Biochemical reactions relevant to hydrogen production
The generation of hydrogen by biological means is not energy intensive compared with the conventional thermochemical techniques, since the operating temperature and pressure are not very high. As raw materials organic waste streams can be used that can be considered as a renewable resource (Vijayaraghavan & Mohd Soom, 2006).The method of the dark fermentation has certain advantages compared with the other biological processes. In contrast to bio-photolysis and photo fermentation, the process needs no solar radiation, but the required energy is supplied by the organic substrates and hence the process is not interrupted during the night. Moreover, the production rate of the H2 of the fermentative bacteria in comparison with the other biological processes is greater (Kumar et al., 2000; Nath et al., 2005).
The different process parameters that are relevant for hydrogen production have been surveyed (Li & Fang, 2007; Wang & Wan, 2009) and include the type of substrate, nutrient concentration, the inoculum, pH, reactor configuration, hydraulic retention time (HRT), organic loading rate (OLR). Carbohydrate-rich substrates are the most suitable for fermentative H2 production systems (Hawkes et al., 2002; Kapdan & Kargi, 2006; Meherkotay & Das, 2008; Ueno et al., 2007) seeded with saccharoclastic microorganisms, They are able to break down organic substances via the Embden-Meyerhof pathway resulting to different metabolic products depending on the type of microorganism and the environmental conditions driving their catabolism (Hallenbeck, 2009).
The relevant microbial groups for the fermentative hydrogen production groups are clostridia and enterobacteria (Hallenbeck, 2005; Hawkes et al., 2007). Both groups were repeatedly experimentally confirmed as major hydrogen producers (Valdez-Vazquez & Poggi-Varaldo, 2009).
Enteric bacteria are gram-negative rods, facultative aerobic, with relatively simple nutrient requirements and can not form spores (Schmauder, 1992). Among the species that can produce H2, are
Clostridia are spore forming, gram-positive bacteria (Schmauder, 1992). Through sporulation they can survive for example dehydration, heat and large changes in pH. Clostridial catabolism includes a variety of reactions and hence fermentation end-products such as acetate, acetone (
Aim of this work was to experimentally study the effect of HRT and OLR on bio-hydrogen production in terms of maximization of H2 yield, so as to optimize substrate utilization efficiency that contributes to the cost effectiveness of the process. Experiments were carried out in large-lab scale reactors of 30 L working volume. In this way, experience in the start-up procedure, selection of only H2 producing microorganisms and the stability of long term continuous operation without methanogenesis of such a set-up could be gained. This can further be used for the scale-up of bio-hydrogen production towards the final aim of commercial implementation. An attempt towards the clarification of the possible metabolic pathways and the involved microorganisms, with along as their possible interactions was also undertaken. The way that these microorganisms behave and interract with each other and their milieu is very important for the design of effective hydrogen producing systems. The understanding of these processes can help the designer to manipulate hydrogen production by the suitable variation of the process parameters. With the use of molecular biological techniques it is possible to acquire better insights into such systems.
2. Materials and methods
2.1. Experimental set-up
Dark fermentation experiments were conducted in two identical 40 L reactors made of borosilicate glass (QVF) with a working volume of 30 L heated at 37 °C ± 2 °C by a heating pipe. The content of the fermenter was homogenized by external recirculation with eccentric screw pumps (Netsch). The pH was regulated by means of a pH glass electrode (Endress & Hauser, Orbisint CPS11) and a pH measuring transducer (Endress & Hauser, Mycom) connected to a programmable controller (Endress & Hauser, Memograph), which controlled 2 dosing pumps (Metrohm, Dosimat) for automatic addition of a sodium hydroxide solution 25% v/v and a hydrochloric acid solution 25% v/v, respectively. The Organic Loading Rate (OLR) was adjusted to the desired level by dosing (Metrohm, Dosimat) with molasses diluted 1:2 w/w and supplemented with nutrients. Every 100 mL of nutrient solution contained the following quantities in g; 1.72 FeSO4∙7H2O, 0.36 CaCl2∙2H2O, 3.78 KCl, 0.17 MgCl2∙6H2O, 11.46 NH4Cl, 1.05 KH2PO4, 0.181 FeCl2∙4H2O, 0.041 NiCl2∙6H20, 0.021 CoCl2∙6H2O, 0.011 ZnCl2, 0.170 KI, 0.177 (NaPO3)6, 0.0085 MnCl2∙4H2O, 0.0085 NH4VO3, 0.0085 CuCl2∙2H2O, 0.0061 Al2(SO4)3∙18H2O, 0.0085 NaMoO4∙2H2O, 0.0085 H3BO3, 0.0085 Na2WO4∙2H2O, Na2SeO3 0.0085, 0.170 cysteine. Depending on the Hydraulic Retention Time (HRT) applied, the following quantities of this solution were added to the molasses solution; 33 mL for HRT > 2 d, 66 mL for 1 d< HRT < 2 d and 123 mL for HRT < 1 d.
