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High-yield biohydrogen production from non-detoxified sugarcane bagasse: Fermentation strategy and mechanism
Accepted Manuscript High-yield biohydrogen production from non-detoxified sugarcane bagasse: Fermentation strategy and mechanism Bin-Bin Hu, Ming-Yuan Li, Yu-Tao Wang, Ming-Jun Zhu PII: DOI: Reference:
S1385-8947(17)31881-8 https://doi.org/10.1016/j.cej.2017.10.157 CEJ 17941
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
25 August 2017 25 October 2017 26 October 2017
Please cite this article as: B-B. Hu, M-Y. Li, Y-T. Wang, M-J. Zhu, High-yield biohydrogen production from nondetoxified sugarcane bagasse: Fermentation strategy and mechanism, Chemical Engineering Journal (2017), doi: https://doi.org/10.1016/j.cej.2017.10.157
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High-yield biohydrogen production from non-detoxified sugarcane bagasse: Fermentation strategy and mechanism Bin-Bin Hua, Ming-Yuan Lib,c, Yu-Tao Wangb,c, Ming-Jun Zhu a,b,c* a
School of Bioscience and Bioengineering, South China University of Technology,
Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People’s Republic of China b
College of Life and Geographic Sciences, Kashgar University, Kashgar 844000,
China c
The Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at
Colleges & Universities under the Department of Education of Xinjiang Uygur Autonomous Region, Kashgar University, Kashgar 844000, China First Author, E-mail address: [email protected]; Tel: +8620 39380636; ∗
Corresponding author, E-mail address: [email protected]; Tel: +8620 39380623;
Fax: +8620 39380601
Abstract: Anaerobic digestion, an attractive process for generation of hydrogen, involves complex microbial processes on decomposition of waste biomass. Here, efficient biohydrogen production (277.4 mM (6.2 L-H2/L)) from non-detoxified sugarcane bagasse (NDSCB) was accomplished through a novel two-stage anaerobic fermentation, which to our knowledge, is the first utilization of NDSCB for biohydrogen fermentation. Interestingly, the hydrogen production of the second stage fermentation (167.8 mM) was significantly higher than that of the first stage fermentation (108.6 mM). Metabolite analysis showed that the metabolic flux had been redirected and Butyrate/Acetate ratio was enhanced in the second stage fermentation. Simulation of the second-stage fermentation condition indicated that acetate and butyrate generated in the first-stage fermentation could significantly enhance the hydrogen production from NDSCB by 51% and 28%, respectively. More interestingly, the stimulatory effect of butyrate was influenced by substrate. The present work provided a comprehensive insight of metabolite on biohydrogen production and an environment-friendly process for lignocellulosic biohydrogen production. Keywords:
Biohydrogen
production;
Two-stage
fermentation;
Metabolite;
Non-detoxified sugarcane bagasse (NDSCB); T. thermosaccharolyticum MJ1
1. Introduction Fossil fuels such as coal, oil and natural gas provide about 95% of the world’s total energy and the demand is increasing over the past decade [1, 2]. Because of overuse of fossil fuels, pollution problems have become more and more serious in our living environment, suggesting the necessity to find an alternative energy source, which is renewable, abundant, clean and cost-effective [1, 3]. One of the great challenges in the coming decade is how to obtain new renewable energy sources that are environmentally friendly and lessen the high dependence on fossil fuels [4]. Lignocellulose composed of cellulose, hemicellulose and lignin is the most abundant raw material on the earth. Due to this advantage, lignocellulosic biomass has been arousing great interest among researchers [5, 6]. Hydrogen has been identified as a clean energy carrier and one of the most ideal alternative fuels due to its combustion in air to form water [7]. In industry, hydrogen has been produced from natural gas (48%) and oil (30%) by steam reforming processes, or other industrial methods such as coal gasification (18%) and water electrolysis (4%) [8, 9]. However, these hydrogen production methods use fossil fuels as the raw material and thus many of the advantages of hydrogen are offset by the raw material. At the same time, the continuous system of hydrogen production is needed for application at industrial scale [10]. Among different technologies of hydrogen production, biohydrogen production shows the greatest potential to replace the traditional methods. Hydrogen produced from biological processes is less energy intensive and more environmentally friendly. Biohydrogen production by dark
fermentation presents a promising way for the sustainable largescale generation of hydrogen in the future hydrogen economy [3]. Compared with other biological production methods, dark fermentation has several advantages: 1) continuously producing hydrogen without the presence of light, 2) more simple and moderate production process, 3) maintaining a higher hydrogen production rate, and 4) utilizing a wide range of low-cost waste materials [4, 7, 11]. Accordingly, hydrogen production by dark fermentation has become more attractive in recent years. The successful conversion of various substrates to biohydrogen depends greatly on the performance of specific microbial species in a complex series of biochemical reactions. The acclimatization of biohydrogen-producing cultures to substrate change is a helpful strategy for enhancing biohydrogen production [12]. After enzymatic hydrolysis, the lignocellulose is hydrolyzed to a mixture of hexose and pentose sugars. Currently, various microorganisms have been reported to produce hydrogen [13]. Compared with hexose-fermenting organisms, only a few co-fermenting organisms have been identified to convert substrate to hydrogen [6]. The co-fermenting thermophilic organisms are considered as a promising candidate for biohydrogen production.
Thermoanaerobacterium
thermosaccharolyticum
(T.
thermosaccharolyticum) is a well-known strain for bio-hydrogen production due to its ability to utilize various kinds of substrates [14-16]. T. thermosaccharolyticum could simultaneously ferment a mixture of hexose and pentose sugars to biohydrogen without obvious delay. Our previous study revealed that T. thermosaccharolyticum MJ1 also possesses an excellent tolerance to acid pretreatment inhibitors and is a
promising candidate for industrial biohydrogen production [17]. Biohydrogen production is usually accompanied with the synthesis of organic acids, which is greatly influenced by metabolites. Acetate, formate, lactate, pyruvate and butyrate are the main soluble end products in broth and closely related with microorganisms. Sreethawong et al. have reported that the increase in nitrogen concentration caused the increased production of volatile fatty acids, directly leading to the decrease of hydrogen production and the increase of butyric acid production accompanying the decrease in acetic acid production [18]. Van Ginkel and Logan have found that acids added to the feed at a concentration of 25 mM decreased hydrogen yields by 13% (acetic) and 22% (butyric), and 60 mM of either acid decreased hydrogen production by 93% [19]. Compared with ethanol, acetate exhibited more significant inhibition on growth and hydrogen production performance of Ethanoligenens harbinese B49; in Clostridium thermocellum ATCC 27405, hydrogen and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen, acetate, and lactate; in Caloramator celer, hydrogen production kinetics was more sensitive to increasing concentration of acetate and formate, with acetate inhibiting the hydrogen production by increasing the ionic strength in the medium [20-22]. The aforementioned results have demonstrated that diverse influences of metabolites in different microorganisms and metabolites could redirect the metabolic pathways. To relieve the recalcitrant structure of lignocellulose, numerous pretreatment methods were exploited to break down the stubborn lignocellulosic structure, such as
dual salts pretreatment, alkaline pretreatment, steam explosion pretreatment, dilute acid pretreatment [23], γ-irradiation pretreatment [24], ozonation pretreatment [25] and acidic-alkaline pretreatment [26]. Among various pretreatment methods, dilute acid pretreatment is an economical pretreatment method widely used in industrial manufacture. As the inhibitors (such as furfural, hydroxymethyl furfural and carboxylic acids) are formed during the pretreatment process, the substrate must be detoxified (such as adsorption and biodegradation), thus increasing the cost of manufacture [27, 28]. Our previous study investigated the application of dilute-acid pretreated sugarcane bagasse hydrolysate in hydrogen production by MJ1 [17]. The residual insoluble substrate (NDSCB) was also an important material for biorefinery. At present, a separate detoxification process was needed to relieve the inhibitory effect of NDSCB, which increased the operation cost and effluent discharge. The full utilization of NDSCB without detoxification was an economical and environmentally friendly technology to increase the competitiveness of biohydrogen production from lignocellulose. In this study, for full use of the residual material obtained in dilute-acid pretreatment process, we investigated the potential of biohydrogen production from the non-detoxified substrate. The underlying mechanism of higher biohydrogen production was also explored. 2. Materials and methods 2.1 Materials and pretreatment Sugarcane bagasse (SCB) was obtained from Guangzhou Sugarcane Industry Research Institute (Guangzhou, China). The non-detoxified sugarcane bagasse
(NDSCB) was obtained from the dilute acid pretreatment process of SCB. The raw SCB was soaked in sulfuric acid solution (1%, g/v) with a solid to liquid ratio of 1:10 (g dry weight to ml) at 121 ℃ for 30 min. The insoluble solid (NDSCB) was directly obtained by vacuum filtration and dried at 50 ℃. Finally, the dried NDSCB was milled and sieved through a 200-mesh (74 µm). The detoxified sugarcane bagasse (DSCB) was obtained from NDSCB after detoxification by distilled water. For detailed instructions, the NDSCB (obtained from 100 g SCB) was mixed fully with 2 L distilled water for 30 min with an intermittent stir. The insoluble solid was directly obtained by vacuum filtration. The operation repeated at least 10 times until the supernatant was neutral (pH 7.0). After vacuum filtration, the DSCB was dried, milled and sieved as substrate. The dried power (both NDSCB and DSCB) was used in the following studies. The commercial cellulase Cellic® CTec2 was kindly provided by Novozymes (China) Investment Co. Ltd. Filter paper activity of cellulase was 90 FPU/mL and pNPG activity of β-glucosidase was 2743 U/mL. 2.2 Microorganism and medium The anaerobic T. thermosaccharolyticum MJ1 isolated previously was used for biohydrogen production and maintained as previously reported [17]. All cultures were inoculated with a volume of 10% (v/v) from freshly prepared cultures at exponential growth phase (about 18 h) in serum bottles. Additionally, MJ1 was anaerobically grown in 100 ml serum bottles for a final working volume of 30 ml. The strain was cultured at 55 ℃ with rotary shaking at 150 rpm.
