Direction of glucose fermentation towards hydrogen or ethanol production through on-line pH control

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Direction of glucose fermentation towards hydrogen or ethanol production through on-line pH control Dogan Karadag*, Jaakko A. Puhakka Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland

article info

abstract

Article history:

The present study investigated the production of hydrogen (H2) and ethanol from glucose

Received 21 June 2010

in an Anaerobic Continuous Stirred Tank Reactor (ACSTR). Effects of hydraulic retention

Received in revised form

time (HRT) and pH on the preference of producing H2 and/or ethanol and other soluble

14 July 2010

metabolic products in an open anaerobic enriched culture were studied. Production rates of

Accepted 23 July 2010

H2 and ethanol increased with the increase of biomass concentration. Open anaerobic

Available online 21 August 2010

fermentation was directed and managed through on-line pH control for the production of H2 or ethanol. Hydrogen was produced by ethanol and acetate-butyrate type fermenta-

Keywords:

tions. pH has strong effect on the H2 or ethanol production by changing fermentation

Hydrogen

pathways. ACSTR produced mainly ethanol at over pH 5.5 whereas highest H2 production

Ethanol

was obtained at pH 5.0. pH 4.9 favored the lactate production and accumulation of lactate

ACSTR

inhibited the biomass concentration in the reactor and the production of H2 and ethanol.

HRT

The microbial community structure quickly responded to pH changes and the Clostridia

pH

dominated in ACSTR during the study. H2 production was maintained mainly by Clostridium

Fermentation

butyricum whereas in the presence of Bacillus coagulans glucose oxidation was directed to lactate production. ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Fermentative biotechnology has a wide range of applications in waste management [1e3]. Organic wastes can be fermented with the concomitant production of methane, hydrogen and soluble metabolites such as organic acids and solvents [4]. In recent years, hydrogen and ethanol fermentation has received increasing research attention as a means of producing renewable fuels. In addition to methane, both are sustainable energy carriers with high energy content. Their generation rates are high and the production is non-energy intensive through fermentation [5].

Hydrogen fermentation is accompanied with co-production of volatile fatty acids (VFAs) and ethanol. H2 production may occur via different fermentation mechanisms. Theoretically 4 mol H2/ mol glucose can be produced during the acetate type fermentation, whereas, butyrate type produces 2 mol H2/mol glucose [6]. In general, fermentative hydrogen production is accomplished with the combination of acetate and butyrate fermantation and the ratio of butyrate to acetate (B/A) closely correlates with the hydrogen yield [7]. Theoretically 2.67 mol of H2 is produced from 1 mol glucose as follows when the B/A ratio is 1 [8]: 3C6 H12 O6 þ 2H2 O/8H2 þ 6CO2 þ 2C2 H4 O2 þ 2C4 H8 O2

(1)

Abbreviations: ACSTR, anaerobic continuous stirred tank reactor; HRT, hydraulic retention time; ORP, oxidationereduction potential; VFA, volatile fatty acid; PCR, polymerase chain reaction; DGGE, denaturing gradient gel electrophoresis; HPY, hydrogen production yield; HPR, hydrogen production rate; EPY, ethanol production yield; EPR, ethanol production rate. * 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 ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.07.139

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In ethanol type fermentation, ethanol and acetic acid products dominate while propionic acid production remains at low levels [9]. During the ethanol type fermentation of glucose, both ethanol and acetate were produced together with H2 and CO2 [10].

0.11 g/L [16]. FeCl2.4H2O was the iron source and 50 mg/L of Na2S2O4 was added to the medium as a reducing agent. Glucose and iron were in the same feed tank and continuously mixed with magnetic stirrer. All other nutrients were added to the second feed tank and both tanks were kept at 4  C.

C6 H12 O6 þ H2 O/2H2 þ 2CO2 þ C2 H4 O2 þ C2 H6 O

2.3.

(2)

Anaerobic Continuous Stirred Tank Reactor (ACSTR) has been used widely for continuous fermentative H2 production due to simple construction, ease of operation and effective mixing [11]. The process is sensitive to environmental conditions such as pH and Hydraulic retention time (HRT). Low hydrogen production rate has been reported in ACSTR at low HRT due to biomass wash-out [12e14]. Rapid changes in pH cause shifts in microbial community structure and metabolic patterns, therefore, pH control is the main instrument for continuous H2 production [15]. Production of desired energy carriers from waste materials through fermentation requires successful management of microbial communities. This study was undertaken to evaluate the possibilities of managing ethanol and H2 production through HRT and pH control in an open Anaerobic Continuous Stirred Tank Reactor (ACSTR). Original sample was obtained from Hisarko¨y Hot Spring in Turkey and anaerobically enriched in serum bottles. Glucose fed ACSTR was operated at 37  C and, metabolic patterns and microbial community dynamics were followed for 60 days on continuous basis.

