Demonstration and optimization of sequential microaerobic dark- and photo-fermentation biohydrogen production by immobilized Rhodobacter capsulatus JP91

Accepted Manuscript Demonstration and optimization of sequential microaerobic dark- and photofermentation biohydrogen production by immobilized Rhodobacter capsulatus JP91 Emrah Sağ ır, Meral Yucel, Patrick C. Hallenbeck PII: DOI: Reference:

S0960-8524(17)31992-2 https://doi.org/10.1016/j.biortech.2017.11.018 BITE 19168

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

9 October 2017 6 November 2017 7 November 2017

Please cite this article as: Sağ ır, E., Yucel, M., Hallenbeck, P.C., Demonstration and optimization of sequential microaerobic dark- and photo-fermentation biohydrogen production by immobilized Rhodobacter capsulatus JP91, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.11.018

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.

Demonstration and optimization of sequential microaerobic dark- and photofermentation biohydrogen production by immobilized Rhodobacter capsulatus JP91 Emrah Sağır1,2, Meral Yucel2,,and Patrick C. Hallenbeck1,3 1

Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, CP6128 Succursale Centre-ville, Montréal, Québec, Canada H3C 3J7

2

Department of Biological Sciences, Middle East Technical University, Ankara 06800, Turkey 3

Life Sciences Research Center, Department of Biology United States Air Force Academy

Keywords: Biohydrogen; Photofermentation; Immobilized Cells; Sequential Process; Hydrogen from Glucose Abstract Hydrogen generation from complex substrates composed of simple sugars has the potential to mitigate future worldwide energy demand. The biohydrogen potential of a sequential microaerobic dark- and photo-fermentative system was investigated using immobilized Rhodobacter capsulatus JP91. Biological hydrogen production from glucose was carried out using a batch process and a bench-scale bioreactor. Response surface methodology with a Box-Behnken design was employed to optimize key parameters such as inoculum concentration, oxygen concentration, and glucose concentration. The maximum hydrogen production (21+0.25 mmol H2/L) and yield (7.8+0.1mol H2/mol glucose) were obtained at 6 mM glucose, 4.5% oxygen and 62.5 v/v % inoculum concentration, demonstrating the feasibility of enhanced hydrogen production by immobilized R. capsulatus JP91 in a 1

sequential system. This is the first time that a sequential process using an immobilized system has been described. This system also achieved the highest hydrogen yield obtained by an immobilized system so far. Keywords: Biohydrogen; immobilized system; sequential fermentation; Rhodobacter capsulatus; Response Surface Methodology

2

1. Introduction The world energy demand is to increase for the foreseeable future, with total energy demand predicted to be 30% higher in 2035 than the present day (https://www.bp.com/content/dam/bp/pdf/energy-economics/energy-outlook-2017/bp-energyoutlook-2017.pdf, accessed 9/17/2017). Alternative energy sources are expected to become increasingly important in filling in the expected shortage in energy production with a predicted contribution of 10% by 2035 (BP). Hydrogen has been put forward as a potentially valuable and promising environmentally friendly energy carrier, but to form part of a real solution, its production must be sustainable. At present the vast majority of hydrogen is produced via fossil fuels. Many consider biohydrogen as a promising way for renewable production. Dark fermentation is a feasible and common route for biohydrogen production as it enables the utilization of a wide range of substrates including agricultural and industrial wastes (Hallenbeck, 2009; Kumar et al., 2017). A great number of feedstocks have been utilized through dark fermentation for hydrogen generation (Argun and Kargi, 2011). Although dark fermentation offers some important advantages, hydrogen yields are theoretically low when this route is used alone, reaching at most 33% of the maximal theoretical yield. Therefore, for maximal substrate utilization, another step is required following the dark fermentation stage. Amongst the various biological hydrogen production routes, photofermentation has gained widespread interest and attention in the past few decades. Therefore, a sequential hydrogen producing system could enhance overall hydrogen yield. The system is also more economically feasible if both of the stages take place in the same bioreactor.

3

Photofermentation is promising as, utilizing captured light energy, a wide variety of substrates can be nearly stoichiometrically converted to hydrogen (Hallenbeck and Liu, 2015). Purple non-sulfur bacteria belong to a group of photosynthetic microorganisms which are able to grow photoheterotrophically and carry out photofermentation under anaerobic conditions (Hallenbeck and Benemann, 2002). Normally, purple non-sulfur bacteria realize hydrogen production by photofermentation under anaerobic light conditions. However, as light conversion efficiencies are relatively low, photofermentative hydrogen production systems are at present too costly to be practical. Thus, there is a great need for the development of sustainable and efficient systems. Hydrogen is produced by the PNSB using nitrogenase, an ATP dependent enzyme that also requires high energy electrons. In photofermentation, the extensive energy demand is met by bacterial photosynthesis. However, Besides, the required energy and reducing power for nitrogenase can also be obtained through microaerobic dark fermentation instead of photofermentation (Fig. 1). Previously it was shown that various organic acids and sugars can be used for hydrogen production through microaerobic dark fermentation (Abo-Hashesh and Hallenbeck, 2012). Recently it was shown that hydrogen production from lactate, normally impossible from a thermodynamic point of view under fermentative conditions, can be carried out using microaerobic conditions with a relatively high yield (1.4±0.1mol H2/mol lactate) (Lazaro et al., 2017). Cell immobilization technique has been applied in a myriad of studies including hydrogen production. Cell immobilization is considered as a superior alternative to suspended cultures due to potential elimination of drawbacks including hurdles in the processing of large scale operations, wash out of biomass, and mixing issues. This technique also enables use of 4

