Enhancement of Hydrogen Production through a Mixed Culture of

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Enhancement of Hydrogen Production through MixedCulture of Enterobacter cloacae and Rhodobacter sphaeroides Yang Zhang, Liejin Guo, and Honghui Yang Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b01173 • Publication Date (Web): 01 Jun 2017 Downloaded from http://pubs.acs.org on June 9, 2017

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Enhancement of Hydrogen Production through Mixed-Culture of Enterobacter cloacae and Rhodobacter sphaeroides Yang Zhang†, Liejin Guo*,†, Honghui Yang†, ‡ †

State Key Laboratory of Multiphase Flow in Power Engineering, Xi’an Jiaotong University, Xi’an, 710049, P.R. China

‡

Department of Environmental Science and Engineering, Xi’an Jiaotong University, Xi’an, 710049, P.R. China

ABSTRACT Biohydrogen production is a hopeful approach to produce hydrogen (H2), which is considered as an alternative clean fuel in the future. In this work, a newly isolated strain YA012 was identified to be Enterobacter cloacae according to the 16S rRNA gene sequence analysis. This strain could generate about 1.5 mol H2/mol glucose under anaerobic condition, and acetic acid, butyric acid and ethanol were its main soluble metabolites. For improving H2 production, a mixed-culture system of Enterobacter cloacae YA012 and Rhodobacter sphaeroides HY01 was constructed, and the influences of important factors on H2 production were researched. When the ratio of dark- to photo-fermentative bacteria (RDP), initial pH, phosphate buffer concentration (PBC) and light intensity were set at 1:600, 7.5, 50 mM and 10 klux, respectively, the H2 production reached optimum. The maximum H2 yield and maximum H2 evolution rate of this mixed-culture were 3.96 mol H2/mol glucose and 105.6 ± 25.1 mL/(Lh), respectively. It’s also found that light intensity and stable pH have a remarkable impact on H2 production by the mixture, besides high PBC is beneficial to maintain pH stability and increase the ratio of acetic acid to butyric acid from E. cloacae. When the cornstalk pretreated by HCl-cellulase hydrolysis was employed as substrate, the H2 yield of mixed-culture was up to 226.3 ± 4.4 mL-H2/g-cornstalk under the optimal conditions, which enhanced by 170% and 38% compared with that of dark- and photo-fermentation

*Corresponding

author, Tel: +86-29-82663895; Fax: +86-2982669033.

Email address: [email protected]. 1

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alone, respectively. The results suggest that mixing dark- and photo-fermentation is a hopeful method to produce H2 from biomass, and our research achievements could be conducive to the development of mixed-culture.

1. INTRODUCTION Hydrogen (H2) is accepted as a hopeful clean fuel for the future with the merits of high calorific value and non-pollution.1 In recent years, biohydrogen production is increasingly receiving continuous attention and becoming a hot research area due to the following several advantages:2-4 (a) Microorganisms for biohydrogen production are easy to be acquired from nature, for instance, dark-fermentative bacteria (DFB), photosynthetic bacteria (PSB), and biophotolysis microorganisms. (b) Biohydrogen production can use many kinds of substrates to produce H2, for instance, industrial effluents and agricultural organics, which are both extensive and inexpensive raw materials. (c) Biohydrogen production is an environmentally friendly process, even can curb environmental pollution. (d) Biohydrogen production can be carried out at outdoor condition and easily controlled. Dark- and photo-fermentative H2 production have been studied for several years among the biohydrogen production methods. Enterobacter cloacae, which is a common dark-fermentative bacterium, can decompose saccharide substrates into H2, volatile fatty acids (VFAs) and alcohols under anaerobic condition.5, 6

Although E. cloacae has the advantage of high H2 evolution rate, more energy flows

to by-products rather than H2, such as VFAs, hindering hydrogenase activity.7 Rhodobacter sphaeroides, which is a versatile purple-non-sulfur photosynthetic bacterium, can efficiently utilize VFAs to produce H2 under anaerobic-illumination condition.8,