2.

Methods and materials

2.1.

Enrichment of hot spring culture

The anaerobic culture was enriched using a water sample from a Hisarko¨y geothermal hot spring located in Balıkesir Province, Turkey. The temperature of the sampling site was 45  C. The enrichments were set up in 120 mL anaerobic serum bottles with 48 mL of medium and 2 ml of the sample [16]. Enrichment medium had the same composition as the ACSTR medium. Bottles were flushed with nitrogen for 5 min to provide anaerobic conditions, capped with a rubber stopper and placed in a shaker at 150 rpm for 2 days. Enrichment was performed at 37  C and the initial pH was 6.8. During the enrichment, H2 and CO2 were monitored in biogas and no methane was produced. After the second enrichment, 150 ml of the enriched culture was transferred to the ACSTR.

2.2.

Experimental setup

Experiments were carried out in an ACSTR of 1 L with a working volume of 0.9 L [17]. The ACSTR was mixed by mechanical stirring at 100 rpm and the temperature was maintained at 37  C by circulating hot water through the water jacket. Automatic titration (Metrohm, 719 S) was used to maintain the pH at desired level using 5 M KOH or 1 M HCl. Nutrient media was divided into two feeding tanks to avoid microbial growth in the feed tubes. Media was prepared using tap water as follow: glucose, 9 g/L (50 mM); NaHCO3, 4 g/L; NH4Cl, 0.6 g/L; NaH2PO4$H2O, 10.7 g/L; Na2H2PO4, 3.2 g/L; K2HPO4 3H2O, 0.125 g/L; MgCl2 6H2O, 0.1 g/L and CaCl2 2H2O,

Continuous operation

ACSTR was filled with 750 ml of medium and sparged with nitrogen to obtain anaerobic conditions. The ACSTR initially inoculated with 150 ml of the enriched culture was operated for 1 day in batch mode and then converted to continuous feed. On day 3, additional 120 mL new inoculum was added. Glucose concentration was kept at 9 g/L throughout the experiments and the ACSTR was continuously operated as open system for 60 days in two different phases. During the first 32 days, HRT was decreased gradually from 10 h to 5 h in 3 steps. During this period, the OLR was increased stepwise from 21.6 g/L/d to 43.2 g/L/d. In the second phase, pH was controlled in the range of 4.9 and 6.0 by adding 5 M KOH or 1 M HCl while HRT was held constant at 5 h. ACSTR was routinely monitored for pH, oxidationereduction potential (ORP), biomass concentration (VSS), H2, ethanol, and other metabolic products.

2.4.

Analyses

ORP and pH were determined by a pH meter (Metrohm, 719 S). Biogas production was monitored using a wet gas meter (Ritter Apparatebau, Bochum, Germany). The composition of biogas 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. Samples for glucose, VFA and alcohol analyses were withdrawn three times per week during the Phase 1 and once per day in the second phase. These analyses were carried out using a Shimadzu LC20AD high performance liquid chromatography (HPLC) with 0.01 N H2SO4 mobile phase. VSS (g/L) was used to estimate the biomass concentration in the ACSTR and measured according to the procedures in Standard Methods [18].

2.5.

Molecular characterization of microbial diversity

Microbial community analysis was performed using DNA extraction and Polymerase chain reactionedenaturing gradient gel electrophoresis (PCReDGGE) of partial 16S rRNA genes followed by their sequencing. DNA was extracted from the samples using MOBIO Power Soil DNA Extraction kit (MOBIO Laboratories). Amplification of partial bacterial 16S rRNA genes of the community DNA, DGGE and analysis of sequence data were performed as previously described [19].

3.

Results and discussion

3.1.

Reactor start-up

Following the first day of ACSTR operation, biogas production started and pH decreased to almost 5.0 due to the

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Fig. 2 e Biomass concentration during continuous operation in ACSTR fed with glucose.

Fig. 1 e Daily variations in (a) pH and ORP, and (b) H2 and ethanol yields in Phase 1 during continuous opeartion of ACSTR fed with glucose.

accumulation of volatile fatty acids. As hydrogen production is inhibited at below pH 5.0 [20], the pH was on-line controlled and maintained at above 5.0. On day 6, pH stabilized and ACSTR was operated thereafter without pH control. Until the day 5, basal media contained 0.5 mg/L iron. H2 production rate and H2 content in biogas remained at 0.35 L/d and 20%, respectively. Increasing the iron concentration from 0.5 to 50 mg/L dramatically increased the HPY (Fig. 1) and, therefore, the nutrient media with 50 mg/L of iron was used.

