Enhancement of bio-hydrogen yield and pH stability in photo fermentation process using dark fermentation effluent as succedaneum

Journal Pre-proofs Enhancement of bio-hydrogen yield and pH stability in photo fermentation process using dark fermentation effluent as succedaneum Yameng Li, Zhiping Zhang, Quanguo Zhang, Nadeem Tahir, Yanyan Jing, Chenxi Xia, Shengnan Zhu, Xueting Zhang PII: DOI: Reference:

S0960-8524(19)31734-1 https://doi.org/10.1016/j.biortech.2019.122504 BITE 122504

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Bioresource Technology

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23 September 2019 21 November 2019 25 November 2019

Please cite this article as: Li, Y., Zhang, Z., Zhang, Q., Tahir, N., Jing, Y., Xia, C., Zhu, S., Zhang, X., Enhancement of bio-hydrogen yield and pH stability in photo fermentation process using dark fermentation effluent as succedaneum, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122504

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Enhancement of bio-hydrogen yield and pH stability in photo fermentation process using dark fermentation effluent as succedaneum Yameng Li1,2, Zhiping Zhang1,3, Quanguo Zhang1*,2, Nadeem Tahir1,3, Yanyan Jing1,3, Chenxi Xia2, Shengnan

Zhu1,3, Xueting Zhang1,2 1Key

Laboratory of New Materials and Facilities for Rural Renewable Energy, MOA of China, Henan Agricultural

University, Zhengzhou 450002, China 2Institute

3Henan

of Agricultural engineering, Huanghe S & T University, Zhengzhou450006, China

International Joint Laboratory of Biomass Energy and Nanomaterials, Zhengzhou 450002, China

*Corresponding

author:

Tel:

+86

13603985394.

Fax:

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63558040.

E-mail

address:

[email protected]

ABSTRACT The photo fermentation hydrogen yield from dark fermentation effluents (DFEs) can be promoted by adding corn straw enzymatic hydrolysate adjusts the nutritional composition of DFEs. As compared with the control group (without enzymatic hydrolysate addition), the effect of adding enzymatic hydrolysate make H2 yield increase from 312.54 to 1287.06 mL H2/g TOC, and maximum hydrogen production rate increase 2.14 to 10.23 mL/h. On the other hand, buffer reagents remained in DFEs make which can replace part sodium citrate buffer to maintain pH stability in synchronized saccharification and photosynthetic fermentation process with corn straw as substrate, the best result was observed at the ration of 1:2 (33 mL DFEs, 67 mL sodium citrate buffer) with the hydrogen yield of 436.30±10 mL, and which can cut down the GHG in the life cycle of hydrogen production. Key words: Bio-hydrogen, simultaneous saccharification fermentative, enzymatic hydrolysate, dark fermentation effluent, buffering capacity, C/N 1 Introduction Hydrogen being clean energy source has shown great potential to replace the fossil fuel with water as 1

by-product when combustion (Lee et al., 2017; Li et al., 2018; Phowan and Danvirutai, 2014). Among various methods of bio-hydrogen production, sequential dark and photo-fermentation is considered to be the most effective path for bio-hydrogen production as volatile fatty acids generated during dark fermentation process are consumed as carbon source for photosynthetic bacteria (PSB) to produce H2 in the presence of light. The highest theoretical yield of sequential operation dark and photo-fermentation is 12 mol H2/mol glucose (Azbar and Cetinkaya Dokgoz, 2010; Rai and Singh, 2016), which is difficult to achieved but it can be optimized by various processes. Dilution of dark fermentation broth, optimization of hydrogen production medium, addition of metal ions were the most commonly used optimization methods(Lo et al., 2011; Özgür and Peksel, 2013) Utilization of DFEs by PSB to produce H2 is more complicated as compare to pure sugars because of various factors such as substrate inhibitions (NH4+), strict environmental control, appropriate concentration of acetic acid and butyric acid and C/N. Some researchers have shown that zeolite can remove NH4+ effectively in the DFEs to eliminate the inhibitor effect of excess NH4+ on PSB (Lee et al., 2011; Wang et al., 2018), and diluting DFEs could get appropriate concentration of acetic acid and butyric acid for PSB to produce H2(Azbar and Cetinkaya Dokgoz, 2010; Liu et al., 2010). Some reports showed that by adding extra organic matters into DFEs can have positive effect on photo fermentation hydrogen production (PFHP) by shortening the delay period of hydrogen production (Azbar and Cetinkaya Dokgoz, 2010; Silva et al., 2016). Silva et al (Silva et al., 2016) found that by adding different type of extra sugars as supplement into DFEs, H2 yield can be improved by limiting the inhibition of bacteria. Azbar et al (Azbar and Cetinkaya Dokgoz, 2010) reported the significant increase in H2 yield