9

Although R. sphaeroides has the superiority in substrate conversion

efficiency, H2 evolution mediated by nitrogenase requires abundant energy, resulting in low H2 evolution rate.10 Therefore, the mutual collaboration and complementation of photo- and dark-fermentation could improve biohydrogen production. The two-step fermentative H2 production has been researched extensively,11, 12 and the cumulative H2 yield (CHY) enhances prominently in contrast with photo- or darkfermentation. Nevertheless, the pretreatment of dark-fermentation effluents and the 2


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integration of photo- and dark-bioreactors restrict the development of two-step fermentation.13,

14

In consequence, researchers have paid more attention on the H2

production through mixed-culture of dark- and photo-fermentation (MDPF). A few reports have shown that PSB cannot compete with DFB for glucose in the mixed-culture system,15, 16 because PSB prefer VFAs to glucose, and the growth rates of DFB are much faster than PSB. Hence, PSB can utilize VFAs from dark-fermentation to enhance total hydrogen production in the mixed-culture. Lee et al. mixed C. butyricum and R. sphaeroides to acquire the H2 evolution rate of 15.9 mL/(Lh), and its steady H2 evolution doesn’t undergo any lag time.17 Chandra and Mohan reported a MDPF that produces 1.4-fold higher CHY than individual dark-fermentation.18 Laurinavichene and Tsygankov achieved the CHY of 4.5 mol H2/mol hexose in the mixed-culture of C. butyricum and R. sphaeroides, which increases by 227% in contrast with darkfermentation alone.16 Takagi et al. showed an immobilized MDPF under the fluctuant illumination to generate more H2 than single dark- or photo-fermentation.19 Jiao et al. mixed C. cellulolyticum and Rhodopseudomonas palustris to improve CHY by 60% in contrast with dark-fermentation alone, and the ultimate cell concentration of Rhodopseudomonas palustris in the mixture is about 1.2-fold augment than that in individual photo-fermentation.20 These data on MDPF show that mixed-culture produces more H2 than monoculture. However, there are few researches on the mixedculture of Enterobacter and PSB,21, 22 and the optimum conditions for MDPF are still unclear. Furthermore, the common substrates, such as glucose, sucrose and starch, are often utilized for researching biohydrogen production through MDPF, whereas there is little research on agricultural wastes or industrial wastes as substrates for biohydrogen production through MDPF. Therefore, combining Enterobacter cloacae YA012, which is a newly isolated bacterium, and Rhodobacter sphaeroides HY0123 was employed for studying H2 production in this work. The influences of the ratio of dark- to photo-fermentative bacteria (RDP), initial pH, phosphate buffer concentration (PBC) and light intensity (LI) on MDPF were investigated. At last, biohydrogen production of different fermentation 3


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systems using cornstalk as substrate was studied under the respective optimum conditions.

2. MATERIAL AND METHODS 2.1 Culture conditions. The medium A (MedA, g/L) used for incubating DFB at 37 °C for 24 h in an anaerobic box, composed of 5 glucose, 3 yeast extract, 10 peptone, 3 NaCl and 3 NaAc (sodium acetate), and the pH was adjusted to 7.0. The modified Sistrom’s minimal medium (MedB),24 in which the carbon source was 10 g/L glucose instead of sodium succinate, was employed for cultivating PSB at 35 °C for 48 h under the dark-aerobic condition, and the pH was adjusted to 7.0. The other different modified Sistrom’s minimal medium (MedC), in which the carbon source and nitrogen source were 10 g/L glucose and 3 g/L yeast extract instead of sodium succinate and ammonium sulfate, respectively, was employed for biohydrogen production by MDPF, and the pH was adjusted to 7.25. 2.2 Screening and identifying for DFB. The H2-producing DFB were acquired from dairy manure. The bacteria were enriched with MedA liquid media and then spread on MedA plates under the anaerobic condition. Single colonies were chosen to re-streak at least three times on MedA plates to ensure the bacteria were pure. A strain named YA012 was picked up for subsequent study. Its 16S rRNA gene was synthesized by PCR using a pair of universal primers, 27F and 1492R.25 The products of PCR were sequenced in Sangon Biotech (Shanghai) Co., Ltd，and then the results were contrasted in Genbank. According to the BLAST data from NCBI, the neighborjoining method was used to construct a phylogenetic tree. 2.3 Biohydrogen production from glucose. A single colony of HY01 or YA012 was incubated in 50 mL sterilized tubes filled with 10 mL medium for pre-culture, the cells were collected by centrifuge and re-suspended with MedC for H2 production. The batch experiments for H2 production were carried out in 60 mL syringes as the reactors filled with 10 mL culture, and the volume of biogas was measured based on the scale mark of syringe per se. These reactors were set in a photo-incubator at 30 °C under illumination. The effects of different factors on MDPF were researched. 4