high cell concentrations through the process, as well as possible reuse (Kumar, 2016). There have been a large number of attempts to immobilize cells including the use of biofilms or through artificial means (Kosourov and Seibert, 2009; Tian et al., 2010; Tsygankov and Kosourov, 2014). Amongst the various cell immobilization methods, cell entrapment has received high attention due to its low price, relative ease and feasibility of its application, and the durability of the entrapment material (Keskin et al., 2011; Xie et al., 2012). A variety of materials have been used as matrices to immobilize cells, including; PVA (Polyvinyl alcohol), glass, latex, clay, agar, agarose, alginate, and carrageenan (Tsygankov and Kosourov, 2014). Agar has been widely used for cell immobilization as it is a biodegradable, natural, non-toxic, durable and cheap material (Song et al., 2011). In one study, a dark fermentative bacterium (Clostridium butyricum) and a photofermentative bacterium (Rhodopseudomonas faecalis RLD-53) were co-immobilized and a hydrogen yield of 5.4 mol H2/mol glucose was achieved (Liu, 2010). Similarly, biohydrogen was produced with the highest hydrogen yield of 7.1 mol H2/mol glucose, by a co-immobilized cultures of Lactobacillus delbrueckii NBRC13953 and Rhodobacter sphaeroides RV (Asada et al., 2006). Glucose containing substrates have been widely used for biohydrogen production via single-stage (Kumar 2000; Ghosh, 2012), sequential (Yokoi et al., 2001; Hitit et al., 2017a) or co-cultures (Xie et al., 2010; Hitit et al., 2017b) of various dark and photofermentative bacteria. These processes need to be improved in order to increase hydrogen yields and production rates. Design of experiments provides a statistical and mathematical map of the experiments to be employed for the optimization of the important parameters (Montgomery, 2009). This technique reveals the effects and interactions of the key variables on the desired responses for the evaluation of optimal conditions. Response surface methodology with the Box-Behnken 5

method is an important statistical tool in the determination of optimal values for critical parameters in an experimental design (Box and Behnken, 1960). A great number optimization studies have focused on key factors such as; initial substrate concentration, inoculum size, pH, temperature, light intensity for various dark and photofermentative bacteria in biohydrogen production (Ghosh and Hallenbeck, 2010; Androga et al., 2014; Hitit et al., 2016). Response surface methodology has been implemented for the optimization of glucose concentration, glutamate concentration, and light intensity for enhanced hydrogen yield (Ghosh et al., 2012). A maximum hydrogen yield of 5.5 mol H2/mol glucose by Rhodobacter capsulatus JP91 in a single-stage photofermentation was demonstrated. More recently, a hydrogen yield of 9.0 mol H2/mol glucose was achieved using Rhodobacter capsulatus JP91 with a photobioreactor operated in continuous mode (Abo-Hashesh et al., 2013). The present work aimed to enhance hydrogen production in a sequential microaerobic dark- and photo-fermentative system by using a single culture of immobilized R. capsulatus JP91 (Fig. 1). For this reason, response surface methodology with a Box-Behnken design was implemented for optimization of inoculum concentration, oxygen concentration, and glucose concentration to improve hydrogen production. One of the goals of the present study was to see whether microaerobic dark fermentation could replace photophosphorylation, the natural mechanism of photofermentation. This would possibly allow higher hydrogen yields to be obtained at a potentially lower cost than a singlestage photofermentative process. Under these conditions, reducing power for nitrogenase can be provided by the microaerobic dark fermentative system via oxidative phosphorylation. In addition, the photofermentative stage also builds up the overall yield by utilizing the organic acids as produced as a result of the glucose consumption in the microaerobic dark 6

fermentation step. Therefore, the overall hydrogen yield might be expected to be higher than with a single-stage dark or photofermentation process.

2. Materials and Methods 2.1 Microorganism and growth conditions The photosynthetic purple non-sulfur bacterium, R. capsulatus JP91, a markerless hupderivative of the wild-type strain (B10) (Colbeau et al., 1990), was provided by Dr. John Willison, and used for hydrogen production through the application of sequential microaerobic dark- and photo-fermentation. R. capsulatus JP91 was cultivated for 3-4 days at 30 0C under continuous illumination (120 W/m2) in a light chamber (Biotronette Mark III, Lab-line Instruments). The cells were grown in RCV- lactate medium until the OD of the grown culture was 1.5 at 600 nm. 2.2 Immobilization procedure The previously grown suspended cultures (OD600 :1.5) were centrifuged at 10,000 rpm for 10 min. Bacterial pellets were re-suspended and adjusted to various concentrations (25-50100 v/v %). Different concentrations of bacterial inoculum were mixed with 10 mL molten agar in a Falcon tube. Then, the mixture was transferred to the 160 mL glass bottle bioreactors. An agar-bacterial complex solidified on the bottom surface of the bioreactor completely after 10 min at 4 0C. The bioreactors were then filled with 30 mL RCV - glucose