The HRT was independent of substrate dosing. It was regulated by the pumping (Prominent, Gamma/L) of tap water and automatic removal of excess mixed liquor by a peristaltic pump (Ismatec, MPC Standard) controlled by a water lever sensor (Endress & Hauser, Liquiphant). The tab water was stored in containers and was daily sparged with N2 in order to reduce the dissolved oxygen concentration bellow 1 mg/L. The produced biogas quantity was measured with a drum-type gas meter (Ritter, TG 05) and registered into the programmable controller. The produced gas was collected in gas bags (Lindte). In Figure 1 the experimental set-up is presented.
2.2. Reactor operation
The inoculum of the reactor has been acquired from the anaerobic digester of the Sewage Treatment Plant for Research and Education (LFKW) of the University of Stuttgart (Germany). It was diluted to 2% to 4% Total Solids (TS) concentration and sieved consecutively through 4 mm and 2 mm mesh size to prevent clogging of the tubing. It was then pretreated for 24 h at 105 °C, in order to kill the methanogenic bacteria. The dried sludge was pulverized and solved into tab water for the start-up of the system. As substrate sugar beet molasses acquired from a sugar factory in south Germany were used. In Table 2 the composition of molasses is presented. For the start-up of the system, the reactor was fed with 450 g of sucrose in a batch mode at pH 6.5 in order to enrich the biomass in H2-producing microorganisms. Upon sucrose depletion continuous operation of the system was started. The pH was reduced to 5.5, a value that has been reported to be the optimum for continuous bio-hydrogen production (Mariakakis et al., 2011). The first phase of continuous operation aimed at the further selection of the biomass for hydrogen producing bacteria by application of high HRT and low OLR. The various experimental conditions tested during the continuous operation are presented in table 3. Their selection was based depending on the experimental progress as described in chapter 3.1. In many cases one of the two reactors had to be re-inoculated with seed sludge acquired by the other reactor. All phases had a minimum duration of 5 times the applied HRT. At phase DVII (table 3) Fe2+ at end-concentration in reactor of 1000 mg/L was added.
|Ntot [mg/kg]||18610||Maltose [mg/L]||--|
|Ptot [mg/kg]||216||Acetate [mg/L]||1020|
|TS [g/kg]||848||Propionate [mg/L]||175|
|VS [%]||892||Butyrate [mg/L]||3062|
|Sucrose [g/kg]||520||D-Glucose [mg/L]||--|
|Lactose [mg/L]||--||Lactate [mg/L]||13736|
2.3. Analytical methods
The analyses of concern were determined according to the german standards (Deutsches Institut fuer Normung, 2002) and performed three times a week. These included; total solids (TS), volatile suspended solids (VSS), chemical oxygen demand (COD), a group parameter used for the detection of carbonaceous matter and nitrogen (in total and soluble form acquired after filtration through membrane with 0.45 μm pore diameter). Glucose, sucrose and lactic acid have been determined spectrophotometrically after enzymatic digestion by test kits according to the manufacturer’s instructions (R-Biopharm). Gas Chromatography was used to analyze organic acids and alcohols. The sample was filtered through a 0.45 µm pore diameter filter and acidified with a 96% H2SO4 solution. Organic acids, ethanol and butanol were detected by GC (Perkin Elmer) mounted with a fused silica capillary (Varian) and using a flame ionization detector. Both the injection and capillary temperatures were set at 280 °C. Biogas composition was determined once daily after up-grading for particulate matter and water vapor removal by a gas analyzer (ABB, AO2020), equipped with an infrared detector for CH4 and CO2 and a thermal conductivity detector for H2.
