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Bio-hydrogen production during acidogenic fermentation in a multistage stirred tank reactor
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 8 ( 2 0 1 3 ) 2 1 8 5 e2 1 9 0
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Bio-hydrogen production during acidogenic fermentation in a multistage stirred tank reactor Estela Tapia-Venegas a,1, Juan Esteban Ramirez a,1, Andre´s Donoso-Bravo a,1, Lorena Jorquera a,1, Jean-Phillipe Steyer b, Gonzalo Ruiz-Filippi a,* a b
Escuela de Ingenierı´a Bioquı´mica, Facultad de Ingenierı´a, Pontificia Universidad Cato´lica de Valparaı´so, General Cruz 34, Valparaiso, Chile INRA, UR050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, Narbonne F-11100, France
article info
abstract
Article history:
The objective of this study was to evaluate the production of hydrogen in a two-stage CSTR
Received 30 August 2012
system e both reactors having the same volume e and compare its performance with
Received in revised form
a conventional one-stage process. The lab-scale two-stage and one-stage systems were
19 October 2012
operated at five pHs and five hydraulic retention time (HRTs). The maximum volumetric
Accepted 16 November 2012
hydrogen productivity and yield obtained with the two-stage system were 5.8 mmol L1 h1
Available online 28 December 2012
and 2.7 mol H2 mol glucose1, respectively, at an HRT of 12 h and pH 5.5. Overall, the twostage system showed, at steady state, a better performance that the one-stage system for
Keywords:
all the evaluated pHs. However, a comparison between the one-stage system, operating at
Bio-hydrogen
6 h of HRT, and the first reactor of the two-stage system at the same HRT did not show any
Anaerobic digestion
significant difference, highlighting the positive impact of having a two-stage process. The
Renewable energy source
determination of the ratio between the experimental measured H2 in the gas phase and the
Plug flow
theoretical H2 generated in the liquid phase (discrepancy factor) indicated that an impor-
Series reactor
tant part of the hydrogen produced in the first reactor was transferred into the second reactor instead of being desorbed in the headspace. Therefore, the improving of hydrogen production in the two-stage system is rather attributed to the increased transfer of hydrogen from liquid to gas than an actual total hydrogen production increase. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
There are several operational variables that influence the hydrogen production by anaerobic digestion among the most important ones: pH, hydraulic retention time (HRT), partial pressure of hydrogen and liquid/gas equilibrium of the system [1e4]. In regards to the HRT (which is the inverse of the dilution rate), it is advisable to use a dilution rate less than 0.17 h1 or greater than 6 h HRT, when using continuous reactors with
suspended biomass, since the maximum specific growth rate (mmax) of hydrogen producers in an anaerobic mixed inoculums is less than that value. Despite this hydraulic constraint, different operational HRT ranges have been used depending on the type of substrates, reactor and inoculum as well as the pretreatment applied to eliminate the methanogens. In any case, it is clear that as HRT increases, the hydrogen production decreases, being the maximum reported value for HRT to produce hydrogen in a continuous operation between 14 and
* Corresponding author. Tel.: þ56 32 2273819. E-mail address: [email protected] (G. Ruiz-Filippi). 1 Tel.: þ56 32 2273819. 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2012.11.077
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17 h [5]. On the other hand, it is usually accepted, although this is still unclear, that hydrogen accumulation, which is related to the partial pressure of hydrogen in the reactor headspace, might inhibit hydrogen producers [6]. In this context, different strategies have been used to minimize this effect, for example, the dilution of the biogas by sparging an inert gas and vacuum application [7e9]. However, these strategies may increase the cost of the process and it is necessary to investigate new and more economically feasible alternatives to reduce the effect of hydrogen partial pressure. One possible option is the use of multistage stirred tank reactors in series which are characterised by having several states across the reactors. This configuration is well suited for processes in which a certain degree of product inhibition may occur [10,11]. Therefore, using two continuous stirred tank reactors (CSTR) in series could, first of all, enhance the selection of hydrogen producers by washing out the methanogens, while at the same time, reducing the hydrogen partial pressure that a one-stirred tank reactor with the same total volume would have. On the other hand, the reactors geometry may also influence the bio-hydrogen reactor performance since the transfer of hydrogen from the liquid to the gas phase depends on the interfacial specific area from the liquid to the gas. The aim of this study was to assess the bio-hydrogen production in a twostage series system in terms of hydrogen yield and volumetric productivity compared to a classical one-stage continuous system.
2.
Materials and method
2.1.
Experimental set-up
Three glass-made reactors were designed and implemented at lab-scale. A conventional one-stage CSTR system with a volume of 4 L and a two-stage system, composed by two CSTR reactors in series of 2 L each, were used. Each reactor was connected to auxiliary equipment: temperature and pH sensors for monitoring, peristaltic pumps for influent feeding and effluent draw off, pH control pump by adding a bicarbonate solution, a mechanical stirrer and a heating jackedtype system. Both systems were maintained at 37 C and operated in parallel.
2.2.
Table 1 e Synthetic wastewater composition (adapted from Bruce et al. [27]). Nutrient Glucose Ammonium bicarbonate Potassium dihydrogen phosphate Magnesium sulphate heptahydrate Ferrous chloride Sodium chloride Sodium molybdenum oxide dihydrate Calcium chloride dihydrate Manganese sulphate monohydrate
Chemical formula
Concentration (g/L)
C6H12O6 NH4HCO3
5 2
KH2PO4
1
MgSO4$7H2O
0.1
FeCl2 NaCl NaMoO4$2H2O
0.00278 0.01 0.01
CaCl2$2H2O
0.01
MnSO4$H2O
0.0094
2.3.
Systems evaluation and comparison
2.3.1.
Determination of the optimal conditions for the systems
Both systems were operated at five HRTs: 6, 8, 10, 12, 14 h aiming to find the best hydraulic conditions in terms of hydrogen yield and productivity. Each reactor of the two-stage system were operated at the same HRT i.e. half of the total one. Each HRT condition was evaluated at five pHs 4.0, 4.5, 5.0, 5.5 and 7.0. The influence of these variables was assessed by using the response surface methodology (RSM) in order to assess the effect of each variable as well as their combined influence. The analysis was carried out using the software Statgraphics Plus.