7

medium (Ghosh et al., 2012). The initial pH of the medium was adjusted to 7.0 using potassium phosphate buffer (0.64 M KH2PO4 and K2HPO4). 2.3 Experimental setup for hydrogen production Hydrogen production was carried out using a sequential dark and photofermentative process. To this end, the first step of the experiment started with dark fermentation and was followed by photofermentation. The experiments were carried out with 160 mL glass bottles. These bioreactors were filled with 10 mL agar-bacteria complex and 30 mL RCV- glucose medium with a head space of 120 mL. The bioreactors were flushed with argon for 10 min in order to achieve the anaerobic environment. Afterwards, the bioreactors were placed in an incubator at 30 0C for the microaerobic dark fermentative process (New Brunswick Scientific Co. Inc) with an agitation of 120 rpm. The dark fermentative stage continued for 3 days. In the second part of the process, the bioreactors were transferred to the environmental chamber and incubated under continuous illumination (120 W/m2) without the addition of any substrate or buffer. The photofermentative step was prolonged for 6 days. Daily samples (2 mL) from the reactors were taken for the analysis of glucose concentration, pH, and OD. 2.4 Experimental design The design of an experiment is a critical tool which determines the overall conclusion of a work. Three independent variables; inoculum concentration (25-100 v/v % = X1), oxygen concentration (1-8 % = X2) and glucose concentration (2-10 mM = X3) were optimized for enhancing hydrogen production and yield. These three variables and their interactions were analyzed and showed on various RSM plots. For optimization of these parameters, Response surface methodology with the Box-Behnken design was employed by using the software 8

Design-Expert (Stat-Ease Inc., USA). The experimental design composed of total 17 runs and the center point was replicated four times for a reliable estimation (Table 1). The overall hydrogen production, hydrogen yield, and glucose consumption were determined as the responses of the experimental design.

2.5 Analytical methods Bacterial cell concentration (OD600) was measured by a spectrophotometer during cultivation (Thermo Scientific Evolution 260 BIO). Hydrogen gas production was measured daily by using a glass syringe to transfer a small aliquot of the head space to a gas chromatograph (Shimadzu GC-8A) equipped with a thermal conductivity detector and a 1 m column packed with molecular sieve 5A with argon as the carrier gas. The oven temperature was maintained at 60 0C and the flow rate was 25 mL/min. The consumption of glucose was determined by the Dinitrosalicylic Acid (DNS) Method (Miller, 1959). Hydrogen yield (mol H2/mol glucose) was calculated as the division of produced hydrogen by the consumed substrate.

3. Results and Discussion 3.1 Hydrogen production Response surface methodology is a powerful, effective and widely used approach in environmental and biological processes to evaluate the effects of different variables and their interactions on the responses (Montgomery, 2009). In this study, hydrogen production and 9

yield were optimized by examining the effects and the interactions of inoculum concentration, substrate concentration, and oxygen concentration on hydrogen production. The RSM contour plots definitely show that hydrogen production was influenced by oxygen concentration, glucose concentration, and inoculum size (Fig 2). A gradual increase in hydrogen production was observed when the initial glucose concentration was increased from 2 mM to 6 mM, and then hydrogen production decreased as glucose was increased to 10 mM (Fig. 2A, C). .). Hydrogen production increased when the oxygen concentration was raised from 1% to 4.5%, and then decreased as the oxygen concentration increased from 4.5% to 8% (Fig. 2B, 2C).Hydrogen production decreased as inoculum concentration decreased from 100 v/v% to 25% v/v% with an apparent maximum near 62.5% (Fig. 2B). The maximum hydrogen production (0.84 mmol) was obtained at 4.5% oxygen, 62.5% inoculum and 6 mM glucose concentration (Figure 2, Table 1). ANOVA analysis of the hydrogen production confirmed that the quadratic polynomial model was statistically significant (F-value: 34.73, pvalue <0.05) (not shown). The perturbation analysis of hydrogen production as a function of inoculum, oxygen and glucose concentration provides some interesting insights (Fig. 3A). Notably, a steep decrease in hydrogen production was observed when the oxygen concentration increased from the central point (4.5%) to 8%. The curve of the inoculum concentration increased dramatically from 25% to the 62.5%, and then a sigmoidal decline was observed from the central point to 100%. The reason for this could be the reduction of light penetration at higher cell concentrations. Similarly, the curve of glucose concentration was parallel to the curve of inoculum concentration. Firstly, a steep increase from 2 mM (code: -1) to 6 mM (code: 0.0) and then a gradual decrease towards 10 mM (code :1). The measure of the goodness of fit (R2, 10

