Renewable Energy 34 (2009) 970975

Contents lists available at ScienceDirect

Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Bio-hydrogen production from waste fermentation: Mixing and static conditions

X. Gomez*, M.J. Cuetos, J.I. Prieto, A. Moran
Chemical Engineering Dept. IRENA, University of Leon, Avda. de Portugal 41, 24071 Leon, Spain

a r t i c l e i n f o
Article history: Received 11 December 2007 Accepted 26 August 2008 Available online 23 October 2008 Keywords: Hydrogen production Dark fermentation Wastes Inoculum Digestion

a b s t r a c t
One of the main disadvantages of the dark fermentation process is the cost associated with the stages needed for obtaining H2 producing microorganisms. Using anaerobic microora in fermentation systems directly is an alternative which is gaining special interest when considering the implementation of largescale plants and the use of wastes as substrate material. The performance of two H2 producing microora obtained from different anaerobic cultures was studied in this paper. Inoculum obtained from a waste sludge digester and from a laboratory digester treating slaughterhouse wastes were used to start up H2 fermentation systems. Inoculum acclimatized to slaughterhouse wastes gave better performance in terms of stability. However, due to the limited availability of this seed material, further work was performed to study the behaviour of the inoculum obtained from the municipal wastewater treatment plant. The process was evaluated under static and mixing conditions. It was found that application of a low organic loading rate favoured the performance of the fermentation systems, and that agitation of the reacting mass could alleviate unsteady performance. Specic H2 production obtained was in the range of 1926 L/kg SVfed with maximum peak production of 3867 L/kg SVfed. Although the performance of the systems was unsteady, recovery could be achieved by suspending the feeding process and controlling the pH in the range of 5.05.5. Testing the recovery capacity of the systems under temperature shocks resulted in total stoppage of H2 production. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Hydrogen has a higher gravimetric energy density than any other known fuel and is compatible with electrochemical and combustion processes for energy conversion without producing the carbon-based emissions that contribute to environmental pollution and climate change [1]. Hydrogen production through dark fermentation has advantages over other processes because of its ability to produce hydrogen continuously from a number of renewable feed-stocks without any input of external energy [2]. In this sense, the production of this clean fuel allows not only the generation of energy from a renewable source, but also the treatment of bio-wastes thus avoiding the impact of their disposal on the environment. These two features make biological hydrogen production a novel and promising approach to meet increasing energy needs as a substitute for fossil fuels [3]. Complete treatment of the waste is obtained by the coupling of a methanogenic stage in a two-phase conguration for the simultaneous production of hydrogen and methane allowing the stabilization of biosolids [47].

* Corresponding author. University of Leon, Institute of Natural Resources, Avda de Portugal n 41, Leon 24071, Spain. Tel.: 34 987 29 18 41; fax: 34 987 29 18 39. E-mail address: xagomb@unileon.es (X. Gomez). 0960-1481/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2008.08.011

It is known that during organic and hydraulic shock loads, methanogenic precursors generate H2, CO2 and volatile fatty acids (VFAs) which accumulate in anaerobic digesters as a result of an imbalance between production and consumption [8]. The conventional anaerobic treatment process can be divided into two distinct stages: acidication and methane production. Each stage is carried out by a number of microorganisms through syntrophic interactions. Acidication produces H2 as a by-product, which in turn is used as an electron donor by many methanogens at the second stage of the process [9]. It should be desirable to obtain an extra bio-fuel such as hydrogen from the conventional anaerobic digestion of wastes. The conversion of the acidication stage to a hydrogen fermentation phase in a two-phase digestion system would allow the rapid scale-up of this biotechnology, making it feasible to harvest H2 at the acidication stage of the anaerobic treatment, leaving the remaining acidied products for further methanogenic treatment [10]. Some of the major drawbacks of the dark fermentation process are the application of a pre-treatment step to obtain an H2producing inoculum, and the continuous addition of alkali to maintain pH within a desired range, since a low pH can inhibit hydrogen-producing microorganisms. Reduction of the amount of alkali needed will be reected in the reduction of process operating costs. Strategies to reduce such needs of alkali for pH control

