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Exploring optimal environmental factors for fermentative hydrogen production from starch using mixed anaerobic microflora
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33 (2008) 1565 – 1572
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journal homepage: www.elsevier.com/locate/ijhydene
Exploring optimal environmental factors for fermentative hydrogen production from starch using mixed anaerobic microflora Kuo-Shing Leea, Yao-Feng Hsub, Yung-Chung Loc, Ping-Jei Linb, Chiu-Yue Lind, Jo-Shu Changc, a
Department of Safety Health and Environmental Engineering, Central Taiwan University of Science and Technology, Taichung, Taiwan Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan c Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan d BioHydrogen Laboratory, Department of Water Resource Engineering, Feng Chia University, Taichung, Taiwan b
art i cle info
ab st rac t
Article history:
Our previous work demonstrated that an acclimated mixed bacterial consortium was able
Received 15 June 2007
to produce H2 from sugar substrates. To reduce the medium cost for more commercially
Received in revised form
viable H2 production, cassava starch was used as the feedstock to produce H2 via dark
10 October 2007
fermentation. Three factors, namely, temperature, pH and starch concentration ðCstarch Þ,
Available online 26 November 2007
were intensively examined for their effects on H2 production activity. The H2 production
Keywords: Fermentative hydrogen production Starch Environmental factors pH Kinetics
kinetics was determined using a Monod-type kinetic model. The results show that mesophilic temperature ð37 CÞ is preferable for H2 production with the H2 -producing sludge used. The H2 production efficiency and the composition of soluble metabolites were found to be highly sensitive to the change in pH, as pH 6.0 seemed to give the best overall H2 production performance. In a non-pH-controlled culture (initial pH ¼ 8:5), ethanol and butyrate were the major soluble metabolites, whereas the predominant metabolites switched to butyrate alone (accounting for 70–80% of total soluble microbial products) when the culture pH was controlled at a fixed level ranging from 5.5 to 7.0. Meanwhile, the maximum H2 production rate occurred when the initial starch concentration was 24 g COD/l. The dependence of H2 production rate on starch concentration could be described by using Monod-type model and the predicted kinetic constants, namely, maximum H2 production rate ðvmax;H2 Þ and Monod constant ðKs Þ, were 1741 ml/h/l and 16.28 g COD/l, respectively. Under the optimal conditions (37 C, pH 6.0, Cstarch ¼ 24 g COD=l), the H2 production rate increased to 1119 ml/h/l, while a high H2 yield of 9.47 mmol H2 /g starch was obtained. This performance appeared to be superior to that obtained from other starch-to-bioH2 systems reported in the literature. & 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
Corresponding author: Tel.: +886 6 2757575 62651, fax: +886 6 2357146.
E-mail address: [email protected] (J.-S. Chang). 0360-3199/$ - see front matter & 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2007.10.019
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1.
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
Introduction
Hydrogen, a clean and efficient energy carrier, is considered a feasible alternative to fossil fuels. Conventional H2 production methods (e.g., thermochemical process) mainly utilize fossil fuels as the raw materials. These processes are not sustainable and environmentally friendly. Therefore, a cost effective and non-polluting means of H2 production is still in great demand [1]. Hydrogen production from organic materials through biological processes, such as photo and dark fermentation, are considered promising and sustainable H2 production methods because they are pollution-free and less energy intensive. Among the biohydrogen production methods, dark fermentation seems more feasible for commercial processes due primarily to the feature of attaining much higher H2 production rates than other bio-hydrogenation systems. Glucose and sucrose are most often used substrates for dark H2 fermentation [2–6] because these simple carbohydrates are readily utilized by microorganism. However, those sugar substrates may be too expensive for commercialized H2 production. From the perspective of energy supply, mass and
Cumulative H2 production (ml)
1600 37 °C 55 °C
1400 1200 1000 800 600 400 200 0 0
10
20
30
40
Time (h) Fig. 1 – Cumulative H2 production profiles for cultures conducted at 37 and 55 C. (Initial pH ¼ 8:5, initial starch concentration ¼ 16 g COD=l; symbols: experimental data, curves: prediction with Gompertz equation.)
33 (2008) 1565 – 1572
low-cost production of H2 from sufficient substrate resources is inevitably required. The starch seems to be a feasible feedstock for large scale H2 production because it is one of the most abundant organic resources produced from plant in nature. In addition, starch also contributes to a marked portion of biological oxygen demands in many types of waste and/or wastewater, which also require appropriate clean-up. Nevertheless, there is little information in the literature regarding the fermentative H2 production from starch [7–9]. Temperature and pH are usually the target factors influencing the performance of bioH2 production from starch. Zhang et al. [7] compared H2 production from starch at 37 and 55 C by mesophilic sludge and found that more starch was converted into H2 at 55 C, but a longer lag time was required. They also examined the effect of initial pH (from 4.0 to 9.0) on fermentative H2 production from starch and obtained the maximum H2 yield of 92 ml/g starch (17% of the theoretical value) at pH 6.0, whereas the maximum specific H2 production rate of 365 ml/d/g volatile suspended solid (VSS) occurred at pH 7.0. Using mixed culture to convert starch into H2 , Khanal et al. [8] discovered that the lowest initial pH (4.5) tested gave the highest H2 production yield (125 ml H2 =g COD starch), but the lowest specific H2 production rate, which was the highest at pH 5.5–5.7. Fang et al. [9] investigated the effect of pH on hydrogen production from rice slurry with H2 producing sludge at a concentration of 5.5 g carbohydrate/l. The results show that the maximum H2 yield of 346 ml/g carbohydrate occurred at pH 4.5, but again operation at this low pH value led to a long lag phase (36 h) for H2 evolution. Thus, the optimal pH for H2 production from starch seems to be more acidic than the optimal pH (5.5) for H2 production from glucose reported by the same group [2]. On the other hand, the knowledge on substrate-dependent H2 -producing kinetics is also the key to a successful operation of a H2 producing bioreactor. Unfortunately, systematic kinetic study on the dependence of H2 production efficiency on substrate (starch) concentration is still lacking [7,9]. As literature shows that the environmental factors and substrate concentration play crucial roles in affecting cell growth and H2 production efficiency [2,4,7–9], this study aimed to understand how temperature, pH and substrate concentration affect H2 production from cassava starch using
Table 1 – Performance of fermentative H2 production at different temperatures T ð CÞ
a
pH
Model simulationa
Substrate concentration (g COD/l) P (ml)
Rm (ml/h)
l (h)
37
Not controlled 0.204
16
1447
477
25.7
55
Not controlled
16
241
83
26.0
Simulated with modified Gompertz equation.
H2 yield (mmol H2 / g starch)
Substrate utilization (%)
Cell yield (g VSS/ g starch)
0.998
5.34
82
1.44
51
0.085
r2
0.997
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2.
Material and methods
2.1. Hydrogen-producing sludge and fermentation medium The seed sludge was obtained from a municipal sewage treatment plant located in central Taiwan. The sludge was thermally pretreated at 952100 C for 1 h. The medium used for H2 fermentation contained 8–32 g COD/l of cassava starch (i.e., 6.75–27 g starch/l; Antik Sempurna S/B, Malaysia) as the sole carbon source and was supplemented with sufficient amounts of inorganic salts. To make the cassava starch disperse evenly in water, the starch was thermally pretreated at 90295 C until aqueous solution become semitransparent.