0.94) showed that a good agreement between predicted and observed values (not shown). The lack of fit F-value (1.25) was found to be insignificant, indicating that the model fits the actual data well, and also predictions could be done by using the experimental data. 3.2 Hydrogen yield A great number of feedstocks, industrial and agricultural wastes possess a high content of glucose, present in various biopolymers. Dark fermentation can convert this glucose to hydrogen at a maximum yield of 33% of the theoretical value (12 mol H2/mol glucose) (Hallenbeck, 2009). As is well known, the effluent of the dark fermentation is mostly comprised of organic acids which can be converted to additional hydrogen by photofermentation, potentially allowing for the complete utilization of glucose. Here hydrogen yield was examined as a response by using Response surface methodology with the BoxBehnken design, which also allows the determination of the effects and the interaction of inoculum concentration, substrate, and oxygen concentration on hydrogen yield. A quadratic model for hydrogen yield confirmed that the model was statistically significant and could be used to make predictions about the response (Hydrogen yield). The model for hydrogen yield was shown to be statistically significant by ANOVA analysis (F-value: 29.48 , p<0.05) (not shown). Hydrogen yield ranged from 0.88 to 7.8 mol H2/mol glucose between the runs. When hydrogen yield was examined as a function of inoculum and glucose concentration, it was observed that the lowest hydrogen yields were obtained at 8% and 2% oxygen, with the highest yields being obtained at an oxygen concentration of 4.5% (Fig. 4A). An initial substrate concentration higher than 6 mM decreased hydrogen production, thereby leading to lower hydrogen yields (Figure 4A, 4C.

11

These results are also in agreement with some previous studies which found that higher substrate concentrations inhibit hydrogen yield (Nath et al., 2008; Keskin and Hallenbeck, 2012). The inoculum concentration is also a critical parameter in biohydrogen production as it plays important role in longevity and sustainability of the bioreactor operations. From the analysis of the RSM plots, it was observed that higher hydrogen yields were obtained as inoculum concentration was brought to 62.5% from 25%. Correspondingly, lower hydrogen yields were seen as the inoculum concentration decrease from 100% to 62.5% (Figures 4A, 4B). This is corroborated by examination of a perturbation plot for hydrogen yield (Fig. 3B), where inoculum concentration had the greatest influence on hydrogen yield among all the parameters. A steep curve was observed when inoculum concentration increased from 25% to the central point (62.5%), then a decline in the curve with further increases up to 100%. The curves indicating the effects of glucose and oxygen concentration were similar and increased towards the central point, and then decreased as their concentration increased. The measure goodness of fit (R2, 0.94) indicated good agreement between observed and predicted values (not shown). The lack of fit F-value (2.78) was not significant, which means that the model fits the experimental data well. Moreover, the optimal hydrogen yield was obtained within the range of the examined parameters. In the present work, a sequential microaerobic dark- and photofermentative process was used to improve hydrogen production and yield, which were previously obtained at lower amounts (0.16 mol H2/mol glucose) by a single microaerobic dark fermentation process (Abo-Hashesh and Hallenbeck, 2012). The highest hydrogen yield achieved in this study was higher than the yield obtained on glucose with an immobilized system (Asada et al., 2006). 12

3.3 Glucose Consumption and pH change Glucose consumption ranged between 64% and 100% among the various runs (Table 1). The highest glucose consumption (100%) was obtained at all the runs with 2 mM initial glucose concentration. A quadratic polynomial model was found to be statistically significant by ANOVA analysis (F value: 67.2, p<0.05) (not shown). However, the consumption percentage decreased as the initial glucose concentration increased from 2 mM to 10 mM. The highest hydrogen production (0.84 mmol) and yield (7.8 mol H2/mol glucose) were produced at 6 mM initial glucose. The percent glucose consumption was between 64% and 69% on 6 mM initial glucose with different inoculum and oxygen concentrations. Although the glucose consumption was in a range of 76%-80% on 10 mM initial glucose, the highest hydrogen yield (4.0 mol H2/mol glucose) was obtained at 100 v/v % and 4.5 % inoculum and oxygen concentrations, respectively. The excess glucose might have been converted to other metabolic products and organic acids. The lack of fit F-value (1.37) was insignificant, indicating that the model fits the actual data well. It can easily be seen from the RSM plots that the initial glucose concentration significantly affects the glucose consumption (Fig. 5A,C). The inoculum and oxygen concentration had slight effects on glucose consumption (Fig 5A, B, C). A perturbation plot illustrates the significant effect of initial glucose concentration on glucose consumption (Fig. 3C). It can be clearly seen that the curve representing glucose concentration has a dramatic decrease as it moves to the central point (6 mM). However, a gradual increase can also be observed when glucose concentration was increased to 10 mM. At the higher glucose concentrations, a larger fraction of the consumed glucose was probably used for other metabolic pathways and cellular processes instead of producing hydrogen, 13