3.2.

Effect of HRT

Fig. 1 shows the daily variations of pH and ORP at different HRTs. With the exception of start-up period, pH was not controlled and it varied between 5.2 and 5.6. ORP ranged between 195 and 365 mV. The ORP decreased when pH was increased and vice versa. Similar results were reported for biohydrogen production by (Mohan et al.) [21]. HPY fluctuated in the range of 0.09e1.08 mol H2/mol glucose (Fig. 1). The process performance was as summarized

in Table 1. The HPY increased slightly with the decreasing of HRT from 10 h to 7.5 h while the HPR considerably increased. The decrease of HRT by 50% resulted in HPR increase by about two fold. Biomass concentration increased from 0.44 g VSS/L to 1.53 g VSS/L with the decreasing of HRT from 10 h to 5 h in Phase 1 (Fig. 2). As the glucose degradation (GD) remained at over 99% all the time, the increasing of HPR in Phase 1 was due to the increase of biomass in the ACSTR. On the other hand, pH shifts in Phase 2 adversely affected biomass concentration. Ethanol production yield (EPY) at different HRTs remained stable and lower than the HPY (Fig. 1). The highest EPY was 0.67 mol EtOH/mol glucose during the start-up period and on day 11 it decreased to 0.39 mol EtOH/mol glucose. Thereafter, EPY slightly increased with decreasing HRT and remained constant at around 0.5 mol EtOH/mol glucose.

3.3.

Effect of pH

In the Phase 2, HRT was maintained constant at 5 h and the effect of pH on H2 and ethanol production was investigated. First, the pH was decreased stepwise from 5.3 to 4.9 and followed by gradual increase to 6.0 (Fig. 3). When pH was decreased from pH 5.3 to pH 5.2, the mean HPY, HPR and the H2 content slightly increased (Table 2). Similarly the biomass concentration increased with the highest value of 1.61 VSS/L at pH 5.2 (Fig. 2). On day 34, a technical interruption in feeding

Table 1 e Mean ACSTR performance fed with glucose and operated at various HRTs. HRT (h) Glucose concentration (mM) GD (%) VSS (g/L) H2 content (%) HPR (mol H2/d) HPY (mol H2/mol glucose) EPR (mol EtOH/d) EPY (mol EtOH/mol glucose)

10

7.5

6.0

5.0

50 >99 0.75 35.6 81.8 0.7 59.4 0.5

50 >99 1.33 38.2 115.1 0.8 82.3 0.5

50 >99 1.43 40.0 157.0 0.8 93.4 0.5

50 >99 1.35 35.0 184.4 0.8 109.0 0.5

Fig. 3 e Effect of pH on hydrogen and ethanol yields in ACSTR fed with glucose.

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Table 2 e Mean ACSTR performance fed with glucose and operated at different pH values and fed with glucose. pH Glucose (mM) GD (%) VSS (g/L) H2 content (%) HPR (mol H2/d) HPY (mol H2/mol glucose) EPR (mol EtOH/d) EPY (mol EtOH/mol glucose)

5.3

5.2

4.9

5.3

5.4

5.5

5.8

6.0

50 >99 1.4 38.1 4.9 0.9 111.0 0.5

50 >99 1.50 41.0 5.0 0.9 97.1 0.4

50 92 1.2 44.8 6.1 1.3 30.0 0.2

50 >99 1.4 45.2 6.7 1.1 47.7 0.2

50 >99 1.1 26.5 6.2 0.5 138.8 0.6

50 >99 0.9 18.6 2.1 0.3 158.8 0.7

50 >99 0.9 17.6 1.0 0.2 214.4 1.0

50 >99 0.8 17.5 0.8 0.1 289.2 1.2

decreased the HPY from 1.0 to 0.7 mol H2/mol glucose but the H2 production was regained rapidly after the normal feeding had been obtained. At pH 4.9, HPY increased rapidly with the highest value of 1.6 mol H2/mol glucose on day 39 with the highest average HPY of 1.3 mol H2/mol glucose. At the same time, HPR and H2 content in the gas continued to increase, while the GD decreased to 92% (Table 2). Further increase of pH during the following days steadily decreased the H2 production and at pH 6.0, the average HPY and HPR dropped to 0.06 mol H2/mol glucose and 1.0 L H2/d, respectively. The results show the dependency of H2 production on pH, and that HPY decreased significantly when the pH was over 5.3. This was contrary to the hydrogen production, ethanol generation was decreased with decreasing pH (Fig. 3). When pH decreased from pH 5.3 to pH 4.9, EPY decreased from 0.48 to 0.19 mol EtOH/mol glucose. Increasing pH from pH 4.9 to pH 5.3 increased ethanol production. Further pH increase to over 5.3 increased considerably the EPY. Average EPY and EPR increased with the increasing of pH with the highest values obtained at pH 6.0. Fig. 3 shows simultaneous hydrogen and