The detailed medium composition has been described in our recent work [10].
2.2.
Hydrogen production in batch fermentors
Three series of batch experiments were conducted in 2 l glass reactors containing 200 ml of thermally treated sludge and
3000 Cumulative H2 production (ml)
mixed anaerobic microflora as the H2 -producers. The batch tests examined the H2 -producing performance under thermophilic ð55 CÞ and mesophilic ð37 CÞ temperatures and non-pH-controlled (initial pH ¼ 8:5) or pH-controlled ðpH ¼ 5:527:0Þ environments. Kinetics of H2 production from starch was also determined by conducting H2 fermentation at a range of starch concentrations (8–32 g COD/l). The experimental data was described by Monod-type kinetic model and the optimal kinetic constants were also estimated. The results obtained from this work are expected to provide fundamental knowledge and useful information for optimal operation and practical application of starch-based dark H2 fermentation systems.
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33 (2008) 1565 – 1572
pH 7.0 pH 6.5 pH 6.0 pH 5.5
2500 2000 1500 1000 500 0 0
5
10
15
20
25
Time (h) Fig. 2 – Cumulative H2 production profiles for cultures conducted at a fixed pH of 5.5, 6.0, 6.5 and 7.0. (Temperature ¼ 37 C, initial starch concentration ¼ 16 g COD=l; symbols: experimental data, curves: prediction with Gompertz equation.)
Table 2 – Soluble metabolites formation along with fermentative H2 production at different temperatures T ð CÞ 37 55
pH
Substrate concentration (g COD/l)
SMP (g COD/l)
TVFA (g COD/l)
EtOH (%)
HAc (%)
HPr (%)
HBu (%)
HVa (%)
Not controlled Not controlled
16
6.84
3.84
43.9
10.9
2.4
42.8
0.0
16
1.82
0.92
49.4
9.3
0.9
40.4
0.0
Initial pH ¼ 8:5, final pH ¼ 6:226:3. HAc: acetic acid; HPr: propionic acid; HBu: normal butyric acid; HVa: normal valeric acid; EtOH: ethanol; SMP: soluble microbial products; TVFA (total volatile fatty acid)¼ HAc þ HBu þ HPr þ HVa.
Table 3 – Performance of fermentative H2 production at different pH T ð CÞ
pH
Model simulationa
Substrate concentration (g COD/l) P (ml)
Rm (ml/h)
l (h)
37
7.0 0.182
16
1441
396
8.9
37 37 37
6.5 6.0 5.5
16 16 16
2246 2771 2970
612 857 511
9.7 9.5 7.2
a
Simulated with modified Gompertz equation.
H2 yield (mmol H2 / g starch)
Substrate utilization (%)
Cell yield (g VSS/ g starch)
0.996
5.17
84
8.12 8.63 9.19
84 97 98
0.153 0.260 0.218
r2
0.998 0.996 0.995
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800 ml medium to undergo batch hydrogen fermentation. In series 1, batch cultures were conducted to compare the H2 production performance at 37 and 55 C with a medium
0 7
containing 16 g COD/l of starch at an initial pH of 8.5. In series 2, the effect of pH was examined and the experiments were conducted at pH ranging from 5.5 to 7.0 with a 0.5 increment. The medium contained 16 g COD/l of starch at 37 C. Series 3 mainly investigated the effect of starch concentration on H2 production. The starch concentration tested ranged from 8 to 32 g COD/l with 8 g COD/l increments at pH 6.0 and 37 C. In all batch tests, the mixed liquor in the reactor was first purged with argon for 5–10 min to ensure an anaerobic condition prior to H2 fermentation experiments. During the course of experiments, the quantity and composition of gas products (mainly, CO2 and H2 ) and soluble microbial products (SMP) were monitored at designated time intervals. The gas volumes measured by a volumetric gas meter (Type TG1; Ritter Inc., Germany) were corrected to a temperature of 25 C and a pressure of 1 atm. The H2 yield was calculated based on the mmol of H2 produced per g of starch consumed. The starch consumption was converted from the difference between initial ðTCi Þ and residual ðTCf Þ amounts of total carbohydrate content in the culture. The substrate utilization was the ratio of starch consumption and initial amount of starch and was calculated by ðTCi TCf Þ=TCi . The calculation of cell yield was similar to that for H2 yield, as the amount (g VSS) of cell mass gained replaces the amount of H2 produced in the calculation.
6
2.3.
5
40
The gas products (mainly H2 and CO2 ) was analyzed by gas chromatography (GC) (GC-14A, Shimadzu, Tokyo, Japan) using a thermal conductivity detector. The volatile fatty acids and ethanol were also detected by GC using a flame ionization detector. The conditions and columns used for GC analysis were identical to those reported previously [11]. Standard Methods [12] were used to determine biomass concentration (in terms of VSS) of samples taken from the bioreactor. The total carbohydrate concentration in the effluent was also measured using Standard Methods [12].
20
2.4.
Biomass (g-VSS/L)
Soluble microbial products (mg COD/L)
Cumulative H2 production (ml)
3000 2500 2000 1500 1000 500 0 8000 EtOH HAc HPr HBu
6000 4000 2000
4 3 100
Starch utilization (%)
33 (2008) 1565 – 1572
80 60
Analytical methods
Kinetic analysis
0 0
5
10
15
20
Time (h) Fig. 3 – Time-course profiles of H2 production, soluble metabolite formation, biomass growth and starch utilization for culture conducted at pH 6.0, 37 C, and an initial starch concentration of 16 g COD/l.
The results of cumulative H2 production in the batch experiments were described by the modified Gompertz equation [13]: Rm e ðl tÞ þ 1 , (1) HðtÞ ¼ P exp exp P
Table 4 – Soluble metabolites formation along with fermentative H2 production at different pH T ð CÞ
pH
Substrate concentration (g COD/l)
SMP (g COD/l)
TVFA (g COD/l)
EtOH (%)
HAc (%)
HPr (%)
HBu (%)
HVa (%)
37 37 37 37
7.0 6.5 6.0 5.5
16 16 16 16
5.73 7.19 9.35 8.57
4.99 6.22 9.13 8.38
12.9 13.5 2.3 2.3
12.8 12.5 13.9 16.8
1.7 0.7 0.6 0.6
72.6 73.3 83.2 80.3
0.0 0.0 0.0 0.0
HAc: acetic acid; HPr: propionic acid; HBu: normal butyric acid; HVa: normal valeric acid; EtOH: ethanol; SMP: soluble microbial products; TVFA (total volatile fatty acid)¼ HAc þ HBu þ HPr þ HVa.
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where H is the cumulative volume of H2 produced (ml), P is the H2 production potential (ml), Rm is the maximum H2 production rate (ml/h), l is the lag-phase time (h), t is the incubation time (h) and e is the expð1Þ ¼ 2:718. The dependence of H2 production rate on limiting substrate (starch) concentration was simulated by a Monod-type kinetic model: vH2 ¼
vmax;H2 Cstarch , Ks þ Cstarch
(2)
where vmax;H2 is the maximum volumetric H2 production rate (ml/l/h), Ks is half-saturation (Monod) constant (g COD/l) and Cstarch is starch concentration (g COD/l).