decreasing hydrogen yields. The correlation (R2, 0.92) between actual and predicted values also indicated a good agreement (not shown). The initial pH of each run was adjusted to 7.0. The pH decreased and finally was between 6.3-6.7 during the microaerobic dark fermentation (Fig. 6). The initial pH started to decrease throughout the dark fermentative step and thereafter increased and maintained at 6.46.5 following the photofermentative step. This is probably due to consumption of the accumulated organic acids produced during the first dark fermentative stage. Therefore, the whole process has been carried out under optimal pH for PNSB by using the sequential system (Sasikala, 1993). It was observed that pH drop was more pronounced when the initial glucose concentration increased from 2 mM to 10 mM. This behavior is likely due to higher amounts of accumulated organic acids in dark fermentation effluent. 3.4 Comparison of hydrogen yields with the previous studies The utilization of glucose containing wastes and feedstocks is considered a potential path for practical biohydrogen production. Therefore, optimization of systems processing glucose should be investigated in detail. In biohydrogen production, attempts to achieve maximum hydrogen yields on glucose were done mostly by two-stage (Nath et al., 2005; Su et al., 2009; Liu et al., 2010;) or co-culture studies (Ding et al., 2009; Xie et al., 2010) (Table 2). Previously, two-stage dark- and photofermentation was also carried out by using Enterobacter cloacae and Rhodobacter sphaeroides to improve the overall yield from glucose (Nath et al., 2008). The hydrogen yields of 3.3 mol H2/mol glucose in dark fermentation stage, and 1.7 mol H2/mol acetic acid in the photofermentation stage were achieved. Su et al. used an orthogonal experimental design to optimize hydrogen production in a combined dark and

14

photofermentative system by using C. butyricum and R. palustris. They enhanced hydrogen yield from 1.5 mol H2/mol glucose, obtained in dark fermentation to 5.4 mol H2/mol glucose through photofermentation by using the dark fermentation effluents (Su et al., 2009). Biohydrogen was produced with a combination of dark and photofermentation by Clostridium butyricum and immobilized Rhodopseudomonas faecalis RLD-53 in batch, resuting in a maximum yield of 5.3 mol H2/mol glucose (Liu et al., 2010). The effects of glucose concentration, initial pH and buffer concentration were investigated in a co-culture study by Xie et al. The maximum hydrogen yield was 3.1 mol H2/mol glucose by co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53 (Xie et al., 2010). In a recent study, the highest hydrogen yield of 6.4 ± 1.3 mol H2/mol glucose was obtained by a co-culture of C. butyricum and R. palustris on a potato starch/glucose medium (Hitit et al., 2017b). The present study achieved the highest hydrogen yield obtained from an immobilized system. This work demonstrates the feasibility of sequential microaerobic dark- and photofermentative hydrogen production for the first time. Further studies are required to build an operable large scale production system. 4. Conclusion The present study demonstrates the improvement of hydrogen yield by the sequential microaerobic dark and photofermentation by immobilized R. capsulatus JP91. The results indicated that inoculum concentration, oxygen concentration, and glucose concentration significantly influenced the overall hydrogen production and yield. Besides, the highest hydrogen yield (7.8 mol H2/mol glucose) was achieved by an immobilized system so far. It

15

can be concluded that sequential system could be a promising solution for improvement of the hydrogen yield from glucose. Therefore, further studies are recommended to demonstrate the feasibility of the large scale operations for biohydrogen production. Acknowledgements This research was supported in part by a NSERC (Natural Sciences and Engineering Research Council (Canada) Discovery Grant to PCH and under a CRADA cooperative agreement (FA7000-16-2-0006) between USAF Academy (Life Sciences Research Center, Department of Biology) and Hallenbeck Associates (PCH). Emrah Sagir thanks the TUBITAK 2214A International Doctoral Research Fellowship Program, Turkey (Project number: 1059B141500983) for support. The views expressed here are those of the authors and do not reflect the official policy or position of the United States Air Force, the Department of Defense, or the U.S. Government.

References 1. Abo-Hashesh, M., Hallenbeck, P. C., 2012. Microaerobic dark fermentative hydrogen production by the photosynthetic bacterium, Rhodobacter capsulatus JP91. Int. J. LowCarbon Tech. 7(2), 97–103. 2. Abo-Hashesh, M., Desaunay, N., Hallenbeck, P. C., 2013. High yield single stage conversion of glucose to hydrogen by photofermentation with continuous cultures of Rhodobacter capsulatus JP91. Bioresour Technol. 128, 513–517. 3. Abo-Hashesh, M., Ghosh, D., Tourigny, A., Taous, A., & Hallenbeck, P. C. (2011). Single