 X. Gomez et al. / Renewable Energy 34 (2009) 970975

971

include recirculation of the methanogenic efuent to the hydrogenproducing phase in the two-stage congurations [4] and cofermentation of wastes [11]. The production of hydrogen through continuous fermentation utilizing a non-sterile substrate with readily available mixed microora would be commercially desirable [12]. The advantage when utilizing anaerobic microora resides in the availability of the inoculum when the process is to be coupled to a two-phase digestion process. Experiences with no pretreatment of the inoculum have been reported as successful [46,1114]. Another option to reduce costs during start-up of the hydrogen fermentation process is the use of biosolid pellets as an inoculum source [15]. The aim of the experimental work reported here was the study of the production of H2 by biological means using anaerobic microora as seed material. No pre-treatment was applied since it was considered impractical in large-scale applications. The fermentation of food waste was studied using mixed microora obtained from two different digestion systems. An assessment of the production of H2 by dark fermentation reactors was performed under static and mixing conditions at two different organic loading rates. 2. Experimental 2.1. Materials and microbial culture The experimental set-up and food waste used as feed were based on previous work [5]. The main characteristics of the substrate are presented in Table 1. Two seed inocula were tested to obtain the hydrogen-producing microora. One inoculum proceeded from a laboratory-scale reactor digesting slaughterhouse waste from a poultry-processing plant (denoted as HI). This system had been working under mesophilic conditions (34  C) for 6 months with a hydraulic retention time (HRT) of 36 days and an average methane content in the biogas produced of 65%. The TS and VS concentrations of the inoculum were 19.5 and 11.0 g/L, respectively. The second inoculum tested proceeded from the sludge digester of the wastewater treatment plant (WWTP) of the city of Leon (denoted as WI), with a TS and VS concentration of 11.3 and 7.0 g/L, respectively. The digester treated a mixture of primary sludge and waste activated sludge. The temperature of the digestion process was 32  C and the average HRT was 26 days. No pretreatment was applied to the seed sludge since this was considered impractical for large-scale applications. The experimental work was divided into two stages. During the rst phase of experimentation Erlenmeyer asks with 100 mL working volume were used. The reactors were kept submerged in a water bath at 34  C and were provided with magnetic stirrers. Two systems were started up using inocula HI and WI. The oxidation reduction potential (ORP) was set at 250 20 mV. This potential