Fig. 4 e Distribution of metabolites in experimental Phases 1 and 2 in ACSTR fed with glucose.

ethanol production in the ACSTR while ethanol production was superior at above pH 5.5. When pH increased to 6.0, HPY decreased to 0.1 showing that ethanol production outcompeted the hydrogen generation.

3.4.

Effect of HRT and pH on soluble metabolites

Formation of hydrogen was accompanied with VFAs and ethanol production and their distribution at different operational conditions was as shown in Fig. 4. Ethanol, acetate, butyrate, formate and lactate were produced with trace amount of propionate throughout the experiment. During the start-up period, ethanol, formate and acetate were the major products. Following the start-up, formate and lactate concentrations decreased and lactate production stopped with the decreasing or HRT. At the same time, acetate and ethanol became major metabolic products. Daily fluctuations of formate had similar pattern as ethanol production, while butyrate concentration remained stable. These results suggest that when the sum of acetate and butyrate increased, H2 production increased. The distribution of metabolites fluctuated with the changes in pH. At pH 5.2 and pH 5.3, acetate and butyrate were the major metabolites while H2 yield increased and ethanol production decreased (Fig. 4). At pH 4.9, acetate and butyrate were again main products although acetate, ethanol and formate concentration significantly decreased. At pH 4.9, lactate production increased sharply up to 23.50 mM concentration until the day 41 and HPY decreased slightly. During the following 7 h, further accumulation of lactate continued and the concentration increased up to 32 mM while HYP and EPY dropped suddenly from 1.4 to 0.7 mol H2/mol glucose and from 0.13 to 0.09 mol EtOH/mol glucose,

Fig. 5 e Relationship between the production of acetate plus butyrate and H2 production yield during continuous operation of ACSTR fed with glucose.

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respectively. Decrease in HYP and EPY was due to the less substrate available to produce H2 and ethanol since much of glucose directed to lactate production. Therefore, accumulation of lactate should be monitored closely during the reactor operation. Increasing the pH to 5.3 stopped lactate production, while acetate, ethanol and formate production increased. Butyrate production continued to increase and HPY was increased to 1.40 mol H2/mol glucose. Further increasing of pH accelerated the production of formate and ethanol while decrease in acetate and butyrate production was accompanied with decreased hydrogen generation.

3.5.

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The fermentation pattern

The ratio of ethanol plus acetate to total soluble metabolites comprised 43%e78% with the average of 64% (Fig. 4). During the period of lactate accumulation, the ratio of ethanol plus acetate decreased from 47% to 30%. However, it again increased to 45% when lactate production had stopped following pH increase. These results show that ethanol type fermentation played a main role in the degradation of glucose by the Hisarko¨y Hot Spring enriched culture. Similarly, Ren et al. and Xing et al. [9,22] found that ethanol and acetate are the major soluble metabolites during the biohydrogen production by ethanol type fermentation.

Fig. 6 e Changes in DGGE profiles of microbial community with HRT (a) and pH (b).

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Table 3 e The closest relatives of DGGE bands in the ACSTR microbial community. Band no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Phylogenetic affiliation

Accession no

% identity

Clostridium butyricum strain RCEB 1 Clostridium butyricum strain EB6 Clostridium ramosum strain CM-C50 Clostridium butyricum strain CM-C86 Clostridium butyricum strain CGS6 Clostridium ramosum strain 113 Uncultured bacterium clone G-23 Uncultured bacterium HAW-RM37 Bacillus coagulans strain ATCC Enterobacter sp. TUT1014 Bacillus coagulans strain 17C5 Bacillus coagulans NRIC 1530 Bacillus coagulans DF2 Bacillus coagulans strain X16 Bacillus coagulans strain SKU 12 Clostridium chartatabidum

EU621841 EU183474 EU869233 EU869239 AY540110 NR029247 DQ443949 FN563278 DQ297928 AB098582 DQ297925 AB362709 DQ914287 FJ177636 EU742138 NR 029239

