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Enhancement of anaerobic hydrogen production by iron and nickel
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 5 ( 2 0 1 0 ) 8 5 5 4 e8 5 6 0
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Enhancement of anaerobic hydrogen production by iron and nickel Dogan Karadag*, Jaakko A. Puhakka Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland
article info
abstract
Article history:
The effects of iron and nickel on hydrogen (H2) production were investigated in a glucose-
Received 10 March 2010
fed anaerobic Continuous Flow Stirred Tank Reactor (ACSTR). Both iron and nickel
Received in revised form
improved the reactor performance and H2 production was enhanced by 71% with the sole
20 April 2010
iron or nickel supplementation. In all cases, H2 production yield was increased by lowering
Accepted 28 April 2010
both ethanol and total metabolites production and increasing butyrate production.
Available online 11 June 2010
Furthermore, iron and nickel slightly increased biomass production while glucose degradation decreased with the supplementation of nickel. Dynamic changes in bacterial
Keywords:
composition as analyzed by 16S rRNA gene-targeted denaturing gradient gel electropho-
Hydrogen
resis (DGGE) revealed that hydrogen was produced mainly by Clostridium butyricum strains
ACSTR
and that nickel addition decreased the microbial diversity. Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu.
Iron Nickel PCR-DGGE Clostridium butyricum
1.
Introduction
Anaerobic hydrogen (H2) production has potential as an alternative fuel to the future energy scarcity. As compared to light driven methods, anaerobic fermentation is technically simpler, less energy intensive and it is not dependent on the availability of the solar energy which makes it more feasible for continuous production [1]. Hydrogenase enzymes catalyze the reduction of proton to H2. Hydrogenase enzymes are classified into [NieFe] and [FeeFe] hydrogenases, according to the metal content at their active site [2]. [NieFe] hydrogenases are widely distributed among bacteria [3] and both nickel and iron have important effects on fermentative H2 yields [4e6]. Most studied the effects of iron on fermentative H2 production have been conducted as batch tests. Some researchers reported enhancement of H2 production by iron supplementation [7e9].
Wang and Wan (2009) [10] reviewed the effects of Fe2þ on anaerobic hydrogen production and reported some inconsistency on the optimal Fe2þ concentration. In these studies, different inoculums, substrates and Fe2þ concentration ranges were used. In addition, temperature also affected the effects of Fe2þ on H2 production and optimum Fe2þ concentration decreased at higher temperature [11]. Cui et al., (2009) [12] and Lee et al. (2009) [4] reported that Fe2þ also affects the fermentation pathways. Butyrate formation increased with the increasing of Fe2þ concentration while ethanol formation was favored at low Fe2þ concentration [7,8]. To our knowledge, only the study by Lee et al. (2009) [4] investigated the effect of Fe2þ on continuous H2 production. They indicated that iron sulphate concentration up to 10.9 mg/l increased hydrogenase activity and H2 production in a membrane bioreactor. Only two studies focused on the effects of Ni2þ on anaerobic H2 production. Wang and Wan (2008) [6] using batch
* Corresponding author. Tel.: þ358 449566652; fax: þ358 331152869. E-mail addresses: [email protected] (D. Karadag), [email protected] (J.A. Puhakka). 0360-3199/$ e see front matter Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu. doi:10.1016/j.ijhydene.2010.04.174
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Fig. 1 e Effects of iron on hydrogen and soluble metabolite production in a glucose-fed ACSTR.
experiments at 35 C found that increasing Ni2þ concentration up to 0.2 mg/l enhanced the H2 production while Ni2þ had little effect on glucose degradation efficiency. Metabolic pathway shifts were reported at different Ni2þ concentrations and higher Ni2þ concentration promoted the growth of H2 producing bacteria. The aim of this study was to investigate the effect of Fe2þ and Ni2þ on continuous H2 production in anaerobic completely stirred tank reactor (ACSTR). The changes in metabolic products were also investigated and the dynamics of the microbial community was revealed by denaturing gradient gel electrophoresis (DGGE) and the polymerase chain reaction (PCR) of 16S rDNA.
2.
Methods and materials
2.1.
Experimental setup
The experiment was carried out in an anaerobic completely stirred 1 L tank reactor (ACSTR) with a working volume of 0.9 L [13]. The stirring rate was 100 rpm and the temperature was maintained at 37 C. Hydraulic retention time was maintained as 5 h. Anaerobic inoculum was the same with our former
study and dominated by Clostridium strains [13]. Automatic titration (Metrohm, 719 S) was used to keep pH at 5.0 with 2 M HCl since optimal pH for continuous H2 production was obtained in the range of 5.0e5.3 in our former study [13]. The ACSTR was routinely monitored for pH, oxidation reduction potential (ORP) and biogas production. The performance of the ACSTR was monitored by measuring the H2 production rate and yield, soluble metabolic products (VFAs and ethanol) and the biomass concentration.
2.2.
Nutrient solution
Synthetic nutrient solution was prepared using tap water and kept at 4 C. Glucose concentration in basic nutrient medium (BNM) was maintained as 9 g/l and the concentrations of other nutrients were the same as in the previous experiments [9]. In addition, BNM was amended with 2 g/l of yeast extract and 0.5 mg/l of iron nickel. FeCl24H2O and NiCl26H20 were used as additional iron and nickel sources, respectively.
2.3.
