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Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles
international journal of hydrogen energy xxx (xxxx) xxx
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Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles Balasubramani Ramprakash a, Aran Incharoensakdi a,b,* a
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand b Academy of Science, Royal Society of Thailand, Bangkok 10300, Thailand
highlights Photogenerated electron from the NPs has ability to interact with bacterial cell. The combination of photocatalyst and biocatalyst enhances hydrogen production. TiO2/MV2þ/E. coli hybrid system produces more hydrogen compared with control. Use of natural sunlight is feasible for photocatalytic biohydrogen production.
article info
abstract
Article history:
Photocatalytic hydrogen production using an inorganic bio-hybrid system can contribute to
Received 17 October 2019
the proficient utilization of light energy, but it would necessitate the development of novel
Received in revised form
approaches for preparing a new hydrogen-producing biocatalyst. In this study, we devel-
23 December 2019
oped a hybrid system to produce hydrogen, whereby the highly efficient light-harvesting
Accepted 2 January 2020
inorganic semiconductor (TiO2) was mixed with Escherichia coli to form a biocatalyst with
Available online xxx
the addition of an electron mediator (MV2þ) under different visible light irradiation. Under
Keywords:
showing maximum production at 2000 W m2, with a 2-fold increase in the hydrogen
Escherichia coli
production compared to that without TiO2. The experiments on the continued cycle of
Hybrid system
hydrogen production revealed that the production could be continued for at least 3 cycles
Hydrogen production
of 5 h incubation for each. A possible pathway utilizing glucose for hydrogen production by
Methyl viologen
the hybrid system was proposed based on the analysis of the levels of metabolites. A
Nanoparticles
feasibility study was also conducted using natural sunlight for hydrogen production by the
this hybrid system, the hydrogen production by E. coli was light intensity-dependent
hybrid system. Overall results demonstrated that whole cells of E. coli could be employed for photocatalytic hydrogen production where the intactness of the E. coli was retained under experimental conditions. © 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. E-mail address: [email protected] (A. Incharoensakdi). https://doi.org/10.1016/j.ijhydene.2020.01.011 0360-3199/© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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Introduction The depletion of hydrocarbon fuel reserves, unstable energy prices and increased emission of environmental pollutant gases during combustion of fossil fuels have triggered a search for a new clean and sustainable energy resources [1,2]. Among the various alternative fuels, hydrogen is projected as one of the promising candidate, both from economic and environmental viewpoints, since it produces only water during combustion [3,4]. Water electrolysis, thermo-chemical method, and biological processes are the major hydrogen production methods [5]. However, still over 85% of current worldwide hydrogen production relies on the use of non-renewable fossil fuels [1]. This causes pollution and harsh conditions to the environment and also it requires high costs for the refining. Moreover, the gasification processes has created negative impact and obstructed the growth of hydrogen industry. In contrast, hydrogen production using biological methods is gaining increasing importance over challenging biofuel technologies due to its favorable characteristics such as higher energy content (120 kJ/g), that is approximately three times higher than that of fossil fuels, environmentally friendly, able to use diverse feedstocks or utilization of light/solar energy through photosynthesis. The biological hydrogen production methods include bio-photolysis, photo-fermentation and dark fermentation using obligate anaerobes, facultative anaerobes and photosynthetic bacteria [4]. Among them fermentative hydrogen production is an attractive route compared to other biological hydrogen production processes, as it can use various carbon sources, and even renewable biomass and industrial wastes as substrates [6,7]. The hydrogenase/nitrogenases present inside the microbial cells act as a catalyst and convert the electrons into hydrogen ions during transduction process [8]. Moreover, microorganisms have the selfreproducibility in nature and cost of the biotic (enzymes) catalyst is relatively low, particularly while using the whole cell microbes. Though, the commercialization of these biological methods has been hindered by its lower volumetric productivity [9]. The emergence of nano-science and nanotechnology has been gaining momentum in the past decade due to its ability to exploit diverse nano-scale materials, having sizes ranging from 1 to 100 nm [10,11]. As a consequence, nanoparticles are employed in various applications such as the agricultural, food, cosmetic, pharmaceutical, and electronic industry [12]. The advantages of nanoparticles have been recognized owing to their small size and their high surface to volume ratio which allows them to interrelate very closely with microorganisms [13]. Previous studies showed that addition of nanosized metals to dark fermentation systems could increase the efficiency of biogas production [14,15]. The use of photocatalytic semiconductor oxides emerges as a successful technology for the development of bio-hybrid materials. TiO2anatase is by far the most extensively used photocatalyst, as a wide band-gap (3.2 eV) semiconductor under UV illumination could generate energy rich electronehole pairs able to interact with the bioactive compounds inside the microbial cell. The combination of nano semiconductor materials with hydrogen forming biocatalyst has attracted more interest on clean