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2.4 Biohydrogen production from cornstalk. Dried cornstalk (CK) was shattered and filtered by 60 meshes, then mixed with water in the ratio of 1:15 (g/g). This mixture was soaked by HCl at a final concentration of 1.5%, and then hydrolyzed at 100 °C for 2 h. The initial pH of the treated mixture was adjusted to 5.0 by NaOH before enzymolysis. A certain amount of cellulase (Sigma-Aldrich, 0.8 U/mg) was added into the mixture in the ratio of 12:1 (U/g-CK), and then reacted at 50 °C for 10 h. After HClcellulase pretreatment, the hydrolysate was centrifuged and the supernatant was collected for hydrogen production. The E. cloacae YA012, R. sphaeroides HY01 and their mixed-culture were used for biohydrogen production from cornstalk, respectively. The medium for H2 production was similar to MedC except for cornstalk hydrolysate of 10 g-CK/L instead of glucose. The 60 mL syringes were employed as bioreactors with 10 mL work volume. 2.5 Analytical measurements. Cell concentration was determined by optical density at 660 nm using an UV-vis spectrophotometer (DU730, Beckman Coulter). The H2 content and the concentrations of VFAs and alcohols were analyzed with gas chromatograph as shown in the reference.26 The light intensity was measured with a photometer (1330A, TES) and pH value was tested with a pH meter (pH240, Corning). The amount of glucose in the culture was analyzed by the Glucose (HK) Assay Kit (Sigma) following the instruction. The reducing sugar yield from the cornstalk was measured with dinitrosalicylic acid reagent (DNS) colorimetric method.27 The modified Gompertz equation (Eq. (1)) was employed to simulate the biohydrogen production process,28 in which H is the cumulative H2 yield, P is the total H2 production potential, Vm is the maximum H2 evolution rate, e is 2.718281828 and T is the lag time of H2 production. All of the measurements were carried out at least three times.  V e  H  P exp  exp  m T  t   1   p  

(1)

3. RESULTS AND DISCUSSION 3.1 Identification of YA012. A phylogenetic tree (Figure 1) illustrates the phylogenetic relationship between YA012 and related bacteria based on 16S rRNA sequences from 5


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Genbank, showing the maximal identity with E. cloacae ECNIH4. In consequence, it’s concluded that the isolated strain YA012 pertains to the species of Enterobacter cloacae. In contrast with a few published papers, different E. cloacae strains form different soluble metabolites but they always contain VFAs and alcohols. The formation of these metabolites is dependent on the metabolic pathway that relies on many factors including microorganism used, substrate, pH, temperature and partial pressure of H2, amongst others.7 In our study, acetic acid (HAc), butyric acid (HBu) and ethyl alcohol (HEt) were the main fermentation products, according with the metabolites formed by E. cloacae IIT-BT 08.29 In addition, it’s also found that lactate, propionate and butanol existed in the soluble metabolites, whereas they were at a negligible level that can be detected. The result indicates that the fermentation type of YA012 is the mixed fermentation based on acetic acid and butyric acid. 3.2 Influence of RDP. When the initial pH 7.25, 20 mM PBC and 0.5 klux LI were set, all CHY values of MDPF were higher than that of individual YA012. The CHY enhanced with the RDP varied from 1:0 to 1:600, and reduced with the RDP varied from 1:700 to 1:1000 (Figure 2). However, compared to the mixed-culture, CHY of YA012 was a little higher during 0-12 h while much lower during the middle and later periods of H2 evolution. It may attribute to YA012 that dominates at the beginning of H2 evolution since the growth rate of YA012 is faster than HY01, then DFB and PSB co-produce H2 with the augment of HY01 concentration. This finding is analogous to the works.20, 30 The highest CHY was 1.97 mol H2/mol glucose when the RDP was 1:600, which enhanced by 41.5% in contrast with individual YA012, and its Vm of 91.3 mL/(Lh) was also the highest in this experiment. However, excessive amount of HY01 inhibits the growth and H2 evolution of YA012, resulting in the decrease in CHY values at RDP of 1:700-1:1000. The concentrations of HAc and HBu (Table 1) in MDPF decreased a lot, indicating that the dark-fermentation products are used by HY01 to produce H2. Furthermore, the concentrations of HAc and HBu were 12.6 mM and 14.9 mM at the RDP of 1:600, which reduced most compared with that of individual YA012, 6


