international journal of hydrogen energy 34 (2009) 61716180

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Optimizing hydrogen production from organic wastewater treatment in batch reactors through experimental and kinetic analysis
Yogesh Sharma, Baikun Li*
Department of Civil and Environmental Engineering University of Connecticut, Storrs, CT 06269, USA

article info
Article history: Received 9 June 2009 Accepted 13 June 2009 Available online 5 July 2009 Keywords: Hydrogen production Anaerobic wastewater treatment Chemical oxygen demand pH Specic hydrogen yield Hydrogen production rate

abstract
Anaerobic hydrogen production from organic wastewater, an emerging biotechnology to generate clean energy resources from wastewater treatment, is critical for environmental and energy sustainability. In this study, hydrogen production, biomass growth and organic substrate degradation were comprehensively examined at different levels of two critical parameters (chemical oxygen demand (COD) and pH). Hydrogen yields had a reverse correlation with COD concentrations. The highest specic hydrogen yield (SHY) of 2.1 mole H2/mole glucose was achieved at the lowest COD of 1 g/L and decreased to 0.7 mole H2/ mole glucose at the highest COD of 20 g/L. The pH of 5.56.0 was optimal for hydrogen production with the SHY of 1.6 mole H2/mole glucose, whereas the acidic pH (4.5) and neutral pH (6.07.0) lowered the hydrogen yields. Under all operational conditions, acetate and butyrate were the main components in the liquid fermentation products. Additionally, a comprehensive kinetic analysis of biomass growth, substrate degradation and hydrogen production was performed. The maximum rates of microbial growth (mm) and substrate utilization (Rsu) were 0.03 g biomass/g biomass/day and 0.25 g glucose/g biomass/day, respectively. The optimum pH for the rate of hydrogen production (RH2 ) and SHY were 5.89 and 5.74 respectively. Based on the kinetic analysis, the highest RH2 and SHY for batchmode anaerobic hydrogen production systems were projected to be 13.7 mL/h and 2.32 mole H2/mole glucose. Published by Elsevier Ltd on behalf of International Association for Hydrogen Energy.

1.

Introduction

Wastewater contains signicant amount of organic substances (i.e. carbohydrates) that can be converted to energy (i.e. methane, hydrogen). Hydrogen is expected to be a substitute for fossil fuel as a clean energy resource, since it only generates water when burning [1]. In the two-phase anaerobic treatment of wastewater, hydrogen is produced in the acidogenesis phase (Reaction (1)), which is much faster and more resistant to environmental shocks than the

following methanogenic phase (Reaction (2)). The fermentation liquid products are mainly volatile fatty acids (VFAs) such as acetic acid, propionic acid and butyric acid that are easily biodegradable. Several bacteria species (i.e. Clostridium pasteurianum) have been found to degrade glucose and produces hydrogen along with acetate and butyrate (Reactions (1) and (3)) [2].

C6H12O6 2H2O / 2CH3COOH 4H2 2CO2

(1)

* Corresponding author. Tel.: 860 486 2339; fax: 860 486 2298 E-mail address: baikun@engr.uconn.edu (B. Li). 0360-3199/$ see front matter Published by Elsevier Ltd on behalf of International Association for Hydrogen Energy. doi:10.1016/j.ijhydene.2009.06.031

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Nomenclature VFAs SHY k X S Ks m RH2 Rm Rsu K1, K2 Ki [H] volatile fatty acids specic hydrogen yield, mole H2/mole glucose maximum specic substrate utilization rate, g glucose/g biomass/day biomass concentration, g/L chemical oxygen demand, g/L half velocity constant, g/L specic growth rate constant, g biomass/ g biomass/day rate of hydrogen production, mL/h maximum rate of hydrogen production, mL/h rate of substrate utilization, g glucose/ g biomass/day, respectively protonation and deprotonation equilibrium constants respectively, mole/L inhibition constant, g/L hydrogen ion concentration, mole/L

hydrogen production, different operational conditions substantially alter the rates of hydrogen production, substrate utilization and biomass growth. There is no kinetic study describing the effects of COD and pH on the rate of hydrogen production (RH2 , mL/h) and specic hydrogen yield (SHY, mole H2/mole glucose) using mixed cultures. There are two-fold objectives of this study to enhance the understanding of hydrogen production from organic wastewater treatment. First, two critical operational parameters (COD and pH) were examined at different levels in batch systems to determine their effects on hydrogen production and substrate removal. The quantitative correlations between hydrogen production and operational parameters were determined. Second, a comprehensive kinetic analysis, elucidating the effect of operational parameters on RH2 , SHY, and substrate removal (Rsu) was performed using Michaelis Menton kinetic analysis. The maximum biomass growth rate (mm) was determined using the Monod kinetic analysis.