was achieved by allowing the reactors to have contact with the atmosphere while stirring, until the required value was attained. The pH level was controlled manually, being kept in the range 5.06.0 by the addition of an alkaline solution during the feeding process and by initializing the pH at 6.0. The alkaline solution was prepared by mixing in a proportion of 1:1:1 (weight) NaHCO3, K2HPO4 and Na2HPO4. The solution thus prepared had a total solid concentration of 60 g/L. The reactors were run on a semi-continuous basis for 20 days being fed once a day with a dilution rate of 1/3. In the second phase, digesters with a working volume of 2 L were used and were started up with inoculum from the WWTP. H2 production and stability were evaluated for two organic loading rates (OLRs) by means of variations in the solid content of the feed. The food waste was prepared with a TS concentration of 3 and 6%. Systems were studied under mixing (MC-3 and MC-6) and static conditions (SC-3 and SC-6). The reactors were run on a semicontinuous basis for 40 days being fed once a day with a dilution rate of 1/3. Modication of the temperature was performed on the systems capable of reaching stable performance in order to study the recovery capacity. Initially the temperature was decreased to 24  C on day 20 for 3 days. Once the production of H2 recovered, a second temperature drop of equal duration was performed. 2.2. Analytical techniques Total Kjeldahl nitrogen (TKN) was measured using APHA standard methods [16] and crude protein calculations were based from Kjeldahl total nitrogen determinations (crude protein 6.25 total N) [17]. Total lipid content was determined using a standard Soxhlet method and ammonia was measured using an ion-selective electrode method [16]. Cellulose, hemicellulose and lignin content were estimated by analysis of neutral detergent bre (NDF), acid detergent bre (ADF) and crude bre [18] on ground samples in duplicate using an Ankom 200 bre analyser. Carbohydrate content was calculated by difference. During the fermentation process the following parameters were monitored: pH, TS, VS, chemical oxygen demand (COD), daily gas production, gas composition and concentration of volatile fatty acids (VFA). TS, VS and pH were determined in accordance with the APHA standard methods [16]. The values for TS concentration in the reactors were corrected to eliminate the effect of solids added to control pH. Daily gas production was measured using a reversible liquid displacement device with a wet-tip counter. All gas production data reported were normalized to a standard temperature (0  C) and pressure (760 mm Hg). COD of the supernatant was measured using a Series C99 multi-parameter photometer from Hanna Instruments after centrifugation of the sample at 3500 g and digestion in the presence of dichromate at 150  C for 2 h using a Hanna C9800 reactor. Biogas composition was analysed using a gas chromatograph (Varian CP 3800 GC) equipped with a thermal conductivity detector. A 4-m long column packed with HayeSep Q 80/100 followed by a 1-m long molecular sieve column were used to separate CH4, CO2, N2, H2 and O2. The carrier gas was argon and the columns were operated at 331 kPa and a temperature of 50  C. Volatile fatty acids (VFA) and ethanol were analysed using a gas chromatograph (Varian CP 3800 GC) equipped with a capillary column (from Supelco) and a ame ionization detector. The carrier gas was helium and the temperature of the injector was 250  C. The temperature of the oven was set at 150  C for 3 min and thereafter increased to 180  C. 3. Results and discussion 3.1. Effect of inoculum source on hydrogen production The initial pH of inocula was 7.9 for HI and 7.2 for WI. Once the feeding process started, the pH steadily decreased, taking a day

Table 1 Main characteristics of substrate fed to the semi-continuous reactors for H2 production Parameter TS (g/L) VS (g/L) pH Ammonia-N (mg/L) Total Kjeldahl nitrogen (%)a Protein (%)a Carbohydrate (%)a Cellulose (%)a Hemicelluloses (%)a Lignin (%)a Lipid content (%)a
a

Value 60.0 1.2 55.2 0.6 4.13 0.1 126.5 8.5 1.56 0.45 9.75 2.8 70.1 4.32 8.4 0.37 8.0 0.45 2.7 0.13 1.07 0.57

Based on dry bases.

972

X. Gomez et al. / Renewable Energy 34 (2009) 970975 Table 2 Performance parameters for H2-producing systems WI and HI Fermentation system SH2P (mL/g SVfed) VSr (g/L) TSr (g/L) %H2 in biogas % Dest. VS COD (g/L) Ethanol (mg/L) WI 27.8 8.4 29.1 1.8 43.6 3.0 1431.5 47.2 4.1 27.8 4.3 1223 65 HI 28.3 4.5 31.2 0.45 52.1 0.33 27.5 2.5 43.5 1.6 33.5 2.3 125 12