2.3 Experimental design 2.3.1 Biohydrogen potential tests Biohydrogen production was investigated by batch fermentation of NDSCB in 120 mL serum bottles. Briefly, each serum bottle contained different concentrations of NDSCB (20, 40, 60, 100 g/L) to replace the carbon source in medium (pH 6.5). The cellulase was added at a loading of 20 FPU/g NDSCB after autoclave. For simultaneous saccharification and fermentation (SSF), the fermentation was carried out for 120 h (initial pH 6.5). For prehydrolysis and simultaneous saccharification and fermentation (PSSF), the prehydrolysis by cellulase was carried out for 72 h at 50 ℃ before fermentation (pH 6.5), then the MJ1 was cultured for another 120 h. As for the second stage fermentation, the pH of fermentation broth was adjusted to 7.0 and the serum bottles were sealed again. Next, the serum bottles were purged three times with 100% nitrogen, and then fermented in a shaker for another 120 h. After fermentation, the biohydrogen production in the headspace was analyzed by gas chromatography. 2.3.2 Influence of metabolite on performance of CTec2 The influence of metabolites was investigated by enzymolysis. The cellulase was added at a loading of 20 FPU/g NDSCB. To test metabolites, NDSCB (100 g/L) was degraded by CTec2 in 50 mM phosphate buffer with four different metabolite combinations (3 g/L acetic acid; 2 g/L butyric acid; 3 g/L acetic acid and 2 g/L butyric acid; 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid) at pH 5.0. The sample without added metabolites was used as control. The samples were taken regularly to determine the reducing sugar.
2.3.3 Mechanism of high biohydrogen production in second stage fermentation The mechanism for the high biohydrogen production in the second stage fermentation was investigated by simulating the fermentation environment. After first stage fermentation, the metabolite accumulated in broth was analyzed. MJ1 cultured in NDSCB medium as before was used as control and the samples initially prepared with four different metabolite combinations (3 g/L acetic acid; 2 g/L butyric acid; 3 g/L acetic acid and 2 g/L butyric acid; 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid) were tested. To evaluate the impact of initial acetate concentration on hydrogen production, acetate in the concentration range of 1 to 10 g/L (0, 1, 2, 3, 5, 7, 10 g/L) were added into the broth. To confirm the influences of metabolite and acetate concentration on hydrogen production, the soluble xylose (25 g/L) was used to replace the NDSCB, and the other fermentation conditions were the same as those for NDSCB. After fermentation (120 h), the biohydrogen production in the headspace was analyzed by gas chromatography. The effects of metabolite and acetate concentration on growth were investigated in 10 ml penicillin bottles containing 4.5 ml medium with 0.5 ml seed. The metabolite combinations and acetate concentrations were the same as described above. The value of OD600 was measured regularly for different samples. 2.4 Analytical methods Cell density was monitored by measuring turbidity at 600 nm (GENESYS™ 10S, Thermo Fisher, United States). Hydrogen was measured with a gas chromatograph (Fuli 9790, Fuli, China) equipped with a thermal conductivity detector (TCD) and a
flame ionization detector (FID) as previously described by Li et al. [29]. The concentrations of soluble sugars, organic acid and ethanol in filtered samples were measured using high performance liquid chromatography (HPLC) (Waters 2414, Waters, United States) equipped with a refractive index detector and Aminex HPX-87H column ((Bio-Rad, Hercules, CA) as previously described by Li et al. [29]. The concentration of reducing sugars was measured by the 3, 5-dinitrosalicylic acid (DNS) method [30]. The total phenolics were measured using a Folin-Ciocalteu method [17]. Phloroglucinol dehydrate was used as a standard. For detail operations, 20 µl of diluted sample was mixed with 100 µl of Folin-Ciocalteu reagent (Sangon Biotech, China) and incubated at room temperature for 5 min in dark conditions. Then 80 µl of 7.5% Na2CO3 was added and mixed. After 2 h incubation at room temperature in the dark, the absorbance was measured at 750 nm with a EnSpire-2300 multimode plate reader (PerkinElmer, USA). SPSS for windows (SPSS Inc. Chicago, version 17.0) was used for all statistical analysis and a value of P < 0.05 was considered statistically significant. 3. Results and discussion 3.1 Hydrogen production from DSCB and NDSCB in first stage fermentation To test the fermentation performance of NDSCB, the well-known lignocellulose biodegradation strain (C. thermocellum DSM1313) was first used for hydrogen production. Unfortunately, the strain could not utilize the NDSCB for growth (data not show). Due to its good fermentation performance of non-detoxified dilute-acid
pretreated lignocellulose hydrolysate, MJ1 was proposed for hydrogen production from NDSCB. To compare the fermentation performance, both DSCB and NDSCB were fermented by MJ1 at different concentrations. After detoxification by washing, the inhibitors content was significant decreased. There was only 55.4 ± 1.1 mg/L total phenolics in 10% DSCB medium, which could be ignored as compared with 10% NDSCB medium (872.6 ± 27.7 mg/L of total phenolics). As for the biodegradation performance of DSCB, the DSM 1313 could efficiently utilize DSCB for biohydrogen production that was diametrically opposed to NDSCB (data not shown). Those results indicated DSCB could be used as a detoxified substrate. Meanwhile, both SSF and PSSF were adopted for the selection of a better fermentation mode. The hydrogen yields from DSCB and NDSCB at different conditions are shown in Fig. 1. They were gradually increased with increasing substrate concentration and showed no significant difference between DSCB and NDSCB, suggesting that NDSCB was suitable for biohydrogen production by MJ1 without the detoxification process. The process cost and waste can be significantly reduced by using NDSCB, and the full utilization of NDSCB
contributed
to
biohydrogen
production
in
an
economical
and
environmentally friendly way. And for all we know, this is perhaps the first report about direct biohydrogen production from pretreated SCB without detoxication by bacterium. The hydrogen productions from NDSCB reached 118.7 mM (SSF) and 108.6 mM (PSSF) with no significant difference between the two modes (P=0.3>0.05), demonstrating that MJ1 could effectively produce biohydrogen in either SSF or PSSF
mode. Ren et al. have investigated hydrogen production by T. thermosaccharolyticum W16 from enzymatic hydrolysate of delignified corn stover (detoxified), and the hydrogen production reached 108.5 mM [31]. The data indicated that MJ1 was a promising candidate for biohydrogen production from NDSCB. 3.2 Hydrogen production from DSCB and NDSCB in second stage fermentation After the first stage fermentation, the reducing sugar in broth could not be detected at a substrate concentration below 4%. However, at a higher substrate concentration, there was a certain amount of reducing sugar in the broth and the contents are shown in Table 1. In the PSSF mode, nearly 10 g/L reducing sugar remained at 10% substrate. To fully utilize the fermentable sugars, we started the second stage fermentation at 10% substrate after adjustment of pH. The hydrogen yields of the second stage fermentation are shown in Fig. 2. Due to the lower reducing sugar content in SSF, the hydrogen production in the second stage was low as well. In PSSF, the hydrogen yields of DSCB and NDSCB reached 169.2 and 167.8 mM respectively, without significant difference between the two materials (P=0.85>0.05), demonstrating that MJ1 could effectively utilize NDSCB for repeated hydrogen production. The hydrogen yields of the second stage fermentation in PSSF were
significantly
enhanced
(10%
DSCB:
P=0.02<0.05;
10%
NDSCB:
P=0.006<0.05), indicating that MJ1 could maintain high activity and effectively produce hydrogen in the second stage fermentation. The total hydrogen production reached 277.4 mM (6.2 L-H2/L; 62.1 ml-H2/g-NDSCB) from the two-stage fermentation. Cheng et al. have investigated a novel co-culture system (Clostridium
thermocellum and Thermoanaerobacterium aotearoense) for hydrogen production from alkaline-pretreated SCB and the final hydrogen production reached 50.05 mM [32]. Ren et al. used T. thermosaccharolyticum W16 to produce hydrogen from the enzymatic hydrolysate of alkaline-pretreated corn stover (30 U/g cellulase and xylanase) and the hydrogen yield reached 108.5 mM [31]. Afterwards, Zhao et al. investigated hydrogen production from the enzymatic hydrolysate of fungal-pretreated corn stover (34 FPU/cellulose) by T. thermosaccharolyticum W16 and the hydrogen yield reached 89.3 ml/g-cornstalk [33]. It can be seen that the hydrogen production in the present study was significantly higher than that of the first two studies, but lower than that of Zhao’s study (62.1 ml-H2/g-NDSCB VS 89.3 ml-H2/g-cornstalk). The higher hydrogen yield obtained by Zhao can be attributed to the weaker resistance of cornstalk, fungal pretreatment method, higher cellulase dosage and low solid content. In the present study, the no-detoxification process was adopted, which decreased both the activity of cellulase and the degradability of NDSCB (data not show). Because of the adjustment of broth pH in air atmosphere, we speculate that MJ1 could maintain the cell viability in air atmosphere for a finite period of time. The recycle of fermentation broth is an efficient method for enhancing lignocellulosic biohydrogen production and substrate utilization efficiency. The higher hydrogen production by this simple procedure showed a novel strategy for economic biohydrogen production. 3.3 Effects of metabolites on CTec2 activity Due to lower residual reducing sugar content in the broth after first stage fermentation, the hydrogen production in the second stage fermentation was
