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Inhibitory effect of ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production
international journal of hydrogen energy 33 (2008) 7013–7019
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Inhibitory effect of ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production Bo Wang, Wei Wan, Jianlong Wang* Laboratory of Environmental Technology, INET, Tsinghua University, Beijing 100084, PR China
article info
abstract
Article history:
The inhibitory effect of added ethanol, acetic acid, propionic acid and butyric acid on
Received 14 August 2008
fermentative hydrogen production by mixed cultures was investigated in batch tests using
Received in revised form
glucose as substrate. The experimental results showed that, at 35 C and initial pH 7.0,
13 September 2008
during the fermentative hydrogen production, the substrate degradation efficiency,
Accepted 13 September 2008
hydrogen production potential, hydrogen yield and hydrogen production rate all trended to
Available online 29 October 2008
decrease with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. The inhibitory effect of added ethanol on fermenta-
Keywords:
tive hydrogen production was smaller than those of added acetic acid, propionic acid and
Biohydrogen
butyric acid. The modified Han–Levenspiel model could describe the inhibitory effects of
Inhibitory effect
added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen
Hydrogen yield
production rate in this study successfully. The modified Logistic model could describe the
Hydrogen production rate
progress of cumulative hydrogen production.
Kinetic model
ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Environmental pollution due to the use of fossil fuels as well as their shortfall makes it necessary to find alternative energy sources that are environmentally friendly and renewable. Hydrogen satisfies the above requirements because it produces only water, when it is combusted as a fuel or converted to electricity. Among various hydrogen production processes, biological method is known to be less energy intensive, for they are carried out at ambient temperature and pressure. Biological method mainly includes photosynthetic hydrogen production and fermentative hydrogen production. The efficiency of photosynthetic hydrogen production is low, and it cannot be operated in the absence of light, while fermentative hydrogen production can produce hydrogen all day long without light using various kinds of substrates such as organic wastes, and has a higher feasibility for industrialization. Thus fermentative hydrogen production is more
feasible and widely used. It is of great significance to produce hydrogen from organic wastes by fermentative hydrogen production, because it cannot only treat the organic wastes, but also generate very clean fuel hydrogen [1–4]. During fermentative hydrogen production, ethanol, acetic acid, propionic acid and butyric acid can be produced as the soluble metabolites [1–4]. On the one hand, the undissociated part of these soluble metabolites can permeate the cell membrane of hydrogen-producing bacteria and then dissociate in the cell, which can disrupt the physiological balance in the cell. Thus, some maintenance energy should be used to restore the physiological balance in the cell, which can reduce the energy used for bacteria growth and then inhibit the bacteria growth in a sense; on the other hand, if the dissociated part of these soluble metabolites is present in the fermentative hydrogen production system at a high concentration, the ionic strength will increase, which may result in the cell lysis of hydrogen-producing bacteria. As a result, at
* Corresponding author. Tel.: þ86 10 62784843; fax: þ86 10 62771150. E-mail address: [email protected] (J. Wang). 0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2008.09.027
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international journal of hydrogen energy 33 (2008) 7013–7019
a high concentration, these soluble metabolites can inhibit hydrogen-producing bacteria growth and then inhibit the fermentative hydrogen production accordingly [5–8]. So far, there have been several studies reporting the inhibitory effects of added butyrate and acetate on fermentative hydrogen production [5–8]. Zheng and Yu reported that the specific hydrogen production rate and the substrate degradation efficiency both decreased with increasing added sodium butyrate concentration from 0 to 228 mmol/L [5]. Van Niel et al. reported that the hydrogen production rate decreased with increasing added sodium acetate concentration from 0 to 300 mmol/L [6]. Chin et al. reported that the hydrogen-producing bacteria growth and hydrogen production potential both decreased with increasing added acetate concentration from 0 to 4 g/L and decreased with increasing added butyrate concentration from 0 to 10 g/L [7]. However, to the best of our knowledge, up to now, there have been no studies reporting the inhibitory effects of added ethanol or propionic acid (propionate) on fermentative hydrogen production. However, in some cases, ethanol can be dominant in the soluble metabolites [9–14], and in other cases, propionic acid can be dominant in the soluble metabolites [12,15,16]. At a high concentration, ethanol and propionic acid can also inhibit hydrogen-producing bacteria growth and then inhibit the fermentative hydrogen production accordingly. Thus, the objective of this study was to investigate the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid ranging from 0 to 300 mmol/L on fermentative hydrogen production by mixed cultures and then compare their inhibitory effects.
2.3.
The water displacement method was used to collect and measure the biogas produced. The fraction of hydrogen in the biogas was determined by a gas chromatograph (Model 122, Shanghai, China) equipped with a thermal conductivity detector (TCD) and a 2 m column packed with 5A molecular sieves. Helium was used as the carrying gas at the flow rate of 12 mL/min. The operating temperatures of the column, detector and injector were 40 C, 80 C and 50 C, respectively. All gas production data reported were standardized to the standard temperature (0 C) and pressure (760 mm Hg).The soluble metabolites were also analyzed by a gas chromatograph (Model 8000, Italy) equipped with a flame ionization detector (FID) and a 2 m column packed with GDX-103 (60/80 mesh). The temperatures of the column, oven and detector were 180 C, 240 C and 210 C, respectively. Nitrogen was used as the carrying gas at the flow rate of 50 mL/min. The pH in the solution was measured by a pH meter (Model 526, Germany). The concentration of glucose after reaction was determined by the DNS colorimetric method [17]. The substrate degradation efficiency was estimated by dividing the amount of glucose consumed by the amount of initial glucose. The hydrogen yield was calculated by dividing the hydrogen production potential by the amount of glucose consumed in each batch test. The fraction of each soluble metabolite produced by fermentation in each batch test was calculated by dividing the concentration of each soluble metabolite by the total concentration (mol/L) of the soluble metabolites.