3.
Results and discussion
3.1.
Effect of temperature on hydrogen production
It is of great importance to identify the temperature preference of the H2 -producing bacterial population used in
Cumulative H2 production (ml)
6000 8 g COD/L 16 g COD/L 24 g COD/L 32 g COD/L
5000 4000 3000
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33 (2008) 1565 – 1572
this study. Thus, two typical mesophilic ð37 CÞ and thermophlic ð55 CÞ temperatures were examined [7,9]. The profiles of cumulative H2 production from starch (16 g COD/l) at the two temperatures and an initial pH of 8.5 are illustrated in Fig. 1, which clearly shows that H2 production at 37 C was much more effective than at 55 C. Fig. 1 and Table 1 show that the H2 evolution data could be simulated satisfactorily ðr2 ¼ 0:99720:998Þ based on modified Gompertz equation (Eq. (1)), and the estimated kinetic parameters, as well as H2 yield, substrate utilization and cell yield, are listed in Table 1. For the culture at 37 C, the P and Rm was 477 ml/h and 5:34 mmol H2 =g starch, respectively, which is nearly six-fold higher than that at 55 C. The H2 yield obtained at 37 C (5:34 mmol H2 =g starch) was also much higher than that obtained at 55 C (1:44 mmol H2 =g starch). Meanwhile, the 37 C culture also gave significantly higher cell yield and substrate utilization efficiency than the culture operated at 55 C, as the substrate utilization efficiency and cell yield decreased from 82% to 51% and 0.204 to 0.085 g VSS/g starch, respectively. This seems to indicate that the bacterial populations in the H2 -producing sludge used in this study were mainly mesophiles. Analysis of soluble metabolite composition shows that irrespective the temperature used, the predominant metabolite was ethanol (EtOH) and butyrate (HBu), while acetate was produced at a much lesser amount. This soluble metabolite composition is very different from that obtained from our previous work, which was operated at a much lower pH (ca. 6.5) using sugar-based media [4–6,10,11,14]. Hence, it is likely that the difference in soluble metabolite formation may be strongly associated with the type of carbon substrate and the operating pH range (Table 2).
2000
3.2.
1000 0 0
5
10
15
20
25
Time (h) Fig. 4 – Cumulative H2 production profiles for cultures conducted at an initial starch concentration of 8, 16, 24 and 32 g COD/l. (Temperature ¼ 37 C, pH ¼ 6:0; symbols: experimental data, curves: prediction with Gompertz equation.)
Effect of pH on hydrogen production
The pH effects on H2 production was examined using the preferable temperature of 37 C. With the pH controlled at a fixed value (5.5–7.0), the lag time for H2 evolution was markedly shortened to within 10 h (Table 3). The pHdependent H2 production profiles (Fig. 2) show that operation at pH 5.5 and 6.0 displayed better H2 production performance than the other two pH values. The cultures operated at pH 5.5 and 6.0 also attained better substrate utilization efficiency
Table 5 – Performance of fermentative H2 production at different substrate (starch) concentrations T ð CÞ
pH
Substrate concentration (g COD/l)
Model simulationa
P (ml)
Rm (ml/h)
l (h)
37
6.0 0.281
8
957
534
8.9
37 37 37
6.0 6.0 6.0
16 24 32
2771 4128 5964
857 1119 1069
9.5 9.5 13.2
a
Simulated with modified Gompertz equation.
H2 yield (mmol H2 / g starch)
Substrate utilization (%)
Cell yield (g VSS/ g starch)
0.997
6.24
93
8.63 9.47 11.25
97 88 80
0.260 0.133 0.177
r2
0.997 0.999 0.997
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Volumetric H2 production rate (ml/l/h)
and cell yield than those for pH 6.5 and 7.0, suggesting that the former two pHs were better conditions for the cells to utilize starch for growth. According to the literature, the optimal pH for H2 production from starch varied from 4.5 to 6.0 with few exceptions (Table 7), and our finding appeared to fall within that optimal pH range. Kinetic data (Table 3) indicate that pH 6.0 gave the best H2 production rate at 857 ml/h, whereas the culture at pH 5.5 attained a slightly better H2 yield (9:19 mmol H2 =g starch) over pH 6.0. The H2 producing performance at pH 5.5 and 6.0 is better than most of the values reported in the literature (Table 7). It is also found that the pH-6.0 culture obtained the highest cell yield of 0.260 g VSS/g starch (Table 3). The high cell yield may lead to more electron flow toward biomass. This might explain why pH 6.0 had a higher H2 production rate, but a lower H2 yield when compared with pH 5.5. Fig. 3 further demonstrates that before H2 evolution occurred (0–9 h), starch began to be utilized for biomass growth, while H2 production started to accelerate along with cell growth from the early exponential phase. This growth-dependent H2 production trend is slightly different from that observed in a pure culture of Clostridium butyricum CGS5, as H2 production did not occur until cell growth reached the mid-exponentialphase [15].
1400 1200 1000 800 600 400 200 0 0
8
16
24
32
40
Starch concentration (g COD/L) Fig. 5 – Dependence of volumetric H2 production rate on initial starch concentration. (Temperature ¼ 37 C, pH ¼ 6:0; symbols: experimental data, curves: prediction with Monod-type kinetic model.)
33 (2008) 1565 – 1572
The soluble metabolite data show that with a controlled pH within 5.5–7.0, production of ethanol, one of major products resulting from pH-uncontrolled cultures (Table 2), decreased dramtically, while HBu became the sole predominant metabolite, accounting for 72–84% of total SMP (Table 4). The shift from ethanol-type fermentation to butyrate-type fermentation appeared to markedly enhance both H2 production rate and production yield (Tables 1 and 3). This is reasonable from the biochemical aspect because butyrate is a favorable metabolite for bioH2 production, whereas solvent (e.g., ethanol) formation is usually unfavorable to H2 production due to consumption of additional free electrons from NADH [1]. Furthermore, the metabolite results also show that the metabolic pathway or bacterial community structure (or both) of the H2 -producing sludge used in this work were quite sensitive to the culture pH. This again raises the importance of pH control in bioreactor operation for bioH2 production using the mixed microflora [7–9,16,17].