obviously lower in SSF than in PSSF. Apart from enzymolysis time, metabolites produced by MJ1 might be the main reason for the lower residual reducing sugar content. The influence of metabolites on enzymolysis was investigated using four metabolite combinations (3 g/L acetic acid; 2 g/L butyric acid; 3 g/L acetic acid and 2 g/L butyric acid; 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid). The different metabolite combinations were added to the enzymatic hydrolysis system separately and the reducing sugar production was used to characterize the influence. As shown in Fig. 3, the reducing sugar production had no distinct difference in the four groups, suggesting that metabolite had no obvious inhibiting effect on activity of CTec2, or that lack of extra enzymolysis process before fermentation might be the main reason for the lower residual reducing sugar content in SSF. 3.4 Metabolite production and B/A ratio in PSSF The hydrogen production in the second stage fermentation was significantly higher, thus the metabolite production was analyzed and the results are shown in Fig. 4. The main metabolites in the broth were formate, acetate, lactate, butyrate and ethanol, with acetate and butyrate occupying a large proportion. The metabolite profiles of two stage fermentation were quite different, which implied the metabolic flux has been changed in second fermentation stage. The total metabolite production was lower in the second stage fermentation than in the first stage fermentation. This result was similar to a previous study in that the increase in total volatile fatty acid concentration directly led to the decrease in hydrogen production [18]. More
interestingly, the butyrate production was higher than the acetate production in the second stage fermentation, which was just the opposite to the result in the first stage fermentation, suggesting the occurrence of a shift in the metabolic pathway that might contribute to higher hydrogen production. No matter what kind of substrate was used (DSCB or NDSCB), the molar ratio of butyrate/acetate (B/A) was much higher in the second stage fermentation than in the first stage fermentation (Fig. 4C). Hydrogen production is generally accompanied with the generation of acetate and butyrate, thus the ratio of B/A is frequently used as an indicator to evaluate the effectiveness of hydrogen production [34, 35]. In the present study, the B/A ratio reached 2.3 in the second stage fermentation (10% NDSCB) while it was only 0.4 in the first stage fermentation. Arooj et al. found that a decrease in acetate concentration caused an increase in B/A ratio, resulting in a high hydrogen yield in an anaerobic continuous stirred-tank reactor [35]. The results indicated that the metabolism of MJ1 was affected by fermentation condition and the higher production of butyrate was conducive to higher hydrogen production. The ratio of B/A increased with increasing final pH (pH 4.98 for second stage fermentation and pH 4.68 for first stage fermentation), which was consistent with a previous study [36]. 3.5 Effect of metabolites and acetate concentration on hydrogen production The metabolite analysis indicated the occurrence of a shift in the metabolic pathway in the second stage fermentation. Perhaps the metabolite produced in the first stage fermentation led to the metabolic pathway shift of MJ1 and thus higher hydrogen production. In order to investigate the effect of metabolite on hydrogen
production, four metabolite combinations were employed for simulating the second stage fermentation condition. The hydrogen yields with different metabolite combinations are shown in Fig. 5A. The hydrogen production was significantly enhanced, especially for Groups Acetate (P=0.002<0.05) and Four (P=0.005<0.05). The hydrogen production showed an increase of 51% when 3 g/L acetate was added. In Group Four, the main enhancer of hydrogen production might be acetate. The different hydrogen yields of the four metabolite combination groups implied the different performances of metabolites in hydrogen production. At the same time, the metabolite profiles were changed when different external metabolite combinations were added to the fermentation broths. The B/A molar ratio was significantly improved except for Group Butyrate with a significant reduction (Fig. S1A). This phenomenon was similar with previous observation that the metabolite profile was changed in two stage fermentation, which further confirmed the metabolic flux was changed by external supplemental metabolites. The production of butyrate in Group Butyrate was similar to that of the control, but the acetate production was much higher, thus leading to significant reduction in the B/A molar ratio. The results were consistent with our expectation that metabolite contributed to higher hydrogen production. The higher final pH might be the buffering capacity of additional metabolite. We speculate that the stimulatory effect of metabolite on hydrogen production was mainly attributed to the buffing capacity and the redirection of the metabolic flux. He et al. found that lactate was generally inhibitory to ethanolic fermentation,
and acetate showed an unexpected stimulatory effect on ethanolic fermentation by Thermoanaerobacter ethanolicus [37]. Metabolic shift in C. thermocellum ATCC 27405, represented by change in final end-product concentration, was also found by Rydzak et al. In the presence of sodium acetate (55 mM), ethanol production increased by 28% and hydrogen production marginally decreased, while the hydrogen production increased marginally by additional formate (27 mM) or lactate (55 mM) [20]. Supplementing culture with increasing amounts of exogenous acetate (up to 40 mM (3.28 g/L)) stimulated hydrogen production in Caloramator celer (increasing the accumulation by 25%), but addition of formate did not significantly affect hydrogen production [22]. The different performances of metabolites in hydrogen production might be determined by the characteristics of different strains. In the present study, acetate and butyrate had a significantly positive effect on hydrogen production by MJ1 (10% NDSCB). Due to its higher stimulatory effect on hydrogen production, the effect of acetate concentration was further investigated. As shown in Fig. 5B, the hydrogen production was gradually increased with increasing acetate concentration. The hydrogen production was significantly enhanced (P=0.008<0.05), even with 1 g/L acetate added, while no significant difference was observed in hydrogen production with the acetate concentration further increased from 3 to 10 g/L. Additionally, the B/A molar ratio was enhanced with acetate added to broth (Fig. S1B). These results confirmed the auxo-action of acetate on hydrogen production by MJ1. The pH was also gradually increased with increasing acetate concentration, and the final pH indicated the higher
buffering capacity of higher acetate concentration. At the same time, the hydrogen production also depended on the metabolic rate and hydrolysis of cellulose. The metabolic rate was significant influenced by pH. The model (effect of pH on metabolic rate) revealed an optimal pH value that maximized the metabolic rate, and the metabolic rate was rapidly declined when pH was far away from the optimal pH [38]. The higher final pH indicated that the supplemental metabolites could maintain a relatively stable pH, leading to higher metabolic rate for MJ1 (hydrogen production). The hydrolysis rate of cellulose influenced the reducing sugar content that affected the metabolic rate of microorganisms. Previous study has researched the autocatalytic degradation kinetics of cellulose in homogeneous acidic medium [39]. The autocatalytic degradation ratio was much lower than enzymatic hydrolysis. Because a pre-enzymatic hydrolysis was adopted, the influence of autocatalytic hydrolysis of cellulose on hydrogen production could be ignored in present work. A previous study has indicated that MJ1 could effectively convert xylose, a soluble monosaccharide, to hydrogen [17]. In the present study, xylose was used to test the function of metabolite in hydrogen production by MJ1. The metabolite combinations used were the same as described above. As shown in Fig. 5C, the hydrogen production was significantly enhanced by acetate (P=0.0006<0.05), but obviously suppressed by butyrate (P=0.01<0.05). For Group Four, the promotion of acetate could not offset the inhibition of butyrate, leading to a significant decrease of hydrogen production (P=0.02<0.05). The effect of butyrate on hydrogen production was influenced by substrate, while acetate always exhibited a stimulatory effect.
