Biohydrogen Production by Dark Fermentation of Starch Using Mixed

Article pubs.acs.org/EF

Biohydrogen Production by Dark Fermentation of Starch Using Mixed Bacterial Cultures of Bacillus sp and Brevumdimonas sp. Meidan Bao, Haijia Su,* and Tianwei Tan Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing, China, 100029 ABSTRACT: The energy use of hydrogen is recognized for its environmental advantages. Its utilization is expected to grow significantly over the coming years, and the development of new production methods to complement or substitute the traditional steam reforming, gasification, or electrolysis will further enhance that growth. The microbial conversion of biomass is considered to be the route with the highest immediate potential for its significant hydrogen yield and low energy requirement. The present research investigates this potential, using starch as the raw material for dark fermentation and by using appropriate microorganisms. Fermentation using a single micro-organism strain is shown to be of limited efficiency for H2 production, with a low H2 content and low yield. The combination of two bacterial strains, belonging to Bacillus sp and Brevumdimonas sp., respectively, and each with a specific action in the H2 production, significantly enhances the biohydrogen production yield. Its H2 yield reached 1.04 mol H2/mol glucose, being twice the yield obtained with pure cultures. The specific hydrogen production rate was up to 400 mL H2/(g biomass h). The end products were mainly butyric and acetic acid, with traces of ethanol. The dark fermentation of starch can be considered to be a butyrate-type fermentation. cassava. Its transformation to cassava flour or starch moreover creates 5−8 m3 of wastewater per 1 kg of processed cassava root: the wastewater is also rich in carbohydrates as reflected in its high COD (chemical oxygen demand) and BOD (biological oxygen demand) (≫10 g/L). The present research hence focuses on starches as primary target feedstock, although research using wastewater is currently ongoing. For the fermentation of carbohydrates into H2, both photo fermentation and dark fermentation have been investigated. Compared with photo fermentation, dark fermentation has the advantages of not requiring direct solar input, of accepting a variety of substrates, and using a very simple reactor technology.3 The efficiency of converting glucose into H2 depends on the fermentation pathway used by the microorganisms. According to reactions 1−4 below, the maximum stoichiometric conversion of 4 mol of H2 per 1 mol of glucose can be achieved when acetic acid (CH3COOH) is the fermentation byproduct. The yield is only 2 mol of H2 if butyric acid (CH3(CH2)2COOH) is the main byproduct. Yields are lower when propionic acid is produced, whereas ethanol, propanol, and lactic acid are characteristic of a zero-H2 pathway.

1. INTRODUCTION Hydrogen (H2) is a highly energetic (286 kJ/mol) and clean fuel since only producing H2O upon combustion.1 It is often referred to as a potential clean energy carrier, although also used in the chemical industry for e.g. hydrogenation, to saturate compounds, to crack hydrocarbons, in manufacturing ammonia, methanol, and synthesis gas. The future main use of hydrogen is likely to be in the transportation sector, to reduce pollution. Vehicles can be powered with hydrogen fuel cells, which are three times more efficient than a petrol-powered engine.2 The commercially usable hydrogen is currently being produced in the petrochemical industry, with nearly 70% obtained by steam reformation of naphtha or natural gas, the balance being produced by coal gasification and water electrolysis.3 These traditional processes are highly energy-intensive, not environmentally friendly, and rely upon using depleting fossil-fuel (mainly petroleum) reserves. The production of hydrogen by exploiting alternative sources is of utmost priority. Biomass, as a product of photosynthesis, is the most versatile nonpetroleum renewable resource that can be utilized for sustainable production of hydrogen. A cost-effective and energy-lean production process can be achieved in which carbohydrate-rich wastes, wastewaters, and other sources such as agricultural crops and their waste byproduct are used for economical hydrogen production.4−8 Although long chain fatty acids and proteins are also reported for H2 production, only fermentation of carbohydrates to H2 is thermodynamically favorable. Starch is rich in carbohydrates and can be hydrolyzed by acid or enzymes into a highly concentrated sugar solution which can be easily utilized by fermentative bacteria for hydrogen production. These bacteria degrade organic compounds, releasing H2 and CO2, while a major part of the initial carbohydrate (glucose) feedstock is transformed into mostly VFAs.9 Corn and cassava starches are abundantly available in China. China has indeed embarked on large-scale production of © 2012 American Chemical Society

