Fermentative Production of Hydrogen from Wheat Bran by Mixed

Jul 29, 2008 - In the present studies, treatment of wheat bran was carried out with the intention to produce hydrogen during the anaerobic degradation...
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Ind. Eng. Chem. Res. 2008, 47, 5812–5818

APPLIED CHEMISTRY Fermentative Production of Hydrogen from Wheat Bran by Mixed Anaerobic Cultures Chunmei Pan,†,‡ Yaoting Fan,†,* and Hongwei Hou† Department of Chemistry, Zhengzhou UniVersity, Zhengzhou, Henan 450052, P. R. China, and Department of Biotechnology, Zhengzhou College of Animal Husbandry Engineering, Zhengzhou, Henan 450011, P. R. China

In the present studies, treatment of wheat bran was carried out with the intention to produce hydrogen during the anaerobic degradation process. The effects of the hydrogen-producing microbial sources, pretreatment condition, substrate concentration, inoculum concentration, and initial pH on the hydrogen production were investigated in batch cultivations. The activated sludge of paper mill in Sui County exhibited a better hydrogen production ability in contrast with the other three natural microbial sources, and the predominant hydrogenproducing bacteria were identified as Clostridium sp. The conventional acid pretreatment of wheat bran was essential for adequate converting wheat bran into biohydrogen. The contents of soluble saccharides in the pretreated wheat bran increased from 0.086 g/g total solid (TS) to 0.392 g/g-TS compared with the raw wheat bran. The maximum hydrogen yield of 128.2 mL/g total volatile solid (TVS) and hydrogen production rate of 2.50 mL/(g-TVS h) were obtained at an initial pH 5.0, 80 g/L of pretreated wheat bran, and 60 g/L of activated sludge of paper mill. The maximum hydrogen content in the biogas was 62% (v/v), and there was no significant methane observed. In addition, biodegradation characteristics of the wheat bran by microorganisms were also discussed. Both butyrate and acetate were main byproduct in the metabolism of hydrogen fermentation. 1. Introduction Hydrogen has been promoted as a potential solution for a variety of complex problems related to energy and the environment. Compared to fossil fuels, hydrogen has the advantages of being renewable, providing clean burning, and producing no green-house gases. Traditionally, hydrogen is mainly generated by hydrocarbon reformation, the reactions of natural gas or light oil fractions with steam at high temperatures, as well as electrolysis of water.1–3 However, these methods are both energy-intensive and unfriendly to environment. From an engineering point of view, biological hydrogen production from renewable waste sources by mixed cultures, known as “green technology”, stands out as an environmentally harmless process due to low cost, easy control, and the possible use of organic solid wastes as substrate.4 In the last two decades, extensive scientific attention has been devoted to the conversion of soluble carbohydrates, such as glucose, sucrose, and starch, 5–7 into hydrogen gas by fermentative microbes. Also, various attempts have been conduced toward generating hydrogen from wastewater like paper mill, food processing, rice winery, and dairy waste.8–11 From environmental engineering standpoint, bioconversion of agricultural wastes and residues, especially cellulosic biomass wastes, into hydrogen gas is of great interest. Our previous study showed that the maximum hydrogen yield treating wheat straw and beer lees were 68.1 mL/g total volatile solid (TVS) and 68.6 mL /g total solid (TS) by mixed cultures hydrogenproducing bacteria, respectively.12,13 However, information on hydrogen production from wheat bran via the mixed anaerobic * To whom correspondence should be addressed. Tel./Fax: +86371-67766017. E-mail: [email protected]. † Zhengzhou University. ‡ Zhengzhou College of Animal Husbandry Engineering.