3.3. Effect of substrate concentration on hydrogen production Understanding reaction kinetics of the biological system of interest is usually a critical step toward optimal control and operation of a bioreactor. Unfortunately, there is little information in the literature regarding the dependence of starch concentration on fermentative H2 production [7,9,17]. Hence, the knowledge on starch-dependent H2 production kinetics should be revealed. Using the preferable temperature ð37 CÞ and pH (6.0) for bioH2 production based on the foregoing results, the H2 production performance of cultures with different initial substrate (starch) concentrations of 8–32 g COD/l was investigated. The cumulative H2 production and H2 yield both increased as the initial starch concentration increased (Fig. 4 and Table 5). The volumetric H2 production rate reached a maximum value of 1119 ml/h/l (and a yield of 9:47 mmol H2 =g starch) when 24 g COD/l of starch was used as the sole carbon source (Fig. 5 and Table 6). The dependence of H2 production on starch concentration was simulated with a Monod-type model (Eq. (2)) with good agreement ðr2 ¼ 0:973Þ. The estimated vmax;H2 and Ks values were 1741 ml/h/l and 16.28 g COD/l, respectively. The substrate utilization efficiency and cell yield peaked at a starch concentration of 16 g COD/l, and then decreased at higher substrate concentrations
Table 6 – Soluble metabolites formation along with fermentative H2 production at different substrate (starch) concentrations T ð CÞ
pH
Substrate concentration (g COD/l)
SMP (g COD/l)
TVFA (g COD/l)
EtOH (%)
HAc (%)
HPr (%)
HBu (%)
HVa (%)
37 37 37 37
6.0 6.0 6.0 6.0
8 16 24 32
3.97 9.35 11.75 16.98
3.86 9.13 11.46 16.32
2.8 2.3 2.5 3.9
14.3 13.9 16.4 14.3
1.2 0.6 0.3 0.4
81.7 83.2 80.8 81.4
0.0 0.0 0.0 0.0
HAc: acetic acid; HPr: propionic acid; HBu: normal butyric acid; HVa: normal valeric acid; EtOH: ethanol; SMP: soluble microbial products; TVFA (total volatile fatty acid)¼ HAc þ HBu þ HPr þ HVa.
Table 7 – Comparison of the performance of fermentative H2 production from starch in comparable studies H2 producer
Culture type
Substrate
Examined temperature ð CÞ
Examined pH
Examined substrate concentration (g/l)
Optimal culture condition
pH
Substrate concentration (g/l)
Max. HPRb (ml/l/h)
Reference
Rice slurry
37, 55
4.0–7.0
2.7–22.1c
37
4.5
5.5c
14.12d
NA
[9]
Compost material Sludge
Batch
Starch
37
4.5–6.5
10
NA
NA
6.05
NA
[8]
CSTR
Soluble starch
37
4.0–7.0
4.25
NA
4.5 (initial) 5.2
NA
NA
67
[16]
Corn starch
35
4.0–9.0
2-32
NA
8.0 (initial)
2
7.92
4
[17]
Starch
37, 55
4.0–9.0
4.6–36.6
55
6.0 (initial) 7.0 (initial) 6.0
24 4.6
4.33 3.76
53 3.1
[7]
(2.73)
5.8
20.3 (24 g COD/l)
9.47
1119
This
27 (32 g COD/l)
11.25
1069
study
Mixed bacteria Sludge
Sewage sludge
ðHRT ¼ 17 hÞ Batch
Batch
Batch
Cassava starch
NA: not available. HY: hydrogen yield. b HPR: hydrogen production rate. c g carbohydrate/l. d mmol H2 =g carbohydrate. a
37, 55
5.5–7.0
6.75–27 (8–32 g COD/l)
37
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Batch
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Anaerobic digester sludge
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Temperature ð CÞ
Max. HYa (mmol H2 / g starch)
1571
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(Table 5). The predicted maximum H2 production rate of 1741 ml/h/l from starch is much high than the reported values (Table 7), indicating the high effectiveness of our H2-producing system. The H2 yield obtained from this work is also comparable or higher than those obtained from the comparable studies (Table 7). Like the results exploring the pH effects, the predominant soluble metabolite was HBu (representing 80–84% of SMP), followed by acetate (representing 13–17% of SMP). Moreover, the metabolite composition was quite similar for cultures with different initial starch concentrations, suggesting that the metabolic pathway was essentially independent of substrate (starch) concentration.
4.
Conclusions
This work showed that a thermally treated mixed microflora originating from municipal sewage sludge can be used to convert starch into H2 effectively via dark H2 fermentation. The investigation on the effect of environmental factors (temperature, pH and starch concentration) shows that the seed sludge preferred mesophilic conditions. The H2 production activity and soluble metabolite formation were very sensitive to the control of culture pH. The H2 production kinetics on starch essentially followed Monod-type kinetic model with a predicted maximum H2 production rate ðvmax;H2 Þ and Monod constant ðKs Þ of 1741 ml/h/l and 16.28 g COD/l, respectively. An excellent H2 -producing performance (1119 ml/h/l and 9:47 mmol H2 =g starch) was obtained when the culture was operated at 37 C, pH 6.0, and an initial starch concentration of 24 g COD/l. The performance is better than most of the reported values in the literature. The outcome of this work demonstrates the feasibility of using the mixed culture system for H2 production from starch feedstock.
Acknowledgments The authors gratefully acknowledge the financial support of the National Science Council, Taiwan (Grant no. NSC94-2211E-166-008). We also thank Ms. M.-J. Lin and H.-P. Chen for their assistance on bioreactor operations. R E F E R E N C E S
[1] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001;26:13–28.
33 (2008) 1565 – 1572
[2] Fang HHP, Liu H. Effect of pH on hydrogen production from glucose by a mixed culture. Bioresource Technol 2002;82:87–93. [3] Cheng SS, Chang SM, Chen ST. Effects of volatile fatty acids to a thermophilic anaerobic hydrogen fermentation process degrading peptone. Water Sci Technol 2002;46:209–14. [4] Chen CC, Lin CY, Chang JS. Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate. Appl Microbiol Biotechnol 2001;57:56–64. [5] Lee KS, Lo YC, Lin PJ, Chang JS. Improving biohydrogen production in a carrier-induced granular sludge bed by altering physical configuration and agitation pattern of the bioreactor. Int J Hydrogen Energy 2006;31(12):1648–57. [6] Wu SY, Hung CH, Lin CN, Chen HW, Lee AS, Chang JS. Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge. Biotechnol Bioeng 2006;93(5):934–46. [7] Zhang T, Liu H, Fang HHP. Biohydrogen production from starch in wastewater under thermophilic condition. J Environ Manage 2003;69:149–56. [8] Khanal SK, Chen WH, Li L, Sung S. Biological hydrogen production: effects of pH and intermediate products. Int J Hydrogen Energy 2004;29(11):1123–31. [9] Fang HHP, Li CL, Zhang T. Acidophilic biohydrogen production from rice slurry. Int J Hydrogen Energy 2006;31:683–92. [10] Lee KS, Lin PJ, Chang JS. Temperature effects on biohydrogen production in a granular sludge bed induced by activated carbon carriers. Int J Hydrogen Energy 2006;31(4):465–72. [11] Lee KS, Wu JF, Lo YS, Lo YC, Lin PJ, Chang JS. Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor. Biotechnol Bioeng 2004;87(5):648–57. [12] APHA. Standard methods for the examination of water and wastewater. New York: American Public Health Association; 1995. [13] Zwietering MH, Jongenburger I, Rombouts FM, Van’t Riet K. Modeling of the bacterial growth curve. Appl Environ Microbiol 1990;56:1875–81. [14] Lee KS, Lo YS, Lo YC, Lin PJ, Chang JS. Operation strategies for biohydrogen production with a high-rate anaerobic granular sludge bed bioreactor. Enzyme Microb Technol 2004;35(6–7):605–12. [15] Chen WM, Tseng ZJ, Lee KS, Chang JS. Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge. Int J Hydrogen Energy 2005;30(10):1063–70. [16] Lay JJ. Modeling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol Bioeng 2000;68(3):269–78. [17] Liu G, Shen J. Effects of culture and medium conditions on hydrogen production from starch using anaerobic bacteria. J Biosci Bioeng 2004;98(4):251–6.