C6H12O6 + 2H 2O → 2CH3COOH + 2CO2 + 4H 2

(1)

C6H12O6 → CH3CH 2CH 2COOH + 2CO2 + 2H 2

(2)

C6H12O6 → CH3CH 2COOH + CO2 + H 2

(3)

C6H12O6 → 2CH3CH 2OH + 2CO2

(4)

The highly negative Gibbs free energies (ΔG°) of reactions 1 and 2, being respectively −206.3 and −254.8 kJ/mol, moreover Received: April 21, 2012 Revised: July 25, 2012 Published: August 2, 2012 5872

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ically characterized (Gram strain, colony color, utilization of carbohydrates, starch hydrolysis) by the methods described in Bergey’s manual.21 The isolates were inoculated in the synthesized medium in which the sole carbon source was glucose, lactose, fructose, and xylose, respectively. The starch hydrolysis test was carried out on the plate to see if the transparent circle was present. The further molecular biological characterization was carried out by 16S rDNA analysis as described in section 2.1.2. 2.1.2. DNA Extraction and 16S rDNA Gene Sequence Analysis. Lysis buffer: 200 mL NaCl 1.17 g pH8.0 Tris-HCl (Tris 12.1 g) SDS 10 g. PBS buffer: 200 mL of 3.12 g dissolved NaH2PO4 2H2O. The pH value was adjusted to 8.0 by NaOH. PCI: phenol:chloroform:isoamyl alcohol = 25:24:1. CI: chloroform:isoamyl alcohol = 24:1. DNA was extracted from bacterial cultures in the following steps: Cell cultures (10 mL) were washed twice in phosphate buffered saline (PBS) after being harvested. The cells were concentrated by centrifugation at 10 000 rpm (10 625g) for 10 min, then mixed with 0.3 mL lysis buffer, 0.3 mL PBS buffer, and 0.6 mL chloroform. The mixture was incubated at 60 °C for 10 min. The supernatant was obtained after being centrifuged at 5000 rpm (5312 g) for 10 min, then filled with an equal volume of PCI. The crude DNA was obtained in the supernatant after centrifugation at 15 000 rpm (15 937g) for 5 min. The above steps were repeated about three times until the protein layer has disappeared. Afterward, 1/2 volume of PCI and 1/2 volume of CI were used for removing the residual protein. After centrifugation at 15 000 rpm (15 937g) for 5 min, an equal volume of CI was added and the supernatant was kept after another centrifugation. Then 0.1 volume of sodium acetate and an equal volume of isopropanol were added into the supernatant, and the solution was stored at −20 °C overnight. DNA precipitate was obtained after centrifugation at 12 000 rpm (12 750g) at 4 °C for 30 min, thereafter washed with 70% ethanol. After evaporative drying at room temperature, 50 μL sterilized ultrapure water were used for DNA dissolution and DNA quality was examined by 0.8% agarose gel electrophoresis. Then, the DNA samples were sent to invitrogen for 16S rDNA gene sequence. The sequences were obtained and BLAST searches were done using the NCBI server. For A1, its sequence is 100% similar to Bacillus thuringiensis (strain X6), Bacillus thuringiensis (X5), Bacillus cereus (strain sc-3-w-3), and Bacillus cereus (strain Aj080319IA-16). For B1, a phylogenetic tree was conducted in the context of 16S rRNA gene sequences from three different bacterial strains: Brevundimonas naejangsanensis (BIO-TAS2-2), Brevundimonas diminuta (XW2a), and Brevundimonas diminuta (NK 2.18-3). 2.2. Experimental Setup and Procedure. Batch experiments were conducted in 1 L bottles with 0.9 L reaction volume. Each reactor was filled with 0.8 L fermentation medium, then inoculated with 100 mL seed solution. The air in the head space of the reactors was removed by passing argon gas through the fermentation medium and the head space to provide anaerobic conditions. The reactors were placed in a thermostatic bath (35 °C) and magnetically stirred to ensure thorough mixing and good mass transfer. 2.3. Medium Composition. The seed medium had the following composition: beef extract (3 g/L), peptone (10 g/L), and NaCl (5 g/ L). The fermentation medium contained 10 g/L untreated starch flour, peptone (1 g/L), and NaCl (5 g/L). The starch consists of 15% water, 80% carbohydrate, 0.4% protein, and 0.3% fat. The medium were sterilized at 115 °C for 20 min. 2.4. Analytical Methods. Samples taken from the liquid phase were analyzed for total sugar concentration, then centrifuged at 10 000 rpm (10 625g) to remove the solids, and the clear supernatants were used for the analysis of soluble sugar concentration, volatile fatty acids and alcohols. Total sugar and soluble sugar concentrations were determined by the 3,5-dinitrosalicylic acid (DNS) method.22 Volatile fatty acids and alcohols were determined by using a gas chromatograph (GC-2010) equipped with a flame ionization detector (FID) using a second-order temperature-programmed method: the initial temperature of the oven was 120 °C for 0.5 min, then rose to 180 °C at a rate of 20 °C/min and maintained for 3 min, subsequently rose to 230 °C at a rate of 30 °C/min, and maintained for 10 min. Temperatures of the injection and detector were 250 °C.