2.4.
2.
Materials and methods
2.1.
Seed sludge
The digested sludge collected from a primary anaerobic digester at Beijing Gaobeidian Sewage Treatment Plant (China) was used as the seed sludge. Heat-shock was used in this study to enrich hydrogen-producing bacteria by heating the seed sludge at 100 C for 15 min [2].
2.2.
Experimental procedures
Batch tests were conducted in 150 mL glass bottles. One liter of the nutrient solution contained NaHCO3, 40000 mg; NH4Cl, 5000 mg; NaH2PO4$2H2O, 5000 mg; K2HPO4$3H2O, 5000 mg; FeSO4$7H2O 15000 mg; MgCl2$6H2O 85 mg; and NiCl2$6H2O 4 mg. Fifteen mL of the pretreated seed sludge, 10 mL nutrient solution, 1 g glucose and certain ethanol, acetic acid, propionic acid or butyric acid were added to each glass bottle. And then the total working volume of the bottles was filled to 100 mL by deionized water, which made the ethanol, acetic acid, propionic acid or butyric acid concentrations in the batch tests range from 0 to 300 mmol/L. The initial pH of the mixed solution in each bottle was adjusted to 7.0 by 1 mol/L HCl or 1 mol/L NaOH. Each bottle was flushed with argon for 3 min to provide anaerobic condition, capped with a rubber stopper, and placed in a reciprocal shaker (reciprocation: 150 strokes/ min). The batch tests were conducted at 35 C.
Analytical methods
Kinetic models used in this study
The modified Logistic model (Eq. (1)) was used to describe the progress of cumulative hydrogen production in the batch tests of this study [18]. H¼
P 1 þ exp½4Rm ðl tÞ=P þ 2
(1)
where H (mL) is the cumulative hydrogen production at the reaction time t (h), P (mL) is the hydrogen production potential, Rm (mL/h) is the maximum hydrogen production rate and l (h) is the lag time. In this study, the modified Logistic model was used to fit the cumulative hydrogen production data obtained from each batch test to obtain H, Rm and l. Once the three parameters were obtained, Eq. (2) was used to calculate the hydrogen production rate in each batch test. R¼
P l þ P=Rm
(2)
In this study, the modified Han–Levenspiel model (Eq. (3)) were used to describe the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production rate in this study [5,6,19]. n C (3) R ¼ Rmax 1 Cmax where R (mL/h) is the hydrogen production rate, Rmax (mL/h) is the maximum hydrogen production rate without adding any inhibitors, C (mmol/L) is the inhibitor concentration, Cmax
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international journal of hydrogen energy 33 (2008) 7013–7019
3.
Results and discussion
3.1. Inhibitory effects of added ethanol and acids on substrate degradation efficiency Fig. 1 shows the inhibitory effects of added ethanol and acids on substrate degradation efficiency. The results showed that during fermentative hydrogen production by mixed cultures, the substrate degradation efficiency in batch tests trended to decrease from 99.0% to 95.7%, 79.9%, 74.5% and 76.5% respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to degrade substrate during the fermentative hydrogen production, and the higher their concentration, the greater their inhibitory effects. Zheng and Yu also drew a similar conclusion when studying the inhibitory effects of added sodium butyrate on fermentative hydrogen production [5]. The inhibitory effects of added ethanol on the ability of mixed cultures to degrade substrate during fermentative hydrogen production were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to 10 mmol/L, the inhibitory effects of the added acetic acid and butyric acid on the ability of mixed cultures to degrade substrate were similar, but were both larger than that of added propionic acid. When the added concentration increased from 50 to 300 mmol/L, the inhibitory effects of the added propionic acid and butyric acid on the ability of mixed cultures to degrade substrate were similar, but were both larger than that of added acetic acid.
3.2. Inhibitory effects of added ethanol and acids on hydrogen production Analysis showed that the biogas produced by the heat-shock pretreated sludge contained only hydrogen and carbon
300
Hydrogen production potential (mL)
(mmol/L) is the inhibitor concentration above which the fermentative hydrogen production stops, and n is a constant.
150
100 Ethanol Acetic acid Propionic acid Butyric acid
50
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 2 – Inhibitory effects of ethanol and acids on hydrogen production potential.
dioxide, without detectable methane. In this study, the coefficients of determination (R2) for all the fittings using modified Logistic model were from 0.976 to 1.000, which indicated that the modified Logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully. Fig. 2 shows the inhibitory effects of added ethanol and acids on hydrogen production potential. The results showed that during fermentative hydrogen production by mixed cultures, the hydrogen production potential in batch tests decreased from 274.2 mL to 118.0 mL, 40.8 mL, 21.5 mL and 25.6 mL respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to produce hydrogen, and the higher their concentration, the greater their inhibitory effects. Van Niel et al. also drew a similar conclusion when studying the inhibitory effects of added sodium acetate on fermentative hydrogen production [6]. The inhibitory effects of added ethanol on the ability of mixed 300
Hydrogen yield (mL/g glucose)
Substrate degradation efficiency (%)
200
0
100
90
80 Ethanol Acetic acid Propionic acid Butyric acid 70
250
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 1 – Inhibitory effects of ethanol and acids on substrate degradation efficiency.
250
200
150
100 Ethanol Acetic acid Propionic acid Butyric acid
50
0
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 3 – Inhibitory effects of ethanol and acids on hydrogen yield.
Hydrogen production rate (mL/h)
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international journal of hydrogen energy 33 (2008) 7013–7019
Table 1 – Fitting results by modified Han–Levenspiel model.