2.3.2.
Experimental running
The experiment sequence was randomised (i.e. randomly selected HRT and pH) in order to minimized the experimental bias. Furthermore, for each condition, the reactors were reseeded with the same original inoculum (non-adapted anaerobic biomass) and were kept until steady state conditions were reached, usually after 3 HRTs of operation, although, the reactors were maintained at each condition for around 40 HRTs.
Wastewater and inocula 2.4.
For the experiments, synthetic glucose-based wastewater and suspended biomass were used (Table 1). For the reactors seeding, granular anaerobic inoculum was taken from a fullscale anaerobic plant treating tobacco wastewater with a concentration of 13 g VSS L1 and an acidogenic and methanogenic activity of 0.17 g COD CH4 gVSS1 d1 and 7.74 g COD C6H12O6 g VSS1 d1. A volume of this inoculum equal to the 25% of the reactor volume was added for each experiment, resulting in an initial concentration of 4 g VSS L1. In order to wash out the methanogenic biomass from the inoculum, a biokinetic strategy based on the use of low HRT (in case of CSTR, HRT is equal to the solid retention time, SRT) was applied (6e14 h).
Analytical methods
For each experiment, measurements of the influent and effluent of each reactor were carried out in order to characterize the system performance. Chemical oxygen demand (COD) was determined using Method 5220C, standard methods [12], and glucose concentration was measured using 3,5-Dinitrosalicylic acid, DNS [13]. Prior to COD and glucose determination, samples were centrifuged at 15,000 rpm for 10 min in order to remove suspended solids. The production of volatile fatty acids (VFA) and ethanol was determined by gas chromatography (Shimadzu GC8 and PerkinElmer 500, respectively). The biomass concentration was measured by determining the volatile suspended solids (VSS) in the reactor
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 8 ( 2 0 1 3 ) 2 1 8 5 e2 1 9 0
[12]. The biogas production was measured by liquid displacement, and the composition of biogas was measured by gas chromatography, using GC, Perkin Elmer Clarus 500, with nitrogen as the carrier. The injector, detector and column temperatures were 120, 170 and 35 C, respectively. The hydrogen composition in the biogas is directly related to the hydrogen partial pressure, it was calculated from fraction of the compound in the gas.
3.
Results and discussion
3.1. Influence of the HRT and pH on the two-stage system performance The hydrogen yield and hydrogen volumetric productivity of the two-stage system at the different HRTs and pHs evaluated are presented in Fig. 1. The obtained yields and productivities were all within the range of 0.2e2.7 mol H2 mol glucose1 and 0.7e5.9 mmol H2 L1 h1, respectively. The best performance of the system, in terms of productivity, was observed at HRT of 12 h and pH 5.5 and an OLR of 0.41 g COD L1 h1 with a value of 5.8 mmol H2 L1 h1, whereas the observed yield was 2.7 mol H2 mol glucose1. Other studies have found a similar effect of the HRT but over a wider range. For example Lin et al. [14] examined HRTs between 6 and 48 h and found that the best productivity was between 6 and 12 h in a CSTR with 20 g COD L1 of glucose. The obtained value of yield in this study is even higher than the one obtained with pure culture of Clostridia sp of 1.7 mol H2 mol glucose1, at an organic loading rate 3.3 g COD L1 h1 or as reported, HRT 0.5 days and 20 g COD L1 in a CSTR reactor [15]. Similar values of productivity have been also obtained with thermophilic microorganisms (58 C) 2.58 mmol H2 L1 h1 at an organic load rate of 0.28 g COD L1 h1 or, as reported, 10 g lactose L1 and dilution rate 0.05 h1 [16].
Fig. 1 e Hydrogen yield (A) and productivity (B) of the twostage system. Graphs as a function of HRT and pH. Bars for the different pHs: grey, 4; soft grey with white dots, 4.5; black, 5; soft grey with white diagonal lines, 5.5 and white, 7.
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With respect to the pH effect, at the optimal HRT of 12 h, both the yield and the productivity present their highest value at pH 5.5. In Fig. 1, it can be observed that both variables exert a similar influence on the yield and the productivity. It is known that the pH plays an important role in determining the type of viable anaerobic fermentation pathways or changes in microbial diversity [17]. The lowest productivities were achieved at pH 7.0 and 4.0, where acetic and butyric acids represented 60% of the total COD, while ethanol and propionic acid comprised the remaining 40%. For pH 5.5 where the percentage of acetic and butyric acids is high, the main biochemical pathway for the production of hydrogen would be the acetate-butyrate, described as one of the most important route to produce hydrogen via fermentation. It has been previously reported that pH between 4.5 and 6 are more suitable to produce hydrogen, because the ethanol production pathway is less stimulated [18,19]. Furthermore, this low hydrogen yield could be explained by the presence of fermentative organisms which present a low hydrogen yield (such as the propionate producers) and by fermenters unable to produce hydrogen that could compete for the same substrate, also at pH 7.0, the acid/ethanol ratio was low; an increase in ethanol production, decreases the production of hydrogen and also has been reported that this point the activity of the hydrogenase enzyme begins to decrease as the pH is increased [5,20]. These changes can be further assessed by analysing the behaviour of the volatile fatty acids (VFAs) throughout the system operation. The concentration of VFA and ethanol, at the steady state of the two-stage system for an HRT of 12 h, are shown in Fig. 2. At a pH of 4 the three main VFAs (acetic, propionic, butyric acid) were present at a similar concentration. Between 4.5 and 5.5 the concentration of propionic remained close to zero, whereas at pH of 7.0 its concentration increased again up to reach a value similar to the butyric acid, high concentrations of propionate decreased acetic concentration [17]. Overall, the acetic acid was the most present VFA under all the pH conditions. At pHs 4.5, 5.0 and 5.5, acetic acid and butyric acid composed close to 95% of the total VFAs produced and, in line with the studies reported in the
Fig. 2 e Volatile fatty acid (VFAs) concentration at HRT of 12 h and five pHs. Grey diamonds: acetic acid; grey squares: butyric acid; black triangles: propionic acid and white circles: ethanol.