thermodynamically favor these reactions to take place. These theoretical yields are different from experimental findings and depend on the substrate, bacteria, and cultivation conditions such as pH and oxidation/reduction potential (ORP). Due to different metabolic pathways of different bacteria, the type and amount of VFAs and the hydrogen yield can vary, mainly depending on the species of the bacteria applied. Pure cultures of dark and photo fermentation are reported for hydrogen production from starch under anaerobic conditions. As dark-fermentative bacteria, the microorganisms belonging to Clostridium sp.such as C. buytricum, C. thermolacticum, C. pasteurianum, and C. bifermentants are most widely used to convert glucose to volatile fatty acids and H2/CO2.5,6,10−12 Hydrogen yields in dark fermentation of carbohydrates are between 1 and 2.8 mol H2/mol glucose.6,13−15 The hydrogen production capacity of the anaerobic facultative bacterial culture Enterobacter aerogenes has also been widely studied.16,17 Hydrogen producing aerobic cultures such as Pseudomonos sp., Aeromonos sp., and Vibrio sp. have been tested in recent years.18 Hydrogen production by Thermotogales species and Bacillus sp. was observed in mesophilic acidogenic cultures.19 Most of these isolates were however unable to use starch but only simply reduce sugars to produce hydrogen, due to the lack of hydrolytic enzymes for starch and therefore necessitating extra equipment for the hydrolysis of starch, prior to fermentation. Even with glucose as the substrate, their H2 yields were limited. The present research therefore applies a dark fermentation technique, using a novel combination of a mixed culture that is capable of using starch as substrate directly, without the need of additional hydrolysis unit and using a single reactor and simple procedure. The mixed strains A1 and B1 that are applied in the present study, and belonging to Bacillus. sp and Brevumdimonas sp., respectively, have not yet been reported for hydrogen production. This microorganism mix could use a wide range of substrate, such as unhydrolyzed starch powder and starch wastewater. The two stages of starch hydrolysis and glucose fermentation for hydrogen production are combined in a single reactor. The hydrogen production potentials of pure culture and mixed culture were compared in terms of cumulative hydrogen production, hydrogen yield, and specific hydrogen production rate. Effects of substrate concentration were also investigated. Volatile fatty acid (VFA) generation and total sugar concentration were measured.

2. MATERIALS AND METHODS 2.1. Microorganisms. The strains A1 and B1 were screened and isolated from sludge, obtained from an anaerobic digestion reactor. Strain A1 was capable of hydrolyzing starch by secreting amylase; while strain B1 was able to ferment glucose into hydrogen with high efficiency. A mixture of pure cultures of A1 and B1 were used as the amylase-producing strain and acidogenic bacteria, respectively. Their positive interaction could be considered as a food chain. The two steps of starch hydrolysis and hydrogen production can be carried out in the same reactor, making the process easier and more economic. The combination moreover makes them able to utilize complex substrate directly, such as cassava, food waste, and sugar-containing wastewater. The bacteria were first grown on the seed medium during 4−5 days at 37 °C. The cultures were then transferred to the fermentation medium to produce the hydrogen. The initial biomass concentration was 0.05 ± 0.01 g/L. 2.1.1. Isolation, Purification, and Phenotypic Identification of A1 and B1. Strain A1 and B1 were isolated from activated sludge. The isolation and purification procedure follows the anaerobic platemessanine method.20 Colonies on the plate were further bacteriolog5873