2.
2.1.
CH3COOH / CO2 CH4 (2)

Material and method

Batch-mode hydrogen production system setup

C6H12O6 / CH3CH2CH2COOH 2H2 2CO2

(3)

Anaerobic hydrogen production is critical for environmental and energy sustainability. In the past two decades, tremendous efforts have been put to investigate a variety of inoculums (i.e. activated sludge, soil, pure cultures) [35], substrates (i.e. wastewater, starch, beet sugar, cellulose) [68], anaerobic fermentation pathways [9,10], and bioreactors (i.e. anaerobic bafed reactors, upow reactors, continuous stirred tank reactors) [1113]. Currently, there are three problems of hydrogen production from anaerobic wastewater treatment. First, the experimental hydrogen production is much lower than the theoretical values. The theoretical specic hydrogen yield (SHY) is 4 mole H2/mole glucose when acetate is the sole fermentation product (Reaction (1)). The yield decreases to 2 mole H2/mole glucose when butyrate is the sole fermentation product (Reaction (3)). But the experimental hydrogen yields are only 0.92.0 mole H2/ mole glucose [6,14]. Second, the accumulation of acidic fermentation products in the solution and hydrogen partial pressure in the headspace is known to inhibit bacterial activities, suppress fermentation and reduce hydrogen yields [2527]. Some engineering parameters (i.e. COD, pH, temperature, and hydraulic retention time) have been studied individually for their effects on hydrogen production [5,9,12]. But the understanding of the hydrogen production under different operational conditions is still very limited. Third, there have been some preliminary kinetic studies of hydrogen production using pure cultures of Enterobacter sp. and Clostridium sp. in batch mode [1524]. A comprehensive kinetic analysis of the effects of engineering parameters (i.e. COD and pH) on hydrogen production, substrate utilization and biomass growth is limited, especially for the mixed cultures (i.e. activated sludge). In a biochemical process such as anaerobic

Serum bottles (volume: 150 mL) (Wheaton Scientic, Millville, NJ) were used as batch reactors in this study. The reactors were lled to 125 mL with a glucose solution as the organic substrate. An inorganic medium (consisting of per L of water: 2.0 g NH4HCO3, 1.0 g KH2PO4, 100 mg MgSO4$7H2O, 10 mg NaCl, 10 mg Na2MoO4$2H2O, 10 mg CaCl2$2H2O, 15 mg MnSO4$7H2O, and 2.78 mg FeCl2) was added to the solution as supplementary micronutrients for bacteria to grow [6]. The fraction of headspace (17% of the bottle volume) was designed to reduce the duration of pressure build-up in the lag period and was consistent with other studies with the similar reactor conguration [6]. The solution was mixed in the serum bottles and then buffered with 0.05 M 2-(N-morpholino) ethanesulfonic acid monohydrate (MES; J.T. Baker, Phillipsburg, NJ). The pH of the solution was adjusted using 1 M KOH. The solution was sparged with nitrogen for 15 min to remove oxygen completely. The bottles were then capped with rubber septum stoppers (Wheaton Scientic, Millville, NJ). The serum bottles were constantly stirred at 250 rpm. The soil collected from an organic farm was used as the inocula. Due to the effectiveness of heat shock treatment in inhibiting methanogens and selecting for hydrogen-producing bacteria, the soil was heated shocked at 100  C for 2 h before being added into the batch reactors [6,28]. The soil concentration in the batch reactors was kept at 20 g/L for all tests.

2.2.