microbes is surprisingly lacking. In many countries, including China, wheat bran is an abundant byproduct of the milling of wheat into white flour. It usually accounts for 14-19% of the grains’ weight.14 On the basis of the data from the Food and Agriculture Organization (FAO), 1.05 billion tonnes of wheat were produced in China in 2007 (world production, 6.03 billion tonnes).15 Correspondingly, there is about 20 million tons of wheat bran generated annually in china. Industrial wheat bran contains the outer coverings, the aleurone layer, and the remnants of the starchy endosperm. It consists mainly of hemicellulose, residual starch, and cellulose16 and has the potential to serve as a low cost attractive feedstock for hydrogen production. For instance, Hawkes et al.17 reported that hydrogen yields of 56 m3 H2/ton dry weight were produced from wheatfeed in batch. It is well-know that the direct conversion of natural cellulose biomass by hydrogen-producing bacteria is considerably hard because of its complicated polymer structure, such as cellulose and hemicellulose. For instance, the maximum hydrogen yield treating raw beer lees and wheat straw were only 5.4 and 2.68 mL H2/g-TS by dairy manure compost, respectively.12,13 According to the best of our knowledge, an effective bioconversion of biomass to hydrogen depends strongly on the pretreatment of raw materials to produce feedstock which can be fermented by the hydrogen-producing bacteria. Various pretreatment methods have been extensively described in order to promote the accessibility of polysaccharides in complex biomass, including mechanical methods such as size reduction through milling; physical methods such as steaming, radiation, and sonication; chemical methods such as alkaline and acid hydrolysis; biological methods such as microbial and enzyme degradation; and a combination of these methods.18,19 However, among these procedures only a few combine the necessary features for

10.1021/ie701789c CCC: $40.75  2008 American Chemical Society Published on Web 07/29/2008

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industrial-scale use, including low capital cost, high yield of reactive carbohydrates, minimal generation of microbial inhibitors, and minimal impact on the environment. By far, most of the pretreatment methods were investigate for ethanol production, and only very few of them were applied to biohydrogen production by anaerobic bacteria. In light of the above background, the availability of abundant amount of wheat bran coupled with acidophilic anaerobic fermentation resulting in hydrogen production is considered to be an ideal means because it not only utilizes renewable resource but also produces the clean and readily usable hydrogen energy in a sustainable fashion. The objective of this research was to investigate the feasibility of producing hydrogen from wheat bran. For this purpose, the effects of the hydrogen-producing microbial sources, pretreatment method, substrate concentration, inoculum concentration, and initial pH on the hydrogen production were investigated by the anaerobic mixed cultures in batch cultivations utilizing wheat bran as substrate. 2. Materials and Methods 2.1. Hydrogen-Producing Microflora. Four natural microbial sources were tested as inoculums for hydrogen fermentation as follows: the activated sludge from Sui County paper mill, the digested sludge from Zhumadian paper mill, and the wheat straw compost and the corn stalk compost from the suburb of Zhengzhou city. Prior to use, the natural samples were baked in the infrared oven for 2 h to suppress as much nonsporeforming hydrogen-consuming bacterial activity as possible while still preserve the activity of the hydrogen-producing sporeforming anaerobes. In this study, the inoculum concentrations were calculated based on the wet weights before the sludge were baked. The predominant hydrogen-producing bacteria were isolated according to the method reported by Pan et al.20 Routine examinations were performed with a light microscope CH-30 (Olympus, Japan). The genomic DNA was extracted from cell pellets, and then the 16S rDNA gene was amplified by PCR as described.21 The 16S rDNA sequence was aligned with others available in GenBank. 2.2. Wheat Bran. The wheat bran used in the experiments were obtained from a flouring mill in the suburb of Zhengzhou city, which is composed of the following components: 33.7% hemicelluloses, 20.1% starch, 8.27% cellulose, 15.03% protein, 3.06% fat, 8.87 ash, and 10.97% water. Total volatile solid (TVS) value of the substrate was 0.8016 wheat bran (from three replicates). The samples were stored in sealed plastic bags at 4 °C prior to use. 2.3. Pretreatment. 2.3.1. Conventional Acid Pretreatment. The wheat bran at a solid loading of 20% (w/w) was mixed with dilute HCl aqueous solution (final concentrations: 0, 0.01, 0.05, 0.10, 0.50, 1.00 M) and kept boiling in a 500 mL beaker for 30 min. The slurry were collected and neutralized to pH 7 with 5 M NaOH solution. Then they were used as the substrates of hydrogen fermentation. 2.3.2. Pressure-Acid Pretreatment. The wheat bran at a solid loading of 20% (w/w) was mixed with 0.01 M HCl aqueous solution and pretreated at 0.27 mPa with residence times of 5, 10, 20 40, and 60 min. The pretreatment vessel (volume 350 mL), made of Teflon to withstand high temperatures and pressures, was filled with 140 g slurry. When the experiment had been completed, the vessel was placed in a cold water bath and not opened until it had cooled down. After pretreatment, the slurry were neutralized to pH 7 with 5 M NaOH solution and used as the substrates for hydrogen fermentation.