by adding

extra L-malic acid into DFEs. Compared with pure sugars and L-malic acid, enzymatic hydrolysate of corn straw has the advantages of low production cost and readily available, which has also high quality carbon source for PSB to produce H2 (Jiang et al., 2016; Keener et al., 2017). Supplementing DFEs with enzymatic hydrolysate can adjust the composition of fermentation substrate and dilute hydrogen production inhibitors, however, the effect of the 2

method on enhancing hydrogen production potential of DFEs has not been reported. On the other hand, buffers and automatically controlled pH are two common approaches to prevent interruption of hydrogen production due to the accumulation of VFAs which causes a sharp decrease in the culture pH during fermentation process (Liu et al., 2019; Zagrodnik and Laniecki, 2015). Sodium citrate buffer is regarded as an appropriate buffer in dark fermentation (Zhang et al., 2017). Buffer solution will not be consumed in DFHP process due to which only plays a buffer role, hence, buffer reagents exist still in DFEs at end of fermentation. Using DFEs to PFHP have been widely studied (Liu et al., 2010; Mishra et al., 2018; Silva et al., 2016). However, the buffer effect of DFEs by replacing sodium citrate buffer on PFHP from corn straw has not been reported, especially in simultaneous saccharification fermentation (SSF) (Öhgren et al., 2007)with the characteristics of coupling enzymatic hydrolysis and fermentation in a reactor, shorter processing period and elimination of sugar inhibition (Li and Chen, 2007; Rodrigues et al., 2016). In the present work, firstly we evaluate the influence of DFEs supplemented the enzymatic hydrolysate on PFHP and characteristics of fermentation broth, and later the effect of DFEs by replacing sodium citrate buffer to maintain the pH stability in photo-fermentation by SSF has been carried out. 2 Materials and methods 2.1 Raw material The corn straw was collected from a local farm (Zhengzhou, Henan, China), which was air-dried, and contents of cellulose, hemi-cellulose and lignin, and moisture were 39.12±0.68%, 30.95±0. 54%, 10.73±0.28%, 4.35±0.21%, respectively (Li et al., 2018). 2.2 Microorganisms and media The dark fermentation bacteria were screened from sludge and livestock manure, the processes of enrichment and culture of mixed strains were described in our previous work (Li et al., 2018), and the dominated species in the 3

mixed

strains

identified

belonged

to

Para

clostridium,

Enterococcus,

Sporanaerobacter

and

Clostridium_sensu_stricto_1 by 16S rDNA [5]. The growth and fermentation medium was described in previous literature(Zhang et al., 2017). The PSB HAU-M1 mixed bacteria was provided by Key Laboratory for Renewable Energy, New Materials, and Equipment of the Ministry of Agriculture, including Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodopseudomonas palustris (Jiang et al., 2016). 2.3 Experimental design Enzymatic hydrolysis experiments were operated in 200 mL glass reactors loaded with 5 g of raw material, 0.75 g cellulase (10 FPU/mg) and 100 mL sodium citrate buffer (pH 4.8), performed for 48 h at 50 ℃ and 150 rpm. Enzymatic hydrolysate was centrifuged at 10000 rpm for 5 min ， the clear supematant was collected (reducing sugars concentration 7.91 g/L). Dark fermentation by SSF experiments: a series of batch experiments were conducted in working volume 150 mL glass reactor, 100 mL sodium citrate buffer (pH 4.8), 5 g corn straw and 0.75 g cellulase (10 FPU/mg), 20 mL fermentation medium and 30 mL inoculum were added, and then cultured at initial pH 5.5 with temperature of 45℃. The pH was adjusted by 5 M H2SO4 and 5 M KOH. PFHP experiments: the DFEs obtained were firstly centrifuged at 10000 rpm for 5 min and filtered to remove all solids, and then the clear supematant and zeolite were added to conical flask at the ratio of 2:1 (v/w) kept at 30℃ for 3 h to remove the excess NH4+, accompanied by irradiation of ultraviolet lamp for sterilization. Before pretreatment DFEs used to PFHP, 10g/L glucose was added into DFEs to fermentation, no hydrogen and glucose consumption were detected during the hydrogen fermentation period indicating that no viable bacteria were in DFEs. The first scenario: batch PFHP experiments were carried out in 150 mL glass reactor with 120 mL working volume loaded with 24 mL inoculum, 16 mL fermentation medium, and 80 mL mixed liquids consisted of 4

