Hydrogen Production via Thermophilic Fermentation of Cornstalk by

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Hydrogen Production via Thermophilic Fermentation of Cornstalk by Clostridium thermocellum Xi-Yu Cheng†,‡ and Chun-Zhao Liu*,†,‡ †

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡ Graduate School of the Chinese Academy of Sciences, Beijing 100093, People’s Republic of China ABSTRACT: An efficient hydrogen production process via thermophilic fermentation of cornstalk was developed by Clostridium thermocellum 7072. The hydrogen fermentation process was successfully scaled from a 125 mL anaerobic bottle to a 100 L continuous stirred-tank reactor, and the hydrogen production from cornstalk was significantly improved in the bioreactor system because of good mixing and mass transfer. The hydrogen yield in the 100 L continuous stirred-tank reactor reached 61.4 mL/g of cornstalk, which was higher than that in the 125 mL anaerobic bottle. The present work indicated that direct microbial conversion of lignocellulosic waste via C. thermocellum was a promising avenue for biohydrogen production.

1. INTRODUCTION The joint challenges of environmental crises and declining traditional energy reserves are driving intensive research focus in alternative energy production.1
4 Hydrogen is widely regarded as one of the most promising energy carriers because of its high efficiency of conversion to usable power, non-polluting nature, and high mass energy density.5
7 Anaerobic fermentation technology, with outstanding advantages, including low-energy input, simple process, and high production rate, has offered a potential way for hydrogen production.8,9 Hydrogen production from various feedstock, such as sugar, wastewater, municipal solid waste, and food waste, has been reported,9
12 and efficient pretreatment methods, including acidification, microwave irradiation, and steam explosion, have been developed for the enhancement of hydrogen production from lignocellulosic waste.3,13,14 Recently, Clostridium thermocelum, a Gram-positive thermophilic anaerobic bacterium that uses cellulose to synthesize hydrogen, has shown great potential for direct hydrogen production from lignocellulosic waste.15
25 Successful hydrogen production was carried out from thermophilic fermentation of microcrystalline cellulose as well as natural cellulosic biomass (e.g., delignified wood fibers, barley hulls, dried distillers grains, and contaminated barley hulls) with C. thermocellum.15,16 The varied ethanol/acid ratios in fermentation of C. thermocellum strains were reported,20
25 which suggested that the ability of hydrogen production by different C. thermocellum strains was different because hydrogen synthesis was closely related to ethanol and organic acid metabolism. However, to date, information regarding hydrogen and other end-product synthesis characteristics of different C. thermocellum strains is still limited. Therefore, there is still a great interest to understand the thermophilic fermentation process for improving biohydrogen production from lignocellulosic waste using C. thermocellum. The objective of the present study was to understand the characterization of C. thermocellum growth, hydrogen production, and end-product metabolism in the thermophilic fermentation of both microcrystalline cellulose and cornstalk. Additionally, a r 2011 American Chemical Society

scale-up on the hydrogen fermentation process in a bioreactor system was carried out.

2. MATERIALS AND METHODS 2.1. Microorganisms and Media. C. thermocellum strains of 1237, 1313, 2360, 4150, and 7072 were obtained from Deutsche SammLung von Mikroorganismen and Zellkulturen GmbH. A 125 mL anaerobic bottle with a working volume of 50 mL was used for inoculum preparation and the following batch experiments. Each bottle was gassed and degassed with 100% nitrogen gas before sterilization. All bottles were air-sealed with butyl rubber stoppers and screw caps and autoclaved at 121 °C for 20 min. A total of 10% (v/v) of exponentially growing cultures (the OD600 value of the inoculum was about 1.0) was transferred into these anaerobic bottles containing 5 g/L microcrystalline cellulose (Sinopharm Chemical Reagent Co., Ltd., China) and CM4 medium, with a slight modification. The composition of this CM4 medium is as follows (per liter of distilled water): 1.5 g of KH2PO4, 3.8 g of K2HPO4 3 3H2O, 1.3 g of (NH4)2SO4, 1.6 g of MgCl2 3 6H2O, 0.013 g of CaCl2, 5.0 g of yeast extract, 1.25 mg of FeSO4 3 7H2O, 1.0 mg of resazurin, and 0.5 g of L-cysteine.20 The final pH of the medium was adjusted to about 7.2 using sodium bicarbonate. C. thermocellum 1237, 1313, 2360, and 4150 were cultured at 60 °C, and C. thermocellum 7072 was cultured at 55 °C. 2.2. Batch Experiments with Microcrystalline Cellulose and Cornstalk. Cornstalk, obtained from Daxing district, Beijing,