2.3.3. Microwave-Assisted Acid Pretreatment. The wheat bran were suspended in 0.01 M HCl aqueous solution with the solid:liquid ratio of 1:5 in a 500 mL beaker and the beaker was placed at the center of a rotating circular glass plate in a domestic microwave oven (Galanz Group Co.Ltd., WD800, 2450 MHz) for microwave treatment. The applied microwave power was 800 W for 3, 6, 9, 12, and 15 min. The pretreated wheat bran were cooled and adjusted to pH 7.0 with 5 M NaOH before hydrogen fermentation. 2.4. Experimental Procedures. The batch experiments were performed with 250 mL serum vials as batch reactors filled to 150 mL of mixture comprising the baked inoculums, the raw or pretreated wheat bran and 5 mL of nutrient stock solution. Each liter of nutrient stock solution containing 80 g of NH4HCO3, 12.4 g of KH2PO4, 0.1 g of MgSO4 · 7H2O, 0.01 g of NaCl, 0.01 g of Na2MoO4 · 2H2O, 0.01 g of CaCl2 · 2H2O, 0.015 g of MnSO4 · 7H2O, and 0.0278 g of FeCl2, which was slightly modified from the work of Lay et al.22 The initial pH values of the medium were adjusted by pH buffer solution. These bottles were filled with nitrogen gas to remove oxygen to keep the anaerobic environment and then capped with rubber stopper. The bottles were positioned in an orbital shaker with a rotation speed of 120 rpm to provide better contact among substrates under mesophilic conditions (36 ( 1 °C). At each time interval, the total biogas volume was measured by releasing the pressure in the bottles using a gas-collecting vessel of displacement method with saturated brine. All the experiments were carried out independently in triplicates. 2.5. Analytical Methods. The concentrations of hydrogen, volatile fatty acids (VFAs), and alcohols were analyzed by gas chromatography (GC, Agilent 4890).12 The acid hydrolysates (arabinose, xylose, mannose, glucose, and cellobiose) were treated with the reagent of trimethylchlorosilane and hexamethyl disiloxane and, then, were analyzed by another GC (Agilent 4890) equipped with a flame ionization detector (FID) and a HP-5 (cross-linked 5% PH ME siloxane) capillary column. The temperatures of the injection port, the detector, and the oven were 260, 290, and a programmed column temperature of 160-280 °C, respectively. Nitrogen was the carrier gas at a flow rate of 40 mL/min. The soluble sugar concentration was estimated using a 3,5-dinitrosalicylic acid (DNS) method.23 The contents of total solid (TS) and TVS were determined at 105 °C in an incubator dry box and 600 °C in a muffle furnace, respectively. Hydrogen gas production was calculated from the headspace measurement of gas composition and the total volume of biogas produced, at each time interval, using the mass balance equation: V ) V0γi +

∑Vγ

(1)

i i

Where, V is the cumulative hydrogen gas volumes at the current (i); V0 is the volume of headspace of vials; Vi is the biogas volume discharged from the vials at the time interval (i); γi is the fraction of hydrogen gas discharged from the vials at the time interval (i). 2.6. Kinetic Modeling. The cumulative volume of hydrogen produced in the batch experiments followed the modified Gompertz equation:

{ [

H ) P exp -exp

]}

Rme (λ - t) + 1 P

(2)

Where, H is the cumulative hydrogen production (mL); λ, the lag time (h), P, the hydrogen production potential (mL), Rm, the maximum hydrogen production rate (mL/h), e, the constant 2.718281828. In this study, the specific hydrogen production

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Figure 2. Micrographs of partial isolated hydrogen-producing bacteria.

