) continuous modes. In the case of the continuous operation, the distribution of volatile fatty acids can be dived in two parts. In the first one, from day 1 to 13, acetic acid was the main metabolite produced followed by the propionic acid. In the second one, from day 14 to 37, it was noticed that there was not production of propionic acid. This was attributed to the fact that methanogenic bacteria were inhibited due to the operation conditions employed. The main soluble metabolite produced was acetic acid while butyric acid production was 21% lower. DGGE analysis demonstrated that the bacterial composition was evolving with the time. Results are being processed at this moment. Although a several fermentation reactions types were observed during all the experiment, as the HRT decreased, the metabolic pathway shifted from the acetate production as the principal soluble metabolite to the butyric production. On the other hand, as the HRT was reduced, the volumetric hydrogen production rate increased from 34 ml/l reactor /h at 24h-HRT to 163 ml/l reactor /h at a HRT of 6 h. However, in the continuous system the hydrogen yield and VHPR were 2.6 ± 0.2 mol H 2 /mol glucose and 75 ml/l reactor /h, respectively. References APHA (1992). Standard methods for the examination of water and wastewater, 18 th edition. Washington, DC, USA. American Public Health Association. Water Environment Federation. Dubois M., Gilles K. A., Hamilton J. K, Rebers, P. A. and Smith, F. (1956) Colorimetric method for determination of Page 4 of 5

5 sugars and related substrates. Anal Chem, 28, Jung K-W, Kim D-H, Kim S-H Shin H-S. (2011) Bioreactor design for continuous dark fermentative hydrogen production. Bioresour. Technol. doi: /j.biortech Lee, D-Y, Li, Y-Y & Noike, T Continuous H 2 production by anaerobic mixed microflora in membrane bioreactor. Bioresour. Technol. 100, Mizuno O., Dindsdale R., Hawkes F. R., Hawkes D. L. and Noike T. (2000) Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresour. Technol. 73, Ong S. L., Hu J. Y., Ng W. J., Lu Z. R. (2002) Granulation Enhancement in Anaerobic Sequencing Batch Reactor Operation. J Environ. Eng.: ASCE 128, Zhang Z-P, Show K-Y, Tay J-H, Liang D. T., Lee D-J, Jiang W-J. (2006) Effect of hydraulic retention time on biohydrogen production and anaerobic microbial community. Proc. Bio. 41, Acknowledgments This research was supported by CONACYT through the project Page 5 of 5