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Enhancing continuous hydrogen gas production by the addition of nitrate into an anaerobic reactor
Process Biochemistry 41 (2006) 1208–1212 www.elsevier.com/locate/procbio
Short communication
Enhancing continuous hydrogen gas production by the addition of nitrate into an anaerobic reactor Jeong Ok Kim a,c, Yong Hwan Kim a,b,*, Sung Ho Yeom d, Bong Keun Song a,*, In Ho Kim c a
Korea Research Institute of Chemical Technology, PO Box 107, Yusong, Taejon 305-600, Republic of Korea Department of Chemical Engineering, Kwangwoon University, Nowon, Seoul 139-701, Republic of Korea c Department of Chemical Engineering, Chungnam National University, Yusong, Taejon 305-764, Republic of Korea d Department of Environmental and Applied Chemical Engineering, Kangnung National University, Gangneung, Gangwondo, Republic of Korea b
Received 29 March 2005; received in revised form 29 October 2005; accepted 23 November 2005
Abstract Hydrogen gas was produced using granular sludge of mixed culture from sewage digester sludge. Methane gas was detected in the biogas even under acidogenic condition with pH 5.5–6.2 and after heat treatment at 95 8C. In order to repress methane production, nitrate known to inhibit methanogenic bacteria was added to an anaerobic reactor. When the feeding concentration of KNO3 into the reactor was 1000 mg/L and more, the yield of hydrogen gas production (H2-mol/glucose-mol) was almost 1.0 and no methane gas was produced. The nitrate was thought to repress methanogenic bacteria remarkably and this was confirmed using a SEM. # 2005 Elsevier Ltd. All rights reserved. Keywords: Granular sludge; Mixed culture; Hydrogen gas; Methane; Heat treatment; Nitrate
1. Introduction Hydrogen has been produced through naphtha thermalcracking or water electrolysis, which requires much energy and emits global warming gases such as carbon dioxide. As the concerns over environment are growing, green process for hydrogen production is widely studied [1]. One of the most promising methods is that the gas is produced from annually renewed biomass (plant resources) [2]. In an anaerobic process using biomass, organic materials are converted to the final product, methane gas. The anaerobic fermentation process is composed of two steps, acid-generation and methane-generation one. Each step has different complex microbial interactions and is established at a different optimal pH condition for the step [3,4]. Researches on the control of pH for hydrogen gas production, the main product in the acid generation step, have been performed [5,6]. Heat treatment, boiling sludge for 15 min, to inhibit the activity of hydrogenotrophic bacteria
which cannot form spores was also performed. [5]. However, these approaches may not be effective in the long-term continuous operation. It is critical to maintain high density of hydrogen-production bacteria in a reactor to produce the gas stably without methane gas production. Two immobilization methods to maintain high concentration of sludge in the reactor were developed in the previous study [7]; biofilm formation on the hydrophilic carriers and granulation using organicinorganic hybrid polymers. Although the immobilization was able to protect sludge loss and to maintain high concentration of bacteria, methanogenic bacteria were not repressed efficiently and methane gas was detected in a biogas, which degraded the quality of hydrogen gas severely. In this research, nitrate was added to repress the activity of methanogenic bacteria and to produce high concentration of hydrogen gas continuously. 2. Materials and methods 2.1. Sludge and synthetic wastewater
* Corresponding author. Tel.: +82 2 940 5675; fax: +82 42 860 7649. E-mail addresses: [email protected] (Y.H. Kim), [email protected] (B.K. Song). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.11.017
Mixed culture was obtained from sewage digester sludge at the city of Daejeon in Korea, of which solid concentration and ash content were around 43,000 mg/L and 20%, respectively. The composition of synthetic wastewater
J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212 was: 0.44 g/L KH2PO4, 1.8 g/L NaCl, 4.7 g/L (NH4)2SO4, 0.09 g/L MgSO47H2O, 0.09 g/L CaCl2, 0.5 g/L CysteinHCl, 4.0 g/L NaHCO3, 20 g/ L glucose. The glucose was a main carbon source and COD:N:P ratio was 100:2.5:0.5. After the sewage sludge was treated at 95 8C for 15 min and cooled down to room temperature, granular sludge was prepared as described in the next section. The granular sludge occupied 25% of working volume of the reactor. The anaerobic reactor harboring granular sludge was made of Pyrex and operated at 37 8C The working volume of the reactor was 2 L. In order to repress the activity of methanogenic bacteria in the granular sludge, the pH in the reactor was adjusted to 5.8–6.2 for acid-production condition by intermittently feeding 1N NaOH.