Figure 1. Cumulative H2 yield with four types of inoculums: ∆ activated sludge of paper mill in Sui County; O digested sludge of paper mill in Zhumadian city; * wheat straw compost; 0 corn stalk compost. Table 1. Kinetic Parameters for Hydrogen Production Using Various Natural Microbial Sources natural microbial sources activated sludge of Sui county paper mill digested sludge of Zhumadian paper mill corn stalk compost wheat straw compost

Rm Ps λ (h) (mL/(g-TVS h)) (mL/g-TVS)

R2

5

0.90

50.6

0.9915

5

0.61

28.6

0.9925

5 5

0.55 0.31

22.6 17.7

0.9708 0.9959

potential (Ps) is defined as mL/g-TVS of wheat bran, and Rm is expressed as mL/(g-TVS h). The values of Ps, Rm, and λ for each batch test were estimated using the solver function in Excel (version 5.0, Microsoft) with a Newtonian algorithm.24 3. Results and Discussion 3.1. Hydrogen-Producing Characteristics of Various Natural Microbial Sources. To evaluate a hydrogen-producing ability of different natural microbial sources from wheat bran, four types of inoculums, namely, activated sludge of paper mill in Sui County, digested sludge of paper mill in Zhumadian city, wheat straw compost, and corn stalk compost, were studied in the batch experiment. Ps and Rm represent the hydrogengenerating ability and the hydrogen-producing bacterial activity on the defined substrate, respectively. Figure 1 depicts the effects of the various inoculum types on hydrogen production at the fixed initial pH 7.0, sludge or compost concentration 100 g/L, and the raw substrate concentration 100 g/L. Table 1 further summarized the estimated kinetic parameters Ps, λ, Rm, and R2, which were computed from the experimental data using eq 2. As shown in Figure 1 and Table 1, there was a regular trend of cumulative hydrogen production with the four types of inoculums. Hydrogen production began immediately after a short lag phase of 5 h, and the hydrogen production rate maintained a high level for about 70 h. After 80 h incubation, the hydrogen generation almost ceased. In addition, the estimate parameters of Ps, Rm, and λ were quite consistent with these experimental data. Obviously, the four type of inoculums selected in this study had different abilities of converting the same substrate into hydrogen. The maximal Ps (50.6 mL/g-TVS) and the maximal Rm (0.90 mL/(g-TVS h)) of the activated sludge of Sui county paper mill were higher than those of the other sludge or