12
Added substance 9
Ethanol Acetic acid Propionic acid Butyric acid
6
Ethanol Acetic acid Propionic acid Butyric acid
3
0
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 4 – Inhibitory effects of ethanol and acids on hydrogen production rate.
Rmax (mL/h)
Cmax(mmol/L)
n
R2
12.5 12.9 11.8 11.2
819.8 471.0 314.3 306.3
1.8 2.9 1.0 0.6
0.995 0.972 0.988 0.926
100 mmol/L and was 300 mmol/L, the inhibitory effects of added acetic acid, propionic acid and butyric acid on the ability of mixed cultures to produce hydrogen were similar. When the added concentration was 200 mmol/L, the inhibitory effects of added acetic acid on the ability of mixed cultures to produce hydrogen were larger than that of added propionic acid, whose inhibitory effects were larger than that of added butyric acid.
3.3. Inhibitory effects of added ethanol and acids on hydrogen production rate cultures to produce hydrogen were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to 100 mmol/L and was 300 mmol/L, the inhibitory effects of added acetic acid, propionic acid and butyric acid on the ability of mixed cultures to produce hydrogen were similar. When the added concentration was 200 mmol/L, the inhibitory effects of added acetic acid on the ability of mixed cultures to produce hydrogen were larger than that of added propionic acid, whose inhibitory effects were larger than that of added butyric acid. Fig. 3 shows the inhibitory effects of added ethanol and acids on hydrogen yield. The results showed that during fermentative hydrogen production by mixed cultures, the hydrogen yield in batch tests decreased from 277.0 mL/g glucose to 196.5 mL/g glucose, 51.1 mL/g glucose, 28.9 mL/g glucose and 33.5 mL/g glucose respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to produce hydrogen, and the higher their concentration, the greater their inhibitory effects. However, Zheng and Yu. reported that the hydrogen yield increased first and then decreased with increasing added sodium butyrate concentration from 0 to 228 mmol/L [5]. The inhibitory effects of added ethanol on the ability of mixed cultures to produce hydrogen were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to
Fig. 4 shows the inhibitory effects of added ethanol and acids on hydrogen production rate. The results showed that during fermentative hydrogen production by mixed cultures, the hydrogen production rate in batch tests decreased from 12.5 mL/h to 5.5 mL/h, 1.2 mL/h, 0.6 mL/h and 0.9 mL/h respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to produce hydrogen, and the higher their concentration, the greater their inhibitory effects. Van Niel et al. also drew a similar conclusion when studying the inhibitory effects of added sodium acetate on fermentative hydrogen production [6]. The inhibitory effects of added ethanol on the ability of mixed cultures to produce hydrogen were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to 50 mmol/L and was 300 mmol/L, the inhibitory effects of added propionic acid and butyric acid on the ability of mixed cultures to produce hydrogen were similar, but were both larger than that of added acetic acid. When the added concentration increased from 100 to 200 mmol/L, the inhibitory effects of added acetic acid on the ability of mixed cultures to produce hydrogen were larger than that of added propionic and butyric acid. The fitting results using modified Han–Levenspiel model are listed in Table 1. The coefficients of determination (R2) of
Table 2 – Fitting results by the modified Han–Levenspiel model in this study and other studies. Added substance Butyric acid Sodium butyrate Acetic acid Sodium acetate Ethanol Propionic acid
Concentration range (mmol/L)
Rmax
Cmax(mmol/L)
n
0–300 0–228 0–300 0–300 0–300 0–300
11.2 mL/h 61.5 mL/(h g VSS) 12.9 mL/h 85.1 mL/h 12.5 mL/h 11.8 mL/h
306.3 232.7 471.0 365.0 819.8 314.3
0.6 0.337 2.9 1.0 1.8 1.0
References This [5] This [6] This This
study study study study
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international journal of hydrogen energy 33 (2008) 7013–7019
all the fittings were from 0.926 to 0.995, which indicated that the modified Han–Levenspiel model could describe the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production rate in this study successfully. According to the fitting results, the added ethanol, acetic acid, propionic acid and butyric acid concentrations above which the fermentative hydrogen production stops were 819.8 mmol/L, 471.0 mmol/L, 314.3 mmol/L and 306.3 mmol/L, respectively, indicating that the inhibitory effects of added butyric acid on the ability of mixed cultures to produce hydrogen were the largest, with added propionic acid next, followed by added acetic acid, whose inhibitory effects were larger than that of added ethanol. So far, Zheng and Yu [5] and Van Niel et al. [6] have reported the fitting of the hydrogen production rate data by the modified Han–Levenspiel model when studying the inhibitory effects of added sodium butyrate and sodium acetate on fermentative hydrogen production. Table 2 summarizes fitting results of the hydrogen production rate data by the modified Han–Levenspiel model in this study and their studies. As shown in Table 2, the fitting results of the inhibitory effects of added acetic acid and butyric acid on fermentative hydrogen production rate by the modified Han–Levenspiel model in this study were different from the fitting results of the inhibitory effects of added sodium butyrate and sodium acetate on fermentative hydrogen production rate by modified Han–Levenspiel model in the studies by Zheng and Yu [5] and Van Niel et al. [6]. The possible reasons for these differences were the differences between this study and their studies in terms of the seed sludge, the substrates, their concentrations, temperatures, pH, the added inhibitor concentration ranges studied and the like. Generally speaking, the added ethanol concentration above which the fermentative hydrogen production stops was the highest, with the added acetic acid (sodium acetate) concentration next, followed by the added propionic acid concentration, which was higher than the added butyric acid (sodium butyrate) concentration, indicating that the inhibitory effects of added butyric acid (sodium butyrate) on the ability of mixed cultures to produce hydrogen were the largest, with added propionic acid next, followed by added acetic acid (sodium acetate), whose inhibitory effects were larger than that of added ethanol. Chin et al. [7] and Van Ginkel and Logan [8] also reported that the inhibitory effects of added butyrate (sodium butyrate) on the ability of
Added ethanol Distribution of the soluble metabolites (%) concentration Ethanol Acetic Propionic Butyric (mmol/L) acid acid acid 25.3 11.8 17.6 14.1 5.6 1.4
53.8 67.5 63.9 50.8 57.4 59.4
3.9 2.6 5.6 5.2 2.6 1.8
Added acetic acid concentration (mmol/L)
0 10 50 100 200 300
Distribution of the soluble metabolites (%) Ethanol
Acetic acid
Propionic acid
Butyric acid
25.2 29.3 29.2 27.3 23.9 7.7
53.6 38.8 36.0 34.5 39.6 30.8
4.3 4.3 5.9 8.1 5.1 8.8
16.9 27.7 28.9 30.1 31.4 52.7
hydrogen-producing bacteria to produce hydrogen were larger than that of added acetate (sodium acetate).