Operational condition

Two critical operational parameters including COD and pH were tested individually. COD concentration varies with wastewater sources (e.g. storm runoff, agricultural wastewater) and pH varies with acidic fermentation products in anaerobic systems. All these variations will affect the populations and activities of hydrogen-producing bacteria. Five levels of COD (120 g/L) and ve levels of pH (4.57.0) were examined with only one parameter being changed under each

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condition, leading to the total eleven operational conditions. A baseline of COD concentration of 5 g/L and pH of 5.5 was selected for this study. All tests were conducted at 30  C.

2.3.

Control bottle tests

used (Reaction (4)) [33]. E represents the active enzyme form, while E and E2 are inactive enzyme forms that are obtained by the protonation and deprotonation of E. The Michaelis pH function (Equation (3)), derived by determining the fraction of active enzyme concentration (E), was used to analyze the dependence of RH2 on pH [33].
H H E % E % E2 H H K1 K2

Since soil contains a variety of organic substances that can be utilized by anaerobic bacteria, a control test without the addition of glucose was conducted under each operational condition in order to identify the contribution of these organic substances to hydrogen production. The actual hydrogen volume generated from the degradation of glucose equals the total hydrogen volume generated in the batch reactors (with glucose being added) minus the hydrogen volume generated in the control bottles (without glucose being added).

(4)

  2 K1 H H =K2 RH2 Rm H

(3)

2.4.

Biogas volume measurement

The biogas was collected periodically from the headspace of the batch reactors [29]. The total volume of biogas was measured by inserting a glass syringe with a capacity of 20 or 100 mL (Perfektum Syringes; Popper & Sons, Inc., NY) into the headspace of the batch reactor and allowing biogas to ow into the syringe against atmospheric pressure. The volume reading was taken when an equilibrium status was achieved inside the syringe.

where Rm is maximum RH2 (mL/h), K1 and K2 are equilibrium constants (moles/L) respectively (Reaction (4)), and [H] is hydrogen ion concentration in the solution (moles/L). To describe the effect of pH on SHY, the model of active ionization state was used in formulation of MichaelisMenton function (Equation (4)).   2 K1 H H =K2 SHY SHYm H (4)

where SHYm is the maximum SHY (mole H2/mole glucose) To illustrate the effect of pH on Rsu, Equation (5) was used.   2 Rsu Rsum H K1 H H =K2 (5)

2.5.

Analytical methods

where Rsum is the maximum Rsu (g glucose/day). The optimum pH for RH2 , SHY, and Rsu was calculated using Equation (6) [33]. h i pH log K1 K2 1=2 (6)

The biogas in the headspace was sampled with a gas tight syringe with a capacity of 50 mL (SGE, Illinois) and analyzed using a gas chromatograph (Agilent 6890N) equipped with a packed column and a thermal conductivity detector. The glucose concentration was measured by performing the phenol-sulfuric acid assay with a spectrophotometer (Cary 50 Bio; Varian, CA) [30]. The pH of the solution in the batch reactors was measured at the end of each test using an Ag/AgCl reference electrode (Accumet AP72). The pH of organic soil was measured according to the standard method [31]. The biomass concentration was measured using the total solids measurement technique according to the standard method [32].

The Monod kinetic analysis of substrate utilization and biomass growth were performed in this study. For substrate utilization kinetics, the Equation (7) was used [34,35]. Rsu k X S=Ks S (7)

where k is maximum Rsu (g substrate/g biomass/day), X is biomass concentration (mg/L), S is COD in solution (mg/L), and Ks is half velocity constant (mg/L). For microbial growth analysis, the Equation (8) was employed. m mm S=Ks S (8)

2.6.

Kinetic analysis

To describe the effect of COD concentrations on RH2 , Andrews inhibition model was used (Equation (1)) [15]. R Rm S= Ks S S2 =Ki (1)

where Rm is the maximum rate of hydrogen production (mL/h), Ks is the half saturation constant (g/L), Ki is the inhibition constant (g/L), and S is COD concentration (g/L). To describe the effect of COD concentrations on SHY, the MichaelisMenton kinetic model was employed [33]. SHY SHYm S=Ks S (2)

where mm is maximum specic growth rate (1/day). All the kinetic analyses were performed using GraphPad software. In addition, the regression analyses were performed by the statistical software MINITAB to illustrate the effects of different operational conditions (COD and pH) on hydrogen production.

3.