16

stage photofermentative hydrogen production from glucose: An attractive alternative to two stage photofermentation or co-culture approaches. Int J Hydrogen Energy 36(21), 13889– 13895. 4. Argun, H., Kargi, F., 2011. Bio-hydrogen production by different operational modes of dark and photo-fermentation : An overview. Int. J. Hydrogen Energy 36(13), 7443–7459. 5. Androga, D.D., Sevinc, P., Koku, H., Yucel, M., Gunduz, U., Eroglu, I., 2014. Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710. Int. J. Hydrogen Energy, 39(6), 2472–2480. 6. Asada, Y., Tokumoto, M., Aihara, Y., Oku, M., Ishmi, K., Wakayama, T., 2006. Hydrogen production by co-cultures of Lactobacillus and a photosynthetic bacterium Rhodobacter sphaeroides RV. Int J Hydrogen Energy 31,1509–1513. 7. BP Energy Outlook, 2017. https://www.bp.com/content/dam/bp/pdf/energyeconomics/energy-outlook-2017/bp-energy-outlook-2017.pdf, accessed 9/17/2017. 8. Box, G.E.P., Behnken, D.W., 1960. Three level design for the study of quantitative variables. Technometrics 2, 455–475. 9. Chen, X., Sun, Y., Xiu, Z.L., Xi, Li., Daijia, Z., 2006. Stoichiometric analysis of biological hydrogen production by fermentative bacteria. Int J Hydrogen Energy 31,539–549. 10. Colbeau, A., Magnin, J.P., Cauvin, B., Champion, T., Vignais, P.M., 1990. Genetic and physical mapping of a hydrogenase gene cluster from Rhodobacter capsulatus. Mol. Gen. Genet. 220 (3), 393–399. 11. Ding, J., Liu, B.F., Ren, N.Q., Xing, D.F., Guo, W.Q., Xu, J.F., 2009. Hydrogen 17

production from glucose by co-culture of Clostridium Butyricum and immobilized Rhodopseudomonas faecalis RLD-53. Int J Hydrogen Energy 34, 3647–3652. 12. Ghosh, D., Sobro, I. F., Hallenbeck, P. C., 2012. Optimization of the hydrogen yield from single-stage photofermentation of glucose by Rhodobacter capsulatus JP91 using response surface methodology. Bioresour Technol. 123, 199–206. 13. Ghosh, D., Hallenbeck, P.C., 2010. Response surface methodology for process parameter optimization of hydrogen yield by the metabolically engineered strain Escherichia coli DJT135. Bioresour Technol. 101:1820-1825. 14. Hallenbeck, P. C., 2009. Fermentative hydrogen production : Principles , progress , and prognosis. Int J Hydrogen Energy, 34(17), 7379–7389. 15. Hallenbeck, P.C., Benemann, J.R., 2002. Biological hydrogen production; fundamentals and limiting processes. Int. J. Hydrogen Energy 27, 1185-1193.

16. Hitit Z.Y., Zampol, C., Hallenbeck, P. C., 2016. Single stage hydrogen production from cellulose through photo-fermentation by a co-culture of Cellulomonas fimi and Rhodopseudomonas palustris. Int J Hydrogen Energy, 42(10): 6556–6566. 17. Hitit Z.Y., Zampol, C., Hallenbeck, P. C., 2017a. Increased hydrogen yield and COD removal from starch / glucose based medium by sequential dark and photo-fermentation using Clostridium butyricum and Rhodopseudomonas palustris. Int J Hydrogen Energy, 42(30), 18832–18843. 18. Hitit Z.Y., Zampol, C., Hallenbeck, P. C., 2017b. Hydrogen production by co-cultures of Clostridium butyricum and Rhodospeudomonas palustris : Optimization of yield using 18

response surface methodology. Int J Hydrogen Energy, 42(10): 6578–6589. 19.Keskin, T., Abo-Hashesh, M., Hallenbeck, P. C., 2011. Photofermentative hydrogen production from wastes. Bioresour Technol. 102(18), 8557–8568. 20. Keskin, T., Hallenbeck, P. C., 2012. Hydrogen production from sugar industry wastes using single-stage photofermentation. Bioresour Technol. 112, 131–136. 21. Kim, M.S., Baek, J.S., Lee, J.K., 2006. Comparison of H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant. Int J Hydrogen Energy 31,121–127. 22. Kosourov, S.N., Seibert, M., 2009. Hydrogen Photoproduction by Nutrient-Deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions. Biotechnol. Bioeng. 102, 50–58. 23. Kumar, N., Das, D., 2000. Enhancement of hydrogen production by Enterobacter cloacae IIT- BT 08. Process Biochem 35, 589–593. 24. Kumar, G., Mudhoo, A., Sivagurunathan, P., Nagarajan, D., 2016. Recent insights into the cell immobilization technology applied for dark fermentative hydrogen production. Bioresour Technol. 219, 725–737. 25. Kumar, G., Sivagurunathan, P., Pugazhendhi, A., Thi, N. B. D., Zhen, G., Chandrasekhar, K., Kadier, A., 2017. A comprehensive overview on light independent fermentative hydrogen production from wastewater feedstock and possible integrative options. Energ Convers Manage. 141, 390–402. 26. Lazaro, C.Z., Hitit, Z.Y., Hallenbeck, P.C., 2017. Optimization of the yield of dark 19