DNA extraction, 16S rDNA of eubacteria and clostridia PCR amplification, DGGE analysis and sequencing were performed as previously described (Mariakakis et al., 2011).
3. Results and discussion
3.1. Reactor operation
In figure 2, the reactor operation parameters, gas production and hydrogen yield performance for the experimental phases DI to DIII are presented.
After the start-up of the reactor for a week, the HRT and OLR were reduced to 4 d and 11.6 g sucrose / (L∙d) respectively. After about 15 d a continuous H2 production could be established (phase DI), which stabilized after approximately 30 d. The average soluble COD concentration in the reactor was app. 60 g/L and the average H2 yield 2.47 mol H2/mol hexose.
On the 41st day of operation the OLR was increased from 11.6 g sucrose / (L∙d) to 24.7 g sucrose / (L∙d) (phase DII), which caused an increase to the residual sucrose concentration in the reactor during the first days. The biomass was not able to metabolize the total amount of substrate. Over the total 35 d of operation of this phase H2 in the produced biogas gradually decreased and after 70 d no H2 was detected. The fact that biogas production was sustained, indicated biological activity, but not towards H2 production. In phase DIII the HRT was reduced to 2 d with the OLR retained unchanged. The concentration of soluble COD decreased gradually from 100 g/L to 60 g/L due to the higher dilution rate. Nevertheless, H2 production could not be restored and the reactor operation was terminated.
After one month of operation the excess sludge from reactor a (R-a) was used to inoculate reactor b (R-b) (phases D1 to D7). In figure 3 the reactor operation parameters, gas production and hydrogen yield performance for the experimental phases D1 to D7 together with the data from phase DI for continuity and comparison reasons are presented. Biogas and hydrogen production started immediately upon seeding of the reactor at HRT of 3 d and OLR of 11.6 g sucrose / (L∙d). Hydrogen production was stable, but the yield was lower than 1 mol H2/mol hexose. The concentration of soluble COD did not exceed 45 g/L. Increase of the OLR at 24.7 g sucrose / (L∙d) lead to steep increase of the soluble COD and COD-sucrose concentration above 120 g/L and 60 g/L respectively and a decrease of the COD concentration of the metabolites. It seems that in the beginning the biomass concentration was not sufficient to metabolize the whole amount of sucrose, which on its turn accumulated in inhibitory levels as demonstrated by the cease of biogas and hydrogen production and the reduction of the COD-organic acids. It has been reported (Hafez et al., 2010) that glucose can become inhibiting for residual concentrations above 20 g/L. This problem could be overcome by the reduction of HRT to 1 d (phases D3 and on). H2 production was restored immediately and was maintained until the termination of the experiment due to time constraints. Biogas and hydrogen production exhibited fluctuations over time. These can be generally attributed to the experimental procedure followed, which affected the actual HRT and OLR. The dosing of water and substrate was stopped everyday for different periods each time, in order to be prepared as described in Materials and methods. Stronger fluctuations were related mostly to the failure of substrate dosing due to tube clogging. In all phases, except phases D4 and D6, the H2 yield was equal or lower than 1 mol H2/mol hexose (table 3). In phase D4 it reached 1.53 mol H2/mol hexose for HRT of 1 d and OLR of 36.1 g sucrose / (L∙d) and in phase D6 1.31 mol H2/mol hexose for for HRT of 0.5 d and OLR of 36.1 g sucrose / (L∙d).