3.4. Effects of added ethanol and acids on the distribution of the soluble metabolites Analysis showed that the major soluble metabolites produced by the mixed cultures in this study were ethanol, acetic acid, propionic acid and butyric acid, which were also reported in other studies [20–26]. Tables 3–6 summarizes the effects of added ethanol, acetic acid, propionic acid and butyric acid on the distribution of the soluble metabolites produced by fermentation, respectively. As shown in Table 3, the fraction of ethanol in the soluble metabolites produced by fermentation trended to decrease with increasing added ethanol concentration from 0 to 300 mmol/L, while the fraction of butyric acid in the soluble metabolites produced by fermentation trended to increase with increasing added ethanol concentration from 0 to 300 mmol/L. As shown in Table 4, the fraction of acetic acid in the soluble metabolites produced by fermentation trended to decrease with increasing added acetic acid concentration from 0 to 300 mmol/L, while the fraction of butyric acid in the soluble metabolites produced by fermentation increased with increasing added acetic acid concentration from 0 to 300 mmol/L. In this study, acetic acid was dominant in the soluble metabolites produced by fermentation with increasing added acetic acid concentration from 0 to 200 mmol/L, while butyric acid was dominant in the soluble metabolites when added acetic acid concentration was 300 mmol/L.
Table 5 – Effects of propionic acid concentration on the distribution of the soluble metabolites.
Table 3 – Effects of ethanol concentration on the distribution of the soluble metabolites.
0 10 50 100 200 300
Table 4 – Effects of acetic acid concentration on the distribution of the soluble metabolites.
17.0 18.2 13.0 29.9 34.4 37.4
Added propionic acid concentration (mmol/L)
0 10 50 100 200 300
Distribution of the soluble metabolites (%) Ethanol
Acetic acid
Propionic acid
Butyric acid
24.9 50.0 15.2 7.7 6.9 5.3
53.0 35.5 58.2 48.8 47.6 45.4
5.4 4.5 4.6 6.6 7.7 19.5
16.7 9.9 22.0 37.0 37.8 29.8
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international journal of hydrogen energy 33 (2008) 7013–7019
Table 6 – Effect of butyric acid concentration on the distribution of the soluble metabolites. Added butyric acid concentration (mmol/L)
Distribution of the soluble metabolites (%) Ethanol Acetic acid
0 10 50 100 200 300
25.3 27.1 28.5 12.7 12.1 13.4
53.8 50.0 40.8 29.5 15.2 26.1
Propionic Butyric acid acid 3.9 11.2 13.6 24.0 34.1 36.3
17.0 11.8 17.0 33.8 38.5 24.3
As shown in Table 5, acetic acid was dominant in the soluble metabolites produced by fermentation with increasing added propionic acid concentration from 50 to 300 mmol/L and was 0, while ethanol was dominant in the soluble metabolites produced by fermentation when added propionic acid concentration was 10 mmol/L. As shown in Table 6, the fraction of propionic acid in the soluble metabolites produced by fermentation increased with increasing added butyric acid concentration from 0 to 300 mmol/L, while the fraction of acetic acid in the soluble metabolites produced by fermentation trended to decrease with increasing added butyric acid concentration from 0 to 300 mmol/L. Acetic acid was dominant in the soluble metabolites produced by fermentation with increasing added butyric acid concentration from 0 to 50 mmol/L, while butyric acid was dominant in the soluble metabolites produced by fermentation with increasing added butyric acid concentration from 100 to 200 mmol/L, and propionic acid was dominant in the soluble metabolites when the added butyric acid concentration was 300 mmol/L. All this showed that the added ethanol, acetic acid, propionic acid and butyric acid could change the distribution of the soluble metabolites produced by fermentation a lot, which may result from the metabolic pathway shift of substrate by hydrogen-producing bacteria. Zheng and Yu also drew a similar conclusion when studying the inhibitory effects of added sodium butyrate on fermentative hydrogen production [5].
4.
and butyric acid on fermentative hydrogen production rate in this study successfully. Modified Logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.
Conclusions
The inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production by mixed cultures were investigated in batch tests using glucose as a substrate. The following conclusions could be drawn. At 35 C and initial pH 7.0, during the fermentative hydrogen production, the substrate degradation efficiency, hydrogen production potential, hydrogen yield and hydrogen production rate all trended to decrease with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. The inhibitory effects of added ethanol on fermentative hydrogen production were smaller than those of added acetic acid, propionic acid and butyric acid. Modified Han–Levenspiel model could describe the inhibitory effects of added ethanol, acetic acid, propionic acid
Acknowledgement We are grateful to the precious comments and careful correction made by anonymous reviewers. We would also like to thank the National Natural Science Foundation of China for financially supporting this research under Contract No. 50325824.