pretreated DFEs and enzymatic hydrolysate with various ratios of 1:1,2:1,3:1,4:1 and 5:1(v/v). The control 1 and 2 tests used 80 mL enzymatic hydrolysate and DFEs as substrate, respectively. The initial pH was adjusted to 7 by 5 M H2SO4 and 5 M KOH. The second scenario: Feasibility of DFEs as buffer, batch experiments were carried out in 200 mL glass reactor with 180 mL working volume loaded with 5 g corn straw and 0.75 g cellulase (10 FPU/mg), 36 mL inoculum, 44 mL fermentation medium, and 100 mL mixed buffer solution consisting of pretreated DFEs and sodium citrate buffer with various ratios of 1:3,1:2,1:1,2:1 and 3:1(v/v), the control test was added 100 mL sodium citrate buffer. The initial pH was adjusted to 6.5 by 5 M H2SO4 and 5 M KOH. All reactors were placed in constant-temperature incubator at 30℃ with light intensity 3000 lx. All the experiments were triplicate to ensure quality of the result. All batch experiments reactors were bubbled with argon gas for 5min to keep anaerobic condition. 2.4 Instruments and methods The relative hydrogen concentration and concentration of VFAs were measured using a GC (6820GC-14B, Agilent, USA). Oxidation-reduction potential (ORP) value of the fermentation medium was measured by ORP meter (Az8851, Guangzhou, China), a pH meter was used to measure the pH value of solution. Total organic carbon (TOC) and total nitrogen (TN) were measured by a total organic carbon analyzer (multi N/C 3100/1, analytikjena, Germany). The OD660 of bacteria was measured using a 721 visible spectrophotometer at 660 nm. 2.5 Model fitting and statistical analyses Gompertz equation is commonly adopted to study the kinetic of batch hydrogen production, the modified Gompertz Eq. (1) as follows:

{

[

P(t) = 𝑃𝑚𝑎𝑥𝑒𝑥𝑝 ―𝑒𝑥𝑝

𝑟𝑚𝑒

𝑃𝑚𝑎𝑥(𝜆

]}

― 𝑡) + 1

(1)

Where, P(t) is the cumulative hydrogen production (mL TS), rm is the maximum hydrogen production rate (mL· h-1 TS), Pmax is the hydrogen production potential (mL TS), λ is the lag phase time (h), t is the incubation time (h), е is 2.718. 5

The kinetic equation of hydrogen production rate (v(t)) as follows Eq (2)： v(𝑡) =

𝑑𝑃(𝑡) 𝑑𝑡

{

[

𝑟𝑚𝑒

= 𝑟𝑚𝑒𝑥𝑝 2 + 𝑃𝑚𝑎𝑥(𝜆 ― 𝑡) ― 𝑒𝑥𝑝

𝑟𝑚𝑒

𝑃𝑚𝑎𝑥(𝜆

]}

― 𝑡) + 1

(2)

The time at which v achieves vmax (tmax) can be calculated as: 𝑃𝑚𝑎𝑥

(3)