China, was dried at 60 °C in an oven. Cornstalk used in all experiments was milled to e1 mm powder using a plant miller. The main composition of cornstalk is as follows (w/w): 31.1% cellulose, 27.0% hemicellulose, 14.4% lignin, and 13.0% total soluble sugar. Batch experiments were carried out using microcrystalline cellulose and the above cornstalk as a carbon source, respectively. The substrate concentration was 5 g/L, and the inoculum volume was 10% volume of the culture medium. Three bottles containing inoculum and CM4 Received: January 10, 2011 Revised: March 21, 2011 Published: March 22, 2011 1714

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Figure 1. CSTR (10 L). (Top) Photograph of the reactor layout. (Bottom) Schematic of the reactor system: (1) pH meter, (2) temperature controller, (3) motor controller, (4) reactor, (5) heater, (6) cornstalk flakes, (7) solid residue outlet, (8) feed inlet, (9) N2 inlet, (10) volume bottle, (11) water-sealed bottle, (12) wet gas meter, (13) sample outlet, and (14) effluent tank. medium without microcrystalline cellulose and cornstalk were incubated at the same temperature to correct the hydrogen production from microcrystalline cellulose and cornstalk. The bottles were manually mixed once per day. The batch experiments were carried out in triplicates.

2.3. Hydrogen Fermentation in a Continuous Stirred-Tank Reactor (CSTR). Cornstalk used in the CSTR experiments was obtained from the Chinese Academy of Agricultural Sciences, Beijing, China. The main composition of cornstalk is as follows (w/w): 24.0% cellulose, 22.6% hemicellulose, 7.2% lignin, and 28.2% total soluble sugar. Cornstalk used in the bioreactor experiments was milled to e1 mm powder using a plant miller. The milled cornstalk was used as a substrate of hydrogen fermentation in a 10 L CSTR designed by our group (Figure 1). A micromotor, controlled by a transducer, stably run at selected speed ranged from 50 to 750 rpm and was installed below the bottom of the reactor. The pH value detection in the CSTR was carried out by a pH sensor (Mettler Toledo 405-DPAS-SC-K8S) and a pH controller (Eutech Rlpha-pH 800, Singapore). The temperature control was carried out by a Pt 100 sensor and a proportional
integral
derivative (PID) temperature controller (Beijing Huibang XMT614, China). A total of 6.5 L of CM4 medium with 30 g/L cornstalk was added to the CSTR, followed by nitrogen sparging for 30 min. The CSTR was inoculated with 10% (v/v) of exponentially growing cultures of C. thermocellum