longer for the HI system to reach the value of 5.5. At this point alkaline solution was added to avoid a further pH reduction. The pH of the systems was restored to 6.0 after each feeding process was accomplished. It has been reported that the pH decrease at the beginning of the feeding was not only due to the VFA accumulation, but also the decrease of the pH by the feed [6,19]. In the present study, due to the low pH of the substrate, the addition of alkali was intended not only for correction of the pH of the reactor contents but also for correction of the pH decrease caused by the feed. The high volume of liquid replaced in every feeding process resulted in a pH drop around 4.8. Once the feeding procedure was completed and pH corrected, the pH of the reactors fell naturally; reaching values around 5.2, and no further corrections were undertaken until the next feeding procedure. During the start-up phase of the fermentative systems CH4 was detected in the biogas produced before the pH reached 5.5 units. CH4 was no longer detected once the pH reached this value and was maintained lower than 6.0. Fig. 1 presents the specic H2 production (SH2P) for reactors treating food wastes. This parameter was calculated based on the volume of H2 produced by VS fed to the system. System HI used start-up inoculum from the anaerobic reactor treating slaughterhouse waste. The WI system used the inoculum obtained from the WWTP digester. The HI system was characterized by stable biogas production with an average H2 concentration of 27.5 0.5% in the biogas. Since the performance of this system was stable, an HRT of 3 days could be set. WI, although incapable of stable biogas production, was able to recover every time after inhibition. The reactivation of the system was achieved by suspending the feeding and controlling the pH in the 5.05.5 range. Whenever H2 production was re-initialized a variable increase of pH to 5.86.5 units was observed. The H2 composition of biogas was around 29 31.5% on the rst day of re-initialization decreasing to 14% on the day before ceasing H2 production. Both systems were characterized by the generation of a layer of oating solids which had to be manually destroyed every day before proceeding with the withdrawal of material from reactors and posterior feeding. In Table 2 the performance parameters of hydrogen production systems are presented. Although performances were similar, WI parameters were calculated based on continuous H2 production periods, since this system was incapable of producing H2 4 days out of 20. For both reactors SH2P values are similar but with a large standard deviation for the WI reactor due to unsteady performance. System HI performed better and presented a lower destruction of VS. VFA concentration was particularly high for system WI (see Fig. 2), and data from this system was not stable. A particularly high concentration of butyric acid can be seen in Fig. 2. Concentration of

evolved VFA is an important parameter for analysing fermentation pathways and may explain the variability of hydrogen production presented in Fig. 1 for system WI. The maximum acetate concentration for this reactor was 3735 mg/L (62.3 mM) and the maximum butyrate concentration was 7683 mg/L (87.3 mM). At the acidied pH range, high dissociated acetate and butyrate concentrations can contribute to the inhibition of the hydrogen fermentation process. Concentrations of 60 mM acetic or butyric acid have been reported as inhibitory with a decrease in H2 production greater than 93% when using glucose as substrate [20]. It is presumed that the VFAs in their undissociated forms can freely permeate the bacteria cell membrane. If undissociated VFAs are pumped into the culture, they will penetrate the plasma membrane and dissociate depending on the pH inside the cell [21]. If a high level of dissociated VFA is present in the culture, the ionic strength in the solution will increase. Such an increase can result in cell lysis. As a result, the inhibitory effect will occur [22]. Inhibitory effects of butyrate on biological H2 production have been also studied by Zheng and Yu [23] obtaining moderate inhibition at butyrate concentrations in the range of 8.3612.54 g/L. Fig. 2 shows that butyric acid in system WI is close to the inhibitory range, explaining the high instability in H2 production. Both systems were characterized by the increasing concentration of caproic acid, its presence being observed from day 6 onwards, increasing in concentration until reaching a stable value

a
VFA (mg/l)

5000 4000 3000 2000 1000 0 0 5 10 15 20

80

WI

HI

Time (days) SH2P (mL-H2/gVSfeed)

60

b 10000
8000

40

VFA (mg/l)

6000 4000 2000 0

20

0 Acetic

10

15 Butyric

20 Caproic

10

12

14

16

18

20

Time (days)
Propionic

Time (days)
Fig. 1. Specic H2 production for systems HI and WI.

Fig. 2. VFA concentration for systems (a) HI and (b) WI.