𝑡𝑚𝑎𝑥 = 𝜆 + 𝑟𝑚·𝑒

3 Results and discussions 3.1 Dark fermentation hydrogen production process The cumulative hydrogen yield of 348.30±10 mL was acquired during dark fermentation hydrogen production by SSF (Fig.1), which was higher than the hydrogen yield (317.75 mL) from asynchronous saccharification and fermentation (Li et al., 2018), the maximum hydrogen production rate of 28.06 mL/h was achieved at 7.24 h (Table 1). It has been observed that acetic acid and butyrate were the main by-products, which are 3652.23±202 mg/L, and 945.22±79 mg/L, respectively. At the end of fermentation, the TOC and TN concentration were 4383.65±117 mg/L and 768±43 mg/L respectively. The accumulation of volatile fatty acids lowers the final pH to 5.18 ±0.06 from initial value pH of 5.5. The kinetic parameters obtained from the Gompertz model (Eq.1) are shown in Table 1. The values of R2 in Table 1 clearly shows good agreement between experimental and predicted values which indicates that hydrogen production data can be fitted very well by Gompertz model. 3.2 Effect of the ratio of DFE and enzymatic hydrolysate on PFHP The physicochemical properties of DFEs can be changed by adding enzymatic hydrolysate. The influence of supplementing DFEs with various volume enzymatic hydrolysate on PFHP is represented in Fig.2a. The mixed substrates showed shorter fermentation time with no hydrogen release for 48 h. The maximum hydrogen yield of 199.39±7 mL was obtained at ratio of 1:1, which is lower than that of the control test 1, nevertheless, the mixed substrates gave higher hydrogen production rate in previous 20 h. Meanwhile, at the ratio 2:1, hydrogen production rate was also higher as compared to the control test 1 in previous 16 h. This can be likely due to the higher TN 6

concentration in mixed substrates which promoted the PSB growth (Fig.2e), and advanced the logarithmic growth phrase (Kim et al., 2012), which can be verified by tmax(Fig.2b). NH4+ is the major part of TN in DFEs ( Wang et al., 2018), higher NH4+ concentration have negative effect on nitrogenase activity, which plays a critical role in metabolism of hydrogen production. Therefore, lower hydrogen yields were achieved from the mixed substrates at ratio 3:1,4:1 and 5:1 with the TN concentration of 977±25, 1002±23 and 1015±26 mg/L (Fig.2c), respectively. The control 2 with the highest TN concentration (1089±21 mg/L) led to the lowest hydrogen yield. Fig.2b illustrates the hydrogen production rate curves fitted by the Eq. (2). For the mixed substrates, the peak period of hydrogen production is concentrated between 6-24 h, where maximum hydrogen production rate of 10.23 mL/h was achieved at 10.23 h with ratio of 1:1. Along with the increase in ratio, the rm (Table 2) decreased as tmax goes shorter (Fig.2b). In comparison to mixed substrates, the rm of the control test 1 came later as bacteria grow slowly under low TN concentration (563±22mg/L). Organic matter is degraded by bacteria to produce ATP to operate metabolism of growth and H2 production (Jurado-Marban et al., 2019), TOC is an index of organic matter content. When the enzymatic hydrolysate was used as substrate, the TOC concentration of 6070.08±55 mg/L was higher than mixed substrates (Fig.2c), meanwhile, the mixed substrates contained higher TN concentration and increased with the increase in ratio. From the relationship between TOC degradation and hydrogen production yield, the control test 1 gave the highest conversion efficiency, 2115.33 ±58.33 mL H2/g TOC(Fig.2d). For the mixed substrates, higher TOC conversion was realized at ratio 1:1(40 mL DFE, 40 mL enzymatic hydrolysate), 1287.06 ±45.18 mL H2/g TOC, but when the mixed ratio changed to 2:1, the 1251.27 ±54.98 mL H2/g TOC was obtained, the difference in TOC conversion between the ratio of 1:1 and 2:1 was not obvious. By continuously decreasing the amount of enzymatic hydrolysate lowered the TOC conversion. In terms of utilization effect of DFEs, the optimal ratio was 2:1. The kinetic parameters obtained from Eq.1 are listed in Table 2. The values of R2 ≥0.9991 clearly shows that experimental values were close to predicted values. Mixed substrates had shorter lag phase time, but the rm was 7