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Fermentation gas was sampled from the head space of the reactors. These samples were used to analyze the gas compositions. The amount of total gas produced was determined by water displacement method with a measurement accuracy of 2 mL, representing an error of 1% at the onset of fermentation to 0.1% at the end. The data were averages of triplicate experiments. To simplify the graphical presentation of the results, and due to the very limited experimental error, error bars were not included in the figures. The composition of the gas was analyzed by gas chromatograph (GC-2014C, Shimadzu, Japan), equipped with a thermal conductivity detector (TCD) and a TDX-01 packed (3 mm inside diameter) stainless steel column (2 m × 3 mm). Argon gas was used as the carrier gas with a flow rate of 30 mL/min. Temperatures of the column oven, injection, and detector were 160, 160, and 180 °C, respectively. The cumulative hydrogen gas production was determined from the following:23

cultures of A1 and B1 produced about 3 times less H2 than the mixed cultures); • that the ratio of strains A1 and B1 was relatively important, with A1/B1 ratios of 1/1 or 2/1 producing about the same amount of H2, while a ratio of 1/2 did not improve the H2 production at all; • that the maximum rate of H2 production of mixed cultures 1/1 and 2/1 was about 3 mL H2/min, against 1.5 mL H2/min for the A1 and B1 strains individually and for the 1/2 ratio. • that the lag time (acclimatization) remains about the same for all experiments, i.e. 20−30 h; • that the H2 production only starts when the pH value decreased to about 5 (the pH thereafter remains fairly stable and the H2 production stopped at pH ∼ 4; the pH is indeed a major factor for fermentative hydrogen production, affecting the activity of hydrogenase as well as of the bacteria). The very low pH value obtained when using only the B1 culture is due to its acidogenic activity. The composition of the microorganisms had a significant effect on hydrogen production.25 Early in the fermentation, A1 played a major role. It can produce amylase to hydrolyze starch into glucose which can be used as substrate by B1. The highest cumulative H2 production was obtained when for A1/B1 = 1/1 and A1/B1 = 2/1. The lowest cumulative H2 production was obtained for A1/B1 = 1/2, even slightly lower than with pure cultures. The low cumulative hydrogen production in the culture of A1/B1 = 1/2 was due to the fact that B1 was dominant in this culture where starch hydrolysis by A1 was lacking during early period of the fermentation process and starch was not completely utilized. The ratio of the two strains A1/B1 = 1/1 was chosen in subsequent experiments. With fermentation time, the pH exhibited a downward trend, dropping almost linearly in the first 24 h, and then declining gradually and gently. The change of the pH is due to the formation of VFAs from starch fermentation and the consumption of the organic acids for H2 production, although clearly the production rate of volatile organic acids was faster than the utilization rate of volatile acids for hydrogen production. The results obtained are consistent with other researches.26,27 3.1.2. Byproducts from the Fermentation. As far as the by products from fermentation are concerned, Figure 2 illustrates the measured values of VFAs and ethanol. As can be seen from Figure 2, the endproducts of A1 in the liquid phase were 17 mg/L ethanol and 439 mg/L acetic acid, while those of B1 were 13 mg/L ethanol and 1046 mg/L propionic acid. The end products of the mixed culture were acetic acid, butyric acid, and a little ethanol. The content of butyric acid in the total acid varied from 45% to 60%. The end products of the mixed culture of A1/B1 = 1/1 were 16 mg/L ethanol, 423 mg/L acetic acid, and 525 mg/L butyric acid. Acetic and butyric acid were dominant end products, which agreed with the literature.26 According to the previous reaction eqs 1−4, it is hence evident that the dominant acetic/butyric products of the mixed culture experiments should provide higher H2 yields than when ethanol/propionic acid are the products in the case of pure cultures. Table 1 summarizes hydrogen yields for the different cultures.