 X. Gomez et al. / Renewable Energy 34 (2009) 970975

973

for system HI, and not following any particular trend for system WI. Ethanol production was observed, with a higher concentration in system WI. Investigations of H2-producing butyrateacetate metabolism point to the pH range 5.25.8 as the optimum over a variety of HRT (632 h) and substrate types (sucrose, starch and beer industry wastes) [24]. With a higher HRT, system HI presented high butyrateacetate concentrations with low concentrations of propionic acid, although at high pH, ethanol was also produced. The ethanol concentration reported for system WI was particularly high reaching a steady value, although this system was incapable of steady performance. Ethanol has been found in systems working at low pH. Optimal operating conditions have been reported [25,26] for the ethanolacetate H2-producing fermentation of molasses at pH 4.04.5, HRT 46 h. Butyrate and ethanol have been dominant end-products on a molar basis working with a granular sludge bed reactor and sucrose as substrate (4.7 g L/L, 18 h HRT) [27]. 3.2. Evaluation under mixing and static conditions. Effect of the OLR From results obtained in Section 3.1 it was observed that inoculum derived from slaughterhouse digestion systems was capable of stable H2 production. However, the availability of this inoculum depends on the existence of a digester treating such waste. If largescale plants are to be installed, inoculum from a sewage sludge digester is to be preferred due to its greater availability. In this section all reactors were started up using inoculum WI. The SH2P for H2 systems under static and mixing conditions is presented in Fig. 3. As in Section 3.1, instability was observed in the production of H2 gas, with the systems being capable of recovery every time the feeding process was suspended and pH was controlled within the range of 5.05.5. Systems fed with food waste at TS contents of 6% (MC-6 and SC-6), presented a longer lag phase. However, whenever the systems recovered, the peak production of gas generated was greater

a
SH2P (mL-H2/gVSfeed)

80 70 60 50 40 30 20 10 0 0 5 10 15 20 25

SC-6

SC-3

30

35

40

Time (days)

b
SH2P (mL-H2/gVSfeed)

80 70 60 50 40 30 20 10 0

MC-6

MC-3

T (MC-3)

40 35 30 20 15 10 5 0 T (C) 25

than that obtained from systems fed with lower OLR (MC-3 and SC-3). The reduction of the OLR from 41.4 to 20.7 g VS/day, although with lower production, allowed the maintenance of longer periods of gas generation. The increase of H2 production with the solid content of the feed has already been reported in previous studies by the authors [11]. However, in that case, the co-fermentation of food wastes with waste blood resulted in a high concentration of CH4 with nil production of H2 in the biogas whenever the TS concentration of the feed was between 3 and 4%. In the present study no CH4 was detected in the biogas produced in any of the systems evaluated. System MC-3 was capable of continuous H2 production for 19 days, with an average H2 content of 26.5 3.5%. In contrast, systems working under static conditions presented an irregular prole of gas production. The reduction of OLR as a strategy to obtain stable operation was not sufcient to attain the objective, although this allowed the system a greater number of days on which H2 was produced. Since it has been reported that removal of H2 from the reactor liquor by sparging with N2 resulted in stable operation and increments of H2 yields [28], any operating conditions which favour the removal of this gas will also favour process stability. The results obtained in this study showed that agitation and reduction of OLR aided the stability of process performance. Solid substrate anaerobic fermentation for the production of H2 using OFMSW has been evaluated under mesophilic and thermophilic conditions, obtaining stable performance at high OLR although reactors were not agitated [14]. The difference from the present study is probably related to inoculum acclimation. Microorganisms used for solid fermentation were obtained from a methanogenic solid substrate anaerobic digester. On day 20 the temperature of system MC-3 was decreased to 24  C for 3 days to determine the capability of recovery of the fermentation process. Lowering the temperature resulted in the cessation of gas production for 7 days. Re-establishing mesophilic conditions and controlling pH along with the suspension of feeding allowed the recovery of H2 production. However, a delay of 4 days was observed in the recovery of the system. A posterior temperature drop caused a subsequent failure of fermentation with no recovery although the same strategies were performed. The performance parameters for fermentation reactors are presented in Table 3. As in the previous case, since no stable conditions were attained, the average H2 production obtained during the periods of H2 generation are shown. Means reported for SH2P were not statistically different (applying one-way ANOVA at the 0.05 level). In this sense, the lower gas production observed in systems with lower OLR was not accompanied by a decrease in the utilization of substrate. The effect of OLR on H2 production has been studied using soluble substrates such as glucose and sucrose [2931], reporting reductions in H2 yield with an increase in OLR. Kim and coworkers [32], in the assessment of H2 production of food waste and sewage sludge as substrates in batch systems, reported an increase