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Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Inhibitory effect of ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production Bo Wang, Wei Wan, Jianlong Wang* Laboratory of Environmental Technology, INET, Tsinghua University, Beijing 100084, PR China
article info
abstract
Article history:
The inhibitory effect of added ethanol, acetic acid, propionic acid and butyric acid on
Received 14 August 2008
fermentative hydrogen production by mixed cultures was investigated in batch tests using
Received in revised form
glucose as substrate. The experimental results showed that, at 35 C and initial pH 7.0,
13 September 2008
during the fermentative hydrogen production, the substrate degradation efficiency,
Accepted 13 September 2008
hydrogen production potential, hydrogen yield and hydrogen production rate all trended to
Available online 29 October 2008
decrease with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. The inhibitory effect of added ethanol on fermenta-
Keywords:
tive hydrogen production was smaller than those of added acetic acid, propionic acid and
Biohydrogen
butyric acid. The modified Han–Levenspiel model could describe the inhibitory effects of
Inhibitory effect
added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen
Hydrogen yield
production rate in this study successfully. The modified Logistic model could describe the
Hydrogen production rate
progress of cumulative hydrogen production.
Kinetic model
ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Environmental pollution due to the use of fossil fuels as well as their shortfall makes it necessary to find alternative energy sources that are environmentally friendly and renewable. Hydrogen satisfies the above requirements because it produces only water, when it is combusted as a fuel or converted to electricity. Among various hydrogen production processes, biological method is known to be less energy intensive, for they are carried out at ambient temperature and pressure. Biological method mainly includes photosynthetic hydrogen production and fermentative hydrogen production. The efficiency of photosynthetic hydrogen production is low, and it cannot be operated in the absence of light, while fermentative hydrogen production can produce hydrogen all day long without light using various kinds of substrates such as organic wastes, and has a higher feasibility for industrialization. Thus fermentative hydrogen production is more
feasible and widely used. It is of great significance to produce hydrogen from organic wastes by fermentative hydrogen production, because it cannot only treat the organic wastes, but also generate very clean fuel hydrogen [1–4]. During fermentative hydrogen production, ethanol, acetic acid, propionic acid and butyric acid can be produced as the soluble metabolites [1–4]. On the one hand, the undissociated part of these soluble metabolites can permeate the cell membrane of hydrogen-producing bacteria and then dissociate in the cell, which can disrupt the physiological balance in the cell. Thus, some maintenance energy should be used to restore the physiological balance in the cell, which can reduce the energy used for bacteria growth and then inhibit the bacteria growth in a sense; on the other hand, if the dissociated part of these soluble metabolites is present in the fermentative hydrogen production system at a high concentration, the ionic strength will increase, which may result in the cell lysis of hydrogen-producing bacteria. As a result, at
* Corresponding author. Tel.: þ86 10 62784843; fax: þ86 10 62771150. E-mail address: [email protected] (J. Wang). 0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2008.09.027
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a high concentration, these soluble metabolites can inhibit hydrogen-producing bacteria growth and then inhibit the fermentative hydrogen production accordingly [5–8]. So far, there have been several studies reporting the inhibitory effects of added butyrate and acetate on fermentative hydrogen production [5–8]. Zheng and Yu reported that the specific hydrogen production rate and the substrate degradation efficiency both decreased with increasing added sodium butyrate concentration from 0 to 228 mmol/L [5]. Van Niel et al. reported that the hydrogen production rate decreased with increasing added sodium acetate concentration from 0 to 300 mmol/L [6]. Chin et al. reported that the hydrogen-producing bacteria growth and hydrogen production potential both decreased with increasing added acetate concentration from 0 to 4 g/L and decreased with increasing added butyrate concentration from 0 to 10 g/L [7]. However, to the best of our knowledge, up to now, there have been no studies reporting the inhibitory effects of added ethanol or propionic acid (propionate) on fermentative hydrogen production. However, in some cases, ethanol can be dominant in the soluble metabolites [9–14], and in other cases, propionic acid can be dominant in the soluble metabolites [12,15,16]. At a high concentration, ethanol and propionic acid can also inhibit hydrogen-producing bacteria growth and then inhibit the fermentative hydrogen production accordingly. Thus, the objective of this study was to investigate the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid ranging from 0 to 300 mmol/L on fermentative hydrogen production by mixed cultures and then compare their inhibitory effects.
2.3.
The water displacement method was used to collect and measure the biogas produced. The fraction of hydrogen in the biogas was determined by a gas chromatograph (Model 122, Shanghai, China) equipped with a thermal conductivity detector (TCD) and a 2 m column packed with 5A molecular sieves. Helium was used as the carrying gas at the flow rate of 12 mL/min. The operating temperatures of the column, detector and injector were 40 C, 80 C and 50 C, respectively. All gas production data reported were standardized to the standard temperature (0 C) and pressure (760 mm Hg).The soluble metabolites were also analyzed by a gas chromatograph (Model 8000, Italy) equipped with a flame ionization detector (FID) and a 2 m column packed with GDX-103 (60/80 mesh). The temperatures of the column, oven and detector were 180 C, 240 C and 210 C, respectively. Nitrogen was used as the carrying gas at the flow rate of 50 mL/min. The pH in the solution was measured by a pH meter (Model 526, Germany). The concentration of glucose after reaction was determined by the DNS colorimetric method [17]. The substrate degradation efficiency was estimated by dividing the amount of glucose consumed by the amount of initial glucose. The hydrogen yield was calculated by dividing the hydrogen production potential by the amount of glucose consumed in each batch test. The fraction of each soluble metabolite produced by fermentation in each batch test was calculated by dividing the concentration of each soluble metabolite by the total concentration (mol/L) of the soluble metabolites.