2.2. Preparation of granular sludge Sludge itself has the negative charge of 26 mV. In order to make the sludge neutral, high molecular cationic polymer (cationic polyacrylamide, M.W.: 15,000,000) of 0.5% (w/w) of dry sewage digester sludge was added to the sludge and stirred at 400 rpm for 2 min. Since the terminals of residual cation may cause detrimental effect on the microorganisms, anionic organic material (anionic silica sol), less than 20 nm in diameter, of 0.7% (w/w) of dry sewage digester sludge was added and stirred at 200 rpm for 2 min. The total time required for forming granules of 1–3 mm in diameter, thus, was just about 5 min. The schematic procedure is shown in Fig. 1 [7].
2.3. Analytical method HRT (hydraulic retention time) control started after 48 h of operation and the HRT was stepwise decreased by increasing the influent after confirming that over 90% of glucose was consumed. The continuous operation started shortly after biogas began to be produced. The effluent from the reactor was taken every 24 h to analyze pH, ORP (oxidation-reduction potential), CODcr (Chemical oxidation demand determined by the oxidative action of K2Cr2O7), MLSS (mixed liquor suspended solids), NO3–N and residual glucose. The pH and ORP were measured using a portable pH meter (Orion, 520A, USA). MLSS representing microorganism growth was measured according to the standard method. CODcr and NO3–N was measured through Hach method (Hach, DR/
1209
2010, USA). Residual glucose was colorimetrically measured using an enzyme kit (Glucose assay kit, Sigma, USA). The amount of biogas produced by the microorganisms was measured according to a water displacement method [7] and the composition of the biogas was analyzed using a GC (Gas Chromatography, DS 6200) equipped with a TCD (Thermal Conductivity Detector) and a HayesepQ column (80/100 mesh). The operating temperature of the GC was 25 8C for the oven, 120 8C for the detector and 90 8C for the injector. Argon gas was used as a carrier gas and its flow rate was 30 mL/min. The granular sludge was washed and fixed at 4 8C for 24 h in the fixing solution composed of 5% glutaraldehyde in anaerobic cacodylate buffer. The sludge was then lyophilized in liquid nitrogen to be cut. After dehydrated using 20, 50, 80 and 90% ethanol sequentially, the cross-section of the granular sludge was observed using a SEM (Scanning Electron Microscope, Philips XL30S FEG, The Netherlands) [8]. An anaerobic CSTR (continuous stirred tank reactor) was used for hydrogen production in this study. The agitation speed was 110 rpm in the reactor. A Utube was attached to the effluent of the reactor for continuous substrate feeding and gas-stop.
3. Results and discussion 3.1. pH control The anaerobic reactor containing the granular sludge was operated for 48 days with the load of 35 kg COD/m3/day under anaerobic condition. In order to maintain organic loads constantly, the influent glucose concentration was decreased as decreasing HRT. The hydrogen production yield (mol-H2/ mol-glucose) using a mixed culture at optimal pH condition of 5.5 was reported to be 2.1 0.1 [3]. However, the results did not mention the operation duration, they just showed glucose conversion, the biogas composition and the yield of hydrogen production at each pH [3]. Although the stable production of
Fig. 1. Schematic procedure of granulation (a) shows the state of initial sludge where filamentous microorganisms and flock forming microorganisms coexist, (b) shows the initial flock after adding cationic polymer to the sludge. The flock is soon disintegrated by the shear force from a mixer and a large portion of cationic polymer not involved in the flock formation remains on the surface of microorganisms as shown in (c). The microorganisms are reflocked by the addition of silica. Also, the silica penetrates into the flock and neutralizes excess positive charge of cationic polymer, which results in the formation of compact flock as shown in (d).