composts, which may be due to the differences in bacterial species in the inoculum.25 It was suggesting that the selection of seeds can dramatically affect the seed microbial community’s ability to produce hydrogen. Three strains of anaerobic bacteria were screened and purified from the batch reactor in order to understand the species of hydrogen-producing bacterial from activated sludge of paper mill in Sui County. The micrographs are illustrated in Figure 2. They had a moderate optimum temperature for growth (36 °C) under strictly anaerobic conditions, which had high ability for hydrogen production from different organic substrates such as glucose, sucrose, maltose, lactose, cellobiose, xylose and pretreated wheat bran. They grew well and active in the pH range of 5.0-7.5. The optimal hydrogen yields (150-276 mL/ g-glucose) were obtained at an initial glucose concentration of 15 g/L and an initial pH 6.5. The nearly full-length sequences of 16S rDNA gene were determined for the isolates. By aligning with the 16S rDNA gene sequences from GenBank releases, the strains exhibited 99% sequence identity with Clostridium sp.. Morphological, physic-biochemical character and comparative sequence analysis of 16S rDNA indicated that these dominated strains belong to Clostridium sp.. The result is expected because the natural microbial sources became hydrogenproducing Clostridium-rich sludge through heating treatment, and it is consistent with the finding of Fan et al.,26 in which Clostridia could be enriched using a heat-shocked treatment to exclude nonspore-formers. These experimental results indicated that the activated sludge of paper mill in Sui County comprised favorable mixed microorganisms which exhibited considerable potential in the conversion of wheat bran into hydrogen. Therefore, the other discussions followed were based on the activated sludge in Sui County. 3.2. Pretreatment of Wheat Bran. In order to better exploitation of raw material for hydrogen production, the effectiveness of pretreatments of substrate on hydrogen production was further discussed. Herein, three pretreatment methods were employed for the hydrolysis of the wheat bran. The processes of hydrogen fermentation were performed at the fixed substrate concentration 100 g/L, pH7.0 and inoculum concentration 100 g/L. 3.2.1. Conventional Acid Pretreatment. The dilute acid hydrolysis process was an effective and inexpensive method of pretreating biomass. Figure 3a depicts the effects of HCl concentration by conventional acid pretreatment method on the hydrogen production. As shown in Figure 3a, the cumulative H2 yield and Rm were significantly affected by HCl concentration. When HCl concentration rose from 0 M to 0.01M, the cumulative H2 yield increased from 50.9 mL/g-TVS to the maximum 80.9 mL/g-TVS, as well as Rm increased from 0.90 to 1.22 mL/(g-TVS h). Thereafter, the cumulative H2 yield and Rm slightly changed with the increase of HCl concentration in the range of 0.01-0.10 M and, then, sharply decreased with further elevating HCl concentration. Acid concentrations equivalent to 0.50 and 1.00 M HCl proved to be too severe. A

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Figure 3. Effects of the pretreatment of substrate on the cumulative H2 yield. (a) Conventional acid pretreatment. (b) Pressure-acid pretreatment. (c) Microwaveassisted acid pretreatment.

concentration of 0.01 M HCl was sufficient to hydrolyze wheat bran for hydrogen production at the hydrolysis temperatures investigated for 30 min. The wheat bran consists mainly of starch, hemicellulose, cellulose, and protein. For us, important substances in wheat bran are starch above all, followed by polysaccharide. Pretreatment with dilute HCl can hydrolyze the natural macromolecule substances (i.e., starch, arabinoxylans, β-glucan, and protein) into directly assimilable micronutrients such as mono- or oligosaccharides, amino acids, and polypeptides. Compared with the raw wheat bran, the contents of soluble saccharides in the wheat bran pretreated by 0.01 M HCl increased from 0.086 to 0.392 g/g-TS. The soluble saccharides in the pretreated wheat bran were mainly composed of arabinose (5.8%), xylose (23.9%), mannose (1.9%), glucose (66.3%), and cellobiose (2.1%). These micromolecular sugars are relatively easy to be assimilated into the hydrogen-producing bacterial cells. In addition, the higher HCl concentration (e.g., 0.50, 1.00 M) was also in favor of the hydrolysis of wheat bran because the total sugar in the pretreated wheat bran reached 0.416 and 0.421 g/gTS, respectively. Whereas, the high Cl- anion concentration could stress fermentative organisms to a point beyond which the efficient utilization of sugars was reduced and hydrogen formation decreased. The cumulative H2 yield was down to only 25.65 mL/g-TVS from the pretreated wheat bran using 1.00 M HCl. This phenomenon was similar to the result reported by Fan et al.,12 in which the hydrogen yield gradually declined as HCl concentration increased from 22.9 mL/g-TVS at HCl concentration of 2.0% to 6.0 mL/g-TVS at HCl concentration of 5.0%. It was indicated that the conventional acid pretreatment under an appropriate HCl concentration was advantaged to convert wheat bran into hydrogen by microorganisms. 3.2.2. Pressure-Acid Pretreatment. Figure 3b illustrates the effects of high-pressure pretreating time on hydrogen production. As can be seen from Figure 3b, the wheat bran pretreated by combined high pressure with acid method showed the higher cumulative H2 yield of 86.3 mL/g-TVS and Rm of 1.29 mL/(gTVS h) than those by conventional acid pretreated wheat bran at longer reaction time of 60 min, although there was no significant difference in hydrogen yields at shorter reaction time in the range of 5-20 min. The total soluble sugar reached 0.354 and 0.442 g/g-TS by the pressure-acid pretreatment of 40 and 60 min, respectively. This means that the increase in reaction time of high pressure-acid pretreatment can strengthen the splitting of the macromolecule substances such as starch and hemicellulose. 3.2.3. Microwave-Assisted Acid Pretreatment. The microwave-assisted acid pretreatment significantly affected the hydrogen production from wheat bran. Figure 3c demonstrated