Reactor operation
The ACSTR was operated continuously for 108 days in three phases. During the first two phases of experiment, the effect
Table 1 e Effects of iron concentration on a glucose-fed ACSTR performance. Feþ2 concentration (mg/l)
H2 Yield (mol H2/mol glucose) H2 (%) Glucose degradation (%) VSS (g/l) Total soluble metabolites (g/l) Eth þ Ace/Total metab. (%)
0.5
10
25
50
100
0.49e0.89 (0.66) 33.6e42.6 (36.8) >99 1.4e1.8 (1.6) 5.3e5.9 (5.5) 68.0e78.2 (72.2)
0.40e1.12 (0.76) 27.0e46.2 (40.0) >99 1.4e1.8 (1.6) 5.2e5.7 (5.4) 58.7e76.1 (67.5)
0.59e1.05 (0.83) 33.6e44.3 (37.7) >99 1.5e1.8 (1.6) 5.2e5.7 (5.4) 52.7e68.8 (62.0)
0.84e1.43 (1.13) 40.7e47.9 (44.7) 84.9e99 (97.2) 1.3e1.8 (1.5) 4.7e5.6 (5.2) 51.3e58.4 (51.3)
0.51e1.21 (1.08) 42.2e48.6 (44.5) >99 1.7e2.1 (1.8) 5.2e5.9 (5.4) 54.5e60.8 (57.9)
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Table 2 e Effects of nickel concentration on a glucose-fed ACSTR performance. Niþ2 concentration (mg/l)
H2 Yield (mol H2/mol glucose) H2 (%) Glucose degradation (%) VSS (g/l) Total soluble metabolites (g/l) Eth þ Ace/Total met. (%)
0.5
10
25
50
0.48e1.09 (0.82) 39.9e44.5 (43.1) 85.3e99.0 (90.5) 0.88e1.42 (1.14) 5.2e6.2 (5.8) 32.8e38.3 (34.9)
0.41e1.21 (1.0) 43.0e49.3 (46.9) 24.5e96.3 (66.7) 1.22e1.39 (1.30) 1.9e5.1 (3.6) 44.3e55.5 (48.8)
0.85e1.88 (1.40) 42.3e46.4 (44.6) 10.0e82.6 (47.5) 1.05e1.57 (1.27) 3.6e5.9 (4.4) 32.7e53.9 (42.9)
1.27e1.48 (1.39) 44.5e47.3 (45.9) 80.2e96.7 (86.5) 1.33e1.47 (1.40) 3.9e4.0 (4.0) 34.8e40.3 (38.2)
of Feþ2 and Ni2þ were investigated separately while combined Fe2þ and Niþ2 effects were studied in the third phase. In the first phase (38 days), the concentrations of Fe2þ were increased from 0.5 to 100 mg/l. During the second phase, ACSTR was operated in 28 days and the effect of Ni2þ was studied in the range of 0.5e50 mg Ni2þ/l. In the third phase (42 days), Fe2þ and Ni2þ were supplemented together and their concentrations were increased from 0.5 to 100 mg/l.
2.4.
Analyses
Biogas production was measured twice per day using a wet gas meter (Ritter Apparatebau, Bochum, Germany). The biogas composition including H2 and CO2 was analyzed by a gas chromatograph (Shimadzu GC-2014) equipped with a thermal conductivity detector. N2 was used as carrier gas and the temperatures of the injector, column and detector were 110 C, 80 C and 110 C, respectively. Liquid samples for residual glucose, biomass concentration, VFAs and ethanol analysis were withdrawn from the ACSTR three times per week. Shimadzu LC-20AD liquid chromatograph with 0.01 N H2SO4 mobile phase was used to measure the VFAs and ethanol. VSS (g/l) was used to estimate the biomass concentration in the bioreactor and was quantified by following the procedures described in Standard Methods [14]. ORP and pH were determined by a pH meter (Metrohm, 719 S). Hydrogen
production yield (HPY) was reported as the amount of H2 produced (mol) per the initial glucose concentration (mol).
2.5.
Microbial community analyses
Liquid samples from ACSTR were centrifugated (7600 rpm, 10 min) and pellets were stored at 20 C for microbial community analysis. DNA was extracted with a VIOGENE Blood and Tissue Genomic DNA kit (Proteogenix SA, Fegersheim, France) according to manufacturer’s instructions. Amplification of partial bacterial 16S rRNA genes of the community DNA and DGGE were performed as previously described by [15] with the exception of using the denaturing gradient from 30% to 70%. Analysis of sequence data was performed as previously described [15].
3.
Results and discussion
3.1.
Effect of iron
In the Phase 1, iron concentration was stepwise increased from 0.5 to 100 mg Fe2þ/l with H2 production yields (HPY), % glucose degradation (GD), biomass concentrations and metabolites as presented in Fig. 1. Biomass concentration, GD and total amounts of metabolites remained stable with the
Fig. 2 e Effects of nickel on hydrogen and soluble metabolite production in a glucose-fed ACSTR.