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J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212
Fig. 2. The profile of biogas production (a) *: hydrogen gas production rate, ~: yield (mol-H2/mol-glucose), –: HRT (b) *: hydrogen, *: methane, !: carbon dioxide, —: HRT.
hydrogen gas and repression of methanogenic bacteria was possible at pH 5.5, long-term continuous operation resulted in the re-growth of methanogenic bacteria, which led to increase of methane content in the biogas [2,3] The hydrogen gas
production, yield, biogas composition during pH-stat operation at different HRTs is shown in Fig. 2. The high methane content of 40–60% was maintained for 37 days of operation despite the pH control suitable for acid-production. After 37 days of
Fig. 3. Effect of nitrate addition on the composition of biogas *: hydrogen, *: methane, !: carbon dioxide, —: HRT.
J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212
operation, methanogenic bacteria were washed out due to as short as 4 h of HRT [9]. As consequence, methane production was decreased and the hydrogen gas content in the biogas increased to 40% as shown in Fig. 2(b). In spite of high hydrogen content in the biogas, the rate of hydrogen gas production was decreased due to low microbial population as shown in Fig. 2(a). The results implied that the control of pH and HRT was not enough to repress the activity of methanogenic bacteria completely. 3.2. Repression of methanogenic bacteria by the addition of nitrate After 50 days of operation, nitrate was added into the reactor for both repressing methane production and obtaining high concentration of hydrogen gas. The methanogenic bacteria are known to be repressed under aerobic condition or semianaerobic condition [10]. Since extreme aerobic condition may also cause negative effect on the acid-generation step, semianaerobic condition is thought to be favorable. Because the ORP value in the reactor gives an idea of oxidation–reduction state of the fermentation, the effect of nitrate on the ORP was also considered (see below). The average values of hydrogen gas production, yield (mol-H2/mol-glucose), MLSS, ORP level at various nitrate concentrations are shown in Table 1. The maximum hydrogen gas production yield was 1.3 at 1000 mg/L of KNO3 in the reactor. The range of fluctuation of hydrogen gas production at various KNO3 concentrations was 30 ml/L/h and no methane was detected after KNO3 addition. The ORP level was 206 mV, higher than that under anaerobic condition, 340 mV, and the ORP level was slightly increased with increasing nitrate concentration. The results implied that the addition of nitrate influenced the ORP level [11]. Recent research showed that even the ORP as high as +420 mV was not able to repress the growth of methanogenes and that methane production by pure Methanosarcina barkeri under semianaerobic condition was not suppressed [12]. Also, the ORP level was maintained at 290 mV when nitrate was added to the anaerobic digester. Therefore, the repression of methane production by the addition nitrate cannot be explained in terms of ORP change. The composition change of biogas due to nitrate addition is shown in Fig. 3. The content of methane gas in the biogas started to be decreased gradually shortly after nitrate addition and reached to 0% 6 days after the addition. Although the concentration of nitrate was constant (1000 mg/L) in the feeding solution, the anaerobic condition was maintained in the reactor with HRT of 16 h.