33 (2008) 1565 – 1572
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijhydene
Exploring optimal environmental factors for fermentative hydrogen production from starch using mixed anaerobic microflora Kuo-Shing Leea, Yao-Feng Hsub, Yung-Chung Loc, Ping-Jei Linb, Chiu-Yue Lind, Jo-Shu Changc, a
Department of Safety Health and Environmental Engineering, Central Taiwan University of Science and Technology, Taichung, Taiwan Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan c Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan d BioHydrogen Laboratory, Department of Water Resource Engineering, Feng Chia University, Taichung, Taiwan b
art i cle info
ab st rac t
Article history:
Our previous work demonstrated that an acclimated mixed bacterial consortium was able
Received 15 June 2007
to produce H2 from sugar substrates. To reduce the medium cost for more commercially
Received in revised form
viable H2 production, cassava starch was used as the feedstock to produce H2 via dark
10 October 2007
fermentation. Three factors, namely, temperature, pH and starch concentration ðCstarch Þ,
Available online 26 November 2007
were intensively examined for their effects on H2 production activity. The H2 production
Keywords: Fermentative hydrogen production Starch Environmental factors pH Kinetics
kinetics was determined using a Monod-type kinetic model. The results show that mesophilic temperature ð37 CÞ is preferable for H2 production with the H2 -producing sludge used. The H2 production efficiency and the composition of soluble metabolites were found to be highly sensitive to the change in pH, as pH 6.0 seemed to give the best overall H2 production performance. In a non-pH-controlled culture (initial pH ¼ 8:5), ethanol and butyrate were the major soluble metabolites, whereas the predominant metabolites switched to butyrate alone (accounting for 70–80% of total soluble microbial products) when the culture pH was controlled at a fixed level ranging from 5.5 to 7.0. Meanwhile, the maximum H2 production rate occurred when the initial starch concentration was 24 g COD/l. The dependence of H2 production rate on starch concentration could be described by using Monod-type model and the predicted kinetic constants, namely, maximum H2 production rate ðvmax;H2 Þ and Monod constant ðKs Þ, were 1741 ml/h/l and 16.28 g COD/l, respectively. Under the optimal conditions (37 C, pH 6.0, Cstarch ¼ 24 g COD=l), the H2 production rate increased to 1119 ml/h/l, while a high H2 yield of 9.47 mmol H2 /g starch was obtained. This performance appeared to be superior to that obtained from other starch-to-bioH2 systems reported in the literature. & 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
Corresponding author: Tel.: +886 6 2757575 62651, fax: +886 6 2357146.
E-mail address: [email protected] (J.-S. Chang). 0360-3199/$ - see front matter & 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2007.10.019
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1.
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
Introduction
Hydrogen, a clean and efficient energy carrier, is considered a feasible alternative to fossil fuels. Conventional H2 production methods (e.g., thermochemical process) mainly utilize fossil fuels as the raw materials. These processes are not sustainable and environmentally friendly. Therefore, a cost effective and non-polluting means of H2 production is still in great demand [1]. Hydrogen production from organic materials through biological processes, such as photo and dark fermentation, are considered promising and sustainable H2 production methods because they are pollution-free and less energy intensive. Among the biohydrogen production methods, dark fermentation seems more feasible for commercial processes due primarily to the feature of attaining much higher H2 production rates than other bio-hydrogenation systems. Glucose and sucrose are most often used substrates for dark H2 fermentation [2–6] because these simple carbohydrates are readily utilized by microorganism. However, those sugar substrates may be too expensive for commercialized H2 production. From the perspective of energy supply, mass and
Cumulative H2 production (ml)
1600 37 °C 55 °C
1400 1200 1000 800 600 400 200 0 0
10
20
30
40
Time (h) Fig. 1 – Cumulative H2 production profiles for cultures conducted at 37 and 55 C. (Initial pH ¼ 8:5, initial starch concentration ¼ 16 g COD=l; symbols: experimental data, curves: prediction with Gompertz equation.)
33 (2008) 1565 – 1572
low-cost production of H2 from sufficient substrate resources is inevitably required. The starch seems to be a feasible feedstock for large scale H2 production because it is one of the most abundant organic resources produced from plant in nature. In addition, starch also contributes to a marked portion of biological oxygen demands in many types of waste and/or wastewater, which also require appropriate clean-up. Nevertheless, there is little information in the literature regarding the fermentative H2 production from starch [7–9]. Temperature and pH are usually the target factors influencing the performance of bioH2 production from starch. Zhang et al. [7] compared H2 production from starch at 37 and 55 C by mesophilic sludge and found that more starch was converted into H2 at 55 C, but a longer lag time was required. They also examined the effect of initial pH (from 4.0 to 9.0) on fermentative H2 production from starch and obtained the maximum H2 yield of 92 ml/g starch (17% of the theoretical value) at pH 6.0, whereas the maximum specific H2 production rate of 365 ml/d/g volatile suspended solid (VSS) occurred at pH 7.0. Using mixed culture to convert starch into H2 , Khanal et al. [8] discovered that the lowest initial pH (4.5) tested gave the highest H2 production yield (125 ml H2 =g COD starch), but the lowest specific H2 production rate, which was the highest at pH 5.5–5.7. Fang et al. [9] investigated the effect of pH on hydrogen production from rice slurry with H2 producing sludge at a concentration of 5.5 g carbohydrate/l. The results show that the maximum H2 yield of 346 ml/g carbohydrate occurred at pH 4.5, but again operation at this low pH value led to a long lag phase (36 h) for H2 evolution. Thus, the optimal pH for H2 production from starch seems to be more acidic than the optimal pH (5.5) for H2 production from glucose reported by the same group [2]. On the other hand, the knowledge on substrate-dependent H2 -producing kinetics is also the key to a successful operation of a H2 producing bioreactor. Unfortunately, systematic kinetic study on the dependence of H2 production efficiency on substrate (starch) concentration is still lacking [7,9]. As literature shows that the environmental factors and substrate concentration play crucial roles in affecting cell growth and H2 production efficiency [2,4,7–9], this study aimed to understand how temperature, pH and substrate concentration affect H2 production from cassava starch using
Table 1 – Performance of fermentative H2 production at different temperatures T ð CÞ
a
pH
Model simulationa
Substrate concentration (g COD/l) P (ml)
Rm (ml/h)
l (h)
37
Not controlled 0.204
16
1447
477
25.7
55
Not controlled
16
241
83
26.0
Simulated with modified Gompertz equation.
H2 yield (mmol H2 / g starch)
Substrate utilization (%)
Cell yield (g VSS/ g starch)
0.998
5.34
82
1.44
51
0.085
r2
0.997
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2.
Material and methods
2.1. Hydrogen-producing sludge and fermentation medium The seed sludge was obtained from a municipal sewage treatment plant located in central Taiwan. The sludge was thermally pretreated at 952100 C for 1 h. The medium used for H2 fermentation contained 8–32 g COD/l of cassava starch (i.e., 6.75–27 g starch/l; Antik Sempurna S/B, Malaysia) as the sole carbon source and was supplemented with sufficient amounts of inorganic salts. To make the cassava starch disperse evenly in water, the starch was thermally pretreated at 90295 C until aqueous solution become semitransparent.