lower than the control test except the ratio 1:1. 3.3 Effect of the ratio of DFEs and sodium citrate buffer on PFHP The influence of DFEs by replacing sodium citrate buffer on pH in photo fermentation process is shown in Fig.3a. In all batch experiments, the pH dropped from initial pH 6.5 to around 5.5 in 12 h, the reasons of the phenomenon may be caused by microbial metabolic activity in order to adopt favorable fermentation environment. Under the conditions with the ratio of 1:3, 1:2 and 1:1 (DFEs: sodium citrate buffer), variation range of pH was similar to that of the control test in 24 h. After 24 h, the pH of the ratio of 2:1 and 3:1 was lower than others, but the final pH went up to around 6.20 due to consumption of volatile fatty acids. It can be seen from Fig.3a that no significant difference was detected in the pH of fermentation broth with different conditions indicating that DFEs had the potential to maintain the pH stability in photo fermentation process. During fermentation process, the ORP varied from -355 to -520 mv (Fig.3a), lower OPR ensured the requirements for the activity of coenzyme and iron redox proteins, the ORP value increased during the anaphase of the fermentation caused by lack of sufficient substrates to produce reductive power. The cumulative H2 yields from corn straw by SSF is shown in Fig.3b. H2 yield was significantly influenced by the addition volume of DFEs, which dropped from 436.30±10 mL to 241.06±8 mL, along with the ratio increased from 1:2 to 3:1. The control test obtained 349.44±11 mL H2, which was lower as compared to ratios of 1:3, 1:2 and 1:1(Fig.3b). This could be most likely due to extraction of energy of DFEs by HAU-M1 to produce hydrogen which indicates that DFEs can be utilized as both buffer solution and carbon source during photo fermentation by SSF. The lowest H2 yield of 241.06±8 ml was obtained at the ratio of 3:1 with shortest fermentation time of 72 h which is much lower as compared to other fermentation time which is 84 h. This can be due to the higher TN and inhibitors concentration which lead bacteria prematurely enter a period of decay and eventually to death (Fig.2e). The highest hydrogen production rate of 11.44 mL/h was obtained at 1:2 at 21.34 h (Fig.3c), along with the ratio 8

increased from 1:2 to 3:1, the rm declined. The tmax increased with the increase in DFE volume as HAU-M1 need a long time to adapt to the environment. During the SSF, the TOC and TN concentration firstly declined and then increased (Fig.5d and e), this can be due to the growth activity and hydrogen production metabolism was vigorous in the early stage of fermentation, in which the consumption rate of organic matter was higher than the rate of formation. After 60 h, the accumulation of hydrogen production inhibitors and bacterial death resulted in a decrease in substrate consumption, therefore, the TOC and TN concentration increased in later period. Nitrogen source is the essential element to microorganism for their activity, microorganism’s composition, growth and reproduction. Carbon source is the energy source to microorganism vital activity. Appropriate C/N ratio is a necessary condition for hydrogen production stability and high substrate conversion. Fig.5f depicts the variation of C/N in the fermentation broth under different conditions, which increased in the first 12 h, and then became stabilized. Compared with the control test, the C/N in experimental group was lower due to addition of DFEs increased the TN concentration. As seen from Fig.5b and f, the highest hydrogen yield was obtained at the ratio of 1:2, in which the C/N of SSF fermentation broth was between 8 and 8.6, meanwhile, the ratio 1:3 with the C/N between8.4 and 9 also showed good hydrogen yield, indicating that the optimum C/N ratio for PFHP by SSF was between 8 and 9 in this paper. The kinetic parameters are listed in Table 3. The values of R2 ≥0.9967 clearly shows the experimental data can be fitted well by Gompertz model. The Pmax of 437.12 mL was obtained in at ratio 1:2, the experimental H2 yield was 436.30mL. The lag phase time increased with the increasing in DFE volume, meanwhile, mixed buffer solution made the tmax late. pH 4.8 sodium citrate buffer contained citric acid (4.83 g/L) and sodium citrate (7.94 g/L). During the life cycle of citric acid and sodium citrate production, some resources (e.g., electricity, water,) were consumed led to a certain amount of greenhouse gas (GHG) was produced. The life cycle GHG emissions of citric acid and sodium 9

citrate were 6.24 kg CO2-eq/kg and 4.50 kg CO2-eq/kg obtained from Simapro 8.5. In the paper, DFEs were found to can replace part of the buffer solution to maintain the operation of PFHP from corn straw with SSF. The optimum alternative volume was 33mL in the total 100mL buffering solution in PFHP process, resulted in cutting down 2.17g CO2-eq GHG in the life cycle of PFHP from corn straw by SSF.

Conclusion DFEs can not only be used as carbon source but also replace part of sodium citrate buffer in PFHP process. The supplementation of DFEs with enzymatic hydrolysate results in improvement of potential H2 yield from mixed substrates in PFHP process, the best adding proportion was 1/3(v/v), corresponding with 1251.27 ±54.98 mL H2/g TOC. On the other hand, DFEs can replace part of sodium citrate buffer utilized in PFHP to maintain pH, as well as reducing the usage of buffer reagents in photo fermentation, which cut down 2.17g CO2-eq GHG in the life cycle of PFHP from corn straw by SSF.