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3.3. The changes in the anaerobic reactor after nitrate addition Since the influent C/N ratio was over 7 and since more than 99% of nitrate was removed in the reactor in this study, it can be supposed that denitrification with the consumption of glucose occurred when nitrate was added. The related theoretical equation for the denitrification was suggested to be [13]: 24NO3 þ 5C6 H12 O6 ! 12N2 þ 30CO2 þ 18H2 O þ 24OH (1) When the maximum concentration of feeding potassium nitrate is 2000 mg/L with HRT 16 h as shown in Fig. 4, 250 mg/h of
Table 1 The characteristics of fermentation for hydrogen gas production at different nitrate concentrations KNO3 (mg/L)
H2 production rate (mL/L/h)
Yield (mol-H2/ mol-glucose)
MLSS (mg/L)
ORP (mV)
0 500 1000 2000
49.7 101.6 132.2 123.4
0.4 0.6 1.0 0.8
4098 2200 2376 3730
221.3 199.4 198.0 168.0
Fig. 4. The observation of H2 producing granule using SEM; (a) overall granule shape, (b) microbial before adding nitrate, and (c) microbial after adding nitrate.
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J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212
potassium nitrate or 2.48 mmol/h of nitrate is fed to the reactor. In order to remove 2.48 mmol/h of nitrate, 0.52 mmol/h of glucose must be consumed according to Eq. (1). Since the feeding rate of glucose is 2.5 g/h, (2 L/16 h) (20 g/L), or 13.8 mmol/h at the same HRT, only 3.8% of feeding glucose is consumed. Also, the increase of CO2 due to nitrate addition is 3.10 mmol/h. Therefore, the concentration changes of glucose and carbon dioxide caused by nitrate addition were not thought to affect the metabolism related to hydrogen production. According to the theoretical hypothesis, the intermediates during denitrification such as NO and N2O may have repressed methanogenic bacteria [12]. The ratio of electron donor to electron acceptor is a very important parameter in the repressive effect of nitrate on methanogenic bacteria. As the ratio is increased, more nitrate is reduced to nitrogen gas and nitrogen oxides inhibiting methannogenic bacteria decrease in the solution. On the contrary, under low ratio condition [12], the intermediates nitrogen oxides in the solution can repress the bacteria. If the hypothesis is correct, the ratio of electron donor to electron acceptor should be decreased to induce the nitrogen oxide intermediates in the reactor and to repress the formation of methane gas [12]. This is how the addition of nitrate was thought to decrease the ratio and to repress methanogenic bacteria in this study. 3.4. Observation of granular sludge using a SEM Granular sludge concentration as high as 2000 mg/L was maintained in the reactor throughout the operation. Since the density of granular sludge was higher than digester sludge, the loss of microorganisms due to low HRT and continuous operation was almost negligible. The granular sludge was taken out after 70 days of operation and observed using a SEM as shown in Fig. 4. The overall figure of the granule with 100 magnifications appeared roughly spherical as shown in Fig. 4(a). The SEM images of granular sludge before and after adding nitrate with 4000 magnification are shown in Fig. 4(b) and (c), respectively. The photograph showed that the microorganisms looking like noodles were predominant in the
granules after nitrate addition, and they are believed to be the hydrogen-producing bacteria [7]. Acknowledgement The present research has been conducted by the Research Grant of Kwangwoon University. References [1] Hawkes FR, Dinsdale R, Hawkes DL, Hussy sI. Sustainable fermentative hydrogen production: challenges for process optimization. Int J Hydrogen Energy 2002;27:1339–47. [2] Ueno Y, Kawai T, Sato S, Otsuka S, Moritimo M. Biological production of hydrogen from cellulose by natural anaerobic microflora. J Ferm Bioeng 1995;79(4):395–7. [3] Fang HHP, Liu H. Effect of pH and hydrogen production from glucose by a mixed culture. Bioresour Technol 2002;82:87–93. [4] Inanc B, Matsui S, Ide S. Proprionic acid accumulation and controlling factors in anaerobic treatment of carbohydrate: effect of H2 and pH,. Water Sci Technol 1996;34(5–6):317–25. [5] Lay JJ. Modelling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol Bioeng 2000;68(3):269–78. [6] Andel