The detailed medium composition has been described in our recent work [10].
2.2.
Hydrogen production in batch fermentors
Three series of batch experiments were conducted in 2 l glass reactors containing 200 ml of thermally treated sludge and
3000 Cumulative H2 production (ml)
mixed anaerobic microflora as the H2 -producers. The batch tests examined the H2 -producing performance under thermophilic ð55 CÞ and mesophilic ð37 CÞ temperatures and non-pH-controlled (initial pH ¼ 8:5) or pH-controlled ðpH ¼ 5:527:0Þ environments. Kinetics of H2 production from starch was also determined by conducting H2 fermentation at a range of starch concentrations (8–32 g COD/l). The experimental data was described by Monod-type kinetic model and the optimal kinetic constants were also estimated. The results obtained from this work are expected to provide fundamental knowledge and useful information for optimal operation and practical application of starch-based dark H2 fermentation systems.
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33 (2008) 1565 – 1572
pH 7.0 pH 6.5 pH 6.0 pH 5.5
2500 2000 1500 1000 500 0 0
5
10
15
20
25
Time (h) Fig. 2 – Cumulative H2 production profiles for cultures conducted at a fixed pH of 5.5, 6.0, 6.5 and 7.0. (Temperature ¼ 37 C, initial starch concentration ¼ 16 g COD=l; symbols: experimental data, curves: prediction with Gompertz equation.)
Table 2 – Soluble metabolites formation along with fermentative H2 production at different temperatures T ð CÞ 37 55
pH
Substrate concentration (g COD/l)
SMP (g COD/l)
TVFA (g COD/l)
EtOH (%)
HAc (%)
HPr (%)
HBu (%)
HVa (%)
Not controlled Not controlled
16
6.84
3.84
43.9
10.9
2.4
42.8
0.0
16
1.82
0.92
49.4
9.3
0.9
40.4
0.0
Initial pH ¼ 8:5, final pH ¼ 6:226:3. HAc: acetic acid; HPr: propionic acid; HBu: normal butyric acid; HVa: normal valeric acid; EtOH: ethanol; SMP: soluble microbial products; TVFA (total volatile fatty acid)¼ HAc þ HBu þ HPr þ HVa.
Table 3 – Performance of fermentative H2 production at different pH T ð CÞ
pH
Model simulationa
Substrate concentration (g COD/l) P (ml)
Rm (ml/h)
l (h)
37
7.0 0.182
16
1441
396
8.9
37 37 37
6.5 6.0 5.5
16 16 16
2246 2771 2970
612 857 511
9.7 9.5 7.2
a
Simulated with modified Gompertz equation.
H2 yield (mmol H2 / g starch)
Substrate utilization (%)
Cell yield (g VSS/ g starch)
0.996
5.17
84
8.12 8.63 9.19
84 97 98
0.153 0.260 0.218
r2
0.998 0.996 0.995
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800 ml medium to undergo batch hydrogen fermentation. In series 1, batch cultures were conducted to compare the H2 production performance at 37 and 55 C with a medium
0 7
containing 16 g COD/l of starch at an initial pH of 8.5. In series 2, the effect of pH was examined and the experiments were conducted at pH ranging from 5.5 to 7.0 with a 0.5 increment. The medium contained 16 g COD/l of starch at 37 C. Series 3 mainly investigated the effect of starch concentration on H2 production. The starch concentration tested ranged from 8 to 32 g COD/l with 8 g COD/l increments at pH 6.0 and 37 C. In all batch tests, the mixed liquor in the reactor was first purged with argon for 5–10 min to ensure an anaerobic condition prior to H2 fermentation experiments. During the course of experiments, the quantity and composition of gas products (mainly, CO2 and H2 ) and soluble microbial products (SMP) were monitored at designated time intervals. The gas volumes measured by a volumetric gas meter (Type TG1; Ritter Inc., Germany) were corrected to a temperature of 25 C and a pressure of 1 atm. The H2 yield was calculated based on the mmol of H2 produced per g of starch consumed. The starch consumption was converted from the difference between initial ðTCi Þ and residual ðTCf Þ amounts of total carbohydrate content in the culture. The substrate utilization was the ratio of starch consumption and initial amount of starch and was calculated by ðTCi TCf Þ=TCi . The calculation of cell yield was similar to that for H2 yield, as the amount (g VSS) of cell mass gained replaces the amount of H2 produced in the calculation.
6
2.3.
5
40
The gas products (mainly H2 and CO2 ) was analyzed by gas chromatography (GC) (GC-14A, Shimadzu, Tokyo, Japan) using a thermal conductivity detector. The volatile fatty acids and ethanol were also detected by GC using a flame ionization detector. The conditions and columns used for GC analysis were identical to those reported previously [11]. Standard Methods [12] were used to determine biomass concentration (in terms of VSS) of samples taken from the bioreactor. The total carbohydrate concentration in the effluent was also measured using Standard Methods [12].
20
2.4.
Biomass (g-VSS/L)
Soluble microbial products (mg COD/L)
Cumulative H2 production (ml)
3000 2500 2000 1500 1000 500 0 8000 EtOH HAc HPr HBu
6000 4000 2000
4 3 100
Starch utilization (%)
33 (2008) 1565 – 1572
80 60
Analytical methods
Kinetic analysis
0 0
5
10
15
20
Time (h) Fig. 3 – Time-course profiles of H2 production, soluble metabolite formation, biomass growth and starch utilization for culture conducted at pH 6.0, 37 C, and an initial starch concentration of 16 g COD/l.
The results of cumulative H2 production in the batch experiments were described by the modified Gompertz equation [13]: Rm e ðl tÞ þ 1 , (1) HðtÞ ¼ P exp exp P
Table 4 – Soluble metabolites formation along with fermentative H2 production at different pH T ð CÞ
pH
Substrate concentration (g COD/l)
SMP (g COD/l)
TVFA (g COD/l)
EtOH (%)
HAc (%)
HPr (%)
HBu (%)
HVa (%)
37 37 37 37
7.0 6.5 6.0 5.5
16 16 16 16
5.73 7.19 9.35 8.57
4.99 6.22 9.13 8.38
12.9 13.5 2.3 2.3
12.8 12.5 13.9 16.8
1.7 0.7 0.6 0.6
72.6 73.3 83.2 80.3
0.0 0.0 0.0 0.0
HAc: acetic acid; HPr: propionic acid; HBu: normal butyric acid; HVa: normal valeric acid; EtOH: ethanol; SMP: soluble microbial products; TVFA (total volatile fatty acid)¼ HAc þ HBu þ HPr þ HVa.
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where H is the cumulative volume of H2 produced (ml), P is the H2 production potential (ml), Rm is the maximum H2 production rate (ml/h), l is the lag-phase time (h), t is the incubation time (h) and e is the expð1Þ ¼ 2:718. The dependence of H2 production rate on limiting substrate (starch) concentration was simulated by a Monod-type kinetic model: vH2 ¼
vmax;H2 Cstarch , Ks þ Cstarch
(2)
where vmax;H2 is the maximum volumetric H2 production rate (ml/l/h), Ks is half-saturation (Monod) constant (g COD/l) and Cstarch is starch concentration (g COD/l).