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7072 and was flushed again by nitrogen gas for 15 min. The motor speed and temperature were controlled at 100 rpm and 55 ( 1 °C, respectively. Pilot-scale hydrogen fermentation was carried out in a 100 L CSTR (KBT-100 Reactor, Korea). The CM4 medium containing 30 g/L milled cornstalk was added to the 100 L CSTR, followed by nitrogen sparging for 30 min. The 100 L CSTR was inoculated with 10% (v/v) exponentially growing cultures of C. thermocellum 7072. The final volume was 60 L. The CSTR was flushed again by nitrogen gas for 15 min after inoculation. The stirring speed and temperature were controlled at 100 rpm and 55 ( 1 °C, respectively. Hydrogen fermentation in 125 mL anaerobic bottles was conducted with the experimental procedures described in section 2.2, except that the substrate concentration was 30 g/L cornstalk collected from the Chinese Academy of Agricultural Sciences. 2.4. Analysis. The optical density of the cultivated cells was measured at 600 nm using an ultraviolet (UV) spectrophotometer (Unico UV2100 spectrophotometer, Dayton, NJ) according to the reported procedures, with slight modifications.26
28 Cell cultures with solid substrate were vibrated and centrifuged at 500 rpm for 10 min, and the resulting supernatant containing cells was used for the measurement of the optical density. Acetate, lactate and ethanol concentrations were determined using a gas chromatograph (Agilent GC 7890, Santa Clara, CA) equipped with a flame ionization detector and a fused-silica capillary column (30 m  0.32 mm  0.25 μm, PB-INNOWax). The temperatures of the injector and detector were 200 and 250 °C, respectively. The oven temperature increased from 60 °C by a ramp-up of 20 °C/min for 6 min and held at a final temperature of 180 °C for 4 min. Nitrogen was used as the carrier gas with a flow rate of 2.6 mL/min. Cellulose, hemicellulose, and lignin contents were measured using the methods described by Goering and Van Soest.29 The contents of hemicellulose, cellulose, and lignin were calculated on the basis of residual total solid (TS) after fermentation. TS was determined according to the standard method described by the American Public Health Association (APHA).30 The lactate concentration was measured using a biosensor (SBA-40C). The gas composition was measured using chromatography (Agilent GC 7890, Santa Clara, CA) fitted with a thermal conductivity detector. The gas chromatograph was equipped with two columns separated by a switching valve as designed by the manufacturer. The first column was a Plot Q polymer column, to separate CO2 and high-molecular-weight compounds, and the second one was a molecular sieve column to separate the low-molecular-weight gas (H2, O2, N2, and CH4). Helium was used as the carrier gas at a flow rate of 23 mL/min. The oven, injector, and detector temperatures were 50, 150, and 250 °C, respectively. Calibration curves generated for the above gas components were linear and reproducible. The gas produced in the anaerobic bottles was collected in gastight bags. The volume of the gas produced from batch fermentation in the anaerobic bottles was measured using a glass syringe.31 The gas volume produced from batch fermentation in the CSTR was measured using a wet gas meter. The hydrogen yield, expressed as moles of H2 produced per mole of glucose equivalents, was calculated according to the method reported in the recent literature.17 In brief, the formula of cellulose was considered as (C6H10O5)n, and the mole amount of glucose equivalents was obtained on the basis of the molecular weight of the cellulose monosaccharide molecule. The statistical analyses were performed using SPSS 8.0 statistical software program, and the significance was statistically evaluated using one-way analysis of variation (ANOVA) with Duncan’s multiple range test at p e 0.05. 2.5. Kinetic Parameters. The cumulative hydrogen production data were fitted to a modified Gompertz equation,32 a suitable model 1715

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Figure 3. Hydrogen production during thermophilic fermentation of microcrystalline cellulose by C. thermocellum strains. Means with different letters are significantly different at p e 0.05 according to Duncan’s test.

3. RESULTS AND DISCUSSION 3.1. Hydrogen Fermentation of Cellulose via C. thermocellum. Yellow insoluble affinity substances were observed in the

Figure 2. (A) Cell growth, (B) cellulose reduction, and (C) pH variation during thermophilic fermentation of microcrystalline cellulose by C. thermocellum strains.

to describe the cumulative hydrogen fermentation process in a batch experiment (
) Rm e ðλ
tÞ þ 1 HðtÞ ¼ P exp
exp P where H(t) is the cumulative hydrogen volume (mL), P is the hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL of H2 L
1 h
1), λ is the lag time (h), and e equals 2.718. The cumulative hydrogen production curve was nonlinearly fitted by the equation with Origin 8.0 pro.

cell cultures of the five C. thermocellum strains when they began to grow in cellulose. As shown in Figure 2A, C. thermocellum 1237, 1313, and 2360 grew quickly after a 12 h lag phase and the OD600 value reached about 1.0
1.3 at 48 h. C. thermocellum 4150 and 7072 began to grow quickly after 24 h, and their OD600 values reached the maximum at 48 and 72 h, respectively. The cellulose degradation ratio (i.e., weight loss during the fermentation) and pH variation corresponded with the cell growth (panels B and C of Figure 2). The cellulose degradation ratio for all strains exceeded more than 95% after 120 h of cultivation, and the final pH for all strains decreased to about 6.4. The hydrogen production paralleled the cellulose degradation in thermophilic fermentation of C. thermocellum, and rapid hydrogen production occurred from 12 to 48 h (Figure 3). The hydrogen yield of C. thermocellum 1237, 1313, 2360, and 4150 reached 84.6, 96.8, 93.6, and 113.2 mL/g of cellulose added, respectively. C. thermocellum 7072 was the most efficient hydrogen producer and gave the highest hydrogen yield of 158.9 mL/g of cellulose added after 120 h among all five strains tested. The molar hydrogen yield in cellulose thermophilic fermentation via C. thermocellum 1237, 1313, 2360, 4150, and 7072 varied from 0.7 to 1.2 mol of H2/mol of glucose equivalents. High levels of ethanol and lactate production were observed in the fermentation broths of C. thermocellum 1237, 1313, and 2360 (Figure 4), and low levels of ethanol and lactate were found in the fermentation both of C. thermocellum 7072 (Figure 4). Many studies showed that a low hydrogen yield was related to high lactate and ethanol production.15,17 The corresponding molar hydrogen yield of C. thermocellum 7072 reached 1.2 mol of H2/mol of glucose equivalent of cellulose, which was comparable to some reported data.15,17 The molar hydrogen yield in thermophilic fermentation of 4.5 g/L R-cellulose via C. thermocellum 27405 reached 0.7 mol of H2/mol of glucose equivalent of cellulose.15 Molar hydrogen yield in thermophilic fermentation of 5 g/L R-cellulose via C. thermocellum JN4 was 0.8 mol of H2/mol of glucose equivalent of cellulose, and the molar hydrogen yield increased to 1.8 mol of H2/mol of glucose equivalent of cellulose 1716