Acknowledgements The work was supported by the National Natural Science Foundation of China (No.51676065 & U1504509), and Key Scientific Research Projects of University in Henan province (18A480004). References Azbar, N., Cetinkaya Dokgoz, F.T., 2010. The effect of dilution and l-malic acid addition on bio-hydrogen production with Rhodopseudomonas palustris from effluent of an acidogenic anaerobic reactor. Int. J. Hydrogen Energy. 35, 5028–5033. doi:10.1016/j.ijhydene.2009.10.044 Jiang, D., Ge, X., Zhang, T., Liu, H., Zhang, Q., 2016. Photo-fermentative hydrogen production from enzymatic hydrolysate of corn stalk pith with a photosynthetic consortium. Int. J. Hydrogen Energy.41, 16778–16785. doi:10.1016/j.ijhydene.2016.07.129 Jurado-Marban, V.H., Tapia-Bustos, M.A., Gonzalez-Garcia, R.A., Salgado-Manjarrez, E., García-Peña, E.I., 2019. 10

Hydrogen production by a mixed photoheterotrophic culture: Correlation between gene expression analysis and physiological behavior. Int. J. Hydrogen Energy.44, 641–651. doi:10.1016/j.ijhydene.2018.10.197 Keener, H., Li, Y., Ge, X., Zhou, X., Zhang, Q., Jiang, D., Chen, Z., 2017. Comparison of sodium hydroxide and calcium hydroxide pretreatments of giant reed for enhanced enzymatic digestibility and methane production. Bioresour. Technol. 244, 1150–1157. doi:10.1016/j.biortech.2017.08.067 Kim, M.S., Kim, D.H., Cha, J., Lee, J.K., 2012. Effect of carbon and nitrogen sources on photo-fermentative H 2 production associated with nitrogenase, uptake hydrogenase activity, and PHB accumulation in Rhodobacter sphaeroides KD131. Bioresour. Technol.116, 179–183. doi:10.1016/j.biortech.2012.04.011 Lee, C.M., Hung, G.J., Yang, C.F., 2011. Hydrogen production by Rhodopseudomonas palustris WP 3-5 in a serial photobioreactor fed with hydrogen fermentation effluent. Bioresour. Technol.102, 8350–8356. doi:10.1016/j.biortech.2011.04.072 Lee, D.J., Wang, Y., Zhang, Z., Lee, D.J., Zhou, X., Jing, Y., Jiang, D., Hu, J., He, C., 2017. Photo-fermentative hydrogen production from crop residue: A mini review. Bioresour. Technol.229, 222–230. doi:10.1016/j.biortech.2017.01.008 Li, D., Chen, H., 2007. Biological hydrogen production from steam-exploded straw by simultaneous saccharification and fermentation. Int. J. Hydrogen Energy.32, 1742–1748. doi:10.1016/j.ijhydene.2006.12.011 Li, Y., Zhang, Z., Zhu, S., Zhang, H., Zhang, Y., Zhang, T., Zhang, Q., 2018. Comparison of bio-hydrogen production yield capacity between asynchronous and simultaneous saccharification and fermentation processes from agricultural residue by mixed anaerobic cultures. Bioresour. Technol. 247, 1210–1214. doi:10.1016/j.biortech.2017.09.053 Liu, B.F., Ren, N.Q., Xie, G.J., Ding, J., Guo, W.Q., Xing, D.F., 2010. Enhanced bio-hydrogen production by the combination of dark- and photo-fermentation in batch culture. Bioresour. Technol.101, 5325–5329. 11