VH2, i = VH2, i − 1 + VwC H2, i + VR, iC H2, i − VR, i − 1C H2, i − 1 where VH2,i and VH2,i−1 are the volumes of cumulative hydrogen gas production (mL) calculated after the ith and the previous measurement; Vw is the total gas volume measured by the water displacement method (mL); CH2,i is the concentration of H2 in the total gas measured by the water displacement method (%); VR,i and VR,i−1 are the volumes of the gas in the head space of the reactor for the ith and previous measurement (mL); CH2,i and CH2,i−1 are the concentration of H2 in the head space of the reactor for the ith and the previous measurement (%). The biomass concentration in the inoculum was determined by centrifuging 20 mL sample at 10 000 rpm (10 625g) for 5 min and drying at 105 °C until constant dry weight.24 The pH of the fermentation medium was monitored by using a pH meter with relevant probe.

3. RESULTS AND DISCUSSION 3.1. Preliminary Investigations: Definition of Parameter Influence. In a preliminary series of experiments, the optimum parameter range was determined. 3.1.1. Comparison of Pure and Mixed Cultures. When comparing pure cultures and mixed cultures, a set of experiments at a substrate concentration of 10 g/L was conducted. Results on the cumulative H2 production and evolution of pH are presented in Figure 1. The lines between data points of H2 production are best fit trend lines only. These results reveal the following: • that the use of mixed strains increases and cooperates in the production of fermenter gas (H2 + CO2) (pure

Figure 1. Variation of H2 production and pH value with time for different cultures. Full symbols refer to H2 production; hollow symbols represent pH values. 5874

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137 mL H2/g starch, respectively. A higher substrate concentration of 15 and 20 g/L resulted in a lower hydrogen yield (93 and 67 mL H2/g starch, respectively), tentatively explained by the inhibition through the VFAs at the high starch loading: a high concentration of VFAs is inhibitory to the bacteria since the undissociated part of the VFA can permeate the cell membrane of the bacteria and disrupt the physiological balance in the cell; the dissociate part of the VFA present in the broth causes a high ionic strength, leading to the cell lysis of the bacteria. Wang et al.28 investigated the inhibitory effect of ethanol, acetic acid, propionic acid, and butyric acid on fermentation hydrogen production by mixed cultures and found that hydrogen production was inhibited by ethanol, acetate, propionic acid, and butyrate at a concentration of 10 mmol/L. When the added concentration was increased to 300 mmol/L, hydrogen yield decreased by 57%, 85%, and 91% for ethanol, acetate, and butyrate, respectively. In terms of cumulative hydrogen production and H2 yield, the optimal substrate concentration was 10 g/L. The end products in the liquid phase of different substrate concentrations at the end of fermentation are depicted in Figure 4. The end products in the liquid phase were mainly

Figure 2. End products in the liquid phase of different cultures at the end of the fermentation.

Table 1. Hydrogen Yields and Hydrogen Contents of Different Cultures culture type pure culture A1 pure culture B1 mixed culture (A1:B1 = 1:1) mixed culture (A1:B1 = 2:1) mixed culture (A1:B1 = 1:2)

H2 yield (mol H2/mol glucose)

H2 content in biogas

0.40 0.36 1.04

19% 53% 60%

0.98

55%

0.28

52%

The reasons for the low value of A1/B1 = 1/2 were already explained in section 3.1.1. 3.1.3. Effect of the Substrate Concentration. The effect of the substrate concentration on the H2 production was examined by varying the amount of starch present, while using an optimum ratio of A1/B1 of 1/1. Figure 3 shows the variation of the H2 production with time for different substrate concentrations. The H2 production was completed within 250 h. The hydrogen yield decreased with the increased substrate concentration from 5 to 20 g/L. The hydrogen yield of 5 and 10 g/L concentration was with 157 and

Figure 4. End products in the liquid phase for different substrate concentrations at the end of the fermentation.