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Fig. 3 e Effects of iron plus nickel addition on hydrogen and soluble metabolite production in a glucose-fed ACSTR.
increasing of iron concentration whereas HPY and distribution of metabolites fluctuated. GD decreased to 84.9% on day 24 due to the technical problems in the feeding. The average biomass concentration was around 1.6 g VSS/l and it slightly increased at the iron concentration of 100 mg/l (Table 1). HPY fluctuated up to 25 mg Fe2þ/l after which it increased steadily within the concentration range of 25e50 mg Fe2þ/l and remained stable at 100 mg Fe2þ/l. The lowest and highest average HPY’s were 0.66 and 1.13 mol H2/mol glucose at 0.5 mg Fe2þ/l and 50 mg Fe2þ/l, respectively. The HPY remained constant with the further increasing of iron concentration. These results indicate that optimum iron concentration was 50 mg Fe2þ/l for H2 production in ACSTR and HPY was enhanced by 71% with this dosage. HPY was accompanied with lactate, acetate, formate, butyrate and ethanol production. Total metabolites were stable around 5.5 g/l while the distribution of metabolites considerably changed during the phase 1. Acetate and butyrate were the major metabolites whereas the ratio of acetate plus ethanol to total metabolites decreased from 72.2% to 51.3% with the increasing of Fe2þ concentration from 0.5 to 50 mg Fe2þ/l (Table 1). Li and Ren (1998) [16] reported that ethanol type fermentation was dominant when the percentage of ethanol plus acetate is over 60%. Therefore, the
main H2 production mechanism in ACSTR was ethanol type fermentation. Acetate concentration decreased gradually at Fe2þ concentrations and HPY was decreased by ethanol production (Fig. 1). Increase in ethanol production was associated with the decrease in HPY. At the same time, there was a positive relationship between HPY and butyrate generation. Similar relationship between butyrate and HPY was also reported by other researchers [17e19]. Only minor amounts of formate were detected during the Phase 1, slight increases in formate were followed by decrease in HPY and increase in ethanol production (Fig. 1) Similar relationships between formate and ethanol were observed in our former study [13]. At lower Fe2þ concentrations H2 production was occupied by mainly ethanol type fermentation. At higher Fe2þ concentrations, the degradation mechanism was a combination of ethanol and acetate-butyrate type fermentations. Similarly, other researchers reported that Fe2þ addition resulted in metabolic shifts [4,20].
3.2.
Effect of nickel
During Phase 2, ACSTR was operated for 10 days with BNM with nickel and iron at 0.5 mg/l. ACSTR performance
Table 3 e Effects of iron plus nickel addition on hydrogen production on a glucose-fed ACSTR performance. Feþ2 and Niþ2 concentration (mg/l)
H2 Yield (mol H2/mol glucose) H2 (%) Glucose degradation (%) VSS (g/L) Total soluble metabolites (g/l) Eth þ Ace/Total met. (%)
0.5
10
25
50
100
0.60e1.11 (0.98) 39.2e43.9 (41.2) 93.1e99.0 (96.2) 1.0e1.3 (1.1) 4.5e5.8 (5.2) 34.6e43.7 (37.9)
1.18e1.48 (1.29) 41.2e45.9 (43.8) 91.4e95.9 (93.8) 1.0e1.1 (1.1) 5.0e5.2 (5.1) 35.7e41.2 (37.9)
0.32e1.55 (1.22) 33.6e48.3 (44.6) 50.1e86.0 (74.2) 0.9e1.1 (1.0) 3.4e4.9 (4.2) 28.2e44.4 (38.0)
1.30e1.53 (1.37) 42.0e44.9 (43.4) 81.3e86.4 (84.5) 1.2e1.6 (1.4) 3.8e4.3 (4.1) 41.5e44.3 (43.0)
1.19e1.42 (1.33) 41.9e46.1 (43.6) 76.2e89.4 (83.1) 1.3e1.4 (1.4) 4.1e4.5 (4.3) 42.0e43.8 (42.9)
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Table 4 e Affiliation of DGGE bands determined in a glucose-fed ACSTR. Band no 1 2 3 4 5 6 7 8 9 10
Phylogenetic affiliation Bacillus coagulans ATCC Bacillus coagulans X16 Clostridium butyricum SD2 Clostridium chartatabidum Clostridium butyricum W4 Clostridium butyricum RCEB 1 Clostridium butyricum CGS6 Clostridium ramosum CMC50 Clostridium butyricum EB6 Clostridium butyricum HAWHC1
Accession no DQ297928 FJ177636 EU477411 NR 029239 DQ831126 EU621841 AY540110 EU869233 EU183474 AY604563
% identity 100 100 99 95 99 100 100 100 100 97
butyrate was the main soluble metabolite followed by acetate and lactate. Average acetate concentration decreased from 2.6 g/l in phase 1 to 2.0 g/l in Phase 2 and ethanol production decreased over 50% with the increase in Ni2þ concentration from 0.5 to 25 mg Ni2þ/l. Similarly, Wang and Wan (2008) [6] reported that ethanol production was negatively affected by nickel addition. Moreover, Ni2þ supplementation positively affected the HPY by lowering ethanol production. In addition, no formate was produced and there was a inverse relationship between lactate accumulation and HPY(Fig. 2). Phase 1 and phase 2 results indicate that hydrogen production by Hisarkoy hot spring enrichment culture contained [NieFe] hydrogenases and HPY was positively affected both iron and nickel.
3.3.