the effect of microwave irradiating time on hydrogen production. As shown in Figure 3c, the cumulative H2 yield of 52.3 mL/ g-TVS and Rm of 1.02 mL/(g-TVS h) occurred at the microwave heating of 3 min, which were similar to those from nonpretreated wheat bran. Simultaneously, the maximal cumulative H2 yield of 92.7 mL/g-TVS and Rm of 1.30 mL/(g-TVS h) were achieved at 9 min. Then, the maximal cumulative H2 yield and Rm sharply declined as irradiating time increased, which may be attributed to the undesired secondary reactions of the hydrolysis process and the decomposition of some useful components in wheat bran on the over irradiating condition. For example, glucose and xylose can be degraded into furfural and hydroxymethylfurfural, respectively. These byproduct of sugar affect fermentation efficiency negatively because some of them are toxic to fermentative microorganisms and inhibit their metabolism. Generally, microwave irradiation could change the ultrastructure of cellulose, degrade hemicellulose, and increase the susceptibility of the substrate.27 In this test, the total soluble sugar in the microwave-assisted acid pretreated wheat bran reached 0.461 g/g-TS at 9 min hydrolysis time. It was suggested that the wheat bran pretreated by microwave-assisted acid had fast hydrolysis rate and its hydrolysate had the higher sugar content, which is suitable for subsequent hydrogen fermentation process. From the above, it can be seen that direct hydrogen fermentation with raw wheat bran gave only hydrogen yield of 50.6 mL/ g-TVS, slightly above 60% of the hydrogen yield obtained with conventional acid hydrolysis. This showed that conventional acid pretreatment was essential to hydrolyze substantially the polysaccharides for further hydrogen fermentation. The maximum cumulative hydrogen yield using microwave-assisted acid pretreatment or pressure-acid pretreatment was slightly higher than that by conventional acid pretreatment. However, the pretreatment technique utilized microwave or high pressure have many disadvantages including highly consumption of energy, complicated operation, strict control of equipment, difficult to realize industrialization, and so on. Therefore, only conventional acid pretreatment had actual feasibility of utilizing wheat bran for hydrogen production and was selected as an appropriate pretreatment method throughout the batch tests. 3.3. Effect of Substrate Concentration on Hydrogen Production. Figure 4 depicted that plots based on eq 2 satisfactorily fit the cumulative hydrogen production data for conventional acid pretreated wheat bran ranging from 40 to 200 g/L at the fixed inoculum concentration 100 g/L and pH 7.0. It was observed that the cumulative H2 yield increased with enhancing the concentration of the substrate in the range of 40-80 g/L. The maximum cumulative H2 yield of 86.3 mL/gTVS occurred at the substrate concentration of 80 g/L.

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Figure 4. Changes of cumulative H2 yield with substrate concentration at five concentration levels from 40 to 200 g/L: ] 40; ∆ 80; * 120; 2160; O 200 g/L.

Figure 6. Changes of cumulative H2 yield with initial pH values at five pH levels from 4.0 to 8.0: ] 4.0; ∆ 5.0; * 6.0; 2 7.0; O 8.0.

Figure 5. Changes of cumulative H2 yield with inoculum concentration at five concentration levels from 30 to 150 g/L: ] 30 g/L; ∆ 60 g/L; *90 g/L; 2 120 g/L; O 150 g/L.

Figure 7. Specific hydrogen production potential and maximum hydrogen production rate at different initial pH levels.