JGV, Zoutberg GR, Crabbendam PM, Breure AM. Glucose fermentation by Clostridium butyricum grown under a self generated gas atmosphere in chemostat culture. Microbiol Biotechnol 1985;23:21–6. [7] Kim JO, Kim YH, Ryu JY, Song BK, Kim IH, Yeom SH. Immobilization methods for continuous hydrogen gas production: biofilm formation versus granulation. Process Biochem 2005;40:1331–7. [8] Macleod FA, Guiot SR, Costerton JW. Layered structure of bacterial produced in an upflow anaerobic sludge bed and filter reactor. Appl Environ Microbiol 1990;56(6):1598–607. [9] Han SK, Shin HS. Biohydrogen production by anaerobic fermentation of food waste. Int J Hydrogen Energy 2004;29:569–77. [10] Wastewater Engineering, Metcalf & Eddy, 3rd ed., McGraw-Hill; 1991. p. 420–5. [11] Clarens M, Bernet N, Delgenes JP, Moletta R. Effect of nitrogen oxides and denitrification by Pseudomonas stutzeri on acetotrophic methanogenesis by Methanosarcina mazei. FEMS Microbiol Ecol 1998;25:271–6. [12] Roy R, Conrad R. Effect of methanogenic precursors (acetate, hydrogen, propionate) on the suppression of methane production by nitrate in anoxic rice field soil. FEMS Microbiol Ecol 1999;28:49–61. [13] Wastewater Engineering, Metcalf & Eddy, 3rd ed., McGraw-Hill; 1991. p. 711–26.
Short communication
Enhancing continuous hydrogen gas production by the addition of nitrate into an anaerobic reactor Jeong Ok Kim a,c, Yong Hwan Kim a,b,*, Sung Ho Yeom d, Bong Keun Song a,*, In Ho Kim c a
Korea Research Institute of Chemical Technology, PO Box 107, Yusong, Taejon 305-600, Republic of Korea Department of Chemical Engineering, Kwangwoon University, Nowon, Seoul 139-701, Republic of Korea c Department of Chemical Engineering, Chungnam National University, Yusong, Taejon 305-764, Republic of Korea d Department of Environmental and Applied Chemical Engineering, Kangnung National University, Gangneung, Gangwondo, Republic of Korea b
Received 29 March 2005; received in revised form 29 October 2005; accepted 23 November 2005
Abstract Hydrogen gas was produced using granular sludge of mixed culture from sewage digester sludge. Methane gas was detected in the biogas even under acidogenic condition with pH 5.5–6.2 and after heat treatment at 95 8C. In order to repress methane production, nitrate known to inhibit methanogenic bacteria was added to an anaerobic reactor. When the feeding concentration of KNO3 into the reactor was 1000 mg/L and more, the yield of hydrogen gas production (H2-mol/glucose-mol) was almost 1.0 and no methane gas was produced. The nitrate was thought to repress methanogenic bacteria remarkably and this was confirmed using a SEM. # 2005 Elsevier Ltd. All rights reserved. Keywords: Granular sludge; Mixed culture; Hydrogen gas; Methane; Heat treatment; Nitrate
1. Introduction Hydrogen has been produced through naphtha thermalcracking or water electrolysis, which requires much energy and emits global warming gases such as carbon dioxide. As the concerns over environment are growing, green process for hydrogen production is widely studied [1]. One of the most promising methods is that the gas is produced from annually renewed biomass (plant resources) [2]. In an anaerobic process using biomass, organic materials are converted to the final product, methane gas. The anaerobic fermentation process is composed of two steps, acid-generation and methane-generation one. Each step has different complex microbial interactions and is established at a different optimal pH condition for the step [3,4]. Researches on the control of pH for hydrogen gas production, the main product in the acid generation step, have been performed [5,6]. Heat treatment, boiling sludge for 15 min, to inhibit the activity of hydrogenotrophic bacteria
which cannot form spores was also performed. [5]. However, these approaches may not be effective in the long-term continuous operation. It is critical to maintain high density of hydrogen-production bacteria in a reactor to produce the gas stably without methane gas production. Two immobilization methods to maintain high concentration of sludge in the reactor were developed in the previous study [7]; biofilm formation on the hydrophilic carriers and granulation using organicinorganic hybrid polymers. Although the immobilization was able to protect sludge loss and to maintain high concentration of bacteria, methanogenic bacteria were not repressed efficiently and methane gas was detected in a biogas, which degraded the quality of hydrogen gas severely. In this research, nitrate was added to repress the activity of methanogenic bacteria and to produce high concentration of hydrogen gas continuously. 2. Materials and methods 2.1. Sludge and synthetic wastewater