Fig. 4 e Dynamic changes of microbial community during the supplementation of iron (a), nickel (b), and iron plus nickel ª to the glucose-fed ACSTR.
parameters fluctuated more than during Phase 1 (Fig. 1). Biomass concentration and H2 percentage remained stable at around 1.3 g VSS/l and 45.0%, respectively (Table 2). HPY varied in the range of 0.82e1.40 mol H2/mol glucose and on day 21 reached the highest value of 1.88 mol H2/mol glucose with 25 mg Ni2þ/l. H2 percentage was similar to the values in phase 1(Fig. 2). However, the most remarkable changes were obtained in GD. Although GD was over 99% during the Fe2þ supplementation, the highest HPY was obtained with the lowest GD at 25 mg Ni2þ/l in phase 2. The increase in HPY with the simultaneous decrease in GD may be related with metabolic pathway shift and the decreased the amount of total soluble metabolites. During the phase 2,
Effect of iron plus nickel
During the Phase 3, iron and nickel were added together and concentration of both was gradually increased from 0.5 to 100 mg/l. The technical problems caused considerable increase in lactate production while VSS, HPY, GDR, acetate and butyrate production decreased sharply (Fig. 3). Biomass concentrations ranged between 1.1 and 1.4 g VSS/l and it increased with the increasing of both ions (Table 3). H2 percentage within the biogas was around by 43% while total metabolites decreased at higher metal concentrations. As compared to the Phase 2, GDR was higher in Phase 3 while it decreased at higher total concentrations probably due to inhibition. The average HPY increased from 0.98 to 1.37 mol H2/mol glucose and increment rate was 40% at 50 mg/l of Ni2þ and Fe2þ concentration. However, average HPY remained constant as 1.33 mol H2/mol glucose with the further increasing of Fe2þ and Ni2þ amounts. In summary, the increase of HPY in phase 3 was 40% and lower than during phases 1 and 2 (% 71). The lowest concentration of total metabolite was produced during the nickel supplementation while GD was also lower and process stability fluctuated in phase 2. Conversely, GD and biomass concentration were the highest and process was more stabile during Phase 1.
3.4.
Microbial community analyses
The microbial community in ACSTR at different Fe2þ and Ni2þ concentrations was analyzed and compared by PCR-DGGE
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 5 ( 2 0 1 0 ) 8 5 5 4 e8 5 6 0
analysis (Fig. 4). Table 4 shows the results of band sequence affiliations. In the first phase, ten bands were seen in ACSTR predominated by Clostridium species. Mesophilic Clostridia are typical H2 producers [21e23]. Clostridia produce H2 from carbohydrates by two different metabolic pathways. Acetate and butyrate are the main metabolites during acidogenesis and ethanol and acetone are produced at solventogenesis. Clostridia shift their metabolism according to environmental conditions [24]. The Clostridium species in Fig. 4 were affiliated with three different strains including Clostridium butyricum, Clostridium ramosum and Clostridium chartatabidu (Table 4). C. butyricum ferments various substrates to H2, CO2, acetate and butyrate, and has been reported in many H2 producing ACSTR’s [25e28]. Bands 4 and 8 have high similarity to C. chartatabidum and C. ramosum. Both Clostridium species have been reported with the fermentation products of acetate, butyrate, hydrogen and ethanol [29]. The metabolite profiles of this study suggest that the dominance of acetate and butyrate was associated with the presence of Clostridium strains. In addition, two different strains of Bacillus coagulans were detected in ACSTR where B. coagulans are spore forming facultative bacteria with the ability of lactate production [30]. The DGGE profiles in Fig. 4 (a) indicate that increasing in iron concentration did not affect the bacterial community. Therefore, the increase in H2 production at higher iron concentrations related to the metabolic pathway shift. Similar results were reported by Koskinen et al. (2008) [31] for H2 production by thermophilic enrichment culture from Icelandic hot spring. Moreover, Lee et al. (2001) [7] and Chen et al. (2005) [32] indicated that iron is crucial for the hydrogen production of Clostridium. The number of DGGE bands in Phase 2 decreased from Phase 1. Throughout the nickel addition Clostridium chartabidum (Band 4) did not thrive in the ACSTR and C. ramosum (Band 8) disappeared at 10 mg Niþ2/l. In our former study, the positive relationship was reported between increase in ethanol production and the presence of C. chartabidum [33]. Therefore, the decrease in ethanol production after Phase 1 could be attributed to the decreased amount of C. chartabidum. The disappearance of Band 8 after Day 7 suggests that nickel concentration over 10 mg Ni/l inhibited C. ramosum. During the Phase 3, decrease in microbial diversity continued. C. butyricum (Band 5) was not found during Phase 3 and B. coagulans (bands 1 and 2) were not present after the increasing of both iron and nickel concentration to over 25 mg/l. For these reasons, the decreasing of glucose degradation rate and increase in H2 production yield after Day 25 was likely related to inhibition of B. coagulans.
4.
Conclusions
The following conclusions can be drawn from this study: 1. Hydrogen production increased about by 71% with the increasing of iron and nickel supplementation and the highest yields were achieved at the concentrations of 50 mg Fe2þ/l and 25 mg Ni2þ/l.
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2. Ethanol production was favorable at lower iron and nickel concentrations while H2 production was positively affected with the increasing of butyrate generation at higher iron and nickel concentrations. 3. Glucose degradation was over 99% in the presence of iron while nickel adversely affected the glucose oxidation. 4. Microbial composition in ACSTR was dominated with Clostridium species and H2 was produced mainly by C. butyricum. 5. Microbial diversity decreased with the nickel supplementation, and ethanol and H2 production was affected mainly by metabolic pathway shifts rather than changes in bacterial community.