During the whole R-b operation, which together with the time period of phase DI reached 180 d, no methane was detected. Methanogenesis could be inhibited through the thermal pre-treatment of the seed sludge and the selected operation parameters were sufficient for hindering the proliferation of the methanogens in the system. In our previous work for which no pre-treatment of the seed sludge was carried out methanogenesis could be only be inhibited for 120 d (Mariakakis et al., 2011). Sucrose in higher concentrations could be detected only in phases D2 and D7 resulting in average substrate degradations of 71.5% and 83.1% respectively. In D2 sucrose accumulated in the beginning of the phase as results of the long HRT and the OLR of 24.7 g sucrose / (L∙d). At D7 sucrose could not be degraded due to the high OLR of 69.6 g sucrose / (L∙d) and the short HRT of 0.25 d.
The excess sludge from phase D3 of R-b was used to re-inoculate R-a (phases DV and DVI). In phase DVII the reactor was inoculated with excess sludge of phase D6. In Figure 4 the reactor operation parameters, gas production and hydrogen yield performance for these experimental phases together with the data from phase D3 for continuity and comparison reasons are presented.
Biogas and hydrogen production started also in these cases immediately after seeding of the reactor. Due to the OLR set at 47.1 g sucrose / (L∙d), the average soluble COD concentration reached 61 g/L. H2 production (phase DV) yieldied 0.43 mol H2/mol hexose. The decrease of the OLR to 36.1 g sucrose / (L∙d) and increase of HRT to 2 d in phase lead to further increase of the soluble COD to an average concentration of 98 g/L. Hydrogen yield was slightly improved and reached 0.63 mol H2/mol hexose. Biogas and hydrogen production during phase DVII were instable mainly due to malfunctions of the substrate dosing. The hydrogen yield for addition of Fe2+ achieved was 1.68 mol H2 / mol hexose, which corresponds to an increase of approximately 28 % in comparison to phase D6.
The addition of Fe2+ enhanced hydrogen production. From table 3 it can be seen that biomass production rate reached the highest value of 161 g/d comparable only with that of phase D7 that was acquired for double as much OLR. For comparison at phase D6, biomass production rate was only 94 g/d. It seems that this nutrient that is important for H2 production as will be explained in chapter 3.2 was limiting for the biomass growth, since it was not included in high enough concentrations in the nutrient solution with which the molasses solution was supplemented.
In Table 4 the hydrogen yield of other works in comparison to the best acquired by this work are presented. In most of the cases slightly better results were acquired by the other researchers. In a semi-scale reactor operated at HRT of 0.25 d and pH 4.5 hydrogen yield up to 1.86 mol H2/mol hexose was achieved (Ren et al., 2006). Lay et al. (2010) maximized the yield for HRT of 0.5 d and pH of 5.5 like in this work, but achieved a somewhat lower yield of 1.35 mol H2 / mol hexose, even though the reactor set-up was considerably smaller permitting for better control of operation. Aceves-Lara et al (2008) operated a 2 2 L CSTR at HRT of 0.25 d and pH of 5.5. The maximum yield acquired was 1.70 mol H2 / mol hexose for an OLR of 24.2 g COD / (L∙d). For OLR as high as 77.2 g COD / (L∙d) the acquired hydrogen yield was much higher than that of the current work (0.84 mol H2 / mol hexose) for the same HRT and OLR of 69.6 g sucrose/(L∙d), which is equivalent to 107 g COD / (L∙d) (Aceves-Lara et al., 2008). It is obvious that the process can be further optimized.