Results and discussion

3.1. Effect of substrate concentration (COD) on specic hydrogen yield (SHY)

Substrate concentration is an important parameter in optimizing the hydrogen yields due to its signicant effects on bacterial growth and enzymatic activity [36]. In the control

where SHYm is the maximum SHY (mole H2/mole glucose), S is COD concentration (g/L), Ks is half saturation constant (g/L). To describe the effect of pH on RH2 , SHY and substrate utilization rate (Rsu), a model of active ionization state was

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tests without the addition of glucose, hydrogen production (w5 mL) was still detected in the batch reactors due to the degradation of organic substances contained in soil. The actual specic hydrogen yields (SHY, mole H2/mole glucose) had a reverse correlation with COD concentrations (Equation (9)) (Fig. 1). The correlation coefcient (R2) of 0.97 indicates the high adequacy of correlation. The p-value for the COD effects was 0.028, supporting the difference of yields at different COD concentrations at 95% condence interval. SHY 2:380 0:1601 COD 0:003956 COD
2

5.5

4.5

pH
4 Initial pH 3.5

(9)

Final pH

The SHY values decreased from 2.1 to 0.7 mole H2/mole glucose and the hydrogen content decreased from 55% to 32% when the glucose concentration increased from 1 to 20 g/L. There are three possible reasons for the reverse relationship between hydrogen production and substrate concentrations. First, the accumulation of liquid fermentation products at higher substrate concentrations resulted in the over-acidication of bacterial cultures and the inhibition of fermentation [37]. A lower substrate concentration reduced the accumulation of the acidic fermentation production, and led to higher specic hydrogen yields. The measurement of volatile fatty acids (VFAs) showed that 7.3 g/L of total VFAs were detected at the highest substrate concentration of 20 g/L, while only 0.2 g/L VFAs were detected at the substrate concentration of 1 g/L. Second, the pH of the fermentation solution substantially decreased with higher substrate concentrations. The initial pH of the solution was set at 5.5 for all substrate concentrations. The pH dropped sharply to 3.5 at the substrate concentration of 20 g/L, whereas the pH slightly decreased to 5.4 at the substrate concentration of 1 g/L (Fig. 2). Third, the total hydrogen production (mole H2) increased at high substrate concentrations, which led to the high partial pressure of hydrogen in the headspace throughout the batch test period. The accumulation of hydrogen in the batch reactors inhibited the fermentation reactions, and thus lowered the specic hydrogen yields [38]. The SHY values obtained in this study (1.62.1 mole H2/ mole glucose) were much higher than the values previously reported (0.92 mole H2/mole glucose at a COD concentration of

3 0 5 10 15 20

COD (g/L)
Fig. 2 The changes of pH at different COD concentrations before and after batch tests.

4 g/L) [6]. The higher SHY in this study might be caused by the higher temperature (30  C) than their studies (26  C). It has been shown that increasing the temperature within mesophilic range has a positive effect on hydrogen yields.

3.2.

Effect of pH on specic hydrogen yield (SHY)

60

Specific Hydrogen Yield ( mole Hydrogen/mole Glucose)

2.35 2.1 1.85 1.6 1.35

Specific Hydrogen Yield Hydrogen Content R2 = 0.97

50 40 30 20

The pH is an important operational parameter for hydrogen production, since it affects anaerobic metabolic pathways and the activities of hydrogenase enzymes [39,40]. The SHY values were the lowest (1.2 mole H2/mole glucose) at the pH of 4.5, which indicated that the acidic condition inhibited the fermentation and hydrogen production. The SHY values substantially increased to 1.55 mole H2/mole glucose at the pH of 5.5 and 6.0. However, the SHY values decreased to 1.3 and 1.25 at the higher pH of 6.5 and 7.0 (Fig. 3). It had been found that under the neutral pH condition, a signicant amount of substrates were consumed by bacterial growth other than hydrogen production [14], which was veried by the higher biomass concentration at higher pH than at lower pH. This could explain the lower hydrogen yield at neutral pH. The correlation between SHY and pH was established by nonlinear regression analysis, yielding a cubic correlation (Equation (10)). The correlation obtained in this study adequately described the hydrogen yield as a function of pH with a correlation coefcient of 0.93 (Fig. 3). SHY 29:61 15:39 pH 2:493 pH2 0:1319 pH3 (10)

Hydrogen Content (%)

1.1 0.85 0.6 0 5 10 15 20 25 10 0

COD (g/L)
Fig. 1 The specic hydrogen yield (SHY, mole hydrogen/ mole glucose) and the hydrogen content in biogas (%) at different COD concentrations (pH: 5.5, temperature: 30 8C).