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respectively. The consumption of HAc and HBu also causes the augment of pH, thereby, the changing trend of ultimate pH is the same as the CHY values, and the ultimate pH at the RDP of 1:600 was maximum. A few papers have shown that ethanol metabolism may lead to the NAD/NADH imbalance and impact the protons transmembrane transport,31, 32 which are closely related with hydrogen production in PSB. Moreover, Xie et al. found that the hydrogen yield by Rhodopseudomonas faecalis increases with the initial ratio of ethanol to acetate (RE/A) enhancing from 0 to 0.6, then reduces with the RE/A enhancing from 0.6 to 1.0, and finally increases again with the RE/A enhancing from 1.0 to 1.6.33 There is no hydrogen produced by R. faecalis only in presence of ethanol in the medium. In our work, the initial ratio of ethanol to HAc and HBu was approximately 0.6, which couldn’t inhibit hydrogen production. The work by Liu et al. showed that the CHY achieves optimum when the ratio of Clostridium butyricum to Rhodopseudomonas faecalis is 1:600.34 However, Fang et al. employed the ratio of C. butyricum to R. sphaeroides of 1:5.9 to acquire the maximum CHY. The bacteria growth has a great effect on hydrogen production.30 In general, bacteria in the logarithmic phase are chosen for H2 production because the bacteria have high metabolic activity during this period. However, the result in our work suggests that no exact rule conforms to the RDP due to the different conditions of H2 evolution and the growth difference between various DFB and PSB. 3.3 Influence of initial pH. The pH of dark-fermentation effluent is much low because of the fermentation products (mainly VFAs), and the optimum pH for darkfermentation is in the acidic range of 4.5–5.5.35, 36 Nevertheless the optimum pH for photo-fermentation is in the weak alkaline range of 7.0-7.5.9, 37 The optimum pH for H2 evolution by HY01 is 7.0-7.25,23 therefore, low pH is one of the serious problems for this mixed-culture system. When the 1:600 RDP, 20 mM PBC and 0.5 klux LI were set, the CHY values under different initial pH are described in Figure 3. As the initial pH enhanced from 7.0 to 7.5, the CHY enhanced from 1620 to 1860 mL/L-culture, and then strongly decreased from 1460-1420 mL/L at pH 8.0-8.5 (Table 2). The Vm improved from 79.9 to 86.0 mL/(Lh) 7


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at pH 7.0-7.5, and reduced from 77.9 to 74.3 mL/(Lh) at pH 8.0-8.5 (Table 2). There was no obvious difference in H2 production between pH 8.0 and 8.5. The augment of initial pH mitigates acidification induced by VFAs, which is conducive to the growth and H2 evolution of PSB. Nevertheless, excess high initial pH is harmful to both bacteria growth and suppresses hydrogenase or nitrogenase activity in the cell,5, 13 thereby resulting in the decline of H2 evolution. In addition, the ultimate pH enhanced with increasing initial pH, but the highest ultimate pH of 6.15 is still disadvantageous for photo-fermentation. Although VFAs are utilized by HY01, the consumption rate of VFAs is still lower than the generation rate, leading to the decline in pH which consequently reduces the growth and H2 evolution of HY01. The highest CHY was 2.12 mol H2/mol glucose at initial pH 7.5, whereas the ultimate pH of 5.45 is quite low. The result indicates that only improving initial pH is not enough for pH regulation in the MDPF, and it requires more attempts. 3.4 Influence of PBC. It’s found that PBC has a great influence on the soluble metabolites and pH stability in the fermentation.38, 39 Moreover, stable pH is more effective for CHY and H2 evolution rate than regulation of initial pH,29 besides HAc is more appropriate for producing H2 by HY01 compared to HBu. Hence, for stabilizing pH and generating more HAc from YA012 fermentation, different PBC values were employed for H2 production when the 1:600 or 1:0 RDP, initial pH 7.5 and 0.5 klux LI were set. When the PBC increased from 20 to 60 mM, the CHY of YA012 reduced from 1.61 to 0.67 mol H2/mol glucose while the ultimate pH enhanced from 4.78 to 6.28 (Table 3), indicating that low PBC is beneficial for H2 evolution by YA012. The high PBC slows down the decrease in pH, and it causes the pH of culture in range of approximately 5.5-7.5, which goes against H2 evolution by DFB. The result also indicates that pH stability improves remarkably due to the phosphate buffering effect.40 Since mitigating acidification leads to the change of fermentation product composition, the ratio of HAc to HBu enhanced from 0.88 to 1.56 at 20-50 mM PBC and reduced to 1.29 at 60 mM PBC. It’s obvious that HAc preponderates in dark-fermentative products 8