Currently, there is no report available concerning the effect of butyrate on hydrogen production. A possible explanation is that the supplementation of butyrate in xylose medium significantly decreased the butyrate production (P=0.02<0.05), but produced no effect on acetate production, implying that the B/A ratio was significantly decreased (Fig. S1C), and the lower B/A ratio was probably an important contributor to the lower hydrogen production. The hydrogen production from xylose was significantly enhanced by acetate similar to that of NDSCB, and thus the effect of acetate concentration in xylose medium was further tested (Fig. 5D). The gradual growth trend of hydrogen production in xylose was similar to that of NDSCB, with the hydrogen production significantly enhanced (P=0.04<0.05) at an acetate concentration exceeding 2 g/L. The variation trend of B/A molar ratio in xylose broth was similar to that of NDSCB (Fig. S1D). These results confirmed the positive role of acetate in hydrogen production and no relationship between enhancement and carbon source. The relationship between the hydrogen production and acetate concentration was researched to reveal the mechanism of acetate on hydrogen production (Fig. S2). A linear model was simulated according to previous kinetic modeling of batch hydrogen production process [40]. There was a linear relationship between hydrogen production and acetate concentration. The hydrogen production could be significantly enhanced by lower acetate concentration and the tendency was mitigated with the increase of acetate concentration. This indicated the hydrogen production has a higher linear relationship with acetate concentration around a certain range. A better correlation
coefficient was obtained in xylose than NDSCB that suggested a good correlation in pure substrate culture. 3.6 Effect of metabolite and acetate concentration on growth of MJ1 Given the important role of metabolite supplementation in hydrogen production, the effect of metabolite on growth of MJ1 was further investigated for a better understanding of the function of the complementary metabolite. The growth curves of MJ1 with different metabolite combinations are shown in Fig. 6A. The supplementation of four groups of metabolites resulted in an enhancement of MJ1 growth. The stimulatory effect of acetate was strengthened with increasing acetate concentration, and the highest cell density was observed at the highest acetate concentration (Fig. 6B). The specific growth rates of MJ1 with different metabolites were increased, leading to significant enhancement of the final biomass. Furthermore, peak cell turbidity (Group Acetate or Group Three or Group Four) had a nearly 50% increase over that of the control. The peak cell turbidity (Group Butyrate) was higher than that of the control, but lower than that of the other groups, which was similar to a previous report about the stimulation of acetate on the growth of Thermoanaerobacter ethanolicus strain 39E, C. thermocellum 27405 and Thermoanaerobacter ethanolicus strain X514 [37]. As pH was an important factor for microbial growth, the greater biomass obtained from treated groups was partly due to the buffering capacity of the additional metabolite (higher final pH), which could create a more suitable environmental pH for microbial survival. The relationship between biomass and hydrogen production for anaerobic
fermentation by MJ1 could be simulated by the Luedeking–Piret model as previous study [40]. The fitted plots were shown in Fig. S3. The high correlation coefficient (R2=0.98) indicated that the Luedeking–Piret model could properly describe the relationship between biomass and hydrogen production in the anaerobic fermentation process. The hydrogen production was dependent on the biomass when acetate added to the fermentation broth.
4. Conclusions In this study, a simple two-stage anaerobic fermentation strategy was established for efficient biohydrogen production from NDSCB. Meanwhile, the promoting mechanism underlying higher hydrogen production in the second stage fermentation was explored. The metabolite generated in the first stage fermentation contributed to higher hydrogen production. Both B/A ratio and MJ1 growth could be promoted by metabolite supplementation. The stimulations of metabolite on hydrogen production were mainly attributed to the buffer capacity and redirection of the metabolic flux. The recycle of fermentation broth is an economical method to enhance biohydrogen production and substrate utilization. The use of NDSCB does not need a separate detoxification process after pretreatment, thus increasing the competitiveness of biohydrogen production from lignocellulose. The results suggest great potential of the two-stage fermentation for environment-friendly and high-yield biohydrogen production in the future.
Supplementary material Supplementary data (Fig.S1-S3) associated with this article can be found in the online
version.
Acknowledgements This work was supported by the National Natural Science Foundation of China [grant nos. 51478190 and 51278200], the Guangdong Provincial Natural Science Foundation Key Project [grant no. 2014A030311014], and the Guangzhou Science and Technology Program [grant no. 201510010288].
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Lists of figures: Fig. 1 Hydrogen production from DSCB and NDSCB in the first stage fermentation in SSF and PSSF modes. Fig. 2 Hydrogen production from DSCB and NDSCB in the second stage fermentation in SSF and PSSF modes. Fig. 3 Reducing sugar production under different enzymolysis conditions. Fig. 4 Metabolite production and B/A ratio in the two-stage fermentation. Fig. 5 Effect of different metabolite combinations and acetate concentrations on hydrogen production. Fig. 6 Growth curves of MJ1 supplemented with different metabolite combinations and acetate concentrations.
Lists of tables Table 1 The residual reducing sugar content in broth after the first stage fermentation.
Tables Table 1 The residual reducing sugar content in broth after the first stage fermentation Reducing sugar content (g/L) Modes 6%DSCB
6%NDSCB
10%DSCB
10%NDSCB
SSF
0.67±0.51
0.67±0.01
2.17±0.19
3.88±0.11
PSSF
2.07±1.39
1.15±0.18
9.37±0.61
9.93±0.89
Values are shown as mean of duplicate ± standard deviation.
DSCB NDSCB
A
120
Hydrogen production (mM)
Hydrogen production (mM)
140 120 100 80 60 40 20
DSCB NDSCB
B
100 80 60 40 20 0
0 2
4
6
10
2
Substrate concentration (%)
4
6
10
Substrate concentration (%)
Fig. 1 Hydrogen production from DSCB and NDSCB in the first stage fermentation in SSF and PSSF modes. A: SSF mode, B: PSSF mode.
PSSF SSF
Hydrogen production (mM)
180 160 140 120 100 80 60 40 20 0 DSCB
NDSCB
Substrates
Fig. 2 Hydrogen production from DSCB and NDSCB in the second stage fermentation in SSF and PSSF modes.
Control Acetate Butyrate Acetate/Butyrate Formate/Lactate/Acetate/Butyrate
Reducing sugars content (g/L)
12 10 8 6 4 2 0 0
10 20 30 40 50 60 70 80 90 100 110 120 130
Time (h)
Fig. 3 Reducing sugar production under different enzymolysis conditions.
Formate Butyrate
A
Acetate Ethanol
Lactate
Formate Butyrate
B
Acetate Ethanol
Lactate
3.5
2
Production (g/L)
Production (g/L)
3.0 2.5 2.0 1.5 1.0
1
0.5
0
0.0 10%DSCB
10%DSCB
10%NDSCB
C
10%NDSCB
Substrate
Substrate
4.0
First
Second
3.5
Molar ratio of B/A
3.0 2.5 2.0 1.5 1.0 0.5 0.0 10%DSCB
10%NDSCB
Substrate
Fig. 4 Metabolite production and B/A ratio in the two-stage fermentation. A: First stage, B: Second stage, C: B/A ratio.
pH
B
6
160 5 140 120
4
100 3 80 60
2
40 1 20 0
0 Acetate
Butyrate
Three
6
160 5 140 120
4
100 3 80 60
2
40 1 20 0
0
Four
0
1
Groups
Relative hydrogen production
D pH
180
6
160 5 140 120
4
100 3 80 60
2
40 1 20 0
0 Control
Acetate
Butyrate
Groups
2
3
5
7
10
Acetate concentration (g/L)
Three
Four
pH Relative hydrogen production (%)
Relative hydrogen production (%)
C
pH
Relative hydrogen production
pH
200
6
180 5
160 140
4
120 100
3
80
pH
Control
Relative hydrogen production
180
pH
Relative hydrogen production
180
pH Relative hydrogen production (%)
Relative hydrogen production (%)
A
2
60 40
1
20 0
0 0
1
2
3
5
7
10
Acetate concentration (g/L)
Fig. 5 Effect of different metabolite combinations and acetate concentrations on hydrogen production. A and B: NDSCB as carbon source; C and D: xylose as carbon source. Acetate: 3 g/L acetic acid; Butyrate: 2 g/L butyric acid; Three: 3 g/L acetic acid and 2 g/L butyric acid; Four: 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid.
Control Three
A 1.8
Acetate Four
Butyrate
0 5
B
2.0
1 7
2 10
3
1.8
1.6
1.6
1.4
1.4
1.2
OD600
OD600
1.2 1.0 0.8 0.6
1.0 0.8 0.6
0.4
0.4
0.2
0.2
0.0
0.0 0
5
10
15
20
25
30
Time (h)
35
40
45
50
0
5
10
15
20
25
30
35
40
45
50
Time (h)
Fig. 6 Growth curves of MJ1 supplemented with different metabolite combinations and acetate concentrations. A: different metabolite combinations; B: different acetate concentrations (g/L).
Highlights Non-detoxified SCB could be directly used for thermophilic hydrogen fermentation. Two-stage fermentation is suitable for high-yield biohydrogen production. The hydrogen production can be significantly enhanced by supplementary metabolite. Supplementation of metabolite can redirect metabolic flux.