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literature, this is correlated with the increase of H2 productivity at these pHs. The hydrogen content of the biogas was between 21% and 63%, depending on the conditions used (pH and HTR). For each experiment, the resulting biogas consisted exclusively of hydrogen (H2) and carbon dioxide (CO2). Methane was not detected at these operating conditions, indicating the absence of methanogens in the system. The use of short HRT has demonstrated to be a proper alternative for archae elimination and avoiding methanogenesis in operations lasting about 30 days. Same methodology has been successfully reported in continuous hydrogen production [21,22].
3.2. One-stage performance and comparison with the two-stage system Fig. 3 shows the results of the one-stage system performance in terms of hydrogen yield and productivity. The obtained yield and productivity values lie between 0.25 and 1.54 mol H2 mol glucose1 and 0.39e3.13 mmol L1 h1, respectively. The best results are observed at an HRT of 12 h and pH of 5.5 when an OLR of 0.41 g COD L1 h1 was applied. The latter is in agreement to those reported values at similar operating conditions [14], Fang et al. [17], reached a hydrogen yield of 2.1 mol H2 mol glucose1 in a CSTR fed with glucose at an OLR of 1.2 g COD L1 h1, however, in this last case, a specific acidogenic inoculum previously adapted for the hydrogen production was used, which may explain the better obtained results. Nevertheless, greater values of these parameters have been achieved with other types of substrates, for instance, Chang et al. [23], who used molasses as the carbon source, attained a yield and productivity of 2.1 mol H2 mol hexose1 and 8.3 mmol H2 L1 h1, respectively, at an OLR of 120 g COD L1 h1. As for the two-stage system, the highest yield was also obtained at pH 5.5 for HRT of 12 h. However at an HRT of 6 h,
the highest values were obtained in a broader pH range (from 4.5 to 5.5). With respect to the productivity, the best values were obtained at the same range (from 4.5 to 5.5) for both HRTs. Some studies have concluded that the maintenance of a moderately acidic pH (around 5e5.5) creates good conditions for hydrogen production and the role of the pH goes beyond being a control parameter during the anaerobic digestion, which is related to the change of metabolic pathways and activity of the enzyme hydrogenase, therefore likely that the observed changes at different pHs is for this reason and pH 5.5 allowed the best way to produce hydrogen [1,24,25]. The change (in percentage) of the yield and productivity obtained between the two-stage and the one-stage systems at HRT of 12 h is presented in Fig. 4. The yield obtained by the two-stage system was higher (36e41%) than the one-stage system for all the evaluated pHs. Productivity was also improved (from 17 to 65%) in the two-stage system. These increases are similar to those improvements obtained by using N2 sparging, however, in our case, a subsequent gas separation and purification of the H2 will not be necessary as the sparging case [22]. To explain these differences, the one-stage system was operated at 6 h in order to compare it with the first reactor of the two-stage system (when operated at a total HRT of 12 h). The results for both systems did not show statistically significant differences (ANOVA test with a 95% level). The VFAs production was also similar in both systems where the acetic and butyric acids represented 95% to the total VFAs produced and an acetic to butyric acid ratio around 1 was observed for all pHs. If the performance of first reactor of the two-stage system is similar to one-stage system at the same HRT, the explanation of the difference, in terms of productivity and yield, can be on the behaviour of second reactor of two-stage system.
3.3. Two-stage series system strategy: partial pressure and hydrogen behaviour The hydrogen partial pressure is sometimes recognized as a quite influential factor in acidogenic systems. In the first reactor of the two-step process, the hydrogen partial pressure
Fig. 3 e Performance of the one-stage system. Bars for the different pHs: grey, 4; soft grey with with vertical and horizontal lines, 4.5; black, 5; soft grey, 5.5 and white, 7.
Fig. 4 e Systems performance variation comparing the two-stage and the one-stage system at different pHs at HRT of 12. Productivity (white bars), yield (grey bars).
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and hydrogen yield were 49 kPa and 4.30 mmol H2 L1 h1 whereas in the second reactor, they reached 63 kPa and 6.45 mmol H2 L1 h1. These values indicate that, in the case of a two-stage process, a high hydrogen partial pressure does not negatively influence the production yield. This unclear influence can be also seen in Fig. 5 where the productivity of both reactors for all the evaluated pH does not present any correlation with the hydrogen partial pressure. In order to have a better insight of the system behaviour, a mass balance of each reactor was carried out. In anaerobic digestion processes, the production of acetic and butyric acids is usually assumed to be coupled with the hydrogen production according to equations (1) and (2)), respectively. Therefore, knowing the concentration of VFA, we estimated the theoretical H2 production in the liquid phase using these stoichiometric expressions. The discrepancy factor Df, which corresponds to the ratio between the measured H2 in the gas phase and the theoretical H2 generated in the liquid phase, can then be calculated. This factor gives an idea of the H2 that is transferred to the gas phase: for instance, a high vaporization of hydrogen would lead to a Df close to 1. þ C6 H12 O6 þ 4H2 O/4H2 þ 2C2 H4 O2 þ 2HCO 3 þ 4H
(1)
þ C6 H12 O6 þ 2H2 O/2H2 þ C4 H8 O2 þ 2HCO 3 þ 3H
(2)
Table 2 shows the general mass balance of each reactor of the two-stage system for the HRT of 12 h (6 h in each reactor). It is worth to point out that the Df for the one stage system are equal to the first reactor of the two-stage system since the biogas produced and the VFAs measured were not statistically different. As observed, the Df of the first reactor is less than 1 for all the pHs which indicates that not all the hydrogen generated in the liquid phase goes to the gas phase. Unlike the first reactor, Df is greater than 1 in most cases in the second reactor, which indicates that more hydrogen than the theoretical value is being produced and transferred into the gas phase. This is explained by the fact that in the second reactor a dual effect takes place: not only hydrogen is produced from consumption of glucose that was not degraded in the first reactor is measured but also hydrogen desorbed from the effluent from the first reactor that enters the second reactor. It is known that the solubility of hydrogen in water is low; however a culture medium with several soluble compounds and microorganism may present a totally different solubility. The medium characteristics may affect the transfer of ionic
Table 2 e General balance of the two-stage series system at the different evaluated pHs (all the values are in mmol hL1 except Df which is dimensionless). Experimental information Acetic acid Reactor 1 2.92 3.81 2.88 4.79 3.44 Reactor 2 0.45 1.15 0.26 0.78 0.45
Df
Butyric acid
H2
pH
H2
1.43 1.41 1.87 2.42 0.52
5.73 6.70 7.61 8.72 5.04
4.0 4.5 5.0 5.5 7.0
8.70 10.43 9.49 14.40 7.92
0.66 0.64 0.80 0.61 0.64
0.07 0.08 0.03 0.95 0.11
1.14 2.02 2.20 12.90 0.81
4.0 4.5 5.0 5.5 7.0
1.06 2.45 0.58 3.44 1.11
1.08 0.82 3.76 3.75 0.73
a Calculated from acetic acid and butyric acid measured (1st and 2nd column).