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at high PBC, and this may attribute to more carbon flux to HAc rather than HBu.41 The CHY of MDPF enhanced as the PBC increased from 20 to 50 mM and reduced a lot at 60 mM (Figure 4), coinciding with the change of HAc/HBu ratio in darkfermentation alone. The highest CHY of 2.52 mol H2/mol glucose was achieved at 50 mM PBC, which was enhanced by 21.4% compared with that at 20 mM (Table 3). Furthermore, the utilization of both HAc and HBu gradually improved while that of HEt changed a little as the PBC increased from 20 to 50 mM. Meanwhile, the utilization rate of HAc and HBu was up to 84% and 64% at 50 mM PBC, respectively, which were the highest in this experiment. The ultimate pH enhanced from 5.38 to 6.46 with the increasing PBC due to the buffering effect. The CHY of MDPF was still much more than individual YA012 at the same condition, and its maximum CHY was acquired at the ultimate pH 6.41, corresponding with Khanna’s research that the HAc/HBu ratio is maximum at pH 6.5 in E. cloacae.29 Several articles have proved that pH plays a key role in dark-fermentation product composition.6,

36

The high PBC is beneficial for

acidification mitigation and pH stability, which facilitates H2 evolution by MDPF. However, excessive PBC might change the osmotic pressure in the cell, subsequently affecting the bacteria growth and H2 evolution. 3.5 Influence of LI. Light energy is absorbed by PSB to synthesize ATP that is prerequisite for nitrogenase activity, and the augment of LI in a certain range can promote enough ATP synthesis, facilitating PSB growth and H2 evolution rate.42, 43 The different LI values were studied in the MDPF when the 1:600 RDP, initial pH 7.5 and 50 mM PBC were set. As presented in Table 4, the CHY increased from 1.96 to 3.96 mol H2/mol glucose while the Vm declined from 121.1 ± 12.3 to 105.6 ± 25.1 mL/(Lh) as the LI raised. The result shows that augment of LI has remarkable enhancement on H2 yield. YA012 growth is much better than HY01 under low LI, therefore, the H 2 production performance displays the DFB characteristic, videlicet high H2 evolution rate and low CHY. As the LI gradually increases, the H2 evolution and growth of HY01 are dominant in the MDPF, as a result, the H2 production performance displays the PSB characteristic, 9