The low pH has been known to suppress the activity of hydrogenase [41,42]. A pH of 5.5 has been reported to be ideal for hydrogen production [14,26], while the hydrogen yield at 6.0 was slightly higher than at the pH of 5.5 in this study. Because different types of bacterial inoculums such as activated sludge, soil and pure culture (i.e. Clostridium sp) were used in these studies, the optimal pH for hydrogen production changed slightly with bacterial inoculums. Nevertheless, this study indicated that for the batch reactors inoculated with soil, a pH of 5.56.0 was optimal for hydrogen production. The initial and nal pH values of the solution were compared under each pH condition (Fig. 4). When the initial

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1.6

65 R2 = 0.93

Specific Hydrogen Yield (mole Hydrogen/mole Glucose)

resulted in higher hydrogen content in biogas than those at the lower pH.

1.5 60 1.4

Hydrogen Content (%)

3.3. The correlation between hydrogen production and glucose degradation

The hydrogen production volume (mL) in the headspace was measured periodically and correlated with the glucose degradation efciency (%) in the solution. In the COD-level tests (pH: 5.5, temperature 30  C), there was a lag period of 25 h in all batch reactors before the biogas production was detected. After the lag period, the hydrogen production increased exponentially (Fig. 5a). It was clearly shown that the initial glucose concentrations determined the duration of exponential phase. For instance, at the low concentrations of 1 and 5 mg/L, the duration of the exponential phase was 30 h. At the middle concentration of 10 mg/L, the duration increased to 55 h. At the high concentration of 1520 g/L, the duration reached 88 h. The increase in the duration period of the exponential phase was caused by the availability of organic substrates at high concentrations. After the exponential phase, the hydrogen production began to decline. At the 262nd hour, the hydrogen production stopped in all reactors. In terms of glucose consumption, at the 72nd hour, 95% of glucose was degraded when the initial glucose concentrations were 1 and 5 g/L, 90% of glucose was degraded when the initial

1.3 55 1.2

1.1 4 4.5 5 5.5 6 6.5 7 7.5

50

pH
Specific Hydrogen Yield Hydrogen Content

Fig. 3 The specic hydrogen yield (SHY, mole hydrogen/ mole glucose) and the hydrogen content in biogas (%) at different pH (COD: 5 g/L, temperature: 30 8C).

pH was neutral (6.57.0), the nal pH was 1.52.0 lower than the initial pH. When the initial pH was 4.5, the nal pH was only 0.1 lower than the initial pH. The difference between the pH values demonstrated that the fermentation reactions produced a signicant amount of acidic liquid products. At the higher pH, the fermentation proceeded thoroughly and generated high amounts of VFAs, which led to the signicant drop of pH in the solution. At the lower pH, the fermentation production was inhibited, which produced less amounts of acidic liquid products and led to the slight drop of pH values. The effects of the acidic liquid products on the pH were well correlated with the SHY values. The hydrogen content of biogas was 5157% at the pH range of 4.56.5 and increased to 63% at highest pH of 7.0 (Fig. 3), with the exception of 51% at the pH of 6.0. The soil tested had the pH of 6.8, which indicated that the hydrogenproducing bacteria were acclimatized to the neutral pH in natural environment. The increased metabolic activities of the hydrogen-producing bacteria at neutral pH may have

a
Hydrogen Volume (mL)/ g COD

30 25 20 15 10 5 0 0 100 200 300 1 5 10 15 20

Time (hours)

b
Glucose Removal Efficiency (%)
7

100 90 80 70 60 50 40 30 20 10 0 0 100 200 300

Final pH

4 4 5 6 7

Time (hours)
Fig. 5 The changes of hydrogen production (mL H2/g COD) (a) and the glucose removal efciency (%) (b) at different COD concentrations during batch test periods.

Initial pH
Fig. 4 The initial and nal pH values in the batch reactors during the pH-level tests.