doi:10.1016/j.biortech.2010.02.024 Liu, H., Zhang, Z., Zhang, Q., Tahir, N., Jing, Y., Li, Y., Lu, C., 2019. Optimization of photo fermentation in corn stalk through phosphate additive. Bioresour. Technol. R. 7, 100278. doi:10.1016/j.biteb.2019.100278 Lo, Y.C., Chen, C.Y., Lee, C.M., Chang, J.S., 2011. Photo fermentative hydrogen production using dominant components (acetate, lactate, and butyrate) in dark fermentation effluents. Int. J. Hydrogen Energy. 36, 14059– 14068. doi:10.1016/j.ijhydene.2011.04.148 Mishra, P., Singh, L., Ab Wahid, Z., Krishnan, S., Rana, S., Amirul Islam, M., Sakinah, M., Ameen, F., Syed, A., 2018. Photohydrogen production from dark-fermented palm oil mill effluent (DPOME) and statistical optimization: Renewable substrate for hydrogen. J. Clean. Prod. 11–17. doi:10.1016/j.jclepro.2018.07.028 Öhgren, K., Bura, R., Lesnicki, G., Saddler, J., Zacchi, G., 2007. A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover. Process Biochem. 42, 834–839. doi:10.1016/j.procbio.2007.02.003 Özgür, E., Peksel, B., 2013. Biohydrogen production from barley straw hydrolysate through sequential dark and photofermentation. J. Clean. Prod. 52, 14–20. doi:10.1016/j.jclepro.2013.02.035 Phowan, P., Danvirutai, P., 2014. Hydrogen production from cassava pulp hydrolysate by mixed seed cultures: Effects of initial pH, substrate and biomass concentrations. Biomass Bioenergy. 64, 1–10. doi:10.1016/j.biombioe.2014.03.057 Rai, P.K., Singh, S.P., 2016. Integrated dark- and photo-fermentation: Recent advances and provisions for improvement. Int. J. Hydrogen Energy.41, 19957–19971. doi:10.1016/j.ijhydene.2016.08.084 Rodrigues, T.H.S., de Barros, E.M., de Sá Brígido, J., da Silva, W.M., Rocha, M.V.P., Gonçalves, L.R.B., 2016. The Bioconversion of Pretreated Cashew Apple Bagasse into Ethanol by SHF and SSF Processes. Appl.Biochem. Biotechnol.178, 1167–1183. doi:10.1007/s12010-015-1936-0 12

Silva, F.T.M., Moreira, L.R., de Souza Ferreira, J., Batista, F.R.X., Cardoso, V.L., 2016. Replacement of sugars to hydrogen production by Rhodobacter capsulatus using dark fermentation effluent as substrate. Bioresour. Technol.200, 72–80. doi:10.1016/j.biortech.2015.10.002 Wang, X., Wu, X., Hu, J., Zhang, A., Chen, D., Yang, H., Ma, X., Guo, L., 2018. Isolation of a Rhodobacter sphaeroides mutant with enhanced hydrogen production capacity from transposon mutagenesis by NH4+ nitrogen resource. Int. J. Hydrogen Energy.43, 13821–13828. doi:10.1016/j.ijhydene.2018.01.179 Zagrodnik, R., Laniecki, M., 2015. The role of pH control on biohydrogen production by single stage hybrid dark- and photo-fermentation. Bioresour. Technol.194, 187–195. doi:10.1016/j.biortech.2015.07.028 Zhang, Z., Li, Y., Zhang, H., He, C., Zhang, Q., 2017. Potential use and the energy conversion efficiency analysis of fermentation effluents from photo and dark fermentative bio-hydrogen production. Bioresour. Technol.245, 884– 889. doi:10.1016/j.biortech.2017.09.037

Highlights 1. Hydrogen yield potential of DFEs can be improved by adding enzymatic hydrolysate. 2. DFEs can replace part sodium citrate buffer to maintain pH stability in PFHP. 3. Using DFEs as buffer can cut down 2.17g CO2-eq GHG in the life cycle of PFHP. 4. Cumulative hydrogen yield of all experiments can be fitted well by Gompertz model.

13

25 300 200 100

3652.23

4000

20

The main metabolites

3500 3000

15

2500 2000 1500 1000

10

945.22

500 0 Acetic acid

0

43

5

Butyrate Propionic acid

0 0

10

20

30

40

50

Hydrogen production rate (mL/h)

30

tmax =7.24 h Concentration (mg/L)

Cumulative hydrogen yield (mL)

400

60

Time (h)

Fig.1 Dark fermentation process by SSF

200 150

Control 1 1:1 2:1 3:1 4:1 5:1 Control 2

100 50 0

10

tmax (h)

250

Hydrogen production rate (mL/h)

Cumulative hydrogen yield (mL)

tmax

(b)

8

Control 1 1:1 2:1 3:1 4:1 5:1 Control 2

4 2

0

10

20

30

40

50

60

70

80

0

10

20

Time (h)

30

40

50

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70

1100 1000 900 800 Initial TOC End TOC

4800

Initial TN End TN

700 600

4400

80

Control 1 1:1

500 2:1

3:1

4:1

400 5:1 Control 2

Time (h)