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Figure 4. (A) Acetate, (B) ethanol, and (C) lactate production during thermophilic fermentation of microcrystalline cellulose by C. thermocellum strains.

Figure 5. (A) Cell growth, (B) cellulose reduction, and (C) pH variation during thermophilic fermentation of cornstalk by C. thermocellum strains.

when C. thermocellum JN4 was co-cultivated with Thermoanaerobacterium thermosaccharolyticum GD17.17 3.2. Hydrogen Fermentation of Cornstalk via C. thermocellum. The cell growth, substrate degradation, and pH variation were measured during themophilic fermentation of cornstalk with the five strains, and the results are shown in Figure 5. Because cornstalk is hard to be biodegraded because of its recalcitrant crystalline structure and physical barrier of lignin,20 slow cell growth and low weight loss of cornstalk were observed in the thermophilic fermentation (panels A and B of Figure 5). The pH variation from 7.4 to 7.1 was related to the cell growth and

cornstalk biodegradation (Figure 5C). As shown in Table 1, the hemicellulose content in the fermentation residue decreased, whereas the lignin content in the fermentation residue increased to some extent after the thermophilic fermentation, and the highest weight loss of 47.2% was observed in the thermophilic fermentation of cornstalk via C. thermocellum 7072 among the five strains tested. As shown in Figure 6, the hydrogen production was parallel to the cornstalk degradation in the thermophilic fermentation via C. thermocellum stains. The hydrogen yield of C. thermocellum 1237, 1313, 2360, and 4150 reached 20.5, 22.6, 23.0, and 32.0 mL/g of cornstalk, respectively. When C. thermocellum 7072 was used, the highest hydrogen yield of 38.8 mL/g of cornstalk was obtained. 1717

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Table 1. Weight Loss and Lignocellulosic Composition at the End of Thermophilic Fermentation of C. thermocellum Strainsa strain

1237 1313

hemicellulose

cellulose

lignin

content (%)

content (%)

content (%)

26.5 ( 0.6 26.6 ( 0.8

30.4 ( 0.3 30.3 ( 1.2

15.8 ( 0.8 15.8 ( 0.5

weight loss (%) 40.8 ( 0.4 42.5 ( 1.8

2360

26.1 ( 1.5

30.6 ( 0.3

15.8 ( 1.2

43.3 ( 1.6

4150

26.3 ( 1.0

31.2 ( 1.2

16.0 ( 0.6

42.1 ( 2.7

7072

26.8 ( 1.3

31.5 ( 0.6

16.5 ( 0.9

47.2 ( 1.4

Lignocellulosic composition of cornstalk is as follows (w/w): 31.1 ( 0.4% cellulose, 27.0 ( 1.1% hemicellulose, and 14.4 ( 0.2% lignin.

a

Figure 6. Hydrogen production during thermophilic fermentation of cornstalk by C. thermocellum strains. Means with different letters are significantly different at p e 0.05 according to Duncan’s test.