acetic and butyric acid, with a small amount of ethanol. The total acid concentration (1.457 g/L) was the highest when the substrate concentration was 20 g/L. The other total acid concentrations were 1.18, 0.957, and 0.84 g/L at the substrate concentrations of 15, 10, and 5 g/L, respectively. 3.1.4. Effect of the C/N Ratio. To examine the effect of different C/N ratios on the H2 production, peptone was used as N source: N might promote the hydrogen production. Different amounts of peptone were added in the medium when the substrate concentration was fixed at 10 g/L. The variation of cumulative H2 production with time for different C/N is shown in Figure 5. The lag time was almost the same, about 24 h. The highest cumulative hydrogen gas production was obtained with 0.2% peptone added in the medium. After the fermentation, the biomass concentration of 0.3% peptone was 20% higher than that of 0.2% peptone. It is inferred that at a high N-level, the H2 yield decreases at a constant substrate concentration since extra peptone helps the bacteria to grow better, and the bacteria will utilize part of the substrate for their own growth rather than for converting substrate into VFAs and hydrogen. Therefore, the culture with

Figure 3. Variation of the H2 production with time for different substrate concentrations. 5875

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Figure 5. Variation of cumulative H2 production with time for different C/N ratios.

Figure 7. Variation of the cumulative gas production and its composition with time.

0.3% peptone resulted in the lowest cumulative hydrogen production. As can be seen in Figure 6, the end products were still acetic acid, butyric acid, and ethanol. The highest butyric acid content

The hydrogen gas content was 58%, and the molar ratio of H2 and CO2 in the total gas was 1.4/1. Figure 8 depicts variation of the cumulative H2 production and VFAs concentration in the liquid phase with time. The end

Figure 8. Variation of the cumulative H2 production and VFAs concentration in liquid phase with time.

Figure 6. End products in the liquid phase for different C/N ratios at the end of the fermentation.

products in the liquid phase were ethanol, acetic, and butyric acid. VFAs were not converted into H2 and CO2, and they were accumulated in the broth at high concentrations leading to the end of fermentation. During the whole fermentation time, the ethanol concentration was low, about 40 mg/L. The concentration of acetic acid was 0.691 g/L at 320 h. 73% of the total acid was butyric acid, being the main volatile fatty acid in the liquid phase, and its final concentration of 1.958 g/L was achieved at the end of the fermentation. The molar ratio of acetic and butyric acid was 1/2. As clearly shown in Figure 8, butyric acid is closely associated with the hydrogen production and proceeds according to a similar trend in the fermentation process. These VFAs (acetic acid and butyric acid) can be widely used in the chemical, food, and pharmaceutical industry as raw materials: butyrate and acetate can be recovered from the broth and purified for further chemical processing. Further research is required and beyond the scope of the present paper. Figure 9 depicts variation of cumulative H2 production, total sugar, and soluble sugar concentrations with time. Changes in total sugar concentration could represent changes in starch concentration (1 g starch ∼ 0.9 g convertible glucose). During

(61%) was obtained with 0.2% peptone. The other two cultures showed the same butyric acid content of 54% however at a lower hydrogen yield. 3.2. Evaluation of the H2 Production. The previous experiments determined the optimum ratio of strains, the optimal substrate concentration, and the peptone content as 1/ 1, 10 g/L, and 0.2%, respectively. Further experiments were carried out at these optimum conditions. Figure 7 illustrates the cumulative gas production and its composition with time. The cumulative hydrogen production increased fairly fast within the first 50 h and reached 1034 mL after 320 h of fermentation. The end of the gas production is caused by the limitation of the activity of the bacteria at a low pH value. pH has a major impact on the activities of H2-producing bacteria because it affects hydrogenase activity. It was reported that the optimal pH for most of the hydrogen-producing mixed flora was in the range of 4.2−5.0.3 A number of studies revealed that the hydrogen-producing bacteria would be extremely inhibited at pH below 4.0.29 The final pH in this work was 3.9, which was consistent with the literature. 5876

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As a result, the theoretical yield was 2.4 mol H2/mol glucose consumed. The experimental hydrogen yield in this study was 1.04 mol H2/mol glucose fed (equal to 1.66 mol H2/mol glucose consumed), lower than the theoretical yield, because of the partial utilization of the carbon source for the growth and maintenance of the bacteria. The experimental results of the present study are compared with literature data in Table 2. Most of the reported studies used hydrolyzed starch or sugar solution as substrate for hydrogen production rather than starch flour. The two stages of starch hydrolysis and glucose fermentation were however combined together in a single reactor in the present research. Other studies reported slightly higher hydrogen yields because of the external addition of nutrients and trace elements such as FeSO4, MgSO4, Na2MoO4, Fe, and vitamins. Fe2+ has indeed been reported to enhance the H2 production when using mixed bacteria.34,35

Figure 9. Variation of cumulative H2 production, total sugar, and soluble sugar concentrations with time.