|Ren et al. (2006)||6.32||0.44||4.5||1480||10||0.34||--|
|Lay et al. (2010)||40||1||5.5||4||130||0.48||60.2|
3.2. Microbial metabolism
From Figures 2 to 4 and Table 3 it can be seen that the major metabolites for all tested parameters were lactic, acetic, and butyric acid together with ethanol. Propionic and hexanoic acid were produced in low quantities and only in same cases. Pentanoic acid and butanol were not detected at all. Acetate and butyrate are typical for H2 production by mixed acid fermentation of sucrose as already mentioned in introduction. Lactate can be produced by either lactic acid bacteria or by bacteria of the genus
During phase DI lactate was produced in less quantity than acetate and butyrate with the later being produced in almost the same proportion indicating a mixed acid fermentation, even though the theoretical yield of 2.6 mol H2 / mol hexose was not reached (equation 4). The increase of the OLR in phases DII and DIII has lead to gradually increasing lactate production rate with simultaneous increase of acetate into a lesser extent and decrease of butyrate. The higher substrate availability forced the biomass to shift its metabolism from mixed acid fermentation to lactate fermentation. The fact that biogas production also declined is an indicator for homolactic fermentation by LAB, during which only lactate is produced (equation 9). In phases DV and DVI lactic acid production could not be solely due to homolactic fermentation, since biogas was produced. Higher lactic acid production than acetic and butyric acid production was observed in the phases for which HRT and OLR were equal or higher than 1 d and 24.7 g sucrose / (L∙d) respectively. The exact mechanism of lactic acid production can be explained either by the co-existence of LAB (Hafez et al., 2009) or by the metabolic shift of the hydrogen producing clostridia (Minton & Clarke, 1989). In all cases though, high lactic acid production was combined to an increase of the OLR (Oh et al. 2004; Kim et al., 2006; Oh et al., 2004; Hafez et al., 2009) and caused a diminution of hydrogen yield. The effect of lactic acid as a metabolite in hydrogen systems has not yet been clarified. It has been reported to be promoting to hydrogen production at low concentrations and inhibiting at high concentrations. In a work (Baghchehsaraee et al., 2009), an increase of the hydrogen yield combined with the complete degradation of the externally added lactic acid in concentrations up to 3 g/L was observed. In another work, Kim et al. (2012) also observed an increase of 22% in hydrogen yield when lactic acid up to 8 g/L was added to batch fermentors operated at pH of 4.5, and a reduction when the concentration was raised at 18 g/L. The corresponding undissociated form of the lactic acid, which is the potential inhibitor (van Ginkel & Logan, 2005) was 21 mmol/L and 45 mmol/L at pH 4.5 according equation 15. In this work, the highest lactic acid concentration was 35 g/L (9 mmol/L undissociated lactic acid at pH 5.5) and was only reached temporarily in the beginning of phase D3 without any obvious long-term negative influence on the hydrogen process, like in phase DIII. It seems that lactic acid was not the inhibition factor, but another substance that was not monitored.
For phases DI to DIII and D1 to D3 during which OLR lower or equal to 24.7 g sucrose / (L∙d) was applied, acetate and ethanol were produced in almost the same rates. A ratio of EtOH:HAc equal to 1:1 has also been proposed for clostridia as described by equation 1 for enteric bacteria, when hydrogen is evolved only through the conversion of Acetyl-CoA to pyruvate, yielding 2 mol H2 / mol hexose (Minton & Clarke, 1989). For higher loadings the observed ethanol production rate diminishes either due to a metabolic shift of the biomass or due to its consumption. Ethanol has also been detected in other hydrogen producing systems operated at pH 5.5 and various HRT and OLR (Gavala et al., 2006; Karadag & Puhakka, 2010a; Kim et al., 2006; Shen et al., 2009) as a product either of enterobacterial (Hallenbeck, 2005), clostridial hydrogen production (Akutsu et al., 2009; Lin & Lay, 2004), or heterolactic bacteria (Kandler, 1983). In the case of clostridia, it has been suggested that ethanol is produced during the late growth phase during which no hydrogen is produced, while H2 production is favored during the exponential growth phase, during which the organic acids are produced (Nath & Das, 2004), yielding at the end of a batch fermentation a ratio of 1:1. By the adjustment of short HRT in CSTR it is possible to maintain the bacterial population in the exponential growth phase. However, the adjustment has to be suitable so that the biomass concentration in the reactor can be also maintained in concentrations that are suitable for high substrate conversion rates.