3.
Results and discussion
3.1.
Effect of temperature on hydrogen production
It is of great importance to identify the temperature preference of the H2 -producing bacterial population used in
Cumulative H2 production (ml)
6000 8 g COD/L 16 g COD/L 24 g COD/L 32 g COD/L
5000 4000 3000
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33 (2008) 1565 – 1572
this study. Thus, two typical mesophilic ð37 CÞ and thermophlic ð55 CÞ temperatures were examined [7,9]. The profiles of cumulative H2 production from starch (16 g COD/l) at the two temperatures and an initial pH of 8.5 are illustrated in Fig. 1, which clearly shows that H2 production at 37 C was much more effective than at 55 C. Fig. 1 and Table 1 show that the H2 evolution data could be simulated satisfactorily ðr2 ¼ 0:99720:998Þ based on modified Gompertz equation (Eq. (1)), and the estimated kinetic parameters, as well as H2 yield, substrate utilization and cell yield, are listed in Table 1. For the culture at 37 C, the P and Rm was 477 ml/h and 5:34 mmol H2 =g starch, respectively, which is nearly six-fold higher than that at 55 C. The H2 yield obtained at 37 C (5:34 mmol H2 =g starch) was also much higher than that obtained at 55 C (1:44 mmol H2 =g starch). Meanwhile, the 37 C culture also gave significantly higher cell yield and substrate utilization efficiency than the culture operated at 55 C, as the substrate utilization efficiency and cell yield decreased from 82% to 51% and 0.204 to 0.085 g VSS/g starch, respectively. This seems to indicate that the bacterial populations in the H2 -producing sludge used in this study were mainly mesophiles. Analysis of soluble metabolite composition shows that irrespective the temperature used, the predominant metabolite was ethanol (EtOH) and butyrate (HBu), while acetate was produced at a much lesser amount. This soluble metabolite composition is very different from that obtained from our previous work, which was operated at a much lower pH (ca. 6.5) using sugar-based media [4–6,10,11,14]. Hence, it is likely that the difference in soluble metabolite formation may be strongly associated with the type of carbon substrate and the operating pH range (Table 2).
2000
3.2.
1000 0 0
5
10
15
20
25
Time (h) Fig. 4 – Cumulative H2 production profiles for cultures conducted at an initial starch concentration of 8, 16, 24 and 32 g COD/l. (Temperature ¼ 37 C, pH ¼ 6:0; symbols: experimental data, curves: prediction with Gompertz equation.)
Effect of pH on hydrogen production
The pH effects on H2 production was examined using the preferable temperature of 37 C. With the pH controlled at a fixed value (5.5–7.0), the lag time for H2 evolution was markedly shortened to within 10 h (Table 3). The pHdependent H2 production profiles (Fig. 2) show that operation at pH 5.5 and 6.0 displayed better H2 production performance than the other two pH values. The cultures operated at pH 5.5 and 6.0 also attained better substrate utilization efficiency
Table 5 – Performance of fermentative H2 production at different substrate (starch) concentrations T ð CÞ
pH
Substrate concentration (g COD/l)
Model simulationa
P (ml)
Rm (ml/h)
l (h)
37
6.0 0.281
8
957
534
8.9
37 37 37
6.0 6.0 6.0
16 24 32
2771 4128 5964
857 1119 1069
9.5 9.5 13.2
a
Simulated with modified Gompertz equation.
H2 yield (mmol H2 / g starch)
Substrate utilization (%)
Cell yield (g VSS/ g starch)
0.997
6.24
93
8.63 9.47 11.25
97 88 80
0.260 0.133 0.177
r2
0.997 0.999 0.997
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Volumetric H2 production rate (ml/l/h)
and cell yield than those for pH 6.5 and 7.0, suggesting that the former two pHs were better conditions for the cells to utilize starch for growth. According to the literature, the optimal pH for H2 production from starch varied from 4.5 to 6.0 with few exceptions (Table 7), and our finding appeared to fall within that optimal pH range. Kinetic data (Table 3) indicate that pH 6.0 gave the best H2 production rate at 857 ml/h, whereas the culture at pH 5.5 attained a slightly better H2 yield (9:19 mmol H2 =g starch) over pH 6.0. The H2 producing performance at pH 5.5 and 6.0 is better than most of the values reported in the literature (Table 7). It is also found that the pH-6.0 culture obtained the highest cell yield of 0.260 g VSS/g starch (Table 3). The high cell yield may lead to more electron flow toward biomass. This might explain why pH 6.0 had a higher H2 production rate, but a lower H2 yield when compared with pH 5.5. Fig. 3 further demonstrates that before H2 evolution occurred (0–9 h), starch began to be utilized for biomass growth, while H2 production started to accelerate along with cell growth from the early exponential phase. This growth-dependent H2 production trend is slightly different from that observed in a pure culture of Clostridium butyricum CGS5, as H2 production did not occur until cell growth reached the mid-exponentialphase [15].
1400 1200 1000 800 600 400 200 0 0
8
16
24
32
40
Starch concentration (g COD/L) Fig. 5 – Dependence of volumetric H2 production rate on initial starch concentration. (Temperature ¼ 37 C, pH ¼ 6:0; symbols: experimental data, curves: prediction with Monod-type kinetic model.)
33 (2008) 1565 – 1572
The soluble metabolite data show that with a controlled pH within 5.5–7.0, production of ethanol, one of major products resulting from pH-uncontrolled cultures (Table 2), decreased dramtically, while HBu became the sole predominant metabolite, accounting for 72–84% of total SMP (Table 4). The shift from ethanol-type fermentation to butyrate-type fermentation appeared to markedly enhance both H2 production rate and production yield (Tables 1 and 3). This is reasonable from the biochemical aspect because butyrate is a favorable metabolite for bioH2 production, whereas solvent (e.g., ethanol) formation is usually unfavorable to H2 production due to consumption of additional free electrons from NADH [1]. Furthermore, the metabolite results also show that the metabolic pathway or bacterial community structure (or both) of the H2 -producing sludge used in this work were quite sensitive to the culture pH. This again raises the importance of pH control in bioreactor operation for bioH2 production using the mixed microflora [7–9,16,17].