strength or the interaction between the solvent and solute which in this case would be the hydrogen [26]. In this context, Frigon et al. [4], assessed the mass transfer of hydrogen from liquid to gas and noted that it is extremely limited in an anaerobic digestion process, when no thermodynamic equilibrium exists between both phases. Likewise, in that study, a lower hydrogen concentration in the liquid was achieved by recirculating biogas into the reactor. The interfacial surface between the liquid and gas was then increased, as could happen in our reactors in series where the diameter of the reactor belonging to the conventional system is lower than that obtained with the system in series. These differences in the hydrogen behaviour in both reactors could demonstrate that the concentration of hydrogen in the gas is a poor indicator of the concentration at which the biomass is subject because the liquid phase presents mass transfer limitations. Although the importance of the partial pressure remains unclear, during the operation of the two-stage system could be observed that the liquidegas transfer of the hydrogen in the first reactor is less than the second reactor, and the hydrogen produced in the second reactor come from both the residual glucose and the desorption of the soluble hydrogen from the first reactor effluent. These results show that yield, considering all the hydrogen produced (gaseous and dissolved) for the two-stage and the one-stage system was in a range of 1.2e2.2 mol H2 mol glucose1 and 1.12e1.70 mol H2 mol glucose1. These results indicate that the enhancement of the two-stage system performance is, at least in part, due to the improvement in the hydrogen transfer from gas to liquid in two-stage system.
4. Fig. 5 e Productivity of both reactors of the two-stage system as a function of the hydrogen partial pressure.
Indirect measurementsa
Conclusion
A two-stage system, composed of two CSTR reactors in series, showed to be a suitable option to improve bio-hydrogen
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production by dark fermentation. The highest hydrogen volumetric productivity was 5.8 mmol H2 L1 h1 with a yield of 2.7 mol H2 mol glucose1 at a pH of 5.5. This system exhibited higher results with regards to the volumetric productivity and yield than a one-stage reactor e of similar total volume e with an increase between 20 and 60% at several operating pHs. However, this enhancement cannot be directly attributed to the effect of the hydrogen partial pressure and the better performance of the two-stage system is rather attributed to an improvement of the hydrogen transfer to the gas phase.
Acknowledgements This study was funded by Fondecyt 1060109 and 1120659.
references
[1] Aceves-Lara CA, Latrille E, Bernet N, Buffie`re P, Steyer JP. A pseudo-stoichiometric dynamic model of anaerobic hydrogen production from molasses. Water Research 2008; 42:2539e50. [2] Hwang M, Jang N, Hyunb S, Kim I. Anaerobic bio-hydrogen production from ethanol fermentation: the role of pH. Journal of Biotechnology 2004;111:297e309. [3] Cubillos G, Arrue R, Tapia E, Jeison R, Chamy R, Rodrigez J, et al. Simultaneous effects of pH and substrate concentration on hydrogen production by acidogenic fermentation. Electronic Journal of Biotechnology 2010;13(1). 0717-3458. [4] Frigon J-C, Guiot SR. Impact of liquid-to-gas hydrogen mass transfer on substrate conversion efficiency of an upflow anaerobic sludge bed and filter reactor. Enzyme and Microbial Technology 1995;17:1080e6. [5] Valdez-Vazquez I, Poggi-Varaldo H. Hydrogen production by fermentative consortia. Renewable and Sustainable Energy Reviews 2009;13:1000e13. [6] Wang B, Wan W, Wang J. Inhibitory effect of ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production. International Journal of Hydrogen Energy 2008;33(23):7013e9. [7] Hawkes FR, Dinsdale R, Hawkes DL, Hussy I. Sustainable fermentative hydrogen production: challenges for process optimisation. International Journal of Hydrogen Energy 2002; 27(11e12):1339e47. [8] Liang T, Cheng S, Wu K. Behavioral study on hydrogen fermentation reactor installed with silicone rubber membrane. International Journal of Hydrogen Energy 2002; 27:1157e65. [9] Bastidas-Oyanedel J, Aceves-Lara C, Ruiz G, Steyer J-Ph. Thermodynamic analysis of energy transfer in acidogenic cultures. Engineering in Life Science 2008;8(5):487e98. [10] Bakonyi P, Nemesto´thy N, Ramirez Juan, RuizFilippi Gonzalo, Be´lafi-Bako´ K. Escherichia coli (XL1-BLUE) for continuous fermentation of bioH2 and its separation by polyimide membrane. International Journal of Hydrogen Energy 2012;37(7):5623e30.