Acknowledgements This research was funded by the Academy of Finland (HYDROGENE Project, no 107425) and Nordic Energy Research (BioH2 project 06-Hydr-C13). Author Karadag gratefully acknowledges the financial support provided by CIMO (Centre for international mobility, Finland).
references
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Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Enhancement of anaerobic hydrogen production by iron and nickel Dogan Karadag*, Jaakko A. Puhakka Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland
article info
abstract
Article history:
The effects of iron and nickel on hydrogen (H2) production were investigated in a glucose-
Received 10 March 2010
fed anaerobic Continuous Flow Stirred Tank Reactor (ACSTR). Both iron and nickel
Received in revised form
improved the reactor performance and H2 production was enhanced by 71% with the sole
20 April 2010
iron or nickel supplementation. In all cases, H2 production yield was increased by lowering
Accepted 28 April 2010
both ethanol and total metabolites production and increasing butyrate production.
Available online 11 June 2010
Furthermore, iron and nickel slightly increased biomass production while glucose degradation decreased with the supplementation of nickel. Dynamic changes in bacterial
Keywords:
composition as analyzed by 16S rRNA gene-targeted denaturing gradient gel electropho-
Hydrogen
resis (DGGE) revealed that hydrogen was produced mainly by Clostridium butyricum strains
ACSTR
and that nickel addition decreased the microbial diversity. Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu.
Iron Nickel PCR-DGGE Clostridium butyricum
1.
Introduction
Anaerobic hydrogen (H2) production has potential as an alternative fuel to the future energy scarcity. As compared to light driven methods, anaerobic fermentation is technically simpler, less energy intensive and it is not dependent on the availability of the solar energy which makes it more feasible for continuous production [1]. Hydrogenase enzymes catalyze the reduction of proton to H2. Hydrogenase enzymes are classified into [NieFe] and [FeeFe] hydrogenases, according to the metal content at their active site [2]. [NieFe] hydrogenases are widely distributed among bacteria [3] and both nickel and iron have important effects on fermentative H2 yields [4e6]. Most studied the effects of iron on fermentative H2 production have been conducted as batch tests. Some researchers reported enhancement of H2 production by iron supplementation [7e9].
Wang and Wan (2009) [10] reviewed the effects of Fe2þ on anaerobic hydrogen production and reported some inconsistency on the optimal Fe2þ concentration. In these studies, different inoculums, substrates and Fe2þ concentration ranges were used. In addition, temperature also affected the effects of Fe2þ on H2 production and optimum Fe2þ concentration decreased at higher temperature [11]. Cui et al., (2009) [12] and Lee et al. (2009) [4] reported that Fe2þ also affects the fermentation pathways. Butyrate formation increased with the increasing of Fe2þ concentration while ethanol formation was favored at low Fe2þ concentration [7,8]. To our knowledge, only the study by Lee et al. (2009) [4] investigated the effect of Fe2þ on continuous H2 production. They indicated that iron sulphate concentration up to 10.9 mg/l increased hydrogenase activity and H2 production in a membrane bioreactor. Only two studies focused on the effects of Ni2þ on anaerobic H2 production. Wang and Wan (2008) [6] using batch
* Corresponding author. Tel.: þ358 449566652; fax: þ358 331152869. E-mail addresses: [email protected] (D. Karadag), [email protected] (J.A. Puhakka). 0360-3199/$ e see front matter Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu. doi:10.1016/j.ijhydene.2010.04.174
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Fig. 1 e Effects of iron on hydrogen and soluble metabolite production in a glucose-fed ACSTR.
experiments at 35 C found that increasing Ni2þ concentration up to 0.2 mg/l enhanced the H2 production while Ni2þ had little effect on glucose degradation efficiency. Metabolic pathway shifts were reported at different Ni2þ concentrations and higher Ni2þ concentration promoted the growth of H2 producing bacteria. The aim of this study was to investigate the effect of Fe2þ and Ni2þ on continuous H2 production in anaerobic completely stirred tank reactor (ACSTR). The changes in metabolic products were also investigated and the dynamics of the microbial community was revealed by denaturing gradient gel electrophoresis (DGGE) and the polymerase chain reaction (PCR) of 16S rDNA.
2.
Methods and materials
2.1.
Experimental setup
The experiment was carried out in an anaerobic completely stirred 1 L tank reactor (ACSTR) with a working volume of 0.9 L [13]. The stirring rate was 100 rpm and the temperature was maintained at 37 C. Hydraulic retention time was maintained as 5 h. Anaerobic inoculum was the same with our former
study and dominated by Clostridium strains [13]. Automatic titration (Metrohm, 719 S) was used to keep pH at 5.0 with 2 M HCl since optimal pH for continuous H2 production was obtained in the range of 5.0e5.3 in our former study [13]. The ACSTR was routinely monitored for pH, oxidation reduction potential (ORP) and biogas production. The performance of the ACSTR was monitored by measuring the H2 production rate and yield, soluble metabolic products (VFAs and ethanol) and the biomass concentration.
2.2.
Nutrient solution
Synthetic nutrient solution was prepared using tap water and kept at 4 C. Glucose concentration in basic nutrient medium (BNM) was maintained as 9 g/l and the concentrations of other nutrients were the same as in the previous experiments [9]. In addition, BNM was amended with 2 g/l of yeast extract and 0.5 mg/l of iron nickel. FeCl24H2O and NiCl26H20 were used as additional iron and nickel sources, respectively.
2.3.