Table 3 Performance parameter of H2-producing systems under mixing and static conditions Fermentation system OLR (g SV/d) H2 yield (L/L*d) SH2P (mL/g SVfed) Max SH2P (mL/g SVfed) %H2 in biogas VSr (g/L) TSr (g/L) % Dest. VS COD (g/L) Ethanol (mg/L) MC-6 41.4 0.46 0.27 22.1 13.2 44.0 1231 24.3 1.9 36.4 1.4 56.1 4.3 28.7 4.5 1202 299 SC-6 41.4 0.46 0.18 22.1 8.8 38.2 827 26.4 2.3 37.0 3.8 52.0 5.1 26.8 5.7 1121 256 MC-3 20.7 0.20 0.10 19.3 9.3 44.0 1333 11.1 2.3 15.6 3.9 59.8 4.2 20.6 2.1 1000 57 SC-3 20.7 0.27 0.16 25.94 15.7 67.8 1432.5 10.7 2.5 15.3 3.5 61.2 5.4 16.2 3.5 521 119

10

15

20

25

30

35

40

Time (days)
Fig. 3. Specic H2 production for systems: (a) under static (SC-6 and SC-3) and (b) mixing (MC-6 and MC-3) conditions. Temperature in (b) refers to modications applied only to system MC-3.

974

X. Gomez et al. / Renewable Energy 34 (2009) 970975

a 35000
30000 25000 20000 15000 10000 5000 0 0 5 10 15 20 25 30 35 40 VFA (mg/L)

b
VFA (mg/L)

25000 20000 15000 10000 5000 0 0 5 10 15 20 25 30 35 40

c
VFA (mg/L)

7000 6000 5000 4000 3000 2000 1000 0 0 5 10 15 20 25 30 35 40

d
VFA (mg/L)

4000 3500 3000 2500 2000 1500 1000 500 0 0 5 10 15 20 25 30 35 40

Time (days) Acetic Propionic Butyric

Time (days) Caproic

Fig. 4. VFA concentration for systems (a) SC-6, (b) SC-3 under static conditions, and (c) MC-6, (d) MC-3 under mixing conditions.

in the potential H2 production when VS concentration of the feed was increased by up to 3%, and then a decrease, as VS concentration further increased, due to product inhibition by H2 and VFA. The difference in results obtained from this study may be rationalized by the different substrates used and conditions of operation, continuous instead of batch. Differences were also observed in the frequency of peaks of H2 production. Reactors with a high OLR (reactors SC-6 and MC-6) presented shorts peaks of high intensity, while reduction of the OLR (reactors MC-3 and SC-3) resulted in longer periods of production. A oating layer of solids appeared in both static systems which may account for the intermittent production of H2. However MC-6 presented peak production although no oating layer of solid was generated during fermentation. In this sense, agitation of the reactor content was not enough to attain stable gas production. However, an increase in the number of periods of H2 production for system MC-6 can be seen in Fig. 3, indicating that the agitation of the system plays an important role in the performance of the process. The irregular production of gas by the fermentation systems was associated with a high variability of VFA data obtained (Fig. 4). MC-3 was the only system capable of attaining stable gas production during the period evaluated at constant temperature. The distinctive feature of this system is the low value obtained for butyric acid concentration. Although this value was not coupled to a greater H2 yield, a continuous production of H2 was nally attained. The modication of temperature produced a lag phase which was accompanied by a reduction in acetic acid concentration but increments in the concentration of butyric acid. Once the system recovered from the temperature drop there was a 3 day H2 production period before proceeding with a new reduction in temperature. This last modication resulted in a lag phase characterized by a constant VFA concentration probably due to the fact that the reactor was no longer being fed and biological activity had ceased. Propionic acid was produced at the moment temperature modications were performed, while the presence of this acid was continuously observed in the other systems. Ethanol was also produced, and as was already noted in Section 3.1, the high pH was