[11] Levenspiel O. Ingenierı´a de las reacciones quı´micas. 3rd ed. Mexico: Limusa Wiley; 2004. p. 101e120. [12] APHA, AWWA, WPCP. Standard methods for the examination of water and wastewater. Washington DC: American Public Health Association; 1995. p. 1193. [13] Miller GL. Use of Dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 1959; 31(3):426e8. [14] Lin CY, Chang RC. Hydrogen production during the anaerobic acidogenic conversion of glucose. Journal of Chemical Technology and Biotechnology 1999;74(6):498e500. [15] Lin C-Y, Chang R-C. Fermentative hydrogen production at ambient temperature. International Journal of Hydrogen Energy 2004;29:715e20. [16] Collet C, Gaudard O, Pe´ringer P, Schwitzgue´bel J-P. Acetate production from lactose by Clostridium thermolacticum and hydrogen-scavenging microorganisms in continuous culturedeffect of hydrogen partial pressure. Journal of Biotechnology 2005;118:328e38. [17] Fang HHP, Liu H. Effect of pH on hydrogen production from glucose by a mixed culture. Bioresource Technology 2002;82: 87e93. [18] Noike T, Mizuno O. Hydrogen fermentation of organic municipal wastes. Water Science Technology 2000;42(12): 155e62. [19] Chen CC, Lin CY, Chang JS. Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate. Applied Microbiology and Biotechnology 2001;57(1e2):56e64. [20] Castello E, Garcia y Santos C, Iglesias T, Paolino G, Wenzel J, Borzacconi L, et al. Feasibility of biohydrogen production from cheese whey using a UASB reactor: links between microbial community and reactor performance. International Journal of Hydrogen Energy 2009;34:5674e82. [21] Chang JJ, Chen W-E, Shih S-Y, Yu S-J, Lay J-J, Wen F-S, et al. Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNAtargeted reverse transcription-PCR. Applied Microbiology and Biotechnology 2006;70:598e604. [22] Hawkes FR, Hussy I, Kyazze G, Dinsdale R, Hawkes DL. Continuous dark fermentative hydrogen production by mesophilic microflora: principles and progress. International Journal of Hydrogen Energy 2007;32:172e84. [23] Chang JJ, Wu JH, Wen FS, Hung KY, Chen YT, Hsiao CL. Molecular monitoring of microbes in a continuous hydrogenproducing system with different hydraulic retention time. International Journal of Hydrogen Energy 2008;33(5):1579e85. [24] Misturini DR, Berne da Costa J, Aquino de Souza E, Ruaro Peralba M, Samios D, Zachia Ayub MA. Comparison of different pretreatment methods for hydrogen production using environmental microbial consortia on residual glycerol from biodiesel. International Journal of Hydrogen Energy 2011;36(8):4814e9. [25] Khanal SK, Chen WH, Li L, Sung SW. Biological hydrogen production: effects of pH and intermediate products. Journal of Hydrogen Energy 2004;29(11):1123e31. [26] Oh SE, Iyer P, Bruns MA, Logan BE. Biological hydrogen production using membrane bioreactor. Biotechnology and Bioengineering 2004;1:119e27. [27] Bruce EL, Sang-Eun OH, Kim INS, VanGinkel S. Biological hydrogen production measured in batch anaerobic respirometers. Environmental Science and Technology 2002; 36:2530e5.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Bio-hydrogen production during acidogenic fermentation in a multistage stirred tank reactor Estela Tapia-Venegas a,1, Juan Esteban Ramirez a,1, Andre´s Donoso-Bravo a,1, Lorena Jorquera a,1, Jean-Phillipe Steyer b, Gonzalo Ruiz-Filippi a,* a b
Escuela de Ingenierı´a Bioquı´mica, Facultad de Ingenierı´a, Pontificia Universidad Cato´lica de Valparaı´so, General Cruz 34, Valparaiso, Chile INRA, UR050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, Narbonne F-11100, France
article info
abstract
Article history:
The objective of this study was to evaluate the production of hydrogen in a two-stage CSTR
Received 30 August 2012
system e both reactors having the same volume e and compare its performance with
Received in revised form
a conventional one-stage process. The lab-scale two-stage and one-stage systems were
19 October 2012
operated at five pHs and five hydraulic retention time (HRTs). The maximum volumetric
Accepted 16 November 2012
hydrogen productivity and yield obtained with the two-stage system were 5.8 mmol L1 h1
Available online 28 December 2012
and 2.7 mol H2 mol glucose1, respectively, at an HRT of 12 h and pH 5.5. Overall, the twostage system showed, at steady state, a better performance that the one-stage system for
Keywords:
all the evaluated pHs. However, a comparison between the one-stage system, operating at
Bio-hydrogen
6 h of HRT, and the first reactor of the two-stage system at the same HRT did not show any
Anaerobic digestion
significant difference, highlighting the positive impact of having a two-stage process. The
Renewable energy source
determination of the ratio between the experimental measured H2 in the gas phase and the
Plug flow
theoretical H2 generated in the liquid phase (discrepancy factor) indicated that an impor-
Series reactor
tant part of the hydrogen produced in the first reactor was transferred into the second reactor instead of being desorbed in the headspace. Therefore, the improving of hydrogen production in the two-stage system is rather attributed to the increased transfer of hydrogen from liquid to gas than an actual total hydrogen production increase. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
There are several operational variables that influence the hydrogen production by anaerobic digestion among the most important ones: pH, hydraulic retention time (HRT), partial pressure of hydrogen and liquid/gas equilibrium of the system [1e4]. In regards to the HRT (which is the inverse of the dilution rate), it is advisable to use a dilution rate less than 0.17 h1 or greater than 6 h HRT, when using continuous reactors with
suspended biomass, since the maximum specific growth rate (mmax) of hydrogen producers in an anaerobic mixed inoculums is less than that value. Despite this hydraulic constraint, different operational HRT ranges have been used depending on the type of substrates, reactor and inoculum as well as the pretreatment applied to eliminate the methanogens. In any case, it is clear that as HRT increases, the hydrogen production decreases, being the maximum reported value for HRT to produce hydrogen in a continuous operation between 14 and
* Corresponding author. Tel.: þ56 32 2273819. E-mail address: [email protected] (G. Ruiz-Filippi). 1 Tel.: þ56 32 2273819. 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2012.11.077
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17 h [5]. On the other hand, it is usually accepted, although this is still unclear, that hydrogen accumulation, which is related to the partial pressure of hydrogen in the reactor headspace, might inhibit hydrogen producers [6]. In this context, different strategies have been used to minimize this effect, for example, the dilution of the biogas by sparging an inert gas and vacuum application [7e9]. However, these strategies may increase the cost of the process and it is necessary to investigate new and more economically feasible alternatives to reduce the effect of hydrogen partial pressure. One possible option is the use of multistage stirred tank reactors in series which are characterised by having several states across the reactors. This configuration is well suited for processes in which a certain degree of product inhibition may occur [10,11]. Therefore, using two continuous stirred tank reactors (CSTR) in series could, first of all, enhance the selection of hydrogen producers by washing out the methanogens, while at the same time, reducing the hydrogen partial pressure that a one-stirred tank reactor with the same total volume would have. On the other hand, the reactors geometry may also influence the bio-hydrogen reactor performance since the transfer of hydrogen from the liquid to the gas phase depends on the interfacial specific area from the liquid to the gas. The aim of this study was to assess the bio-hydrogen production in a twostage series system in terms of hydrogen yield and volumetric productivity compared to a classical one-stage continuous system.