Thereafter, a further increase of substrate concentration resulted in subsequent reduction of hydrogen yield, e.g., while the concentration of the substrate increased from 80 to 120, 160, and 200 g/L, the corresponding cumulative hydrogen yield declined from 86.3 to 66.5, 59.1, and 53.1 mL/g-TVS, respectively. This suggested that the substrate concentration plays a crucial role in hydrogen production. An increase of substrate concentration could enhance the hydrogen producing efficiency, but the higher substrate concentration would result in excesssubstrate inhibition, simultaneous acid/pH inhibition, and increased hydrogen partial pressures. Moreover, an exorbitant partial pressure level of hydrogen in the headspace of reactor would depress hydrogen synthesis and make metabolic pathways shift to production of more reduced products such as lactate, ethanol, acetone, butanol, or alanine.13 3.4. Effect of Inoculum Concentration on Hydrogen Production. The effect of inoculum concentration on cumulative H2 yield was investigated by varying the inoculum concentration between 30 and 150 g/L keeping the other operative conditions (pH 7.0, substrate concentration 80 g/L) constant. As shown in Figure 5, hydrogen was generated from about 10-12 h after inoculation. The cumulative H2 yield increased remarkable with increasing the inoculum concentration from 76.0 mL/g-TVS at the inoculum concentration of 30 g/L to 99.5 mL/g-TVS at the inoculum concentration of 60 g/L. Thereafter, the cumulative H2 yield decreased steadily from 99.5 to 68.9 mL/g-TVS as the inoculum concentration enhanced from 60 to 150 g/L.

Maximum cumulative H2 yield of 99.5 mL/g-TVS and the maximal Rm of 2.1 mL/(g-TVS h) occurred at the inoculum concentration of 60 g/L. The results implied that the inoculum concentration was also an important factor in hydrogen fermentation from wheat bran. The inoculum from activated sludge was composed of microorganisms and organic substances which may contain toxic substance. Higher concentration of inoculum increased the biomass accumulation and, then, caused the microorganism use nutrients and produce wastes at everincreasing rates. Subsequently, an essential nutrient run out and toxic products accumulated, which resulted in the inhibition of hydrogen production. The results also indicated that the appropriate inoculum concentration was in favor of hydrogen production. 3.5. Effect of Initial pH on Hydrogen Production. In order to determine the optimum initial pH of the medium for maximum hydrogen production, various initial pH values of 4, 5, 6, 7, and 8 were investigated at the fixed inoculum concentration of 80 g/L and sludge concentration of 60 g/L. As shown in Figure 6, hydrogen was produced from about 9.5-13 h at the initial pH 4.0-8.0 after inoculation. The hydrogen yield increased with the increase of culture time before reaching the maximum value. To better understand the pH effect on hydrogen production, Ps and Rm obtained from the Gompertz model were plotted against the corresponding initial pH values as shown in Figure 7. Ps and Rm showed similar trends in the batches. While the initial pH level rose from 4.0 to 5.0, the cumulative H2 yield

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inoculum, and substrate amounts were 5.0, 60, and 80 g/L, respectively. The cumulative hydrogen production was measured with eq 1 and simulated with eq 2. With the modified Gompertz equation, Ps and Rm were estimated as 128.2 mL/g-TVS and 2.50 mL/(g-TVS h), respectively. As shown in Figure 8a, Hydrogen production began immediately after a lag phase of 10 h and the hydrogen production rate maintained a high level at 26-36 h. The hydrogen content in the biogas increased sharply since the onset of hydrogen production and reached a maximum of 62% after cultivation for 32 h. Then after the 87 h incubation time, hydrogen concentration and cumulative hydrogen have no significant change. Furthermore, there is no methane detected in the biogas in all runs. Hydrogen production was usually accompanied with the formation of VFAs and alcohol. As can be seen from Figure 8b and c, both butyrate and acetate were two main byproduct in the batch reactors accounted for about 85% of the total VFAs (TVFAs), followed by little amounts of ethanol and butanol. The concentration of TVFAs increased sharply accompanied with hydrogen production and reached a top value of 17.66 g/L at the exponential phase. The maximum alcohols of 0.781 g/L were observed during the stationary phase. The biodegradation characteristics of the pretreated wheat bran were in close agreement with the most of previous reports based on the studies of fermentation producing hydrogen from sucrose and starch, in which VFAs and alcohols had similar trends.12 4. Conclusions