not sufcient to shift metabolism either to ethanolacetate or ethanolbutyrate type fermentation. The feeding process to the systems was as irregular as the production of H2, since they were fed only during the periods of gas production. Systems working at 6% TS concentrations in the feed experienced an increase in pH up to 6.3 units in every recovery phase, as described in the previous case. However, for system with a lower OLR, although experiencing an increase in pH, this was less steep, reaching values up to 5.7 units. Based on the SH2P obtained from the fermentation systems, hydrogen production may be estimated for a city, for example Madrid (Spain). With an average production of 2.7 106 t MSW/ year a gas production of 2.4 107 m3/year may be expected, accounting for an energy production of 3 105 GJ/year. When fuel cells are incorporated in automation (1.3 kg H2/100 km), the mass of fuel obtained from the fermentation of wastes is sufced for 16,500 vehicles (medium size) running 10,000 km/year [33].

4. Conclusions From results obtained it was observed that inoculum obtained from the digestion system treating slaughterhouse waste presented stable H2 production, while the inoculum obtained from the WWTP digester presented a lower fermentation performance. However, when using this latter inoculum as the source of H2-producing microora, the performance (in terms of stability) of fermentation systems could be improved by reducing the organic loading rate (OLR) and by means of agitation. In this sense, under the conditions evaluated, agitation favoured stability. The reactor at low OLR provided with agitation was capable of producing H2 for a continuous period. Thus, when using the inoculum from the WWTP to start a large-scale reactor, agitation is an important factor to take into consideration in the design of the system for improving performance. A temperature drop in the system working with low OLR and under agitation resulted in a failure in the production of H2. Recovery of the system could be accomplished. However after a second temperature drop the system could not recover.

 X. Gomez et al. / Renewable Energy 34 (2009) 970975

975

Acknowledgements This work was possible thanks to the nancial support of Endesa Corporation by the Novare Project and the collaboration of the WWTP of Leon-SALEAL (mancomunidad municipal para el sanea miento integral de Leon y su alfoz). References
[1] Levin D, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 2004;29(2):17385. [2] Chen W-H, Chen S-Y, Khanal SK, Sung S. Kinetic study of biological hydrogen production by anaerobic fermentation. Int J Hydrogen Energy 2006;31(15):21708. [3] Kapdan IK, Kargi F. Bio-hydrogen production from waste materials. Enzyme Microb Technol 2006;38(5):56982. [4] Kraemer T, Bagley D. Continuous fermentative hydrogen production using a two-phase reactor system with recycle. Environ Sci Technol 2005;39(10):381925. [5] Gomez X, Moran A, Cuetos MJ, Sanchez ME. The production of hydrogen by dark fermentation of municipal solid wastes and slaughterhouse waste: a twophase process. J Power Sourc 2006;157(2):72732. [6] Liu D, Zeng RJ, Angelikadi I. Hydrogen methane production from household solid waste in two-stage fermentation process. Water Res 2006;40(11):22306. [7] Ueno Y, Tatara M, Fukui H, Makiuchi T, Goto M, Sode K. Production of hydrogen and methane from organic solid wastes by phase-separation of anaerobic process. Bioresour Technol 2007;98(9):18615. [8] Voolapalli RK, Stuckey DC. Hydrogen production in anaerobic reactors during shock loads inuence of formate production and H2 kinetics. Water Res 2001;35(7):183141. [9] Vijayaraghavan K, Amin M, Soom M. Trends in bio-hydrogen generation a review. Environ Sci 2006;3(4):25571. [10] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DC, Noike T. Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresour Technol 2000;73(1):5965. [11] Cuetos MJ, Gomez X, Escapa A, Moran A. Evaluation and simultaneous optimization of bio-hydrogen production using 32 factorial design and the desirability function. J Power Sources 2007;169(10):1319. [12] Hussy I, Hawkes FR, Dinsdale R, Hawkes DL. Continuous fermentative hydrogen production from sucrose and sugarbeet. Int J Hydrogen Energy 2005;30(5):47183. [13] Shizas I, Bagley DM. Fermentative hydrogen production in a system using anaerobic digester sludge without heat treatment as a biomass source. Water Sci Technol 2005;52(1-2):13944. [14] Valdez-Vazquez I, Ros-Leal E, Esparza-Garca F, Cecchi F, Poggi-Varaldo HM. Semi-continuous solid substrate anaerobic reactors for H2 production from [15]