2.
Materials and method
2.1.
Experimental set-up
Three glass-made reactors were designed and implemented at lab-scale. A conventional one-stage CSTR system with a volume of 4 L and a two-stage system, composed by two CSTR reactors in series of 2 L each, were used. Each reactor was connected to auxiliary equipment: temperature and pH sensors for monitoring, peristaltic pumps for influent feeding and effluent draw off, pH control pump by adding a bicarbonate solution, a mechanical stirrer and a heating jackedtype system. Both systems were maintained at 37 C and operated in parallel.
2.2.
Table 1 e Synthetic wastewater composition (adapted from Bruce et al. [27]). Nutrient Glucose Ammonium bicarbonate Potassium dihydrogen phosphate Magnesium sulphate heptahydrate Ferrous chloride Sodium chloride Sodium molybdenum oxide dihydrate Calcium chloride dihydrate Manganese sulphate monohydrate
Chemical formula
Concentration (g/L)
C6H12O6 NH4HCO3
5 2
KH2PO4
1
MgSO4$7H2O
0.1
FeCl2 NaCl NaMoO4$2H2O
0.00278 0.01 0.01
CaCl2$2H2O
0.01
MnSO4$H2O
0.0094
2.3.
Systems evaluation and comparison
2.3.1.
Determination of the optimal conditions for the systems
Both systems were operated at five HRTs: 6, 8, 10, 12, 14 h aiming to find the best hydraulic conditions in terms of hydrogen yield and productivity. Each reactor of the two-stage system were operated at the same HRT i.e. half of the total one. Each HRT condition was evaluated at five pHs 4.0, 4.5, 5.0, 5.5 and 7.0. The influence of these variables was assessed by using the response surface methodology (RSM) in order to assess the effect of each variable as well as their combined influence. The analysis was carried out using the software Statgraphics Plus.
2.3.2.
Experimental running
The experiment sequence was randomised (i.e. randomly selected HRT and pH) in order to minimized the experimental bias. Furthermore, for each condition, the reactors were reseeded with the same original inoculum (non-adapted anaerobic biomass) and were kept until steady state conditions were reached, usually after 3 HRTs of operation, although, the reactors were maintained at each condition for around 40 HRTs.
Wastewater and inocula 2.4.
For the experiments, synthetic glucose-based wastewater and suspended biomass were used (Table 1). For the reactors seeding, granular anaerobic inoculum was taken from a fullscale anaerobic plant treating tobacco wastewater with a concentration of 13 g VSS L1 and an acidogenic and methanogenic activity of 0.17 g COD CH4 gVSS1 d1 and 7.74 g COD C6H12O6 g VSS1 d1. A volume of this inoculum equal to the 25% of the reactor volume was added for each experiment, resulting in an initial concentration of 4 g VSS L1. In order to wash out the methanogenic biomass from the inoculum, a biokinetic strategy based on the use of low HRT (in case of CSTR, HRT is equal to the solid retention time, SRT) was applied (6e14 h).
Analytical methods
For each experiment, measurements of the influent and effluent of each reactor were carried out in order to characterize the system performance. Chemical oxygen demand (COD) was determined using Method 5220C, standard methods [12], and glucose concentration was measured using 3,5-Dinitrosalicylic acid, DNS [13]. Prior to COD and glucose determination, samples were centrifuged at 15,000 rpm for 10 min in order to remove suspended solids. The production of volatile fatty acids (VFA) and ethanol was determined by gas chromatography (Shimadzu GC8 and PerkinElmer 500, respectively). The biomass concentration was measured by determining the volatile suspended solids (VSS) in the reactor
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[12]. The biogas production was measured by liquid displacement, and the composition of biogas was measured by gas chromatography, using GC, Perkin Elmer Clarus 500, with nitrogen as the carrier. The injector, detector and column temperatures were 120, 170 and 35 C, respectively. The hydrogen composition in the biogas is directly related to the hydrogen partial pressure, it was calculated from fraction of the compound in the gas.
3.
Results and discussion
3.1. Influence of the HRT and pH on the two-stage system performance The hydrogen yield and hydrogen volumetric productivity of the two-stage system at the different HRTs and pHs evaluated are presented in Fig. 1. The obtained yields and productivities were all within the range of 0.2e2.7 mol H2 mol glucose1 and 0.7e5.9 mmol H2 L1 h1, respectively. The best performance of the system, in terms of productivity, was observed at HRT of 12 h and pH 5.5 and an OLR of 0.41 g COD L1 h1 with a value of 5.8 mmol H2 L1 h1, whereas the observed yield was 2.7 mol H2 mol glucose1. Other studies have found a similar effect of the HRT but over a wider range. For example Lin et al. [14] examined HRTs between 6 and 48 h and found that the best productivity was between 6 and 12 h in a CSTR with 20 g COD L1 of glucose. The obtained value of yield in this study is even higher than the one obtained with pure culture of Clostridia sp of 1.7 mol H2 mol glucose1, at an organic loading rate 3.3 g COD L1 h1 or as reported, HRT 0.5 days and 20 g COD L1 in a CSTR reactor [15]. Similar values of productivity have been also obtained with thermophilic microorganisms (58 C) 2.58 mmol H2 L1 h1 at an organic load rate of 0.28 g COD L1 h1 or, as reported, 10 g lactose L1 and dilution rate 0.05 h1 [16].