2.4.
2.
Materials and methods
2.1.
Seed sludge
The digested sludge collected from a primary anaerobic digester at Beijing Gaobeidian Sewage Treatment Plant (China) was used as the seed sludge. Heat-shock was used in this study to enrich hydrogen-producing bacteria by heating the seed sludge at 100 C for 15 min [2].
2.2.
Experimental procedures
Batch tests were conducted in 150 mL glass bottles. One liter of the nutrient solution contained NaHCO3, 40000 mg; NH4Cl, 5000 mg; NaH2PO4$2H2O, 5000 mg; K2HPO4$3H2O, 5000 mg; FeSO4$7H2O 15000 mg; MgCl2$6H2O 85 mg; and NiCl2$6H2O 4 mg. Fifteen mL of the pretreated seed sludge, 10 mL nutrient solution, 1 g glucose and certain ethanol, acetic acid, propionic acid or butyric acid were added to each glass bottle. And then the total working volume of the bottles was filled to 100 mL by deionized water, which made the ethanol, acetic acid, propionic acid or butyric acid concentrations in the batch tests range from 0 to 300 mmol/L. The initial pH of the mixed solution in each bottle was adjusted to 7.0 by 1 mol/L HCl or 1 mol/L NaOH. Each bottle was flushed with argon for 3 min to provide anaerobic condition, capped with a rubber stopper, and placed in a reciprocal shaker (reciprocation: 150 strokes/ min). The batch tests were conducted at 35 C.
Analytical methods
Kinetic models used in this study
The modified Logistic model (Eq. (1)) was used to describe the progress of cumulative hydrogen production in the batch tests of this study [18]. H¼
P 1 þ exp½4Rm ðl tÞ=P þ 2
(1)
where H (mL) is the cumulative hydrogen production at the reaction time t (h), P (mL) is the hydrogen production potential, Rm (mL/h) is the maximum hydrogen production rate and l (h) is the lag time. In this study, the modified Logistic model was used to fit the cumulative hydrogen production data obtained from each batch test to obtain H, Rm and l. Once the three parameters were obtained, Eq. (2) was used to calculate the hydrogen production rate in each batch test. R¼
P l þ P=Rm
(2)
In this study, the modified Han–Levenspiel model (Eq. (3)) were used to describe the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production rate in this study [5,6,19]. n C (3) R ¼ Rmax 1 Cmax where R (mL/h) is the hydrogen production rate, Rmax (mL/h) is the maximum hydrogen production rate without adding any inhibitors, C (mmol/L) is the inhibitor concentration, Cmax
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3.
Results and discussion
3.1. Inhibitory effects of added ethanol and acids on substrate degradation efficiency Fig. 1 shows the inhibitory effects of added ethanol and acids on substrate degradation efficiency. The results showed that during fermentative hydrogen production by mixed cultures, the substrate degradation efficiency in batch tests trended to decrease from 99.0% to 95.7%, 79.9%, 74.5% and 76.5% respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to degrade substrate during the fermentative hydrogen production, and the higher their concentration, the greater their inhibitory effects. Zheng and Yu also drew a similar conclusion when studying the inhibitory effects of added sodium butyrate on fermentative hydrogen production [5]. The inhibitory effects of added ethanol on the ability of mixed cultures to degrade substrate during fermentative hydrogen production were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to 10 mmol/L, the inhibitory effects of the added acetic acid and butyric acid on the ability of mixed cultures to degrade substrate were similar, but were both larger than that of added propionic acid. When the added concentration increased from 50 to 300 mmol/L, the inhibitory effects of the added propionic acid and butyric acid on the ability of mixed cultures to degrade substrate were similar, but were both larger than that of added acetic acid.
3.2. Inhibitory effects of added ethanol and acids on hydrogen production Analysis showed that the biogas produced by the heat-shock pretreated sludge contained only hydrogen and carbon
300
Hydrogen production potential (mL)
(mmol/L) is the inhibitor concentration above which the fermentative hydrogen production stops, and n is a constant.
150
100 Ethanol Acetic acid Propionic acid Butyric acid
50
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 2 – Inhibitory effects of ethanol and acids on hydrogen production potential.
dioxide, without detectable methane. In this study, the coefficients of determination (R2) for all the fittings using modified Logistic model were from 0.976 to 1.000, which indicated that the modified Logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully. Fig. 2 shows the inhibitory effects of added ethanol and acids on hydrogen production potential. The results showed that during fermentative hydrogen production by mixed cultures, the hydrogen production potential in batch tests decreased from 274.2 mL to 118.0 mL, 40.8 mL, 21.5 mL and 25.6 mL respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to produce hydrogen, and the higher their concentration, the greater their inhibitory effects. Van Niel et al. also drew a similar conclusion when studying the inhibitory effects of added sodium acetate on fermentative hydrogen production [6]. The inhibitory effects of added ethanol on the ability of mixed 300
Hydrogen yield (mL/g glucose)
Substrate degradation efficiency (%)
200
0
100
90
80 Ethanol Acetic acid Propionic acid Butyric acid 70
250
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 1 – Inhibitory effects of ethanol and acids on substrate degradation efficiency.
250
200
150
100 Ethanol Acetic acid Propionic acid Butyric acid
50
0
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 3 – Inhibitory effects of ethanol and acids on hydrogen yield.
Hydrogen production rate (mL/h)
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Table 1 – Fitting results by modified Han–Levenspiel model.
12
Added substance 9
Ethanol Acetic acid Propionic acid Butyric acid
6
Ethanol Acetic acid Propionic acid Butyric acid
3
0
0
10
50
100
200
300
Added ethanol and acid concentration (mmol/L) Fig. 4 – Inhibitory effects of ethanol and acids on hydrogen production rate.