Propionic acid was produced in phases D3 and D4. Sucrose fermentation to propionic acid is a sink for hydrogen and according to equation 6 for each mol of propionic acid produced 1 mol of hydrogen is consumed. The derived hydrogen consumptions for phases D3 and D4 correspond to 15% and 19% of the total produced hydrogen respectively.
Hexanoic acid was mainly produced during phases DI, DII, DVI and D2. The HRT of all these phases was equal to or longer than 2 d. The production of hexanoic acid can only be explained by a possible secondary fermentation of
The addition of Fe2+ in phase DVII did not influence the production rates of acetic acid in comparison to phase D6. On the other hand, the production rate of lactic acid was significantly reduced from 1230 mmol/d to 329 mmol/d corresponding to approximately 72% and ethanol was not produced anymore. Parallel, the production rate of butyrate increased approximately by 27%. The overall bacterial metabolism was shifted to butyrate fermentation and biomass growth as described in 3.1. This is an indication that lactic acid and ethanol production in the phases with relative short HRT (<1 d) was mainly due to the clostridial metabolism. They contributed more than 2/3 to the total lactic acid production. Iron is very important to hydrogen production, which is produced when the simple reaction of Eq. 16 takes place. This reaction is catalyzed in clostridia by a dimetallic iron only [FeFe]-hydrogenase, which receives protons by the reduced form either of ferredoxin or of NADH (Vignais & Billoud, 2007). Under iron limitation the activity of hydrogenase is also limited (Valdez-Vazquez & Poggi-Varaldo, 2009), pyruvate can not be degraded through the pathways leading to hydrogen, but fermentation is shifted towards lactic acid production (Minton & Clarke, 1989).
3.3. Microbial population
3.3.1. Species description and influence of HRT and OLR
The sample times for the phylogenetic analysis and the affiliated dominant species present in each experimental phase are indicated in figures 2 to 4. In figures 5 and 6 the PCR-DGGE profiles of the
In the seed sludge three species of
Also in the case of phases DVI, D1 and D2 (tables 6 and 7) the symbiosis of lactic acid together with hydrogen producing bacteria could be observed resulting to high lactic acid production rates and medium to low hydrogen yields. In all cases, the employed HRT was equal or higher to 2 d. It seems that long HRT contribute to the dominance of lactic acid over hydrogen producing bacteria. In the case of phases D3 to D7, high production rates of lactic acid could still be observed, but they were lower than that of acetic and butyric acid, while medium to higher hydrogen yields could be achieved. The HRT applied was equal to or lower than 1 d. Under these conditions the hydrogen producing metabolism could dominate over the lactic acid metabolism. After phase D3 and on
subsp. bulgaricus strain CH3
|16; 18; 19; 20; 22||84-95||GU470903|
|10; 21||90; 91||AB548940|
|14||Marine bacterium strain SJ-BF7||84||AM260710|
|23; 29; 32||94-100||AB687551|
|24; 39; 43; 44; 45; 50; 51; 52||Clostridium ljungdahlii|
type strain DSM13528T
|25; 26; 28; 30; 31; 40; 41; 42; 46; 47||Clostridium tyrobutyricum|
|48; 49||97; 95||FR734082|
3.3.2. Effect of Fe2+ addition
The addition of Fe2+ at phase DVII had an influence on the bacterial population in comparison to the seed sludge (phase D6).
In an investigation (Karadag & Puhakka, 2010b) about the influence of Fe2+ concentration in the range of 0.5 mg/L to 100 mg/L on a CSTR system fed with glucose and operated at HRT of 0.208 d, OLR of 43.2 g glucose /(L∙d) and pH 5 an increase of 71% in hydrogen yield for Fe2+ concentration of 50 mg/L was achieved, followed by a fermentation shift from ethanol type to butyric acid type like in this work. In two other works (Wang & Wan, 2008) and (Lee et al., 2001) investigating the influence of Fe2+ concentration in batch experiments with vials, optimum concentrations of 350 mg/L and 352.8 mg/L were detected respectively. It seems that there is potential for the optimization of the quantity added to the system.