[16] [17] [18]

[19] [20] [21] [22]

[23] [24]

[25] [26]

[27]

[28]

[29] [30]

[31]

[32]

[33]

organic waste: mesophilic versus thermophilic regime. Int J Hydrogen Energy 2005;30(13-14):138391. Kalogo Y, Bagley DM. Fermentative hydrogen gas production using biosolids pellets as the inoculum source. Bioresour Technol 2008;99(3):5406. doi:10.1016/j.biortech.2007.01.004. Apha-Awwa-Wpcf. Standard Methods for the Examination of Water and Wastewater. New York: American Public Health Association; 1989. Casal JA, Vermaat JE, Wiegman F. A test of two methods for plant protein determination using duckweed. Aquat Bot 2000;67(1):617. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary ber, neutral detergent ber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;74:358397. Han S-K, Shin H-S. Biohydrogen production by anaerobic fermentation of food waste. Int J Hydrogen Energy 2004;29:56977. Van Ginkel S, Logan BE. Inhibition of biohydrogen production by undissociated acetic and butyric acids. Environ Sci Technol 2005;39(23):93516. Gottschalk G. Bacterial metabolism. 2nd ed. New York: Springer; 1986. Niel EWJ, Claassen PAM, Stams AJM. Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus. Biotechnol Bioeng 2003;81(3):25562. Zheng X-J, Yu H-Q. Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic cultures. J Environ Manage 2005;74(1):6570. Hawkes FR, Hussy I, Kyazze G, Dinsdale R, Hawkes DL. Continuous dark fermentative hydrogen production by mesophilic microora: principles and progress. Int J Hydrogen Energy 2007;32(2):17284. Ren N, Wang B, Huang J-C. Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotechnol Bioeng 1997;54(5):42833. Ren NQ, Li YF, Wang AJ, Li JZ, Ding J, Zadsar M. Hydrogen production by fermentation: review of a new approach to environmentally safe energy production. Aquat Ecosystem Health Manage 2006;9(1):3942. Mu Y, Yu H-Q. Biological hydrogen production in a UASB reactor with granules I: physicochemical characteristics of hydrogen-producing granules. Biotechnol Bioeng 2006;94(5):9807. Hussy I, Hawkes FR, Dinsdale R, Hawkes DL. Continuous fermentative hydrogen production from a wheat starch co-product by mixed microora. Biotechnol Bioeng 2003;84(6):61926. Van Ginkel SW, Logan B. Increased biological hydrogen production with reduced organic loading. Water Res 2005;39(16):381926. Kim S-H, Han S-K, Shin H-S. Effect of substrate concentration on hydrogen production and 16S rDNA-based analysis of the microbial community in a continuous fermenter. Process Biochem 2006;41(1):199207. Kyazze G, Martinez-Perez N, Dinsdale R, Premier GC, Hawkes FR, Guwy AJ, et al. Inuence of substrate concentration on the stability and yield of continuous biohydrogen production. Biotechnol Bioeng 2006;93(5):9719. Kim S-H, Han S-K, Shin H-S. Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge. Int J Hydrogen Energy 2004;29(15):160716. von Helmolt R, Eberle U. Fuel cell vehicles: status. J Power Sources 2007;165(2):83343.