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videlicet low H2 evolution rate and high CHY. In addition, the H2 production performance at 8 klux was almost the same as 10 klux, implying that 10 klux LI might be the limitation and higher LI could hinder the H2 evolution. The ultimate pH enhanced from 6.35 to 6.78 that is very close to the optimal pH for HY01. The increase in VFAs consumption results in the augment of ultimate pH with the increasing LI, according with the changing trend of CHY. In addition, the PSB in the inner space of bioreactor can’t obtain enough light illumination, because high pigment content of PSB in the outer space of bioreactor hinders the penetration of incident light, thereby limiting the whole hydrogen production performance, which is the light shielding effect. Our previous work showed that the CHY of HY01 is maximum at approximately 5 klux.23 The shading caused by DFB reproduction intensifies the light shielding effect, therefore, PSB requires more light energy to synthesize enough ATP for hydrogen production. It may be the reason that the optimal LI of this mixed-culture system dramatically increased. 3.6 Hydrogen production from cornstalk. In China, it generates cornstalk of about 220 million tons per year.44 Cornstalk as a kind of agricultural wastes causes bioenergy waste and environmental pollution using traditional treatment method. Biohydrogen production from cornstalk not only generates clean fuel (H2), but also reduces the pollution. The reducing sugar obtained from cornstalk through HCl-cellulase hydrolysis, can be used to produce H2 in the dark- or photo-fermentation process. Up to date, there has been no report on biohydrogen production from cornstalk through MDPF. As shown in Figure 5, the CHY of MDPF was much higher than that of dark- or photo-fermentation alone, and the CHY of dark-fermentation was the lowest. Furthermore, dark-fermentative H2 production finished within 48 h, which was shorter than photo-fermentation (72 h) and mixed-culture fermentation (84 h). It suggests that mixed-culture of DFB and PSB can improve the substrate utilization compare to darkor photo-fermentation. The data related with hydrogen production by the three systems from cornstalk are listed in Table 5. Under pH 7.0, 20 mM PBC and 37 °C, the darkfermentation by YA012 produced 117.3 ± 6.1 mL-H2/g-CK (1173.3 ± 61.1 mL/L) and 10


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its Vm was 50.9 ± 1.9 mL/(Lh). Under pH 7.0, 20 mM PBC and 6000 lux, the photofermentation by HY01 produced 226.3 ± 4.4 mL-H2/g-CK (2263.0 ± 43.8 mL/L) and its Vm was 76.3 ± 5.1 mL/(Lh). The photo-fermentation can produce more H2 using saccharides as substrate than dark-fermentation due to the different metabolic pathways related with hydrogen production. Under the above optimal conditions, the mixedculture of YA012 and HY01 produced 312.5 ± 7.4 mL-H2/g-CK (3125.0 ± 73.5 mL/L), which increased by 170% and 38% compared with that of dark- and photo-fermentation, respectively. The Vm of MDPF was 91.6 ± 3.3 mL/(Lh) that improved by 20% compared with photo-fermentation, indicating that mixture of DFB and PSB could not inhibit the H2 evolution rate. The main soluble metabolites were still HAc, HBu and HEt, while their concentrations were all low, especially the HEt concentration. This may attribute to low reducing sugar concentration (0.38 g/g-CK) and the complicated substrate composition, videlicet cornstalk hydrolysate that may affect the metabolic pathways of DFB or PSB, thereby causing more energy flux to HAc and HBu rather than HEt. The ultimate pH of dark-fermentation was minimal due to the high concentrations of HAc and HBu. Since PSB can use a few VFAs produced in the photo-fermentation from cornstalk,45 the main soluble metabolites concentrations of photo-fermentation were lower than these of dark-fermentation and subsequently its ultimate pH was higher. Consuming VFAs formed by YA012 to produce H2 results in the high ultimate pH of 6.85 in MDPF, which is more than the minimal suitable pH for HY01. It’s the main reason that mixed-culture produces maximum H2 yield in the three systems. The result suggests that the MDPF is a more promising method to produce H2 from agricultural wastes in contrast with dark- or photo-fermentation.

4. CONCLUSIONS In this work, a newly isolated strain YA012 from dairy manure was identified to be Enterobacter cloacae, which could produce 1.5 mol H2/mol glucose under anaerobic condition. To enhance the hydrogen production, the influences of different factors on H2 production through mixing E. cloacae YA012 and R. sphaeroides HY01 were investigated. This mixed-culture system achieved the maximum CHY of 3.96 mol 11


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H2/mol glucose and the Vm of 105.6 ± 25.1 mL/(Lh) under the 1:600 RDP, initial pH 7.5, 50 mM PBC and 10 klux LI. This study showed that pH and light intensity have a great effect on hydrogen production by the mixed-culture. Moreover, this mixed-culture system obtained the CHY of 312.5 ± 7.4 mL-H2/g-CK and the Vm of 91.6 ± 3.3 mL/(Lh) using the cornstalk through HCl-cellulase hydrolysis as substrate, which were much higher than dark- and photo-fermentation, respectively. The results indicate that the combination of dark- and photo-fermentation can enhance hydrogen production efficiently.

ACKNOWLEDGEMENTS The authors would like to thank the National Natural Science Foundation of China for the Youth (No. 51308452), the National Basic Research Program of China (No. 2012CB215303) and the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20120201120058) for their support for this work.