Reactor operation
The ACSTR was operated continuously for 108 days in three phases. During the first two phases of experiment, the effect
Table 1 e Effects of iron concentration on a glucose-fed ACSTR performance. Feþ2 concentration (mg/l)
H2 Yield (mol H2/mol glucose) H2 (%) Glucose degradation (%) VSS (g/l) Total soluble metabolites (g/l) Eth þ Ace/Total metab. (%)
0.5
10
25
50
100
0.49e0.89 (0.66) 33.6e42.6 (36.8) >99 1.4e1.8 (1.6) 5.3e5.9 (5.5) 68.0e78.2 (72.2)
0.40e1.12 (0.76) 27.0e46.2 (40.0) >99 1.4e1.8 (1.6) 5.2e5.7 (5.4) 58.7e76.1 (67.5)
0.59e1.05 (0.83) 33.6e44.3 (37.7) >99 1.5e1.8 (1.6) 5.2e5.7 (5.4) 52.7e68.8 (62.0)
0.84e1.43 (1.13) 40.7e47.9 (44.7) 84.9e99 (97.2) 1.3e1.8 (1.5) 4.7e5.6 (5.2) 51.3e58.4 (51.3)
0.51e1.21 (1.08) 42.2e48.6 (44.5) >99 1.7e2.1 (1.8) 5.2e5.9 (5.4) 54.5e60.8 (57.9)
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Table 2 e Effects of nickel concentration on a glucose-fed ACSTR performance. Niþ2 concentration (mg/l)
H2 Yield (mol H2/mol glucose) H2 (%) Glucose degradation (%) VSS (g/l) Total soluble metabolites (g/l) Eth þ Ace/Total met. (%)
0.5
10
25
50
0.48e1.09 (0.82) 39.9e44.5 (43.1) 85.3e99.0 (90.5) 0.88e1.42 (1.14) 5.2e6.2 (5.8) 32.8e38.3 (34.9)
0.41e1.21 (1.0) 43.0e49.3 (46.9) 24.5e96.3 (66.7) 1.22e1.39 (1.30) 1.9e5.1 (3.6) 44.3e55.5 (48.8)
0.85e1.88 (1.40) 42.3e46.4 (44.6) 10.0e82.6 (47.5) 1.05e1.57 (1.27) 3.6e5.9 (4.4) 32.7e53.9 (42.9)
1.27e1.48 (1.39) 44.5e47.3 (45.9) 80.2e96.7 (86.5) 1.33e1.47 (1.40) 3.9e4.0 (4.0) 34.8e40.3 (38.2)
of Feþ2 and Ni2þ were investigated separately while combined Fe2þ and Niþ2 effects were studied in the third phase. In the first phase (38 days), the concentrations of Fe2þ were increased from 0.5 to 100 mg/l. During the second phase, ACSTR was operated in 28 days and the effect of Ni2þ was studied in the range of 0.5e50 mg Ni2þ/l. In the third phase (42 days), Fe2þ and Ni2þ were supplemented together and their concentrations were increased from 0.5 to 100 mg/l.
2.4.
Analyses
Biogas production was measured twice per day using a wet gas meter (Ritter Apparatebau, Bochum, Germany). The biogas composition including H2 and CO2 was analyzed by a gas chromatograph (Shimadzu GC-2014) equipped with a thermal conductivity detector. N2 was used as carrier gas and the temperatures of the injector, column and detector were 110 C, 80 C and 110 C, respectively. Liquid samples for residual glucose, biomass concentration, VFAs and ethanol analysis were withdrawn from the ACSTR three times per week. Shimadzu LC-20AD liquid chromatograph with 0.01 N H2SO4 mobile phase was used to measure the VFAs and ethanol. VSS (g/l) was used to estimate the biomass concentration in the bioreactor and was quantified by following the procedures described in Standard Methods [14]. ORP and pH were determined by a pH meter (Metrohm, 719 S). Hydrogen
production yield (HPY) was reported as the amount of H2 produced (mol) per the initial glucose concentration (mol).
2.5.
Microbial community analyses
Liquid samples from ACSTR were centrifugated (7600 rpm, 10 min) and pellets were stored at 20 C for microbial community analysis. DNA was extracted with a VIOGENE Blood and Tissue Genomic DNA kit (Proteogenix SA, Fegersheim, France) according to manufacturer’s instructions. Amplification of partial bacterial 16S rRNA genes of the community DNA and DGGE were performed as previously described by [15] with the exception of using the denaturing gradient from 30% to 70%. Analysis of sequence data was performed as previously described [15].
3.
Results and discussion
3.1.
Effect of iron
In the Phase 1, iron concentration was stepwise increased from 0.5 to 100 mg Fe2þ/l with H2 production yields (HPY), % glucose degradation (GD), biomass concentrations and metabolites as presented in Fig. 1. Biomass concentration, GD and total amounts of metabolites remained stable with the
Fig. 2 e Effects of nickel on hydrogen and soluble metabolite production in a glucose-fed ACSTR.