Fig. 1 e Hydrogen yield (A) and productivity (B) of the twostage system. Graphs as a function of HRT and pH. Bars for the different pHs: grey, 4; soft grey with white dots, 4.5; black, 5; soft grey with white diagonal lines, 5.5 and white, 7.
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With respect to the pH effect, at the optimal HRT of 12 h, both the yield and the productivity present their highest value at pH 5.5. In Fig. 1, it can be observed that both variables exert a similar influence on the yield and the productivity. It is known that the pH plays an important role in determining the type of viable anaerobic fermentation pathways or changes in microbial diversity [17]. The lowest productivities were achieved at pH 7.0 and 4.0, where acetic and butyric acids represented 60% of the total COD, while ethanol and propionic acid comprised the remaining 40%. For pH 5.5 where the percentage of acetic and butyric acids is high, the main biochemical pathway for the production of hydrogen would be the acetate-butyrate, described as one of the most important route to produce hydrogen via fermentation. It has been previously reported that pH between 4.5 and 6 are more suitable to produce hydrogen, because the ethanol production pathway is less stimulated [18,19]. Furthermore, this low hydrogen yield could be explained by the presence of fermentative organisms which present a low hydrogen yield (such as the propionate producers) and by fermenters unable to produce hydrogen that could compete for the same substrate, also at pH 7.0, the acid/ethanol ratio was low; an increase in ethanol production, decreases the production of hydrogen and also has been reported that this point the activity of the hydrogenase enzyme begins to decrease as the pH is increased [5,20]. These changes can be further assessed by analysing the behaviour of the volatile fatty acids (VFAs) throughout the system operation. The concentration of VFA and ethanol, at the steady state of the two-stage system for an HRT of 12 h, are shown in Fig. 2. At a pH of 4 the three main VFAs (acetic, propionic, butyric acid) were present at a similar concentration. Between 4.5 and 5.5 the concentration of propionic remained close to zero, whereas at pH of 7.0 its concentration increased again up to reach a value similar to the butyric acid, high concentrations of propionate decreased acetic concentration [17]. Overall, the acetic acid was the most present VFA under all the pH conditions. At pHs 4.5, 5.0 and 5.5, acetic acid and butyric acid composed close to 95% of the total VFAs produced and, in line with the studies reported in the
Fig. 2 e Volatile fatty acid (VFAs) concentration at HRT of 12 h and five pHs. Grey diamonds: acetic acid; grey squares: butyric acid; black triangles: propionic acid and white circles: ethanol.
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literature, this is correlated with the increase of H2 productivity at these pHs. The hydrogen content of the biogas was between 21% and 63%, depending on the conditions used (pH and HTR). For each experiment, the resulting biogas consisted exclusively of hydrogen (H2) and carbon dioxide (CO2). Methane was not detected at these operating conditions, indicating the absence of methanogens in the system. The use of short HRT has demonstrated to be a proper alternative for archae elimination and avoiding methanogenesis in operations lasting about 30 days. Same methodology has been successfully reported in continuous hydrogen production [21,22].
3.2. One-stage performance and comparison with the two-stage system Fig. 3 shows the results of the one-stage system performance in terms of hydrogen yield and productivity. The obtained yield and productivity values lie between 0.25 and 1.54 mol H2 mol glucose1 and 0.39e3.13 mmol L1 h1, respectively. The best results are observed at an HRT of 12 h and pH of 5.5 when an OLR of 0.41 g COD L1 h1 was applied. The latter is in agreement to those reported values at similar operating conditions [14], Fang et al. [17], reached a hydrogen yield of 2.1 mol H2 mol glucose1 in a CSTR fed with glucose at an OLR of 1.2 g COD L1 h1, however, in this last case, a specific acidogenic inoculum previously adapted for the hydrogen production was used, which may explain the better obtained results. Nevertheless, greater values of these parameters have been achieved with other types of substrates, for instance, Chang et al. [23], who used molasses as the carbon source, attained a yield and productivity of 2.1 mol H2 mol hexose1 and 8.3 mmol H2 L1 h1, respectively, at an OLR of 120 g COD L1 h1. As for the two-stage system, the highest yield was also obtained at pH 5.5 for HRT of 12 h. However at an HRT of 6 h,
the highest values were obtained in a broader pH range (from 4.5 to 5.5). With respect to the productivity, the best values were obtained at the same range (from 4.5 to 5.5) for both HRTs. Some studies have concluded that the maintenance of a moderately acidic pH (around 5e5.5) creates good conditions for hydrogen production and the role of the pH goes beyond being a control parameter during the anaerobic digestion, which is related to the change of metabolic pathways and activity of the enzyme hydrogenase, therefore likely that the observed changes at different pHs is for this reason and pH 5.5 allowed the best way to produce hydrogen [1,24,25]. The change (in percentage) of the yield and productivity obtained between the two-stage and the one-stage systems at HRT of 12 h is presented in Fig. 4. The yield obtained by the two-stage system was higher (36e41%) than the one-stage system for all the evaluated pHs. Productivity was also improved (from 17 to 65%) in the two-stage system. These increases are similar to those improvements obtained by using N2 sparging, however, in our case, a subsequent gas separation and purification of the H2 will not be necessary as the sparging case [22]. To explain these differences, the one-stage system was operated at 6 h in order to compare it with the first reactor of the two-stage system (when operated at a total HRT of 12 h). The results for both systems did not show statistically significant differences (ANOVA test with a 95% level). The VFAs production was also similar in both systems where the acetic and butyric acids represented 95% to the total VFAs produced and an acetic to butyric acid ratio around 1 was observed for all pHs. If the performance of first reactor of the two-stage system is similar to one-stage system at the same HRT, the explanation of the difference, in terms of productivity and yield, can be on the behaviour of second reactor of two-stage system.