4. CONCLUSIONS A novel combination of micro-organisms that have not been reported for hydrogen production was applied in this study. Untreated starch powder can be used for the fermentative hydrogen production at 35 °C, when using mixed cultures of bacteria. Optimum operating conditions were determined (substrate concentration, C/N, the ratio of the two strains). The mixed culture performed better than the pure culture, and the ratio A1/B1 = 1/1 was shown to be of highest efficiency. A maximum H2 yield of 1.04 mol H2/mol glucose was obtained at the optimal substrate concentration 10 g/L, with 0.2% peptone. The SHPR was 400 mL H2/(g biomass h). Butyric acid was closely associated with the hydrogen production. Since the hydrogen production is suggested to improve further by increasing the inoculum volume or by adding external nutrients and trace elements, research is ongoing, together with repeat experiments using cassava starch, cassava wastewater, and other waste sources of carbohydrates. The results will be presented in a follow-up paper.

the onset 24 h, glucose was present in the liquid phase from starch hydrolysis at first, and was thereafter consumed without generating hydrogen gas. First, starch was hydrolyzed into glucose by the bacteria which could produce amylase, at the meantime glucose was mainly used for cell growth and VFAs formation. Hydrogen production started from 24 h, the lag time. In the rest of the fermentation process, starch (total sugar concentration) still decreased, and the glucose concentration was almost the same. It showed that starch hydrolysis was continued, and glucose was utilized for hydrogen production. The total sugar concentration decreased gradually from nearly 4.8 to 0.2 g/L indicating effective starch hydrolysis and utilization. Glucose was an intermediary product in the starch fermentation to VFAs and hydrogen. Therefore, soluble sugar concentration increased from an initial value of 0.01 g/L to nearly 0.16 g/L in the beginning 20 h due to starch hydrolysis, then decreased to less than 0.01 g/L due to conversion to VFAs and hydrogen production. It remained almost constant at a low concentration of 7 mg/L for the rest of the fermentation period suggesting that the rate of glucose produced from starch hydrolysis was almost equal to that of glucose consumed for VFAs and hydrogen formation. The cumulative hydrogen production, hydrogen yield, and specific hydrogen production rate were 1034 mL, 1.04 mol H2/ mol glucose, and 400 mL H2/(g biomass h), respectively. On the basis of the molar ratio of VFAs (B/A ratio = 2), the hydrogen production reaction was determined as follows:

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 86-010-64452756. Fax: 86-010-64414268. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

5C6H12O6 + 2H 2O → 4CH3CH 2CH 2COOH + 2CH3COOH + 12H 2

Notes

+ 10CO2

The authors declare no competing financial interest.

Table 2. Comparison of Hydrogen Yield from Starch Substrates by Different Cultures culture Clostridium butyricum and Rhodopseudomonas faecalis RLD-53 Clostridium butyricum CGS2 Clostridium beijerinkii +Rhodobacter sphaeroides-RV anaerobic sludge sewage sludge mixed culture

operation mode

substrate

substrate conc. (g/L)

H2 yield (mol H2/mol glucose)

ref

batch

glucose

9

1.98

26

continuous continuous batch continuous batch

hydrolyzed starch waste wheat powder wheat starch corn starch corn starch

26 5 10 20 10

1.5 0.6 1.14 0.92 1.04

30 31 32 33 this study

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ACKNOWLEDGMENTS The authors thank the following institutions for their support: the National Natural Science Foundation of China (20876008, 21076009), the (863) High Technology Project (2008AA062401), and New Century Excellent Talents in University (NCET-100212).

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dx.doi.org/10.1021/ef300666m | Energy Fuels 2012, 26, 5872−5878