* Corresponding author. Tel.: +82 2 940 5675; fax: +82 42 860 7649. E-mail addresses: [email protected] (Y.H. Kim), [email protected] (B.K. Song). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.11.017
Mixed culture was obtained from sewage digester sludge at the city of Daejeon in Korea, of which solid concentration and ash content were around 43,000 mg/L and 20%, respectively. The composition of synthetic wastewater
J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212 was: 0.44 g/L KH2PO4, 1.8 g/L NaCl, 4.7 g/L (NH4)2SO4, 0.09 g/L MgSO47H2O, 0.09 g/L CaCl2, 0.5 g/L CysteinHCl, 4.0 g/L NaHCO3, 20 g/ L glucose. The glucose was a main carbon source and COD:N:P ratio was 100:2.5:0.5. After the sewage sludge was treated at 95 8C for 15 min and cooled down to room temperature, granular sludge was prepared as described in the next section. The granular sludge occupied 25% of working volume of the reactor. The anaerobic reactor harboring granular sludge was made of Pyrex and operated at 37 8C The working volume of the reactor was 2 L. In order to repress the activity of methanogenic bacteria in the granular sludge, the pH in the reactor was adjusted to 5.8–6.2 for acid-production condition by intermittently feeding 1N NaOH.
2.2. Preparation of granular sludge Sludge itself has the negative charge of 26 mV. In order to make the sludge neutral, high molecular cationic polymer (cationic polyacrylamide, M.W.: 15,000,000) of 0.5% (w/w) of dry sewage digester sludge was added to the sludge and stirred at 400 rpm for 2 min. Since the terminals of residual cation may cause detrimental effect on the microorganisms, anionic organic material (anionic silica sol), less than 20 nm in diameter, of 0.7% (w/w) of dry sewage digester sludge was added and stirred at 200 rpm for 2 min. The total time required for forming granules of 1–3 mm in diameter, thus, was just about 5 min. The schematic procedure is shown in Fig. 1 [7].
2.3. Analytical method HRT (hydraulic retention time) control started after 48 h of operation and the HRT was stepwise decreased by increasing the influent after confirming that over 90% of glucose was consumed. The continuous operation started shortly after biogas began to be produced. The effluent from the reactor was taken every 24 h to analyze pH, ORP (oxidation-reduction potential), CODcr (Chemical oxidation demand determined by the oxidative action of K2Cr2O7), MLSS (mixed liquor suspended solids), NO3–N and residual glucose. The pH and ORP were measured using a portable pH meter (Orion, 520A, USA). MLSS representing microorganism growth was measured according to the standard method. CODcr and NO3–N was measured through Hach method (Hach, DR/
1209
2010, USA). Residual glucose was colorimetrically measured using an enzyme kit (Glucose assay kit, Sigma, USA). The amount of biogas produced by the microorganisms was measured according to a water displacement method [7] and the composition of the biogas was analyzed using a GC (Gas Chromatography, DS 6200) equipped with a TCD (Thermal Conductivity Detector) and a HayesepQ column (80/100 mesh). The operating temperature of the GC was 25 8C for the oven, 120 8C for the detector and 90 8C for the injector. Argon gas was used as a carrier gas and its flow rate was 30 mL/min. The granular sludge was washed and fixed at 4 8C for 24 h in the fixing solution composed of 5% glutaraldehyde in anaerobic cacodylate buffer. The sludge was then lyophilized in liquid nitrogen to be cut. After dehydrated using 20, 50, 80 and 90% ethanol sequentially, the cross-section of the granular sludge was observed using a SEM (Scanning Electron Microscope, Philips XL30S FEG, The Netherlands) [8]. An anaerobic CSTR (continuous stirred tank reactor) was used for hydrogen production in this study. The agitation speed was 110 rpm in the reactor. A Utube was attached to the effluent of the reactor for continuous substrate feeding and gas-stop.