3.3. Two-stage series system strategy: partial pressure and hydrogen behaviour The hydrogen partial pressure is sometimes recognized as a quite influential factor in acidogenic systems. In the first reactor of the two-step process, the hydrogen partial pressure
Fig. 3 e Performance of the one-stage system. Bars for the different pHs: grey, 4; soft grey with with vertical and horizontal lines, 4.5; black, 5; soft grey, 5.5 and white, 7.
Fig. 4 e Systems performance variation comparing the two-stage and the one-stage system at different pHs at HRT of 12. Productivity (white bars), yield (grey bars).
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and hydrogen yield were 49 kPa and 4.30 mmol H2 L1 h1 whereas in the second reactor, they reached 63 kPa and 6.45 mmol H2 L1 h1. These values indicate that, in the case of a two-stage process, a high hydrogen partial pressure does not negatively influence the production yield. This unclear influence can be also seen in Fig. 5 where the productivity of both reactors for all the evaluated pH does not present any correlation with the hydrogen partial pressure. In order to have a better insight of the system behaviour, a mass balance of each reactor was carried out. In anaerobic digestion processes, the production of acetic and butyric acids is usually assumed to be coupled with the hydrogen production according to equations (1) and (2)), respectively. Therefore, knowing the concentration of VFA, we estimated the theoretical H2 production in the liquid phase using these stoichiometric expressions. The discrepancy factor Df, which corresponds to the ratio between the measured H2 in the gas phase and the theoretical H2 generated in the liquid phase, can then be calculated. This factor gives an idea of the H2 that is transferred to the gas phase: for instance, a high vaporization of hydrogen would lead to a Df close to 1. þ C6 H12 O6 þ 4H2 O/4H2 þ 2C2 H4 O2 þ 2HCO 3 þ 4H
(1)
þ C6 H12 O6 þ 2H2 O/2H2 þ C4 H8 O2 þ 2HCO 3 þ 3H
(2)
Table 2 shows the general mass balance of each reactor of the two-stage system for the HRT of 12 h (6 h in each reactor). It is worth to point out that the Df for the one stage system are equal to the first reactor of the two-stage system since the biogas produced and the VFAs measured were not statistically different. As observed, the Df of the first reactor is less than 1 for all the pHs which indicates that not all the hydrogen generated in the liquid phase goes to the gas phase. Unlike the first reactor, Df is greater than 1 in most cases in the second reactor, which indicates that more hydrogen than the theoretical value is being produced and transferred into the gas phase. This is explained by the fact that in the second reactor a dual effect takes place: not only hydrogen is produced from consumption of glucose that was not degraded in the first reactor is measured but also hydrogen desorbed from the effluent from the first reactor that enters the second reactor. It is known that the solubility of hydrogen in water is low; however a culture medium with several soluble compounds and microorganism may present a totally different solubility. The medium characteristics may affect the transfer of ionic
Table 2 e General balance of the two-stage series system at the different evaluated pHs (all the values are in mmol hL1 except Df which is dimensionless). Experimental information Acetic acid Reactor 1 2.92 3.81 2.88 4.79 3.44 Reactor 2 0.45 1.15 0.26 0.78 0.45
Df
Butyric acid
H2
pH
H2
1.43 1.41 1.87 2.42 0.52
5.73 6.70 7.61 8.72 5.04
4.0 4.5 5.0 5.5 7.0
8.70 10.43 9.49 14.40 7.92
0.66 0.64 0.80 0.61 0.64
0.07 0.08 0.03 0.95 0.11
1.14 2.02 2.20 12.90 0.81
4.0 4.5 5.0 5.5 7.0
1.06 2.45 0.58 3.44 1.11
1.08 0.82 3.76 3.75 0.73
a Calculated from acetic acid and butyric acid measured (1st and 2nd column).
strength or the interaction between the solvent and solute which in this case would be the hydrogen [26]. In this context, Frigon et al. [4], assessed the mass transfer of hydrogen from liquid to gas and noted that it is extremely limited in an anaerobic digestion process, when no thermodynamic equilibrium exists between both phases. Likewise, in that study, a lower hydrogen concentration in the liquid was achieved by recirculating biogas into the reactor. The interfacial surface between the liquid and gas was then increased, as could happen in our reactors in series where the diameter of the reactor belonging to the conventional system is lower than that obtained with the system in series. These differences in the hydrogen behaviour in both reactors could demonstrate that the concentration of hydrogen in the gas is a poor indicator of the concentration at which the biomass is subject because the liquid phase presents mass transfer limitations. Although the importance of the partial pressure remains unclear, during the operation of the two-stage system could be observed that the liquidegas transfer of the hydrogen in the first reactor is less than the second reactor, and the hydrogen produced in the second reactor come from both the residual glucose and the desorption of the soluble hydrogen from the first reactor effluent. These results show that yield, considering all the hydrogen produced (gaseous and dissolved) for the two-stage and the one-stage system was in a range of 1.2e2.2 mol H2 mol glucose1 and 1.12e1.70 mol H2 mol glucose1. These results indicate that the enhancement of the two-stage system performance is, at least in part, due to the improvement in the hydrogen transfer from gas to liquid in two-stage system.
4. Fig. 5 e Productivity of both reactors of the two-stage system as a function of the hydrogen partial pressure.
Indirect measurementsa
Conclusion
A two-stage system, composed of two CSTR reactors in series, showed to be a suitable option to improve bio-hydrogen
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production by dark fermentation. The highest hydrogen volumetric productivity was 5.8 mmol H2 L1 h1 with a yield of 2.7 mol H2 mol glucose1 at a pH of 5.5. This system exhibited higher results with regards to the volumetric productivity and yield than a one-stage reactor e of similar total volume e with an increase between 20 and 60% at several operating pHs. However, this enhancement cannot be directly attributed to the effect of the hydrogen partial pressure and the better performance of the two-stage system is rather attributed to an improvement of the hydrogen transfer to the gas phase.
Acknowledgements This study was funded by Fondecyt 1060109 and 1120659.
references
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