As shown in Figure 7, acetate and ethanol were the major end products in the fermentation broth of C. thermocellum. The molar ratio of ethanol/acetate during the thermophilic fermentation of cornstalk was lower than that in the thermophilic fermentation of microcrystalline cellulose. This might be due to the fact that the substrate change from microcrystalline cellulose to natural cellulosic substrates made a shift from the ethanol to the acetate pathway.16 Lactate production in all cultures of the five strains was below 100.0 mg/L, and C. thermocellum 7072 produced the least lactate in the thermophilic fermentation of cornstalk. 3.3. Hydrogen Fermentation of Cornstalk via C. thermocellum 7072 in CSTR. Hydrogen production via thermophilic fermentation of 30 g/L cornstalk was scaled from a 125 mL anaerobic bottle to a 10 L CSTR and then a 100 L CSTR. Because of the better mixing conditions in the two reactors, a higher hydrogen production rate and a shorter lag-phase period were observed in the two CSTRs than in the anaerobic bottles (Figure 8). Table 2 represents the kinetic parameters of hydrogen production from cornstalk in a 125 mL anaerobic bottle, a 10 L CSTR, and a 100 L CSTR. The maximal hydrogen production rate in both a 10 L CSTR and a 100 L CSTR was significantly higher than that in a 125 mL anaerobic bottle. The hydrogen yield in 10 and 100 L CSTRs reached 58.3 and 61.4 mL/g of cornstalk, respectively. To the best of our knowledge, this is the first report on the successful hydrogen production from raw cornstalk in a 100 L bioreactor via C. thermocellum. The hydrogen yield from the thermophilic fermentation of cornstalk by C. thermocellum 7072 in the 100 L CSTR was comparable to some reported data on hydrogen

Figure 7. (A) Acetate, (B) ethanol, and (C) lactate production during thermophilic fermentation of cornstalk by C. thermocellum strains.

fermentation of both steam-exploded cornstalk (SECS) and biological-treated cornstalk in anaerobic bottles.1,33 Hydrogen was produced by simultaneous saccharification and fermentation from SECS using Clostridium butyricum, and the hydrogen yield reached 68 mL/g of SECS.33 Clostridium paraputrificum was introduced to the hydrolysis stage of cornstalk under thermophilic (55 °C) and acidic (pH 5.0) conditions, and the hydrogen yield reached 63.7 mL/g of cornstalk.1 The weight loss of 63.5% was observed in the 100 L SCTR, which was higher than that in the 125 mL anaerobic bottle (Table 2). Acetate and ethanol were major end products of fermentation by C. thermocellum for cornstalk, and the ratio of ethanol/acetate is lower in both 10 and 100 L CSTRs than in 1718

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Figure 8. Cumulative hydrogen volume and (D) end products from thermophilic fermentation of cornstalk in (A) 125 mL anaerobic bottle, (B) 10 L CSTR, and (C) 100 L CSTR.

Table 2. Thermophilic Hydrogen Fermentation of Cornstalk in a 125 mL Anaerobic Bottle, a 10 CSTR, and a 100 L CSTR kinetic parameters of hydrogen fermentation fermentation system

hydrogen yield (mL of H2/g of cornstalk)

weight loss (%)

a

P (mL)

Rm (mL of H2 L
1 h
1)b

λ (h)

R2

125 mL bottle

50.1

52.7

1461.6

201.4

4.4

0.997

10 L CSTR 100 L CSTR

58.3 61.4

61.5 63.5

10676.5 93503.6

767.5 739.9

2.0 2.2

0.977 0.965

a

P is defined as the hydrogen production potential per liter of cultures (mL). b Rm is defined as the maximum hydrogen production rate (mL of H2 L
1 h
1).

125 mL anaerobic bottles (Figure 8D). A higher ethanol concentration was associated with a lower hydrogen yield. In anaerobic bottles without stirring, the produced hydrogen gas was easily entrapped or adsorbed onto cornstalk particles. As a result, the highly localized hydrogen concentration enhanced ethanol biosynthesis and decreased hydrogen production. In the CSTR system, gas products automatically discharged out of the bioreactor system and slow stirring was beneficial to hydrogen emission from liquid broth to the gas phase. Therefore, hydrogen partial pressure decreased, and hydrogen production was enhanced.

4. CONCLUSION The above data proved useful in not only a better understanding of the characterization of C. thermocellum growth, hydrogen production, and end-product synthesis in the thermophilic fermentation of both microcrystalline cellulose and cornstalk

but also developing a promising avenue for biohydrogen production through the direct microbial conversion of lignocellulosic waste by the selected strain of C. thermocellum 7072. Successful hydrogen production was carried out in a pilotscale reactor, where a higher hydrogen production rate was obtained when compared to the fermentation of cornstalk in anaerobic bottles. Together, these results established the most important conditions to explore for future process development of thermophilic hydrogen fermentation via C. thermocellum.

’ AUTHOR INFORMATION Corresponding Author

*Address: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Telephone and Fax: þ86-10-82622280. E-mail: [email protected]. 1719

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’ ACKNOWLEDGMENT This work is financially supported by the National Basic Research Program (973 Program) of China (2007CB714301) and the Knowledge Innovation Program of the Chinese Academy of Sciences (KGCX2-YW-337 and KSCX1-YW-11D1).

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