3.3. Effect of substrate concentration on hydrogen production Understanding reaction kinetics of the biological system of interest is usually a critical step toward optimal control and operation of a bioreactor. Unfortunately, there is little information in the literature regarding the dependence of starch concentration on fermentative H2 production [7,9,17]. Hence, the knowledge on starch-dependent H2 production kinetics should be revealed. Using the preferable temperature ð37 CÞ and pH (6.0) for bioH2 production based on the foregoing results, the H2 production performance of cultures with different initial substrate (starch) concentrations of 8–32 g COD/l was investigated. The cumulative H2 production and H2 yield both increased as the initial starch concentration increased (Fig. 4 and Table 5). The volumetric H2 production rate reached a maximum value of 1119 ml/h/l (and a yield of 9:47 mmol H2 =g starch) when 24 g COD/l of starch was used as the sole carbon source (Fig. 5 and Table 6). The dependence of H2 production on starch concentration was simulated with a Monod-type model (Eq. (2)) with good agreement ðr2 ¼ 0:973Þ. The estimated vmax;H2 and Ks values were 1741 ml/h/l and 16.28 g COD/l, respectively. The substrate utilization efficiency and cell yield peaked at a starch concentration of 16 g COD/l, and then decreased at higher substrate concentrations
Table 6 – Soluble metabolites formation along with fermentative H2 production at different substrate (starch) concentrations T ð CÞ
pH
Substrate concentration (g COD/l)
SMP (g COD/l)
TVFA (g COD/l)
EtOH (%)
HAc (%)
HPr (%)
HBu (%)
HVa (%)
37 37 37 37
6.0 6.0 6.0 6.0
8 16 24 32
3.97 9.35 11.75 16.98
3.86 9.13 11.46 16.32
2.8 2.3 2.5 3.9
14.3 13.9 16.4 14.3
1.2 0.6 0.3 0.4
81.7 83.2 80.8 81.4
0.0 0.0 0.0 0.0
HAc: acetic acid; HPr: propionic acid; HBu: normal butyric acid; HVa: normal valeric acid; EtOH: ethanol; SMP: soluble microbial products; TVFA (total volatile fatty acid)¼ HAc þ HBu þ HPr þ HVa.
Table 7 – Comparison of the performance of fermentative H2 production from starch in comparable studies H2 producer
Culture type
Substrate
Examined temperature ð CÞ
Examined pH
Examined substrate concentration (g/l)
Optimal culture condition
pH
Substrate concentration (g/l)
Max. HPRb (ml/l/h)
Reference
Rice slurry
37, 55
4.0–7.0
2.7–22.1c
37
4.5
5.5c
14.12d
NA
[9]
Compost material Sludge
Batch
Starch
37
4.5–6.5
10
NA
NA
6.05
NA
[8]
CSTR
Soluble starch
37
4.0–7.0
4.25
NA
4.5 (initial) 5.2
NA
NA
67
[16]
Corn starch
35
4.0–9.0
2-32
NA
8.0 (initial)
2
7.92
4
[17]
Starch
37, 55
4.0–9.0
4.6–36.6
55
6.0 (initial) 7.0 (initial) 6.0
24 4.6
4.33 3.76
53 3.1
[7]
(2.73)
5.8
20.3 (24 g COD/l)
9.47
1119
This
27 (32 g COD/l)
11.25
1069
study
Mixed bacteria Sludge
Sewage sludge
ðHRT ¼ 17 hÞ Batch
Batch
Batch
Cassava starch
NA: not available. HY: hydrogen yield. b HPR: hydrogen production rate. c g carbohydrate/l. d mmol H2 =g carbohydrate. a
37, 55
5.5–7.0
6.75–27 (8–32 g COD/l)
37
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Batch
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Anaerobic digester sludge
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Temperature ð CÞ
Max. HYa (mmol H2 / g starch)
1571
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(Table 5). The predicted maximum H2 production rate of 1741 ml/h/l from starch is much high than the reported values (Table 7), indicating the high effectiveness of our H2-producing system. The H2 yield obtained from this work is also comparable or higher than those obtained from the comparable studies (Table 7). Like the results exploring the pH effects, the predominant soluble metabolite was HBu (representing 80–84% of SMP), followed by acetate (representing 13–17% of SMP). Moreover, the metabolite composition was quite similar for cultures with different initial starch concentrations, suggesting that the metabolic pathway was essentially independent of substrate (starch) concentration.
4.
Conclusions
This work showed that a thermally treated mixed microflora originating from municipal sewage sludge can be used to convert starch into H2 effectively via dark H2 fermentation. The investigation on the effect of environmental factors (temperature, pH and starch concentration) shows that the seed sludge preferred mesophilic conditions. The H2 production activity and soluble metabolite formation were very sensitive to the control of culture pH. The H2 production kinetics on starch essentially followed Monod-type kinetic model with a predicted maximum H2 production rate ðvmax;H2 Þ and Monod constant ðKs Þ of 1741 ml/h/l and 16.28 g COD/l, respectively. An excellent H2 -producing performance (1119 ml/h/l and 9:47 mmol H2 =g starch) was obtained when the culture was operated at 37 C, pH 6.0, and an initial starch concentration of 24 g COD/l. The performance is better than most of the reported values in the literature. The outcome of this work demonstrates the feasibility of using the mixed culture system for H2 production from starch feedstock.
Acknowledgments The authors gratefully acknowledge the financial support of the National Science Council, Taiwan (Grant no. NSC94-2211E-166-008). We also thank Ms. M.-J. Lin and H.-P. Chen for their assistance on bioreactor operations. R E F E R E N C E S
[1] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001;26:13–28.
33 (2008) 1565 – 1572
[2] Fang HHP, Liu H. Effect of pH on hydrogen production from glucose by a mixed culture. Bioresource Technol 2002;82:87–93. [3] Cheng SS, Chang SM, Chen ST. Effects of volatile fatty acids to a thermophilic anaerobic hydrogen fermentation process degrading peptone. Water Sci Technol 2002;46:209–14. [4] Chen CC, Lin CY, Chang JS. Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate. Appl Microbiol Biotechnol 2001;57:56–64. [5] Lee KS, Lo YC, Lin PJ, Chang JS. Improving biohydrogen production in a carrier-induced granular sludge bed by altering physical configuration and agitation pattern of the bioreactor. Int J Hydrogen Energy 2006;31(12):1648–57. [6] Wu SY, Hung CH, Lin CN, Chen HW, Lee AS, Chang JS. Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge. Biotechnol Bioeng 2006;93(5):934–46. [7] Zhang T, Liu H, Fang HHP. Biohydrogen production from starch in wastewater under thermophilic condition. J Environ Manage 2003;69:149–56. [8] Khanal SK, Chen WH, Li L, Sung S. Biological hydrogen production: effects of pH and intermediate products. Int J Hydrogen Energy 2004;29(11):1123–31. [9] Fang HHP, Li CL, Zhang T. Acidophilic biohydrogen production from rice slurry. Int J Hydrogen Energy 2006;31:683–92. [10] Lee KS, Lin PJ, Chang JS. Temperature effects on biohydrogen production in a granular sludge bed induced by activated carbon carriers. Int J Hydrogen Energy 2006;31(4):465–72. [11] Lee KS, Wu JF, Lo YS, Lo YC, Lin PJ, Chang JS. Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor. Biotechnol Bioeng 2004;87(5):648–57. [12] APHA. Standard methods for the examination of water and wastewater. New York: American Public Health Association; 1995. [13] Zwietering MH, Jongenburger I, Rombouts FM, Van’t Riet K. Modeling of the bacterial growth curve. Appl Environ Microbiol 1990;56:1875–81. [14] Lee KS, Lo YS, Lo YC, Lin PJ, Chang JS. Operation strategies for biohydrogen production with a high-rate anaerobic granular sludge bed bioreactor. Enzyme Microb Technol 2004;35(6–7):605–12. [15] Chen WM, Tseng ZJ, Lee KS, Chang JS. Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge. Int J Hydrogen Energy 2005;30(10):1063–70. [16] Lay JJ. Modeling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol Bioeng 2000;68(3):269–78. [17] Liu G, Shen J. Effects of culture and medium conditions on hydrogen production from starch using anaerobic bacteria. J Biosci Bioeng 2004;98(4):251–6.