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Figure captions：

Figure 1. Phylogenetic tree constructed with the neighbor-joining method shows the relationship between YA012 and related strains according to 16S rRNA genes from Genbank. Figure 2. Cumulative hydrogen production of mixed-culture under different RDP. Figure 3. Cumulative hydrogen production of mixed-culture under different initial pH. Figure 4. Cumulative hydrogen production of mixed-culture under different PBC values. Figure 5. Cumulative hydrogen production from cornstalk through three different fermentation systems.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Table 1. Kinetic parameters, soluble metabolites concentrations and ultimate pH of H2 production under different RDP. Vm (mL L-1

CHY

h-1) 1:0

RDP

(mol

Soluble metabolites concentrations (mM)

Ultimate

H2/mol glucose)

HAc

HBu

HEt

pH

79.9 ± 7.6

1.53

25.7

28.9

30.7

4.39

1:500

85.8 ± 8.6

1.88

13.9

15.6

26.8

4.54

1:600

91.3 ± 8.6

1.97

12.6

14.9

25.6

5.06

1:700

79.4 ± 8.2

1.67

13.2

16.2

26.3

4.92

1:800

76.6 ± 7.9

1.64

14.1

16.8

26.8

4.81

1:1000

70.1 ± 6.6

1.62

14.5

17.2

27.1

4.69

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Table 2. Kinetic parameters and ultimate pH of H2 production under different initial pH. Initial pH

P (mL H2 L-1)

Vm (mL L-1 h-1)

CHY (mol H2/mol

Ultimate pH

glucose) 7.0

1678.6 ± 64.7

79.9 ± 7.3

1.88

4.58

7.25

1806.1 ± 83.6

82.6 ± 8.7

1.97

5.01

7.5

1956.9 ± 90.8

86.0 ± 8.6

2.12

5.45

8.0

1527.4 ± 59.8

77.9 ± 7.9

1.74

5.64

8.5

1484.5 ± 50.5

74.3 ± 6.4

1.72

6.15

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Table 3. The CHY values, soluble metabolites concentrations and ultimate pH of individual dark-fermentation and mixed-culture under different PBC values. Mixed-culture Soluble metabolites concentrations (mM) HAc HBu HEt

CHY (mol H2/mol glucose)

Ultimate pH

8.3

11.5

22.6

2.07

5.38

7.8

9.5

20.5

2.23

7.2

8.8

18.6

4.5

6.6

5.8

6.5

PBC (mM)

Dark-fermentation Soluble metabolites concentrations (mM) HAc

HBu

HEt

HAc/HBu


20

22.5

25.6

24.3

0.88

1.61

4.78

5.83

30

25.8

23.4

22.9

1.10

1.23

5.43

2.30

6.19

40

27.6

21.6

20.8

1.28

0.94

5.89

16.2

2.52

6.41

50

28.5

18.3

18.6

1.56

0.76

6.18

15.5

2.14

6.46

60

26.4

20.5

17.5

1.29

0.67

6.28

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Ultimate pH

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Table 4. Kinetic parameters and ultimate pH of H2 production under different LI values. Light intensity (klux)

Vm (mL L-1 h-1)


Ultimate pH

3 ± 0.2

121.1 ± 12.3

1.96

6.35

5 ± 0.3

117.1 ± 10.9

2.50

6.45

8 ± 0.5

108.8 ± 22.5

3.94

6.75

10 ± 0.8

105.6 ± 25.1

3.96

6.78

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Table 5. Kinetic parameters，soluble metabolites concentrations and ultimate pH of H2 production from cornstalk. Inoculum

Vm (mL L-1 h-1)

CHY (mL-H2/g-CK)

Soluble

metabolites

concentrations (mM)

Ultimate pH

HAc

HBu

HEt

YA012

50.9 ± 1.9

117.3 ± 6.1

15.5

18.1

4.0

5.01

HY01

76.3 ± 5.1

226.3 ± 4.4

10.0

10.8

0.35

5.53

Mixed-culture

91.6 ± 3.3

312.5 ± 7.4

3.8

6.2

2.8

6.85

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Enhancement of Hydrogen Production through a Mixed Culture of

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