microaerobic production of hydrogen from lactate by Rhodospeudomonas palustris, Bioresour. Technol. doi: 10.1016/j.biortech.2017.08.207. 27. Li, R.Y., Fang, H.H.P., 2008. Hydrogen production characteristics of photoheterotrophic Rubrivivax gelatinosus L31. Int J Hydrogen Energy 33, 974-980. 28. Liu, B.F., Ren, N.Q., Tang, J., Ding, J., Liu, W.Z., Xu, J.F., 2010. Bio- hydrogen production by mixed culture of photo- and dark- fermentation bacteria. Int J Hydrogen Energy 35(7), 2858–2562. 29. Masepohl, B., Hallenbeck, P.C., 2010. Nitrogen and molybdenum control of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus. In: Hallenbeck, P.C. (Ed.), Recent Advances in Phototrophic Prokaryotes, pp. 49–70 30. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428. 31. Miyake, J., Mao, X.Y., Kawamura, S., 1984. Hydrogen photoproduction from glucose by a co-culture of a photosynthetic bacteria and Clostridium butyricum. J Ferm Technol 62(6), 531–535. 32. Montgomery DC., 2009. Design and analysis of experiments. 7th ed. New Jersey, Wiley Hoboken. 33. Nath, K., Kumar, A., Das, D., 2005. Hydrogen production by Rhodobacter sphaeroides strain O.U.001 using spent media of Enterobacter cloacae strain DM11. Appl Microbiol Biotechnol 68(4), 533–541.

20

34. Nath, K., Muthukumar, M., Kumar, A., Das, D., 2008. Kinetics of two- stage fermentation process for the production of hydrogen. Int J Hydrogen Energy 33,1195–1203. 35. Sasikala, K., Ramana, C.V., Rao, P.R., Kovaks, K.L., 1993. Anoxygenic phototrophic bacteria: physiology and advances in hydrogen production technology. Adv. Appl. Microbiol. 38, 211–295. 36. Song, W., Rashid, N., Choi, W., Lee, K., 2011. Biohydrogen production by immobilized Chlorella sp. Using cycles of oxygenic photosynthesis and anaerobiosis. Bioresour Technol. 102, 8676–8681. 37. Su, H., Cheng, J., Zhou, J., Song, W., Cen, K., 2009. Combination of dark- and photofermentation to enhance hydrogen production and energy conversion efficiency. Int J Hydrogen Energy 34, 8846–8853. 38. Tao, Y., He, Y., Wu, Y., Liu, F., Li, X., Zong, W., 2008. Characteristics of a new photosynthetic bacterial strain for hydrogen production and its application in wastewater treatment. Int J Hydrogen Energy 33, 963–973. 39. Tian, X., Liao, Q., Zhu, X., Wang, Y.Z., Zhang, P., Li, J., Wang, H., 2010. Characteristics of a biofilm photobioreactor as applied to photo-hydrogen production. Bioresour. Technol. 101, 977–983. 40. Tsygankov, A., Kosourov, S., 2014. Immobilization of photosynthetic microorganisms for efficient hydrogen production. In: Zannoni, D., De Philippis, R. (Eds.) Microbial Bioenergy: Hydrogen Production. Springer, p. 321–47.

21

41. Xie, G.J., Feng, L.B., Ren, N.Q., Ding, J., Liu, C., Xing, D.F., 2010. Control strategies for hydrogen production through co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53. Int J Hydrogen Energy 35:1929–1935. 42. Xie, G.J., Liu, B.F., Ding, J., Xing, D.F., Ren, H.Y., Guo, W.Q., Ren, N.Q., 2012. Enhanced photo-H2 production by Rhodopseudomonas faecalis RLD-53 immobilization on activated carbon fibers. Biomass Bioenerg 44, 122–129. 43. Yokoi, H., Saitsu, A., Uchida, H., Hirose, J., Hayashi, S., Takasaki, Y., 2001. Microbial hydrogen production from sweet potato starch residue. J Biosci Bioeng. 91, 58–63.

Figure Captions

Fig. 1 Biohydrogen production metabolism of a purple non-sulfur bacterium (R. capsulatus JP91) in the sequential microaerobic dark- and photofermentation. Glucose is broken down by glycolysis for energy (ATP) and reducing equivalent (NADH) requirements. Organic acids are produced as byproducts of the glucose utilization throughout the microaerobic dark fermentation stage. The produced organic acids are consumed in the photofermentation stage under a continuous light illumination at 30 0C. In the dark fermentative stage, TCA cycle produces maximum ATP and NADH in response to oxygen availability via the oxidative phosphorylation. The protons coming from the glucose degradation and central metabolism are transferred to the nitrogenase, which reduces the protons and generates molecular hydrogen. As the light excites the PS unit, the protons moves to the ATPase, which produces the energy (ATP) for nitrogenase activity. In this stage, the required energy is compensated by 22