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concentration was 10 g/L, and 7080% of glucose was degraded when the initial concentration was 1520 g/L (Fig. 5b). Although the glucose removal efciency decreased at higher concentrations, the amounts of glucose removed were actually higher. For example, 0.994.75 g/L glucose was removed when the initial concentrations were 15 g/L, whereas 1214 g/ L glucose was removed when the initial concentrations were 1520 g/L. In the pH-level tests (COD: 5 g/L, temperature: 30  C), the rate of hydrogen production increased as the pH was increased from 4.5 to 7.0 (Fig. 6a). The pH of organic soil tested in this study was 6.8, indicating that the bacteria in the soil could acclimatize quickly at neutral pH and produce hydrogen at a faster rate. This quick acclimation at neutral pH resulted in a shorter duration of exponential phase (22 h) at the pH of 6.07.0 than those (30 h) at lower pH of 4.55.5. In terms of glucose degradation, over 97% of glucose was degraded at the pH of 7.0 after 43 h (Fig. 6b), whereas only 25% of glucose was degraded at the pH of 4.5 during the same test period. This clearly indicated that the metabolic activities of bacteria in soil were faster at neutral pH than at lower pH. After 72 h, 9599% of glucose was degraded in all reactors. No glucose was detected at the end of experiment at 262 h.

3.4.

The kinetic analysis of hydrogen production

In order to improve hydrogen production, it is critical to determine the effects of operational conditions on hydrogen production rate (RH2 ) and specic hydrogen yield (SHY). The

kinetic analysis of the effects of COD concentrations on hydrogen production indicated that RH2 increased at the COD of 15 g/L, but decreased at higher COD of 1020 g/L (Fig. 7a). This inhibition of hydrogen production at high COD was effectively demonstrated by Andrews inhibition model (Equation (1)). The maximum rate of hydrogen production (Rm) was 13.7 mL/h, which corresponded well to the reported rate of 15.1 mL/h (with glucose as the substrate and activated sludge as inoculum, [43]). Increasing the COD within the range of 5 g/L < COD < 10 g/L would increase the RH2 up to 13.7 mL/h. Beyond that point, the RH2 would start to decrease due to substrate inhibition. The Ks and Ki were 12.23 and 3.24 g/L respectively. A high correlation coefcient of 0.97 suggested the adequacy of Andrews model in describing the inhibitory effect of COD on hydrogen production. The experimental results indicated that SHY had a reverse relationship with COD concentrations, increasing from 0.7 moles H2/mole glucose at 20 g/L to 2.1 moles H2/mole glucose at 1 g/L (Fig. 1). The SHY was plotted against 1/S according to MichaelisMenton kinetics, with the projected maximum SHY of 2.32 moles H2/mole glucose (Fig. 7b). This demonstrated that decreasing the COD concentration below the lowest COD tested (1 g/L) would result in a further increase in SHY reaching a maximum of 2.32 mole H2/mole glucose. At this point, the SHY is limited by other operational parameters such as pH, temperature, stirring speed as well as biological properties of the system. Therefore, the manipulation of COD alone is not

a
Hydrogen Volume (mL)/ g COD

25 20 7 15 10 5 0 0 100 200 300 0 0 5 10 15 20 25 6.5 6 5.5 4.5

RH2 (mL/hour)

3 R2 = 0.97 2

Time (hours)

COD (g/L)

b
Glucose Removal Efficiency (%)

100 90 80 70 60 50 40 30 20 10 0 0 100 200 300

b
Specific Hydrogen Yield (mole H2/mole glucose)

2.5 2.0 1.5 1.0 0.5 0.0 0.0 0.5 1.0 1.5 R2 = 0.99

Time (hours)
Fig. 6 The changes of hydrogen production (mL H2/g COD) (a) and the glucose removal efciency (%) (b) at different pH during batch test periods.

1/COD (L/g)
Fig. 7 The kinetic analysis of COD effect on hydrogen production rate (RH2 ) (a) and specic hydrogen yield (SHY) (b).