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Fig. 3 e Effects of iron plus nickel addition on hydrogen and soluble metabolite production in a glucose-fed ACSTR.
increasing of iron concentration whereas HPY and distribution of metabolites fluctuated. GD decreased to 84.9% on day 24 due to the technical problems in the feeding. The average biomass concentration was around 1.6 g VSS/l and it slightly increased at the iron concentration of 100 mg/l (Table 1). HPY fluctuated up to 25 mg Fe2þ/l after which it increased steadily within the concentration range of 25e50 mg Fe2þ/l and remained stable at 100 mg Fe2þ/l. The lowest and highest average HPY’s were 0.66 and 1.13 mol H2/mol glucose at 0.5 mg Fe2þ/l and 50 mg Fe2þ/l, respectively. The HPY remained constant with the further increasing of iron concentration. These results indicate that optimum iron concentration was 50 mg Fe2þ/l for H2 production in ACSTR and HPY was enhanced by 71% with this dosage. HPY was accompanied with lactate, acetate, formate, butyrate and ethanol production. Total metabolites were stable around 5.5 g/l while the distribution of metabolites considerably changed during the phase 1. Acetate and butyrate were the major metabolites whereas the ratio of acetate plus ethanol to total metabolites decreased from 72.2% to 51.3% with the increasing of Fe2þ concentration from 0.5 to 50 mg Fe2þ/l (Table 1). Li and Ren (1998) [16] reported that ethanol type fermentation was dominant when the percentage of ethanol plus acetate is over 60%. Therefore, the
main H2 production mechanism in ACSTR was ethanol type fermentation. Acetate concentration decreased gradually at Fe2þ concentrations and HPY was decreased by ethanol production (Fig. 1). Increase in ethanol production was associated with the decrease in HPY. At the same time, there was a positive relationship between HPY and butyrate generation. Similar relationship between butyrate and HPY was also reported by other researchers [17e19]. Only minor amounts of formate were detected during the Phase 1, slight increases in formate were followed by decrease in HPY and increase in ethanol production (Fig. 1) Similar relationships between formate and ethanol were observed in our former study [13]. At lower Fe2þ concentrations H2 production was occupied by mainly ethanol type fermentation. At higher Fe2þ concentrations, the degradation mechanism was a combination of ethanol and acetate-butyrate type fermentations. Similarly, other researchers reported that Fe2þ addition resulted in metabolic shifts [4,20].
3.2.
Effect of nickel
During Phase 2, ACSTR was operated for 10 days with BNM with nickel and iron at 0.5 mg/l. ACSTR performance
Table 3 e Effects of iron plus nickel addition on hydrogen production on a glucose-fed ACSTR performance. Feþ2 and Niþ2 concentration (mg/l)
H2 Yield (mol H2/mol glucose) H2 (%) Glucose degradation (%) VSS (g/L) Total soluble metabolites (g/l) Eth þ Ace/Total met. (%)
0.5
10
25
50
100
0.60e1.11 (0.98) 39.2e43.9 (41.2) 93.1e99.0 (96.2) 1.0e1.3 (1.1) 4.5e5.8 (5.2) 34.6e43.7 (37.9)
1.18e1.48 (1.29) 41.2e45.9 (43.8) 91.4e95.9 (93.8) 1.0e1.1 (1.1) 5.0e5.2 (5.1) 35.7e41.2 (37.9)
0.32e1.55 (1.22) 33.6e48.3 (44.6) 50.1e86.0 (74.2) 0.9e1.1 (1.0) 3.4e4.9 (4.2) 28.2e44.4 (38.0)
1.30e1.53 (1.37) 42.0e44.9 (43.4) 81.3e86.4 (84.5) 1.2e1.6 (1.4) 3.8e4.3 (4.1) 41.5e44.3 (43.0)
1.19e1.42 (1.33) 41.9e46.1 (43.6) 76.2e89.4 (83.1) 1.3e1.4 (1.4) 4.1e4.5 (4.3) 42.0e43.8 (42.9)
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Table 4 e Affiliation of DGGE bands determined in a glucose-fed ACSTR. Band no 1 2 3 4 5 6 7 8 9 10
Phylogenetic affiliation Bacillus coagulans ATCC Bacillus coagulans X16 Clostridium butyricum SD2 Clostridium chartatabidum Clostridium butyricum W4 Clostridium butyricum RCEB 1 Clostridium butyricum CGS6 Clostridium ramosum CMC50 Clostridium butyricum EB6 Clostridium butyricum HAWHC1
Accession no DQ297928 FJ177636 EU477411 NR 029239 DQ831126 EU621841 AY540110 EU869233 EU183474 AY604563
% identity 100 100 99 95 99 100 100 100 100 97
butyrate was the main soluble metabolite followed by acetate and lactate. Average acetate concentration decreased from 2.6 g/l in phase 1 to 2.0 g/l in Phase 2 and ethanol production decreased over 50% with the increase in Ni2þ concentration from 0.5 to 25 mg Ni2þ/l. Similarly, Wang and Wan (2008) [6] reported that ethanol production was negatively affected by nickel addition. Moreover, Ni2þ supplementation positively affected the HPY by lowering ethanol production. In addition, no formate was produced and there was a inverse relationship between lactate accumulation and HPY(Fig. 2). Phase 1 and phase 2 results indicate that hydrogen production by Hisarkoy hot spring enrichment culture contained [NieFe] hydrogenases and HPY was positively affected both iron and nickel.
3.3.