photophosphorylation instead of oxidative phosphorylation. Abbreviations: PS unit= photosystem unit; TCA= tricarboxylic acid; ATP= Adenosine triphosphate; NADH= Nicotinamide adenine dinucleotide. Fig. 2 Three dimensional RSM plots of hydrogen production (µmol). A, Hydrogen production a function of glucose concentration and inoculum concentration at a constant oxygen concentration of 4.5%; B, Hydrogen production a function of inoculum concentration and oxygen concentration at a constant glucose concentration of 6 mM; C, Hydrogen production as a function of oxygen concentration and glucose concentration at a constant inoculum concentration of 62.5 v/v %. Fig. 3 Perturbation analyses of (A) hydrogen production (mmol), (B) hydrogen yield (mol H2/mol glucose), and (C) glucose consumption (%). The responses are the results of changes in inoculum concentration (curve A), glucose concentration (curve B), and oxygen concentration (curve C). Fig. 4 Three dimensional RSM plots of hydrogen yield (mol H2/mol glucose). A, Hydrogen yield as a function of glucose concentration and inoculum concentration at fixed oxygen concentration of 4.5%; B, Hydrogen yield as a function of inoculum concentration and oxygen concentration at fixed glucose concentration of 6 mM; C, Hydrogen yield as a function of oxygen concentration and glucose concentrations at fixed inoculum concentration of 62.5 v/v%. Fig. 5 RSM plots of glucose consumption (%) as a function of inoculum concentration, oxygen concentration and glucose concentration. A, Glucose consumption in response to changes in inoculum concentration and glucose concentration at constant oxygen 23

concentration of 4.5%; B, Glucose consumption in response to changes in inoculum concentration and oxygen concentration at constant glucose concentration of 6 mM; C, Glucose consumption in response to changes in oxygen concentration and glucose concentration at constant inoculum concentration of 62.5 v/v %. Fig. 6 Changes in pH of different runs throughout the microaerobic dark and photofermentation process. pH change of reactors (R1, R3, R7, R11) on 2 mM glucose (A), (R2, R4, R15, R17) on 6 mM glucose (B), and (R6, R8, R12, R13) on 10 mM glucose initial glucose concentration.

24

25

26

27

28

29

30

Table 1 Experimental design table for optimization of hydrogen production, hydrogen yield and glucose consumption with three independent variables.

Run

Inoculum concentration

Codeda Actualb 1 0 62.5 2 -1 25 3 +1 100 4 0 62.5 d 5 0 62.5 6 0 62.5 7 -1 25 8 +1 100 9 -1 25 10 +1 100 11 0 62.5 12 -1 25 13 0 62.5 14d 0 62.5 15 +1 100 d 16 0 62.5 d 17 0 62.5 a Coded factor values b

Oxygen concentration Codeda -1 -1 0 0 0 +1 0 0 +1 -1 +1 0 -1 0 +1 0 0

Actualb 1 1 4.5 4.5 4.5 8 4.5 4.5 8 1 8 4.5 1 4.5 8 4.5 4.5

Actual factor values

c

The values are the average of the three runs

d

Center points

31

Glucose conc. Codeda -1 0 -1 0 0 +1 -1 +1 0 0 -1 +1 +1 0 0 0 0

Actualb 2 6 2 6 6 10 2 10 6 6 2 10 10 6 6 6 6

H2 Yieldc (Y) mol/mol

Hydrogen Productionc mmol

6.50 1.25 5.50 6.72 7.45 1.37 2.66 4.00 0.88 5.71 3.50 1.96 2.94 6.98 2.30 7.63 7.80

0.20 0.14 0.17 0.72 0.75 0.22 0.08 0.62 0.09 0.60 0.11 0.30 0.50 0.71 0.24 0.84 0.82

Gluco consump %

100 68 100 67 64 80 100 77 65 66 100 76 85 64 65 69 65

Table 2 Bioydrogen production by single-stage, two-stage or co-culture of dark and photofermentation on pure glucose.

Microorganisms

Process

E.coli DJT135 Enterobacter cloacae Clostridium Butyricum Rhodobacter capsulatus JP91 Rhodobacter capsulatus JP91 Rubrivivax gelatinosus L31

Dark fermentation Dark fermentation Dark fermentation Dark fermentation Photofermentation Photofermentation

Hydrogen Yield (mol H2/mol glucose) 1.5 2.2 3.26 0.16 9.0 0.9

Rhodobacter capsulatus JP91 Rhodobacter capsulatus JP91 Rhodobacter sphaeroides KD131

Photofermentation Photofermentation Photofermentation

5.5 3.0 1.5

Gho Abo-Ha Kim

Rhodobacter sphaeroides ZX5

Photofermentation

6.5

Tao

Two-stage Two-stage Two-stage Two-stage Co-culture Co-culture

5.3 5.4 6.7 5.5 7.0 3.1

Nat Liu Nat Su Miya Xie

Co-culture

4.1

Din

Co-culture

7.1

Asad

Enterobacter cloacae and Rhodobacter sphaeroides Clostridium Butyricum Rhodopseudomonas faecalis Enterobacter cloacae Rhodobacter sphaeroides Clostridium butyricum Rhodopseudomonas palustris Clostridium butyricum Rhodobacter sphaeroides Ethanoligenens harbinense Rhodopseudomonas faecalis Clostridium Butyricum Rhodopseudomonas faecalis RLD-53 Lactobacillus delbrueckii Rhodobacter sphaeroides

32

Ref

Gho Kum Che Abo-Ha Abo-Ha Li

HIGHLIGHTS • • • •

Sequential microaerobic dark- and photo-fermentative system investigated H2 from glucose using a batch process and a bench-scale bioreactor H2 by immobilized R. capsulatus JP91 in a sequential system demonstrated Maximum H2 production obtained at 6 mM glucose, 4.5% O2, 62.5 v/v % inoculum

33