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sufcient to achieve theoretical SHY of 4 mole H2/mole glucose. The transformed MichaelisMenton kinetic model was found to be adequate with a correlation coefcient of 0.99. The effects of pH on RH2 were modeled by Michaelis pH function (Equation (3)) (Fig. 8a). Through a non-linear regression of RH2 versus [H], the equilibrium constants K1 and K2 were found to be 4.26 108 and 3.96 105 mole/L, respectively. A high correlation coefcient of 0.85 illustrated the suitability of Michaelis pH function for analysis of RH2 . Based on Equation (6), the optimum pH of 5.89 was calculated for RH2 . This pH value was signicantly lower than the reported pH of 6.35 for hydrogen production rate from starch hydrolysate [44]. This difference in optimum pH can be attributed to use of pure culture of Enterobacter aerogenes in that study compared to mixed culture in this study. The effects of pH on SHY were modeled by Michaelis pH function (Equation (4)). Through a non-linear regression of SHY versus [H], the equilibrium constants K1 and K2 were 3.07 108 and 3.5 105 mole/L respectively with a correlation coefcient of 0.78 (Fig. 8b). The optimum pH for SHY was 5.74 (Equation (6)), which was not signicantly different than the optimum pH of 5.89 for RH2 .

3.5.

The kinetic analysis of substrate utilization

The kinetic analysis of substrate utilization rate (Rsu) was performed for different COD concentrations and pH. The Rsu

for different COD concentrations was modeled using the Monod kinetics (Equation (7)). The Rsu was calculated at each COD concentration (S ) after 43 h (1.79 days, as shown in Fig. 5a), at which point the substrate utilization was in the exponential phase for all COD concentrations. In the next experimental data point (72 h), the reactor with the COD concentration of 1 g/L had already entered the stationary phase (Fig. 5a). To calculate Rsu at different COD concentrations, it was critical to employ the duration period that all the reactors were in the same phase of substrate degradation (Exponential Phase, 43 h). Through a non-linear regression between (Rsu/X ) and S, the values of Ks (8 g/L) and k (0.25 g glucose/g biomass/day) were determined. The correlation coefcient R2 was 0.99 (Fig. 9a), suggesting the adequacy of substrate utilization model in this study. The Rsu for different pH was modeled using the Michaelis pH function (Equation (5)) (Fig. 9b). The Rsu was calculated at each pH value in the exponential phase (43 h). The Rsu increased with increase in pH from 4.5 to 7.0. A non-linear regression of Rsu versus [H] yielded the equilibrium constants K1 and K2 of 1.8 1012 and 8.2 106 mole/L respectively (Equation (5)). Based on Equation (6), an optimum pH of 8.4 was calculated for Rsu. In this model, the effect of pH range of 4.57.0 on Rsu was simulated. The experimental results showed that although Rsu increased with pH within the range of 4.57.0, the SHY started to

a
3.0

0.20

RH2 (mL/hour)

0.15

Rsu /X (day-1)

2.5

R2 = 0.85

R2 = 0.99 0.10

2.0

0.05

0.00001

0.00002

0.00003 0.00 0 5 10 15 20 25

[H+] (mole/L)

b
Specific Hydrogen Yield (mole H2/mole glucose)

COD (g/L)
1.8

b
1.6 R2 = 0.78

Rsu (g glucose/day)

1.4

4 R2 = 0.97 2

1.2

1.0 0.00000

0.00001

0.00002

0.00003

0.00004 0 0.00000 0.00001 0.00002 0.00003 0.00004

[H+]

(mole/L)

Fig. 8 The kinetic analysis of pH effect on hydrogen production rate (RH2 ) (a) and specic hydrogen yield (SHY) (b).

[H+] (mole/L)
Fig. 9 The kinetic analysis of substrate utilization rate (Rsu) (a) at different COD concentrations, (b) at different pH.