Fig. 4 e Dynamic changes of microbial community during the supplementation of iron (a), nickel (b), and iron plus nickel ª to the glucose-fed ACSTR.
parameters fluctuated more than during Phase 1 (Fig. 1). Biomass concentration and H2 percentage remained stable at around 1.3 g VSS/l and 45.0%, respectively (Table 2). HPY varied in the range of 0.82e1.40 mol H2/mol glucose and on day 21 reached the highest value of 1.88 mol H2/mol glucose with 25 mg Ni2þ/l. H2 percentage was similar to the values in phase 1(Fig. 2). However, the most remarkable changes were obtained in GD. Although GD was over 99% during the Fe2þ supplementation, the highest HPY was obtained with the lowest GD at 25 mg Ni2þ/l in phase 2. The increase in HPY with the simultaneous decrease in GD may be related with metabolic pathway shift and the decreased the amount of total soluble metabolites. During the phase 2,
Effect of iron plus nickel
During the Phase 3, iron and nickel were added together and concentration of both was gradually increased from 0.5 to 100 mg/l. The technical problems caused considerable increase in lactate production while VSS, HPY, GDR, acetate and butyrate production decreased sharply (Fig. 3). Biomass concentrations ranged between 1.1 and 1.4 g VSS/l and it increased with the increasing of both ions (Table 3). H2 percentage within the biogas was around by 43% while total metabolites decreased at higher metal concentrations. As compared to the Phase 2, GDR was higher in Phase 3 while it decreased at higher total concentrations probably due to inhibition. The average HPY increased from 0.98 to 1.37 mol H2/mol glucose and increment rate was 40% at 50 mg/l of Ni2þ and Fe2þ concentration. However, average HPY remained constant as 1.33 mol H2/mol glucose with the further increasing of Fe2þ and Ni2þ amounts. In summary, the increase of HPY in phase 3 was 40% and lower than during phases 1 and 2 (% 71). The lowest concentration of total metabolite was produced during the nickel supplementation while GD was also lower and process stability fluctuated in phase 2. Conversely, GD and biomass concentration were the highest and process was more stabile during Phase 1.
3.4.
Microbial community analyses
The microbial community in ACSTR at different Fe2þ and Ni2þ concentrations was analyzed and compared by PCR-DGGE
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 5 ( 2 0 1 0 ) 8 5 5 4 e8 5 6 0
analysis (Fig. 4). Table 4 shows the results of band sequence affiliations. In the first phase, ten bands were seen in ACSTR predominated by Clostridium species. Mesophilic Clostridia are typical H2 producers [21e23]. Clostridia produce H2 from carbohydrates by two different metabolic pathways. Acetate and butyrate are the main metabolites during acidogenesis and ethanol and acetone are produced at solventogenesis. Clostridia shift their metabolism according to environmental conditions [24]. The Clostridium species in Fig. 4 were affiliated with three different strains including Clostridium butyricum, Clostridium ramosum and Clostridium chartatabidu (Table 4). C. butyricum ferments various substrates to H2, CO2, acetate and butyrate, and has been reported in many H2 producing ACSTR’s [25e28]. Bands 4 and 8 have high similarity to C. chartatabidum and C. ramosum. Both Clostridium species have been reported with the fermentation products of acetate, butyrate, hydrogen and ethanol [29]. The metabolite profiles of this study suggest that the dominance of acetate and butyrate was associated with the presence of Clostridium strains. In addition, two different strains of Bacillus coagulans were detected in ACSTR where B. coagulans are spore forming facultative bacteria with the ability of lactate production [30]. The DGGE profiles in Fig. 4 (a) indicate that increasing in iron concentration did not affect the bacterial community. Therefore, the increase in H2 production at higher iron concentrations related to the metabolic pathway shift. Similar results were reported by Koskinen et al. (2008) [31] for H2 production by thermophilic enrichment culture from Icelandic hot spring. Moreover, Lee et al. (2001) [7] and Chen et al. (2005) [32] indicated that iron is crucial for the hydrogen production of Clostridium. The number of DGGE bands in Phase 2 decreased from Phase 1. Throughout the nickel addition Clostridium chartabidum (Band 4) did not thrive in the ACSTR and C. ramosum (Band 8) disappeared at 10 mg Niþ2/l. In our former study, the positive relationship was reported between increase in ethanol production and the presence of C. chartabidum [33]. Therefore, the decrease in ethanol production after Phase 1 could be attributed to the decreased amount of C. chartabidum. The disappearance of Band 8 after Day 7 suggests that nickel concentration over 10 mg Ni/l inhibited C. ramosum. During the Phase 3, decrease in microbial diversity continued. C. butyricum (Band 5) was not found during Phase 3 and B. coagulans (bands 1 and 2) were not present after the increasing of both iron and nickel concentration to over 25 mg/l. For these reasons, the decreasing of glucose degradation rate and increase in H2 production yield after Day 25 was likely related to inhibition of B. coagulans.
4.
Conclusions
The following conclusions can be drawn from this study: 1. Hydrogen production increased about by 71% with the increasing of iron and nickel supplementation and the highest yields were achieved at the concentrations of 50 mg Fe2þ/l and 25 mg Ni2þ/l.
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2. Ethanol production was favorable at lower iron and nickel concentrations while H2 production was positively affected with the increasing of butyrate generation at higher iron and nickel concentrations. 3. Glucose degradation was over 99% in the presence of iron while nickel adversely affected the glucose oxidation. 4. Microbial composition in ACSTR was dominated with Clostridium species and H2 was produced mainly by C. butyricum. 5. Microbial diversity decreased with the nickel supplementation, and ethanol and H2 production was affected mainly by metabolic pathway shifts rather than changes in bacterial community.
Acknowledgements This research was funded by the Academy of Finland (HYDROGENE Project, no 107425) and Nordic Energy Research (BioH2 project 06-Hydr-C13). Author Karadag gratefully acknowledges the financial support provided by CIMO (Centre for international mobility, Finland).
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