(d)

0.9

(e)

0.8

2000

0.7 0.6

1500

OD660

Hydrogen yield (mL/g TOC)

5600

2 1 trol :1 :1 :1 :1 :1 trol Con 1 2 3 4 5Con 5200

6

(c)

6000

0

-10 2500

1200

24 22 20 18 16 14 12 10 8 6

1000

0.5 0.4 0.3 0.2

500

0.1 0

Control 1 1:1

2:1

3:1

4:1

5:1 Control 2

0.0

l l1 InitiaContro 1:1

2 :1

3 :1

4 :1

5:1Control

2

Fig. 2 The effect of the ratio of DFEs and enzymatic hydrolysate on (a) cumulative hydrogen yield; (b) hydrogen production rate ;(c) variation of TOC and TN ;(d) conversion of TOC; (e) growth of bacteria

14

TN (mg/L)

12

(a)

TOC (mg/L)

300

400

-400

5.6

ORP (mv)

-350

300 200

-450

5.2

100 -500

4.8

Control 1:3 1:2 1:1 2:1 3:1

0 20

30

(d)

5000

60 50 40 Time (h)

70

80

20

0

900

Control 1:3 1:2 1:1 2:1 3:1

(e)

TN (mg/L)

4000

3500

10

20

30

60 50 40 Time (h)

70

80

90

12 10 8

Control 1:3 1:2 1:1 2:1 3:1

27

tmax

26 25 24 23 22 21 20 19

6

Control1:3

1:2

1:1

2:1

3:1

4 2 0 20

0

40 Time (h)

80

60

(f)

10

700

9

600

8

500

7

400

6

300 3000

(c)

11

Control 1:3 1:2 1:1 2:1 3:1

800

4500

80

60

40 Time (h)

14

-2

C/N

10

-550 90

Hydrogen production rate (mL/h)

-300

6.0

pH

16 (b)

(a)

Cumulative hydrogen yield (mL)

6.4

TOC (mg/L)

500

-250 Control 1:3 1:2 1:1 2:1 3:1

tmax (h)

6.8

Control 1:3 1:1 2:1 3:1

5 10

20

30

40

50

Time (h)

60

70

80

90

10

20

30

40

60 50 Time (h)

70

1:2

80

90

Fig.3 The effect of the ratio of DFE and sodium citrate buffer on(a) variation of pH and ORP; (b) cumulative hydrogen yield; (c) hydrogen production rate; (d) variation of TOC ;(e) variation of TOC ;(f) variation of C/N

15

Table 1 Kinetic parameters and characteristics of fermentation broth Pmax (mL)

365.6 2

rm (mL·h-1)

λ(h)

R2

tmax (h)

TOC (mg/L)

28.06

2.45

0.9958

7.24

4383.65±11 7

TN (mg/ L) 786±43

Final pH 5.18±0.06

Table 2 Gompertz equation constants at various mixed ratio Pmax (mL)

rm(mL·h-1)

λ(h)

R2

tmax (h)

Control 1

256.69

8.27

12.00

0.9966

23.42

1:1

198.87

10.23

6.57

0.9990

13.72

2:1

159.50

7.63

3.10

0.9997

10.79

3:1

90.74

4.46

2.87

0.9989

10.36

4:1

50.75

3.09

2.65

0.9989

8.69

5:1

46.21

2.91

2.54

0.9990

8.38

Control 2

32.87

2.14

2.43

0.9990

8.08

Table 3 Gompertz equation constants at various conditions Pmax (mL)

rm(mL·h-1)

λ(h)

R2

tmax (h)

Control

354.73

8.19

3.40

0.9937

19.34

1:3

413.91

9.35

4.52

0.9987

20.81

1:2

437.12

11.44

7.28

0.9984

21.34

1:1

362.81

8.73

7.62

0.9991

22.91

2:1

310.30

7.21

8.17

0.9972

24.00

3:1

247.96

6.66

10.44

0.9985

24.14

16

Author contributions: Quanguo Zhang, Yanyan Jing and Zhiping Zhang conceived the idea; Yameng Li, Chenxi Xia, Shengnan Zhu and Xueting Zhang performed research; Yameng Li and Nadeem Tahir analyzed data; all authors contributed to the writing and revisions.

Conflict of interest We declared that we have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

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