3. Results and discussion 3.1. pH control The anaerobic reactor containing the granular sludge was operated for 48 days with the load of 35 kg COD/m3/day under anaerobic condition. In order to maintain organic loads constantly, the influent glucose concentration was decreased as decreasing HRT. The hydrogen production yield (mol-H2/ mol-glucose) using a mixed culture at optimal pH condition of 5.5 was reported to be 2.1 0.1 [3]. However, the results did not mention the operation duration, they just showed glucose conversion, the biogas composition and the yield of hydrogen production at each pH [3]. Although the stable production of
Fig. 1. Schematic procedure of granulation (a) shows the state of initial sludge where filamentous microorganisms and flock forming microorganisms coexist, (b) shows the initial flock after adding cationic polymer to the sludge. The flock is soon disintegrated by the shear force from a mixer and a large portion of cationic polymer not involved in the flock formation remains on the surface of microorganisms as shown in (c). The microorganisms are reflocked by the addition of silica. Also, the silica penetrates into the flock and neutralizes excess positive charge of cationic polymer, which results in the formation of compact flock as shown in (d).
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J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212
Fig. 2. The profile of biogas production (a) *: hydrogen gas production rate, ~: yield (mol-H2/mol-glucose), –: HRT (b) *: hydrogen, *: methane, !: carbon dioxide, —: HRT.
hydrogen gas and repression of methanogenic bacteria was possible at pH 5.5, long-term continuous operation resulted in the re-growth of methanogenic bacteria, which led to increase of methane content in the biogas [2,3] The hydrogen gas
production, yield, biogas composition during pH-stat operation at different HRTs is shown in Fig. 2. The high methane content of 40–60% was maintained for 37 days of operation despite the pH control suitable for acid-production. After 37 days of
Fig. 3. Effect of nitrate addition on the composition of biogas *: hydrogen, *: methane, !: carbon dioxide, —: HRT.
J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212
operation, methanogenic bacteria were washed out due to as short as 4 h of HRT [9]. As consequence, methane production was decreased and the hydrogen gas content in the biogas increased to 40% as shown in Fig. 2(b). In spite of high hydrogen content in the biogas, the rate of hydrogen gas production was decreased due to low microbial population as shown in Fig. 2(a). The results implied that the control of pH and HRT was not enough to repress the activity of methanogenic bacteria completely. 3.2. Repression of methanogenic bacteria by the addition of nitrate After 50 days of operation, nitrate was added into the reactor for both repressing methane production and obtaining high concentration of hydrogen gas. The methanogenic bacteria are known to be repressed under aerobic condition or semianaerobic condition [10]. Since extreme aerobic condition may also cause negative effect on the acid-generation step, semianaerobic condition is thought to be favorable. Because the ORP value in the reactor gives an idea of oxidation–reduction state of the fermentation, the effect of nitrate on the ORP was also considered (see below). The average values of hydrogen gas production, yield (mol-H2/mol-glucose), MLSS, ORP level at various nitrate concentrations are shown in Table 1. The maximum hydrogen gas production yield was 1.3 at 1000 mg/L of KNO3 in the reactor. The range of fluctuation of hydrogen gas production at various KNO3 concentrations was 30 ml/L/h and no methane was detected after KNO3 addition. The ORP level was 206 mV, higher than that under anaerobic condition, 340 mV, and the ORP level was slightly increased with increasing nitrate concentration. The results implied that the addition of nitrate influenced the ORP level [11]. Recent research showed that even the ORP as high as +420 mV was not able to repress the growth of methanogenes and that methane production by pure Methanosarcina barkeri under semianaerobic condition was not suppressed [12]. Also, the ORP level was maintained at 290 mV when nitrate was added to the anaerobic digester. Therefore, the repression of methane production by the addition nitrate cannot be explained in terms of ORP change. The composition change of biogas due to nitrate addition is shown in Fig. 3. The content of methane gas in the biogas started to be decreased gradually shortly after nitrate addition and reached to 0% 6 days after the addition. Although the concentration of nitrate was constant (1000 mg/L) in the feeding solution, the anaerobic condition was maintained in the reactor with HRT of 16 h.