Rmax (mL/h)
Cmax(mmol/L)
n
R2
12.5 12.9 11.8 11.2
819.8 471.0 314.3 306.3
1.8 2.9 1.0 0.6
0.995 0.972 0.988 0.926
100 mmol/L and was 300 mmol/L, the inhibitory effects of added acetic acid, propionic acid and butyric acid on the ability of mixed cultures to produce hydrogen were similar. When the added concentration was 200 mmol/L, the inhibitory effects of added acetic acid on the ability of mixed cultures to produce hydrogen were larger than that of added propionic acid, whose inhibitory effects were larger than that of added butyric acid.
3.3. Inhibitory effects of added ethanol and acids on hydrogen production rate cultures to produce hydrogen were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to 100 mmol/L and was 300 mmol/L, the inhibitory effects of added acetic acid, propionic acid and butyric acid on the ability of mixed cultures to produce hydrogen were similar. When the added concentration was 200 mmol/L, the inhibitory effects of added acetic acid on the ability of mixed cultures to produce hydrogen were larger than that of added propionic acid, whose inhibitory effects were larger than that of added butyric acid. Fig. 3 shows the inhibitory effects of added ethanol and acids on hydrogen yield. The results showed that during fermentative hydrogen production by mixed cultures, the hydrogen yield in batch tests decreased from 277.0 mL/g glucose to 196.5 mL/g glucose, 51.1 mL/g glucose, 28.9 mL/g glucose and 33.5 mL/g glucose respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to produce hydrogen, and the higher their concentration, the greater their inhibitory effects. However, Zheng and Yu. reported that the hydrogen yield increased first and then decreased with increasing added sodium butyrate concentration from 0 to 228 mmol/L [5]. The inhibitory effects of added ethanol on the ability of mixed cultures to produce hydrogen were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to
Fig. 4 shows the inhibitory effects of added ethanol and acids on hydrogen production rate. The results showed that during fermentative hydrogen production by mixed cultures, the hydrogen production rate in batch tests decreased from 12.5 mL/h to 5.5 mL/h, 1.2 mL/h, 0.6 mL/h and 0.9 mL/h respectively, with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. This demonstrated that the added ethanol, acetic acid, propionic acid and butyric acid could inhibit the ability of mixed cultures to produce hydrogen, and the higher their concentration, the greater their inhibitory effects. Van Niel et al. also drew a similar conclusion when studying the inhibitory effects of added sodium acetate on fermentative hydrogen production [6]. The inhibitory effects of added ethanol on the ability of mixed cultures to produce hydrogen were smaller than those of added acetic acid, propionic acid and butyric acid. When the added concentration increased from 0 to 50 mmol/L and was 300 mmol/L, the inhibitory effects of added propionic acid and butyric acid on the ability of mixed cultures to produce hydrogen were similar, but were both larger than that of added acetic acid. When the added concentration increased from 100 to 200 mmol/L, the inhibitory effects of added acetic acid on the ability of mixed cultures to produce hydrogen were larger than that of added propionic and butyric acid. The fitting results using modified Han–Levenspiel model are listed in Table 1. The coefficients of determination (R2) of
Table 2 – Fitting results by the modified Han–Levenspiel model in this study and other studies. Added substance Butyric acid Sodium butyrate Acetic acid Sodium acetate Ethanol Propionic acid
Concentration range (mmol/L)
Rmax
Cmax(mmol/L)
n
0–300 0–228 0–300 0–300 0–300 0–300
11.2 mL/h 61.5 mL/(h g VSS) 12.9 mL/h 85.1 mL/h 12.5 mL/h 11.8 mL/h
306.3 232.7 471.0 365.0 819.8 314.3
0.6 0.337 2.9 1.0 1.8 1.0
References This [5] This [6] This This
study study study study
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all the fittings were from 0.926 to 0.995, which indicated that the modified Han–Levenspiel model could describe the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production rate in this study successfully. According to the fitting results, the added ethanol, acetic acid, propionic acid and butyric acid concentrations above which the fermentative hydrogen production stops were 819.8 mmol/L, 471.0 mmol/L, 314.3 mmol/L and 306.3 mmol/L, respectively, indicating that the inhibitory effects of added butyric acid on the ability of mixed cultures to produce hydrogen were the largest, with added propionic acid next, followed by added acetic acid, whose inhibitory effects were larger than that of added ethanol. So far, Zheng and Yu [5] and Van Niel et al. [6] have reported the fitting of the hydrogen production rate data by the modified Han–Levenspiel model when studying the inhibitory effects of added sodium butyrate and sodium acetate on fermentative hydrogen production. Table 2 summarizes fitting results of the hydrogen production rate data by the modified Han–Levenspiel model in this study and their studies. As shown in Table 2, the fitting results of the inhibitory effects of added acetic acid and butyric acid on fermentative hydrogen production rate by the modified Han–Levenspiel model in this study were different from the fitting results of the inhibitory effects of added sodium butyrate and sodium acetate on fermentative hydrogen production rate by modified Han–Levenspiel model in the studies by Zheng and Yu [5] and Van Niel et al. [6]. The possible reasons for these differences were the differences between this study and their studies in terms of the seed sludge, the substrates, their concentrations, temperatures, pH, the added inhibitor concentration ranges studied and the like. Generally speaking, the added ethanol concentration above which the fermentative hydrogen production stops was the highest, with the added acetic acid (sodium acetate) concentration next, followed by the added propionic acid concentration, which was higher than the added butyric acid (sodium butyrate) concentration, indicating that the inhibitory effects of added butyric acid (sodium butyrate) on the ability of mixed cultures to produce hydrogen were the largest, with added propionic acid next, followed by added acetic acid (sodium acetate), whose inhibitory effects were larger than that of added ethanol. Chin et al. [7] and Van Ginkel and Logan [8] also reported that the inhibitory effects of added butyrate (sodium butyrate) on the ability of
Added ethanol Distribution of the soluble metabolites (%) concentration Ethanol Acetic Propionic Butyric (mmol/L) acid acid acid 25.3 11.8 17.6 14.1 5.6 1.4
53.8 67.5 63.9 50.8 57.4 59.4
3.9 2.6 5.6 5.2 2.6 1.8
Added acetic acid concentration (mmol/L)
0 10 50 100 200 300
Distribution of the soluble metabolites (%) Ethanol
Acetic acid
Propionic acid
Butyric acid
25.2 29.3 29.2 27.3 23.9 7.7
53.6 38.8 36.0 34.5 39.6 30.8
4.3 4.3 5.9 8.1 5.1 8.8
16.9 27.7 28.9 30.1 31.4 52.7
hydrogen-producing bacteria to produce hydrogen were larger than that of added acetate (sodium acetate).