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decrease at pH > 6 (Fig. 3). It was expected that at the pH higher than 7.0, the unsuitable conditions for hydrogenic bacterial activities might decrease the Rsu. Therefore if the effects of pH > 7 on Rsu were to be incorporated in the kinetic model, the optimum pH would have been lower than the obtained optimum pH of 8.4. The correlation coefcient, R2 of 0.97 demonstrated the suitability of Michaelis pH function to describe Rsu as a function of pH. It should be noted that the optimum pH (8.4) for Rsu is signicantly higher than optimum pH (5.75.8) for RH2 and SHY. This discrepancy could be caused by the shift of substrate removal and hydrogen production at different pH. The activities of hydrogen-producing bacteria decreased at higher pH (pH > 6.0), but the metabolic activities of various anaerobic bacteria in the mixed culture were viable up to pH of 7.08.4, which result in high substrate consumption and high biomass growth at higher pH.

the existence of the organic matters in soil may lead to the underestimation of mm. In addition, the Ks value of 16.1 g/L indicated that half of maximum growth rate could be achieved at a COD concentration of 16.1 g/L. Therefore, the COD concentrations higher than 16.1 g/L would result in a higher growth of biomass and a decline of hydrogen yields, which was well corresponded with the experimental hydrogen production results (Fig. 1). Mazijat et al. reported the inhibition of biomass growth at substrate concentration of 5.4 g/L (as food, F ) [46]. The biomass concentration in their study was 1 g/L (as microorganisms, M ), which yielded a ratio of food to microorganism (F/M ) of 5.4. This high F/M ratio may result in the inhibition of biomass growth. In this study, the substrate concentrations ranged from 1.0 to 20 g/L with the inoculum concentration consistent at 20 g/L, which yielded the F:M ratios at 0.051.0. At these low F:M ratios, the biomass growth were adequately described by the Monod kinetics.

3.6.

The kinetic analysis of biomass growth

The kinetic analysis for the specic growth rate of biomass (m) was conducted in the batch reactors. The COD-level tests showed the SHY decreased at higher COD concentrations (Fig. 1), which could be caused by substrate degradation by biomass growth rather than hydrogen production. Through the non-linear regression of m versus COD concentration (S ), the values of mm (0.03 day1) and Ks (16.1 g/L) were obtained (Fig. 10). The biomass growth rate increased with COD concentrations, which testied the lower SHY at high COD (Fig. 1). The high value of correlation coefcient (R2: 0.99) suggested that Monod kinetics can be adequately used to evaluate the microbial growth, substrate utilization and hydrogen production. The mm obtained was much lower than the value (0.3 day1) for methanogenic degradation of propylene glycol [45]. The inoculum used in this study was soil, which is rich in organic matters. The control tests without the addition of glucose indicated that the organic matters in soil was degraded and resulted in biogas production (Data not shown). Since m (mg/L/d/mg/L) was obtained by dividing biomass growth rate (mg/L/d) with the initial biomass concentration (mg/L), the higher value of initial biomass with

4.

Conclusions

0.015

0.010

(day-1)

The study comprehensively investigated the enhancement of hydrogen production from organic substrates in batch reactors under different operational conditions (COD and pH). A kinetic analysis of biomass growth, substrate utilization and hydrogen production was conducted. Two major conclusions were drawn in this study. First, the hydrogen production rate and (RH2 ) and specic hydrogen yields (SHY) were clearly affected by different operational parameters. The COD-level tests showed that the SHY values were reversely related with COD concentrations, and decreased from the highest yield of 2.1 moles H2/mole of glucose at COD of 1.0 mg/L to 0.7 moles at COD of 20 g/L. The pH-level tests showed that the pH of 5.56 was ideal for hydrogen production with the SHY of 1.6 moles H2/mole of glucose. Second, the kinetic analysis reveals the correlation between hydrogen production, substrate degradation and biomass growth. The optimal pH for hydrogen production and substrate degradation was different with the optimal pH of 5.89 and 5.74 for rate of RH2 and SHY, and of 8.4 for substrate degradation. The simulated maximum RH2 was 13.7 mL/h, and the simulated maximum SHY was 2.32 mole H2/mole glucose. The maximum rates of glucose utilization (Rsu) and specic microbial growth (m) were 0.25 g glucose/g biomass/day and 0.03 g biomass/g biomass/day, respectively.

R2 = 0.99 0.005

Acknowledgement
0.000 0 5 10 15 20 25

COD (g/L)
Fig. 10 The kinetic analysis of microbial growth rate (m) at different COD concentrations.

This project was funded by the USGS and Connecticut Water Resource Center. Part of the student scholarship was supported by the Multidisciplinary Graduate Research Grant of Center of Environmental Science and Engineering (CESE). The assistance of Dr. Kenneth Noll in terms of anaerobic microbiology was appreciated.

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