1211
3.3. The changes in the anaerobic reactor after nitrate addition Since the influent C/N ratio was over 7 and since more than 99% of nitrate was removed in the reactor in this study, it can be supposed that denitrification with the consumption of glucose occurred when nitrate was added. The related theoretical equation for the denitrification was suggested to be [13]: 24NO3 þ 5C6 H12 O6 ! 12N2 þ 30CO2 þ 18H2 O þ 24OH (1) When the maximum concentration of feeding potassium nitrate is 2000 mg/L with HRT 16 h as shown in Fig. 4, 250 mg/h of
Table 1 The characteristics of fermentation for hydrogen gas production at different nitrate concentrations KNO3 (mg/L)
H2 production rate (mL/L/h)
Yield (mol-H2/ mol-glucose)
MLSS (mg/L)
ORP (mV)
0 500 1000 2000
49.7 101.6 132.2 123.4
0.4 0.6 1.0 0.8
4098 2200 2376 3730
221.3 199.4 198.0 168.0
Fig. 4. The observation of H2 producing granule using SEM; (a) overall granule shape, (b) microbial before adding nitrate, and (c) microbial after adding nitrate.
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J.O. Kim et al. / Process Biochemistry 41 (2006) 1208–1212
potassium nitrate or 2.48 mmol/h of nitrate is fed to the reactor. In order to remove 2.48 mmol/h of nitrate, 0.52 mmol/h of glucose must be consumed according to Eq. (1). Since the feeding rate of glucose is 2.5 g/h, (2 L/16 h) (20 g/L), or 13.8 mmol/h at the same HRT, only 3.8% of feeding glucose is consumed. Also, the increase of CO2 due to nitrate addition is 3.10 mmol/h. Therefore, the concentration changes of glucose and carbon dioxide caused by nitrate addition were not thought to affect the metabolism related to hydrogen production. According to the theoretical hypothesis, the intermediates during denitrification such as NO and N2O may have repressed methanogenic bacteria [12]. The ratio of electron donor to electron acceptor is a very important parameter in the repressive effect of nitrate on methanogenic bacteria. As the ratio is increased, more nitrate is reduced to nitrogen gas and nitrogen oxides inhibiting methannogenic bacteria decrease in the solution. On the contrary, under low ratio condition [12], the intermediates nitrogen oxides in the solution can repress the bacteria. If the hypothesis is correct, the ratio of electron donor to electron acceptor should be decreased to induce the nitrogen oxide intermediates in the reactor and to repress the formation of methane gas [12]. This is how the addition of nitrate was thought to decrease the ratio and to repress methanogenic bacteria in this study. 3.4. Observation of granular sludge using a SEM Granular sludge concentration as high as 2000 mg/L was maintained in the reactor throughout the operation. Since the density of granular sludge was higher than digester sludge, the loss of microorganisms due to low HRT and continuous operation was almost negligible. The granular sludge was taken out after 70 days of operation and observed using a SEM as shown in Fig. 4. The overall figure of the granule with 100 magnifications appeared roughly spherical as shown in Fig. 4(a). The SEM images of granular sludge before and after adding nitrate with 4000 magnification are shown in Fig. 4(b) and (c), respectively. The photograph showed that the microorganisms looking like noodles were predominant in the
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