S1385-8947(17)31881-8 https://doi.org/10.1016/j.cej.2017.10.157 CEJ 17941
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
25 August 2017 25 October 2017 26 October 2017
Please cite this article as: B-B. Hu, M-Y. Li, Y-T. Wang, M-J. Zhu, High-yield biohydrogen production from nondetoxified sugarcane bagasse: Fermentation strategy and mechanism, Chemical Engineering Journal (2017), doi: https://doi.org/10.1016/j.cej.2017.10.157
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
High-yield biohydrogen production from non-detoxified sugarcane bagasse: Fermentation strategy and mechanism Bin-Bin Hua, Ming-Yuan Lib,c, Yu-Tao Wangb,c, Ming-Jun Zhu a,b,c* a
School of Bioscience and Bioengineering, South China University of Technology,
Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People’s Republic of China b
College of Life and Geographic Sciences, Kashgar University, Kashgar 844000,
China c
The Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at
Colleges & Universities under the Department of Education of Xinjiang Uygur Autonomous Region, Kashgar University, Kashgar 844000, China First Author, E-mail address: [email protected]; Tel: +8620 39380636; ∗
Corresponding author, E-mail address: [email protected]; Tel: +8620 39380623;
Fax: +8620 39380601
Abstract: Anaerobic digestion, an attractive process for generation of hydrogen, involves complex microbial processes on decomposition of waste biomass. Here, efficient biohydrogen production (277.4 mM (6.2 L-H2/L)) from non-detoxified sugarcane bagasse (NDSCB) was accomplished through a novel two-stage anaerobic fermentation, which to our knowledge, is the first utilization of NDSCB for biohydrogen fermentation. Interestingly, the hydrogen production of the second stage fermentation (167.8 mM) was significantly higher than that of the first stage fermentation (108.6 mM). Metabolite analysis showed that the metabolic flux had been redirected and Butyrate/Acetate ratio was enhanced in the second stage fermentation. Simulation of the second-stage fermentation condition indicated that acetate and butyrate generated in the first-stage fermentation could significantly enhance the hydrogen production from NDSCB by 51% and 28%, respectively. More interestingly, the stimulatory effect of butyrate was influenced by substrate. The present work provided a comprehensive insight of metabolite on biohydrogen production and an environment-friendly process for lignocellulosic biohydrogen production. Keywords:
Biohydrogen
production;
Two-stage
fermentation;
Metabolite;
Non-detoxified sugarcane bagasse (NDSCB); T. thermosaccharolyticum MJ1
1. Introduction Fossil fuels such as coal, oil and natural gas provide about 95% of the world’s total energy and the demand is increasing over the past decade [1, 2]. Because of overuse of fossil fuels, pollution problems have become more and more serious in our living environment, suggesting the necessity to find an alternative energy source, which is renewable, abundant, clean and cost-effective [1, 3]. One of the great challenges in the coming decade is how to obtain new renewable energy sources that are environmentally friendly and lessen the high dependence on fossil fuels [4]. Lignocellulose composed of cellulose, hemicellulose and lignin is the most abundant raw material on the earth. Due to this advantage, lignocellulosic biomass has been arousing great interest among researchers [5, 6]. Hydrogen has been identified as a clean energy carrier and one of the most ideal alternative fuels due to its combustion in air to form water [7]. In industry, hydrogen has been produced from natural gas (48%) and oil (30%) by steam reforming processes, or other industrial methods such as coal gasification (18%) and water electrolysis (4%) [8, 9]. However, these hydrogen production methods use fossil fuels as the raw material and thus many of the advantages of hydrogen are offset by the raw material. At the same time, the continuous system of hydrogen production is needed for application at industrial scale [10]. Among different technologies of hydrogen production, biohydrogen production shows the greatest potential to replace the traditional methods. Hydrogen produced from biological processes is less energy intensive and more environmentally friendly. Biohydrogen production by dark
fermentation presents a promising way for the sustainable largescale generation of hydrogen in the future hydrogen economy [3]. Compared with other biological production methods, dark fermentation has several advantages: 1) continuously producing hydrogen without the presence of light, 2) more simple and moderate production process, 3) maintaining a higher hydrogen production rate, and 4) utilizing a wide range of low-cost waste materials [4, 7, 11]. Accordingly, hydrogen production by dark fermentation has become more attractive in recent years. The successful conversion of various substrates to biohydrogen depends greatly on the performance of specific microbial species in a complex series of biochemical reactions. The acclimatization of biohydrogen-producing cultures to substrate change is a helpful strategy for enhancing biohydrogen production [12]. After enzymatic hydrolysis, the lignocellulose is hydrolyzed to a mixture of hexose and pentose sugars. Currently, various microorganisms have been reported to produce hydrogen [13]. Compared with hexose-fermenting organisms, only a few co-fermenting organisms have been identified to convert substrate to hydrogen [6]. The co-fermenting thermophilic organisms are considered as a promising candidate for biohydrogen production.
Thermoanaerobacterium
thermosaccharolyticum
(T.
thermosaccharolyticum) is a well-known strain for bio-hydrogen production due to its ability to utilize various kinds of substrates [14-16]. T. thermosaccharolyticum could simultaneously ferment a mixture of hexose and pentose sugars to biohydrogen without obvious delay. Our previous study revealed that T. thermosaccharolyticum MJ1 also possesses an excellent tolerance to acid pretreatment inhibitors and is a
promising candidate for industrial biohydrogen production [17]. Biohydrogen production is usually accompanied with the synthesis of organic acids, which is greatly influenced by metabolites. Acetate, formate, lactate, pyruvate and butyrate are the main soluble end products in broth and closely related with microorganisms. Sreethawong et al. have reported that the increase in nitrogen concentration caused the increased production of volatile fatty acids, directly leading to the decrease of hydrogen production and the increase of butyric acid production accompanying the decrease in acetic acid production [18]. Van Ginkel and Logan have found that acids added to the feed at a concentration of 25 mM decreased hydrogen yields by 13% (acetic) and 22% (butyric), and 60 mM of either acid decreased hydrogen production by 93% [19]. Compared with ethanol, acetate exhibited more significant inhibition on growth and hydrogen production performance of Ethanoligenens harbinese B49; in Clostridium thermocellum ATCC 27405, hydrogen and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen, acetate, and lactate; in Caloramator celer, hydrogen production kinetics was more sensitive to increasing concentration of acetate and formate, with acetate inhibiting the hydrogen production by increasing the ionic strength in the medium [20-22]. The aforementioned results have demonstrated that diverse influences of metabolites in different microorganisms and metabolites could redirect the metabolic pathways. To relieve the recalcitrant structure of lignocellulose, numerous pretreatment methods were exploited to break down the stubborn lignocellulosic structure, such as
dual salts pretreatment, alkaline pretreatment, steam explosion pretreatment, dilute acid pretreatment [23], γ-irradiation pretreatment [24], ozonation pretreatment [25] and acidic-alkaline pretreatment [26]. Among various pretreatment methods, dilute acid pretreatment is an economical pretreatment method widely used in industrial manufacture. As the inhibitors (such as furfural, hydroxymethyl furfural and carboxylic acids) are formed during the pretreatment process, the substrate must be detoxified (such as adsorption and biodegradation), thus increasing the cost of manufacture [27, 28]. Our previous study investigated the application of dilute-acid pretreated sugarcane bagasse hydrolysate in hydrogen production by MJ1 [17]. The residual insoluble substrate (NDSCB) was also an important material for biorefinery. At present, a separate detoxification process was needed to relieve the inhibitory effect of NDSCB, which increased the operation cost and effluent discharge. The full utilization of NDSCB without detoxification was an economical and environmentally friendly technology to increase the competitiveness of biohydrogen production from lignocellulose. In this study, for full use of the residual material obtained in dilute-acid pretreatment process, we investigated the potential of biohydrogen production from the non-detoxified substrate. The underlying mechanism of higher biohydrogen production was also explored. 2. Materials and methods 2.1 Materials and pretreatment Sugarcane bagasse (SCB) was obtained from Guangzhou Sugarcane Industry Research Institute (Guangzhou, China). The non-detoxified sugarcane bagasse
(NDSCB) was obtained from the dilute acid pretreatment process of SCB. The raw SCB was soaked in sulfuric acid solution (1%, g/v) with a solid to liquid ratio of 1:10 (g dry weight to ml) at 121 ℃ for 30 min. The insoluble solid (NDSCB) was directly obtained by vacuum filtration and dried at 50 ℃. Finally, the dried NDSCB was milled and sieved through a 200-mesh (74 µm). The detoxified sugarcane bagasse (DSCB) was obtained from NDSCB after detoxification by distilled water. For detailed instructions, the NDSCB (obtained from 100 g SCB) was mixed fully with 2 L distilled water for 30 min with an intermittent stir. The insoluble solid was directly obtained by vacuum filtration. The operation repeated at least 10 times until the supernatant was neutral (pH 7.0). After vacuum filtration, the DSCB was dried, milled and sieved as substrate. The dried power (both NDSCB and DSCB) was used in the following studies. The commercial cellulase Cellic® CTec2 was kindly provided by Novozymes (China) Investment Co. Ltd. Filter paper activity of cellulase was 90 FPU/mL and pNPG activity of β-glucosidase was 2743 U/mL. 2.2 Microorganism and medium The anaerobic T. thermosaccharolyticum MJ1 isolated previously was used for biohydrogen production and maintained as previously reported [17]. All cultures were inoculated with a volume of 10% (v/v) from freshly prepared cultures at exponential growth phase (about 18 h) in serum bottles. Additionally, MJ1 was anaerobically grown in 100 ml serum bottles for a final working volume of 30 ml. The strain was cultured at 55 ℃ with rotary shaking at 150 rpm.