3.4. Effects of added ethanol and acids on the distribution of the soluble metabolites Analysis showed that the major soluble metabolites produced by the mixed cultures in this study were ethanol, acetic acid, propionic acid and butyric acid, which were also reported in other studies [20–26]. Tables 3–6 summarizes the effects of added ethanol, acetic acid, propionic acid and butyric acid on the distribution of the soluble metabolites produced by fermentation, respectively. As shown in Table 3, the fraction of ethanol in the soluble metabolites produced by fermentation trended to decrease with increasing added ethanol concentration from 0 to 300 mmol/L, while the fraction of butyric acid in the soluble metabolites produced by fermentation trended to increase with increasing added ethanol concentration from 0 to 300 mmol/L. As shown in Table 4, the fraction of acetic acid in the soluble metabolites produced by fermentation trended to decrease with increasing added acetic acid concentration from 0 to 300 mmol/L, while the fraction of butyric acid in the soluble metabolites produced by fermentation increased with increasing added acetic acid concentration from 0 to 300 mmol/L. In this study, acetic acid was dominant in the soluble metabolites produced by fermentation with increasing added acetic acid concentration from 0 to 200 mmol/L, while butyric acid was dominant in the soluble metabolites when added acetic acid concentration was 300 mmol/L.
Table 5 – Effects of propionic acid concentration on the distribution of the soluble metabolites.
Table 3 – Effects of ethanol concentration on the distribution of the soluble metabolites.
0 10 50 100 200 300
Table 4 – Effects of acetic acid concentration on the distribution of the soluble metabolites.
17.0 18.2 13.0 29.9 34.4 37.4
Added propionic acid concentration (mmol/L)
0 10 50 100 200 300
Distribution of the soluble metabolites (%) Ethanol
Acetic acid
Propionic acid
Butyric acid
24.9 50.0 15.2 7.7 6.9 5.3
53.0 35.5 58.2 48.8 47.6 45.4
5.4 4.5 4.6 6.6 7.7 19.5
16.7 9.9 22.0 37.0 37.8 29.8
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Table 6 – Effect of butyric acid concentration on the distribution of the soluble metabolites. Added butyric acid concentration (mmol/L)
Distribution of the soluble metabolites (%) Ethanol Acetic acid
0 10 50 100 200 300
25.3 27.1 28.5 12.7 12.1 13.4
53.8 50.0 40.8 29.5 15.2 26.1
Propionic Butyric acid acid 3.9 11.2 13.6 24.0 34.1 36.3
17.0 11.8 17.0 33.8 38.5 24.3
As shown in Table 5, acetic acid was dominant in the soluble metabolites produced by fermentation with increasing added propionic acid concentration from 50 to 300 mmol/L and was 0, while ethanol was dominant in the soluble metabolites produced by fermentation when added propionic acid concentration was 10 mmol/L. As shown in Table 6, the fraction of propionic acid in the soluble metabolites produced by fermentation increased with increasing added butyric acid concentration from 0 to 300 mmol/L, while the fraction of acetic acid in the soluble metabolites produced by fermentation trended to decrease with increasing added butyric acid concentration from 0 to 300 mmol/L. Acetic acid was dominant in the soluble metabolites produced by fermentation with increasing added butyric acid concentration from 0 to 50 mmol/L, while butyric acid was dominant in the soluble metabolites produced by fermentation with increasing added butyric acid concentration from 100 to 200 mmol/L, and propionic acid was dominant in the soluble metabolites when the added butyric acid concentration was 300 mmol/L. All this showed that the added ethanol, acetic acid, propionic acid and butyric acid could change the distribution of the soluble metabolites produced by fermentation a lot, which may result from the metabolic pathway shift of substrate by hydrogen-producing bacteria. Zheng and Yu also drew a similar conclusion when studying the inhibitory effects of added sodium butyrate on fermentative hydrogen production [5].
4.
and butyric acid on fermentative hydrogen production rate in this study successfully. Modified Logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.
Conclusions
The inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production by mixed cultures were investigated in batch tests using glucose as a substrate. The following conclusions could be drawn. At 35 C and initial pH 7.0, during the fermentative hydrogen production, the substrate degradation efficiency, hydrogen production potential, hydrogen yield and hydrogen production rate all trended to decrease with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. The inhibitory effects of added ethanol on fermentative hydrogen production were smaller than those of added acetic acid, propionic acid and butyric acid. Modified Han–Levenspiel model could describe the inhibitory effects of added ethanol, acetic acid, propionic acid
Acknowledgement We are grateful to the precious comments and careful correction made by anonymous reviewers. We would also like to thank the National Natural Science Foundation of China for financially supporting this research under Contract No. 50325824.
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