Figure 8. Time-course profile of hydrogen fermentation under optimized conditions: ∆ cumulative H2 yield; [ H2 content; 0 butyrate; 9 acetate; O propionate; ] ethanol; 2 1-butanol; × 2-butanol.

increased from 89.4 to 128.2 mL/g-TVS. Thereafter, the cumulative H2 yield obviously declined from 128.2 to 60.7 mL/ g-TVS with a further increase from pH 5.0 to pH 8.0. The cumulative H2 yield at pH 5.0 was nearly doubled as compared with that obtained at pH 8.0. The Rm also reached its peak at initial pH 5.0. At higher or lower pH, the Rm decreased significantly. It should be noticed that the final pH in anaerobic hydrogen production was around 3.5-4.5 regardless of initial pH. In natural ecosystem, the bacteria can grow at the pH ranging from 4.0 to 11.0, but we can see that the hydrogenproducing microorganisms had a good behavior at pH 5.0, which was in agreement with our previous research.26 It was shown from the above results that the initial pH considerably affected the hydrogen productivity because it may directly affect the hydrogenase activity28 and/or the metabolism pathway.29 In addition, appropriate initial pH control could suppress the hydrogen-consuming activity of the anaerobic microflora.30 3.6. Characteristics of Hydrogen Production under Optimized Conditions. Figure 8 demonstrates the time course profiles and characteristics of hydrogen fermentation under the optimized conditions, which contained (a) cumulative hydrogen yield and hydrogen content, (b) VFAs, and (c) alcohol. The batch fermentation was conducted using activated sludge of paper mill in Sui County as seed microorganisms and conventional acid pretreated wheat bran as substrate. The initial pH,

The feasibility of hydrogen generation utilizing wheat bran as substrate by mixed culture was demonstrated in the batch tests. Experimental results indicated that the hydrogen-producing microbial sources, pretreatment condition, substrate concentration, inoculum concentration, and initial pH level all had an individual significant influence on biohydrogen production. The predominant hydrogen-producing bacteria from activated sludge of paper mill in Sui County were identified as Clostridium sp. The maximum hydrogen yield of 128.2 mL/g-TVS and hydrogen production rate of 2.50 mL/(g-TVS h) occurred at initial pH 5.0, 80 g/L of pretreated wheat bran and 60 g/L of activated sludge of paper mill. During the conversion of wheat bran into hydrogen, the biohydrogen production was usually accompanied with the formation of the volatile fatty acids (VFAs) as main metabolic byproduct such as butyrate and acetate. Acknowledgment This work was supported by the China National Key Basic ResearchSpecialFunds(Nos.2006CB708407and2005CB214501), the National Natural Science Foundation of China (Nos. 90610001 and 20471053), and the Energy & Technology Program from Zhengzhou University. Literature Cited (1) Hart, D. D. Hydrogen power: the commercial future of “the ultimate fuel”; Financial Times Energy Publishing: London, 1997. (2) Fang, H. H. P.; Li, C. L. Acidophilic biohydrogen production from rice slurry. Int. J. Hydrogen Energy 2006, 31, 683. (3) Venkata Mohan, S.; Mohanakrishna, G.; Ramanaiah, S. V.; Sarma, P. N. Simultaneous biohydrogen production and wastewater treatment in Biofilm configured anaerobic periodic discontinuous batch reactor using distillery wastewater. Int. J. Hydrogen Energy 2008, 33, 550. (4) He, D.; Bultel, Y.; Magnin, J. P.; Roux, C.; Willison, J. C. Hydrogen photosynthesis by Rhodobacter capsulatus and its coupling to a PEM fuel cell. J. Power Sources 2005, 141, 19.

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ReceiVed for reView December 31, 2007 ReVised manuscript receiVed June 13, 2008 Accepted June 14, 2008 IE701789C