2.3 Experimental design 2.3.1 Biohydrogen potential tests Biohydrogen production was investigated by batch fermentation of NDSCB in 120 mL serum bottles. Briefly, each serum bottle contained different concentrations of NDSCB (20, 40, 60, 100 g/L) to replace the carbon source in medium (pH 6.5). The cellulase was added at a loading of 20 FPU/g NDSCB after autoclave. For simultaneous saccharification and fermentation (SSF), the fermentation was carried out for 120 h (initial pH 6.5). For prehydrolysis and simultaneous saccharification and fermentation (PSSF), the prehydrolysis by cellulase was carried out for 72 h at 50 ℃ before fermentation (pH 6.5), then the MJ1 was cultured for another 120 h. As for the second stage fermentation, the pH of fermentation broth was adjusted to 7.0 and the serum bottles were sealed again. Next, the serum bottles were purged three times with 100% nitrogen, and then fermented in a shaker for another 120 h. After fermentation, the biohydrogen production in the headspace was analyzed by gas chromatography. 2.3.2 Influence of metabolite on performance of CTec2 The influence of metabolites was investigated by enzymolysis. The cellulase was added at a loading of 20 FPU/g NDSCB. To test metabolites, NDSCB (100 g/L) was degraded by CTec2 in 50 mM phosphate buffer with four different metabolite combinations (3 g/L acetic acid; 2 g/L butyric acid; 3 g/L acetic acid and 2 g/L butyric acid; 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid) at pH 5.0. The sample without added metabolites was used as control. The samples were taken regularly to determine the reducing sugar.
2.3.3 Mechanism of high biohydrogen production in second stage fermentation The mechanism for the high biohydrogen production in the second stage fermentation was investigated by simulating the fermentation environment. After first stage fermentation, the metabolite accumulated in broth was analyzed. MJ1 cultured in NDSCB medium as before was used as control and the samples initially prepared with four different metabolite combinations (3 g/L acetic acid; 2 g/L butyric acid; 3 g/L acetic acid and 2 g/L butyric acid; 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid) were tested. To evaluate the impact of initial acetate concentration on hydrogen production, acetate in the concentration range of 1 to 10 g/L (0, 1, 2, 3, 5, 7, 10 g/L) were added into the broth. To confirm the influences of metabolite and acetate concentration on hydrogen production, the soluble xylose (25 g/L) was used to replace the NDSCB, and the other fermentation conditions were the same as those for NDSCB. After fermentation (120 h), the biohydrogen production in the headspace was analyzed by gas chromatography. The effects of metabolite and acetate concentration on growth were investigated in 10 ml penicillin bottles containing 4.5 ml medium with 0.5 ml seed. The metabolite combinations and acetate concentrations were the same as described above. The value of OD600 was measured regularly for different samples. 2.4 Analytical methods Cell density was monitored by measuring turbidity at 600 nm (GENESYS™ 10S, Thermo Fisher, United States). Hydrogen was measured with a gas chromatograph (Fuli 9790, Fuli, China) equipped with a thermal conductivity detector (TCD) and a
flame ionization detector (FID) as previously described by Li et al. [29]. The concentrations of soluble sugars, organic acid and ethanol in filtered samples were measured using high performance liquid chromatography (HPLC) (Waters 2414, Waters, United States) equipped with a refractive index detector and Aminex HPX-87H column ((Bio-Rad, Hercules, CA) as previously described by Li et al. [29]. The concentration of reducing sugars was measured by the 3, 5-dinitrosalicylic acid (DNS) method [30]. The total phenolics were measured using a Folin-Ciocalteu method [17]. Phloroglucinol dehydrate was used as a standard. For detail operations, 20 µl of diluted sample was mixed with 100 µl of Folin-Ciocalteu reagent (Sangon Biotech, China) and incubated at room temperature for 5 min in dark conditions. Then 80 µl of 7.5% Na2CO3 was added and mixed. After 2 h incubation at room temperature in the dark, the absorbance was measured at 750 nm with a EnSpire-2300 multimode plate reader (PerkinElmer, USA). SPSS for windows (SPSS Inc. Chicago, version 17.0) was used for all statistical analysis and a value of P < 0.05 was considered statistically significant. 3. Results and discussion 3.1 Hydrogen production from DSCB and NDSCB in first stage fermentation To test the fermentation performance of NDSCB, the well-known lignocellulose biodegradation strain (C. thermocellum DSM1313) was first used for hydrogen production. Unfortunately, the strain could not utilize the NDSCB for growth (data not show). Due to its good fermentation performance of non-detoxified dilute-acid
pretreated lignocellulose hydrolysate, MJ1 was proposed for hydrogen production from NDSCB. To compare the fermentation performance, both DSCB and NDSCB were fermented by MJ1 at different concentrations. After detoxification by washing, the inhibitors content was significant decreased. There was only 55.4 ± 1.1 mg/L total phenolics in 10% DSCB medium, which could be ignored as compared with 10% NDSCB medium (872.6 ± 27.7 mg/L of total phenolics). As for the biodegradation performance of DSCB, the DSM 1313 could efficiently utilize DSCB for biohydrogen production that was diametrically opposed to NDSCB (data not shown). Those results indicated DSCB could be used as a detoxified substrate. Meanwhile, both SSF and PSSF were adopted for the selection of a better fermentation mode. The hydrogen yields from DSCB and NDSCB at different conditions are shown in Fig. 1. They were gradually increased with increasing substrate concentration and showed no significant difference between DSCB and NDSCB, suggesting that NDSCB was suitable for biohydrogen production by MJ1 without the detoxification process. The process cost and waste can be significantly reduced by using NDSCB, and the full utilization of NDSCB
contributed
to
biohydrogen
production
in
an
economical
and
environmentally friendly way. And for all we know, this is perhaps the first report about direct biohydrogen production from pretreated SCB without detoxication by bacterium. The hydrogen productions from NDSCB reached 118.7 mM (SSF) and 108.6 mM (PSSF) with no significant difference between the two modes (P=0.3>0.05), demonstrating that MJ1 could effectively produce biohydrogen in either SSF or PSSF
mode. Ren et al. have investigated hydrogen production by T. thermosaccharolyticum W16 from enzymatic hydrolysate of delignified corn stover (detoxified), and the hydrogen production reached 108.5 mM [31]. The data indicated that MJ1 was a promising candidate for biohydrogen production from NDSCB. 3.2 Hydrogen production from DSCB and NDSCB in second stage fermentation After the first stage fermentation, the reducing sugar in broth could not be detected at a substrate concentration below 4%. However, at a higher substrate concentration, there was a certain amount of reducing sugar in the broth and the contents are shown in Table 1. In the PSSF mode, nearly 10 g/L reducing sugar remained at 10% substrate. To fully utilize the fermentable sugars, we started the second stage fermentation at 10% substrate after adjustment of pH. The hydrogen yields of the second stage fermentation are shown in Fig. 2. Due to the lower reducing sugar content in SSF, the hydrogen production in the second stage was low as well. In PSSF, the hydrogen yields of DSCB and NDSCB reached 169.2 and 167.8 mM respectively, without significant difference between the two materials (P=0.85>0.05), demonstrating that MJ1 could effectively utilize NDSCB for repeated hydrogen production. The hydrogen yields of the second stage fermentation in PSSF were
significantly
enhanced
(10%
DSCB:
P=0.02<0.05;
10%
NDSCB:
P=0.006<0.05), indicating that MJ1 could maintain high activity and effectively produce hydrogen in the second stage fermentation. The total hydrogen production reached 277.4 mM (6.2 L-H2/L; 62.1 ml-H2/g-NDSCB) from the two-stage fermentation. Cheng et al. have investigated a novel co-culture system (Clostridium
thermocellum and Thermoanaerobacterium aotearoense) for hydrogen production from alkaline-pretreated SCB and the final hydrogen production reached 50.05 mM [32]. Ren et al. used T. thermosaccharolyticum W16 to produce hydrogen from the enzymatic hydrolysate of alkaline-pretreated corn stover (30 U/g cellulase and xylanase) and the hydrogen yield reached 108.5 mM [31]. Afterwards, Zhao et al. investigated hydrogen production from the enzymatic hydrolysate of fungal-pretreated corn stover (34 FPU/cellulose) by T. thermosaccharolyticum W16 and the hydrogen yield reached 89.3 ml/g-cornstalk [33]. It can be seen that the hydrogen production in the present study was significantly higher than that of the first two studies, but lower than that of Zhao’s study (62.1 ml-H2/g-NDSCB VS 89.3 ml-H2/g-cornstalk). The higher hydrogen yield obtained by Zhao can be attributed to the weaker resistance of cornstalk, fungal pretreatment method, higher cellulase dosage and low solid content. In the present study, the no-detoxification process was adopted, which decreased both the activity of cellulase and the degradability of NDSCB (data not show). Because of the adjustment of broth pH in air atmosphere, we speculate that MJ1 could maintain the cell viability in air atmosphere for a finite period of time. The recycle of fermentation broth is an efficient method for enhancing lignocellulosic biohydrogen production and substrate utilization efficiency. The higher hydrogen production by this simple procedure showed a novel strategy for economic biohydrogen production. 3.3 Effects of metabolites on CTec2 activity Due to lower residual reducing sugar content in the broth after first stage fermentation, the hydrogen production in the second stage fermentation was