100 99 100 100 100 99 83 99 100 100 100 100 83 100 100 95

A maximum of 1.6 mol H2/mol glucose was obtained through anaerobic fermentation. Hydrogen production remained low during the ethanol production. The pathways and H2 production efficiency were closely related to pH and hydrogen yield decreased with increases in the pH. Simultaneous ethanol and hydrogen production occurred in the range of pH 5.0 to pH 5.5 and ethanol production was superior at above pH 5.5. Hydrogen production was mainly depended on the production of acetate plus butyrate (Fig. 5). Butyrate production was stable and the average ratio of acetate plus butyrate to total soluble metabolites remained around 60% until the day 38. However, this ratio increased up to 88.5% with the decreasing the pH to 4.9. H2 was produced only by acetateebutyrate type fermentation between day 39e41, when no formate was observed. With the exception of the high lactate accumulation period, the highest H2 production achieved was in the range of 1.33e1.61 mol H2/mol glucose when B/A ratio was around 1. Based on these results, it is proposed that H2 production occurred by the cooperation of acetate-butyrate and ethanol fermentation as described in Eqs. (1) and (2). Acetateebutyrate fermentation dominated at pH 5.0 while at pH over 5.5 fermentation pattern shifted to ethanol. Similarly, Cheong et al. [23] studied the effect of pH and HRT on the performance of a mesophilic anaerobic bioreactor using mixed culture dominated with Clostridia and found that biohydrogen was produced with the combination of ethanol and acetate-butyrate type fermentations. They also indicated that H2 production through acetateebutyrate type fermentation depended on pH changes in the reactor. In summary, the fermentation patterns were controlled mainly by the pH while HRT did not influence on the production mechanism. Consequently, control of pH directs the open continuous fermentation process to ethanol or hydrogen production.

3.6.

Dynamics of bacterial community composition

Fig. 6 shows the DGGE pattern of the partial 16S rRNA genes amplified from the bioreactor samples under different HRTs and pHs. The identified microorganisms were as given in Table 3. After inoculation of the ACSTR, two Clostridium

species including Clostridium butyricum (bands 1, 2, 4 and 5) and Clostridium ramosum (bands 3 and 6) were dominant throughout the experiment at different HRTs. These Clostridium species are H2 producers and reported in many studies [19,24]. During the start-up, a lactic acid producing bacterium Bacillus coagulans (band 9) was present while it disappeared after reaching stable reactor conditions. B. coagulans is a grampositive, rod-shaped and spore-forming facultative anaerobic bacterium and produces lactate at both mesopilic and thermophilic temperatures [25,26]. During the stable hydrogen and ethanol production the bacterial community remained stable at different operational HRTs. In the second phase, C. ramosum (band 6) was present thoroughout the experiment while C. butyricum was not seen after pH increased over 5.5. These results show that hydrogen production in ACSTR was mainly by C. butyricium and long term maintainance of H2 production by C. butyricum was depended pH. The pH dependency of biohydrogen production by C. butyricium was reported elsewhere [27,28] (Cai et al.; Chong et al.). A major shift in DGGE bands occurred at pH 4.9. The increase in lactate production was associated with six new DGGE bands affiliated with B. coagulans (band 9 and bands 11e15). After the increasing pH to 5.3, B. coagulans (bands 13e15) was not present and no lactate was produced. The pH affected the microbial diversity in the ACSTR and the change in production mechanisms above pH 5.5 could be related to the shift of bacterial community from C. butyricum to Clostridium chartatabidum (band 16). C. chartatabidum is strictly anaerobic spore-former and produces acetate, butyrate, hydrogen and ethanol from glucose [29].

4.

Conclusions

The performance of ACSTR was evaluated through the hydrogen and ethanol production using hot spring enriched culture. HRT strongly affects H2 and ethanol yields. Glucose fermentation is accomplished by the cooperation of ethanol and acetate-butyrate type fermentations. pH has remarkable effects on the distribution of soluble metabolic products and

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desired products can be optimized through on-line pH control. Ethanol production is superior at above pH 5.5 while H2 production is the highest at pH 5.0. On the other hand, the microbial community composition readily responses the pH changes in the ACSTR. Hydrogen production is maintained mainly by C. butyricum while lactic acid production is associated with the presence of B. coagulans.

[14]

[15]

Acknowledgments This research was funded by the Academy of Finland (HYDROGENE Project, no 107425), Nordic Energy Research (BioH2 project 06-Hydr-C13).

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