3.3.3. Symbioses in fermentative hydrogen production systems
There are only a few works that have investigated the bacterial population and the influence of HRT and OLR on hydrogen producing systems by molasses. Ren et al. (2007) studied the influence of pH on the microbial population structure of bio-hydrogen production by molasses in a 2.5 L CSTR seeded with sewage solids and operated at HRT of 0.25 d, OLR between 7 g COD / (L∙d) and 30 g COD / (L∙d) and at temperature of 35 °C. At pH between 5.5 and 6 mixed ethanol-butyrate fermentation was observed. In this work no ethanol could be detected for HRT of 0.25 d. In the reactor a co-existence of clostridia and LAB was observed like in this work. The bacterial population was dominated by
The dominance of the lactic acid bacteria
bacterium clone CA38
In an other investigation (Hung et al., 2011b), it was also suggested that the facultative anaerobes of
In any case, this symbiosis reduces the available substrate for hydrogen production. Furhtermore, in this work, it was not beneficial to hydrogen production in the phases with long HRTs, but deteriorated or even completely inhibited it. Lactobacilli possess the potential of inhibiting other microorganisms by different mechanisms. Their fermentation products consist mainly of lactic and acetic acid, which reduce the pH which on its side reduce their dissociation degree. In the presence of oxygen many species such as
|Marine bacterium SJ-BF7|
In most cases, heat treatment of the seed sludge was applied, but it was not sufficient to inhibit the growth of lactic acid bacteria, although they can not form spores and it has been demonstrated
Hydrogen production by molasses could be successfully carried out in large lab-scale reactors for a period longer than 180 d and under variable combinations of OLR and HRT. The maximum H2 yield obtained was 1.53 mol H2/mol hexose for HRt of 1 d and OLR of 36.1 g sucrose/(L∙d). Improvement of the hydrogen production yield of 28%was achieved by the addition of Fe2+ to an end concentration of 1000 mg/L. In figure 7 the acquired hydrogen yields of all phases as a function of the operation parameters HRT and OLR, along with a suitable range of combination of these parameters, as determined in the current work, are presented. Combinations resulting to COD concentrations higher than 50 g/L (phase D3), was showed to be inhibitory to H2 production. Reason was not the undissociated form of acids, but most probably the production and accumulation of bacteriocidal or bacteriostatic substances, excreted by the LAB. The second line indicates the combination for which the process becomes unfavorable in terms of substrate utilization efficiency and hence can not be considered as cost effective. The applied seed sludge pre-treatment and reactor start-up methods were successful in enriching the biomass in hydrogen producing microorganisms and killing methanogenic microorganisms that are detrimental to H2 production. Nevertheless, H2 production has been carried out parallel to lactic acid metabolism, which was driven either by the presence of LAB, or by the hydrogen producing clostridia due to iron Fe2+ limitation. A co-existence of clostridium species with lactic acid bacteria seems to be unavoidable, even for extensive pre-treatment of the seed sludge. Lactic acid bacteria influence the system primarily by consuming the substrate available for hydrogen production. The co-existence of clostridia and LAB though, seems to become beneficial to hydrogen production at HRTs in the range of 0.5 d to 1 d by supplying certain clostridia genera that are capable of performing secondary fermentation with substrate and/or by removing the residual dissolved oxygen from the system and hence establishing an appropriate milieu for the growth of clostridia. For the successful technical implementation of hydrogen production, for which process control is complex and oxygen in trace concentrations is to be expected, this symbiosis may be regarded as pre-requisite. The exact extent, to which the LAB contribution is beneficial and not adverse, requires the quantification of the biomass for the allocation of the metabolic products to specific microbial species and the monitoring of the concentrations of possible inhibiting substances.
This work was supported by the German Federal Ministry of Education and Research (BMBF), EnBW AG, Purolite Deutschland GmbH and RBS wave GmbH, Grant number 03SF0351. We would like to warmly thank Ms. Kerstin Matthies of the Karlsruhe Institute of Technology for the microbial population analyses.