obviously lower in SSF than in PSSF. Apart from enzymolysis time, metabolites produced by MJ1 might be the main reason for the lower residual reducing sugar content. The influence of metabolites on enzymolysis was investigated using four metabolite combinations (3 g/L acetic acid; 2 g/L butyric acid; 3 g/L acetic acid and 2 g/L butyric acid; 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid). The different metabolite combinations were added to the enzymatic hydrolysis system separately and the reducing sugar production was used to characterize the influence. As shown in Fig. 3, the reducing sugar production had no distinct difference in the four groups, suggesting that metabolite had no obvious inhibiting effect on activity of CTec2, or that lack of extra enzymolysis process before fermentation might be the main reason for the lower residual reducing sugar content in SSF. 3.4 Metabolite production and B/A ratio in PSSF The hydrogen production in the second stage fermentation was significantly higher, thus the metabolite production was analyzed and the results are shown in Fig. 4. The main metabolites in the broth were formate, acetate, lactate, butyrate and ethanol, with acetate and butyrate occupying a large proportion. The metabolite profiles of two stage fermentation were quite different, which implied the metabolic flux has been changed in second fermentation stage. The total metabolite production was lower in the second stage fermentation than in the first stage fermentation. This result was similar to a previous study in that the increase in total volatile fatty acid concentration directly led to the decrease in hydrogen production [18]. More
interestingly, the butyrate production was higher than the acetate production in the second stage fermentation, which was just the opposite to the result in the first stage fermentation, suggesting the occurrence of a shift in the metabolic pathway that might contribute to higher hydrogen production. No matter what kind of substrate was used (DSCB or NDSCB), the molar ratio of butyrate/acetate (B/A) was much higher in the second stage fermentation than in the first stage fermentation (Fig. 4C). Hydrogen production is generally accompanied with the generation of acetate and butyrate, thus the ratio of B/A is frequently used as an indicator to evaluate the effectiveness of hydrogen production [34, 35]. In the present study, the B/A ratio reached 2.3 in the second stage fermentation (10% NDSCB) while it was only 0.4 in the first stage fermentation. Arooj et al. found that a decrease in acetate concentration caused an increase in B/A ratio, resulting in a high hydrogen yield in an anaerobic continuous stirred-tank reactor [35]. The results indicated that the metabolism of MJ1 was affected by fermentation condition and the higher production of butyrate was conducive to higher hydrogen production. The ratio of B/A increased with increasing final pH (pH 4.98 for second stage fermentation and pH 4.68 for first stage fermentation), which was consistent with a previous study [36]. 3.5 Effect of metabolites and acetate concentration on hydrogen production The metabolite analysis indicated the occurrence of a shift in the metabolic pathway in the second stage fermentation. Perhaps the metabolite produced in the first stage fermentation led to the metabolic pathway shift of MJ1 and thus higher hydrogen production. In order to investigate the effect of metabolite on hydrogen
production, four metabolite combinations were employed for simulating the second stage fermentation condition. The hydrogen yields with different metabolite combinations are shown in Fig. 5A. The hydrogen production was significantly enhanced, especially for Groups Acetate (P=0.002<0.05) and Four (P=0.005<0.05). The hydrogen production showed an increase of 51% when 3 g/L acetate was added. In Group Four, the main enhancer of hydrogen production might be acetate. The different hydrogen yields of the four metabolite combination groups implied the different performances of metabolites in hydrogen production. At the same time, the metabolite profiles were changed when different external metabolite combinations were added to the fermentation broths. The B/A molar ratio was significantly improved except for Group Butyrate with a significant reduction (Fig. S1A). This phenomenon was similar with previous observation that the metabolite profile was changed in two stage fermentation, which further confirmed the metabolic flux was changed by external supplemental metabolites. The production of butyrate in Group Butyrate was similar to that of the control, but the acetate production was much higher, thus leading to significant reduction in the B/A molar ratio. The results were consistent with our expectation that metabolite contributed to higher hydrogen production. The higher final pH might be the buffering capacity of additional metabolite. We speculate that the stimulatory effect of metabolite on hydrogen production was mainly attributed to the buffing capacity and the redirection of the metabolic flux. He et al. found that lactate was generally inhibitory to ethanolic fermentation,
and acetate showed an unexpected stimulatory effect on ethanolic fermentation by Thermoanaerobacter ethanolicus [37]. Metabolic shift in C. thermocellum ATCC 27405, represented by change in final end-product concentration, was also found by Rydzak et al. In the presence of sodium acetate (55 mM), ethanol production increased by 28% and hydrogen production marginally decreased, while the hydrogen production increased marginally by additional formate (27 mM) or lactate (55 mM) [20]. Supplementing culture with increasing amounts of exogenous acetate (up to 40 mM (3.28 g/L)) stimulated hydrogen production in Caloramator celer (increasing the accumulation by 25%), but addition of formate did not significantly affect hydrogen production [22]. The different performances of metabolites in hydrogen production might be determined by the characteristics of different strains. In the present study, acetate and butyrate had a significantly positive effect on hydrogen production by MJ1 (10% NDSCB). Due to its higher stimulatory effect on hydrogen production, the effect of acetate concentration was further investigated. As shown in Fig. 5B, the hydrogen production was gradually increased with increasing acetate concentration. The hydrogen production was significantly enhanced (P=0.008<0.05), even with 1 g/L acetate added, while no significant difference was observed in hydrogen production with the acetate concentration further increased from 3 to 10 g/L. Additionally, the B/A molar ratio was enhanced with acetate added to broth (Fig. S1B). These results confirmed the auxo-action of acetate on hydrogen production by MJ1. The pH was also gradually increased with increasing acetate concentration, and the final pH indicated the higher
buffering capacity of higher acetate concentration. At the same time, the hydrogen production also depended on the metabolic rate and hydrolysis of cellulose. The metabolic rate was significant influenced by pH. The model (effect of pH on metabolic rate) revealed an optimal pH value that maximized the metabolic rate, and the metabolic rate was rapidly declined when pH was far away from the optimal pH [38]. The higher final pH indicated that the supplemental metabolites could maintain a relatively stable pH, leading to higher metabolic rate for MJ1 (hydrogen production). The hydrolysis rate of cellulose influenced the reducing sugar content that affected the metabolic rate of microorganisms. Previous study has researched the autocatalytic degradation kinetics of cellulose in homogeneous acidic medium [39]. The autocatalytic degradation ratio was much lower than enzymatic hydrolysis. Because a pre-enzymatic hydrolysis was adopted, the influence of autocatalytic hydrolysis of cellulose on hydrogen production could be ignored in present work. A previous study has indicated that MJ1 could effectively convert xylose, a soluble monosaccharide, to hydrogen [17]. In the present study, xylose was used to test the function of metabolite in hydrogen production by MJ1. The metabolite combinations used were the same as described above. As shown in Fig. 5C, the hydrogen production was significantly enhanced by acetate (P=0.0006<0.05), but obviously suppressed by butyrate (P=0.01<0.05). For Group Four, the promotion of acetate could not offset the inhibition of butyrate, leading to a significant decrease of hydrogen production (P=0.02<0.05). The effect of butyrate on hydrogen production was influenced by substrate, while acetate always exhibited a stimulatory effect.
Currently, there is no report available concerning the effect of butyrate on hydrogen production. A possible explanation is that the supplementation of butyrate in xylose medium significantly decreased the butyrate production (P=0.02<0.05), but produced no effect on acetate production, implying that the B/A ratio was significantly decreased (Fig. S1C), and the lower B/A ratio was probably an important contributor to the lower hydrogen production. The hydrogen production from xylose was significantly enhanced by acetate similar to that of NDSCB, and thus the effect of acetate concentration in xylose medium was further tested (Fig. 5D). The gradual growth trend of hydrogen production in xylose was similar to that of NDSCB, with the hydrogen production significantly enhanced (P=0.04<0.05) at an acetate concentration exceeding 2 g/L. The variation trend of B/A molar ratio in xylose broth was similar to that of NDSCB (Fig. S1D). These results confirmed the positive role of acetate in hydrogen production and no relationship between enhancement and carbon source. The relationship between the hydrogen production and acetate concentration was researched to reveal the mechanism of acetate on hydrogen production (Fig. S2). A linear model was simulated according to previous kinetic modeling of batch hydrogen production process [40]. There was a linear relationship between hydrogen production and acetate concentration. The hydrogen production could be significantly enhanced by lower acetate concentration and the tendency was mitigated with the increase of acetate concentration. This indicated the hydrogen production has a higher linear relationship with acetate concentration around a certain range. A better correlation
coefficient was obtained in xylose than NDSCB that suggested a good correlation in pure substrate culture. 3.6 Effect of metabolite and acetate concentration on growth of MJ1 Given the important role of metabolite supplementation in hydrogen production, the effect of metabolite on growth of MJ1 was further investigated for a better understanding of the function of the complementary metabolite. The growth curves of MJ1 with different metabolite combinations are shown in Fig. 6A. The supplementation of four groups of metabolites resulted in an enhancement of MJ1 growth. The stimulatory effect of acetate was strengthened with increasing acetate concentration, and the highest cell density was observed at the highest acetate concentration (Fig. 6B). The specific growth rates of MJ1 with different metabolites were increased, leading to significant enhancement of the final biomass. Furthermore, peak cell turbidity (Group Acetate or Group Three or Group Four) had a nearly 50% increase over that of the control. The peak cell turbidity (Group Butyrate) was higher than that of the control, but lower than that of the other groups, which was similar to a previous report about the stimulation of acetate on the growth of Thermoanaerobacter ethanolicus strain 39E, C. thermocellum 27405 and Thermoanaerobacter ethanolicus strain X514 [37]. As pH was an important factor for microbial growth, the greater biomass obtained from treated groups was partly due to the buffering capacity of the additional metabolite (higher final pH), which could create a more suitable environmental pH for microbial survival. The relationship between biomass and hydrogen production for anaerobic
fermentation by MJ1 could be simulated by the Luedeking–Piret model as previous study [40]. The fitted plots were shown in Fig. S3. The high correlation coefficient (R2=0.98) indicated that the Luedeking–Piret model could properly describe the relationship between biomass and hydrogen production in the anaerobic fermentation process. The hydrogen production was dependent on the biomass when acetate added to the fermentation broth.
4. Conclusions In this study, a simple two-stage anaerobic fermentation strategy was established for efficient biohydrogen production from NDSCB. Meanwhile, the promoting mechanism underlying higher hydrogen production in the second stage fermentation was explored. The metabolite generated in the first stage fermentation contributed to higher hydrogen production. Both B/A ratio and MJ1 growth could be promoted by metabolite supplementation. The stimulations of metabolite on hydrogen production were mainly attributed to the buffer capacity and redirection of the metabolic flux. The recycle of fermentation broth is an economical method to enhance biohydrogen production and substrate utilization. The use of NDSCB does not need a separate detoxification process after pretreatment, thus increasing the competitiveness of biohydrogen production from lignocellulose. The results suggest great potential of the two-stage fermentation for environment-friendly and high-yield biohydrogen production in the future.
Supplementary material Supplementary data (Fig.S1-S3) associated with this article can be found in the online
version.
Acknowledgements This work was supported by the National Natural Science Foundation of China [grant nos. 51478190 and 51278200], the Guangdong Provincial Natural Science Foundation Key Project [grant no. 2014A030311014], and the Guangzhou Science and Technology Program [grant no. 201510010288].
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Lists of figures: Fig. 1 Hydrogen production from DSCB and NDSCB in the first stage fermentation in SSF and PSSF modes. Fig. 2 Hydrogen production from DSCB and NDSCB in the second stage fermentation in SSF and PSSF modes. Fig. 3 Reducing sugar production under different enzymolysis conditions. Fig. 4 Metabolite production and B/A ratio in the two-stage fermentation. Fig. 5 Effect of different metabolite combinations and acetate concentrations on hydrogen production. Fig. 6 Growth curves of MJ1 supplemented with different metabolite combinations and acetate concentrations.
Lists of tables Table 1 The residual reducing sugar content in broth after the first stage fermentation.
Tables Table 1 The residual reducing sugar content in broth after the first stage fermentation Reducing sugar content (g/L) Modes 6%DSCB
6%NDSCB
10%DSCB
10%NDSCB
SSF
0.67±0.51
0.67±0.01
2.17±0.19
3.88±0.11
PSSF
2.07±1.39
1.15±0.18
9.37±0.61
9.93±0.89
Values are shown as mean of duplicate ± standard deviation.
DSCB NDSCB
A
120
Hydrogen production (mM)
Hydrogen production (mM)
140 120 100 80 60 40 20
DSCB NDSCB
B
100 80 60 40 20 0
0 2
4
6
10
2
Substrate concentration (%)
4
6
10
Substrate concentration (%)
Fig. 1 Hydrogen production from DSCB and NDSCB in the first stage fermentation in SSF and PSSF modes. A: SSF mode, B: PSSF mode.
PSSF SSF
Hydrogen production (mM)
180 160 140 120 100 80 60 40 20 0 DSCB
NDSCB
Substrates
Fig. 2 Hydrogen production from DSCB and NDSCB in the second stage fermentation in SSF and PSSF modes.
Control Acetate Butyrate Acetate/Butyrate Formate/Lactate/Acetate/Butyrate
Reducing sugars content (g/L)
12 10 8 6 4 2 0 0
10 20 30 40 50 60 70 80 90 100 110 120 130
Time (h)
Fig. 3 Reducing sugar production under different enzymolysis conditions.
Formate Butyrate
A
Acetate Ethanol
Lactate
Formate Butyrate
B
Acetate Ethanol
Lactate
3.5
2
Production (g/L)
Production (g/L)
3.0 2.5 2.0 1.5 1.0
1
0.5
0
0.0 10%DSCB
10%DSCB
10%NDSCB
C
10%NDSCB
Substrate
Substrate
4.0
First
Second
3.5
Molar ratio of B/A
3.0 2.5 2.0 1.5 1.0 0.5 0.0 10%DSCB
10%NDSCB
Substrate
Fig. 4 Metabolite production and B/A ratio in the two-stage fermentation. A: First stage, B: Second stage, C: B/A ratio.
pH
B
6
160 5 140 120
4
100 3 80 60
2
40 1 20 0
0 Acetate
Butyrate
Three
6
160 5 140 120
4
100 3 80 60
2
40 1 20 0
0
Four
0
1
Groups
Relative hydrogen production
D pH
180
6
160 5 140 120
4
100 3 80 60
2
40 1 20 0
0 Control
Acetate
Butyrate
Groups
2
3
5
7
10
Acetate concentration (g/L)
Three
Four
pH Relative hydrogen production (%)
Relative hydrogen production (%)
C
pH
Relative hydrogen production
pH
200
6
180 5
160 140
4
120 100
3
80
pH
Control
Relative hydrogen production
180
pH
Relative hydrogen production
180
pH Relative hydrogen production (%)
Relative hydrogen production (%)
A
2
60 40
1
20 0
0 0
1
2
3
5
7
10
Acetate concentration (g/L)
Fig. 5 Effect of different metabolite combinations and acetate concentrations on hydrogen production. A and B: NDSCB as carbon source; C and D: xylose as carbon source. Acetate: 3 g/L acetic acid; Butyrate: 2 g/L butyric acid; Three: 3 g/L acetic acid and 2 g/L butyric acid; Four: 0.5 g/L formic acid, 0.2 g/L lactic acid, 3 g/L acetic acid and 2 g/L butyric acid.
Control Three
A 1.8
Acetate Four
Butyrate
0 5
B
2.0
1 7
2 10
3
1.8
1.6
1.6
1.4
1.4
1.2
OD600
OD600
1.2 1.0 0.8 0.6
1.0 0.8 0.6
0.4
0.4
0.2
0.2
0.0
0.0 0
5
10
15
20
25
30
Time (h)
35
40
45
50
0
5
10
15
20
25
30
35
40
45
50
Time (h)
Fig. 6 Growth curves of MJ1 supplemented with different metabolite combinations and acetate concentrations. A: different metabolite combinations; B: different acetate concentrations (g/L).
Highlights Non-detoxified SCB could be directly used for thermophilic hydrogen fermentation. Two-stage fermentation is suitable for high-yield biohydrogen production. The hydrogen production can be significantly enhanced by supplementary metabolite. Supplementation of metabolite can redirect metabolic flux.