hydrogen production in recent years in the form of inorganicebio hybrid approaches. Such systems add the great values in hydrogen production using light/solar energy. The well-known hydrogen forming biocatalyst, the hydrogenase, catalyzes the reduction of proton to form hydrogen. The addition of Cu and CuSO4 nanoparticles had a negative effect on the production of biohydrogen in Clostridium acetobutylicum NCIM 2337 and Enterobacter cloacae 811101 [16]. On the other hand, the addition of iron nanoparticles increased the biohydrogen yield by 20% in the mixed co-cultures of Rhodobacter sphaeroides NMBL02 and Escherichia coli NMBL04 [17]. In another study, TiO2 and Fe nanoparticles were shown to have a stimulatory effect on hydrogen production by Clostridium pasteurianum [18]. Recently, photocatalytic hydrogen production under visible light irradiation was achieved by dye sensitized TiO2 nanoparticles with [NieFeeSe]-hydrogenase [13], and another study with CdS nanorods and CdTe nanocrystals capped with mercaptopropionic acid combined with a recombinant Clostridial [FeeFe] hydrogenase [19]. These inorganic semiconductors and biocatalytic systems demonstrate the successful photocatalytic hydrogen production. However, the most important disadvantage of this system is that the lower growth rate of the microorganisms was observed during biocatalyst preparation. Hence, the needs of an efficient biocatalyst that can be obtain by means of simple manipulations and have a potential for high hydrogen productivity. Such a kind of biocatalyst could facilitate the construction of practical inorganic bio-hybrid system for the photocatalytic hydrogen production. The utilization of the most extensively studied bacterium E. coli, the laboratory workhorse, as biocatalyst is a promising method. Under the stimulating conditions E. coli has the capability to interact with nanoparticles on its outer cell surface [20]. Being a facultative anaerobe, its synthesis of an endogenous [NieFe]-hydrogenase is an added advantage of using this E. coli cell [21]. This endogenous [NieFe]-hydrogenase is a major component of the FHL (formate hydrogenlyase) complex, which produces hydrogen directly. There is no need for introducing exogenous hydrogenases to the E. coli cells by genetic engineering. In this study, we developed a hybrid system (TiO2/MV2þ/E. coli) and investigated the possibilities of using E. coli endogenous [NieFe] hydrogenases as biocatalyst and the photogenerated electrons from TiO2 along with electron mediator (MV2þ) on the E. coli cell surface intended for the enhancement of biohydrogen production. The basic mechanism of improved hydrogen production was also studied. Finally, the possibility of using natural sunlight (solar energy) to produce hydrogen by the hybrid system was also demonstrated.
Materials and methods TiO2 optimization The optimum TiO2 concentration was studied in MOPS (3-(Nmorpholino) propanesulfonic acid) medium [22]. E. coli K-12 strain was purchased from E. coli genetic stock centre (CGST), CT, USA. Initially E. coli cells were pre-cultured by aerobic cultivation at 37 C, 150 rpm for 6 h in 5 mL of LB Miller medium. Subsequently, the cell pellet after centrifugation of
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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200 mL of the pre-cultures at 12000 g/10 min was resuspended with 50 mL MOPS medium supplemented with 1 mM cysteine and 6 mM glucose. The final concentrations of 0.125, 0.250, 0.375, 0.500, 0.625 and 0.750 mM of TiO2 and 5 mM of MV2þ were added to parallel groups at the beginning of the inoculation. Control groups without MV2þ were also studied for comparison. All the experimental and control groups were incubated at 37 C, the cell density during the incubation period was monitored spectrophotometrically. At set time intervals, the OD at 600 nm of the culture was recorded using a UVevis spectrophotometer.
Quantification of pyruvate, lactate, formate, and NADH/ NAD
Construction and characterization of the hybrid system
For microscopic observation of E. coli whole cell, a light microscope (Seek, Melbourne, Australia) was used. The reaction mixture consists of TiO2/MV2þ/E. coli for photocatalytic hydrogen production. The samples were collected and directly subjected to microscopic observation before and after light irradiation.
In the glovebox, 50 mL of reaction mixture containing 0.375 mM of TiO2 anatase, 150 mM NaCl, 100 mM Tris-HCl (pH 7), 6 mM glucose, 100 mM ascorbic acid, 5 mM MV2þ and biocatalyst (0.1 g dry cell E. coli) was prepared in a quartz reaction bottle sealed by a rubber septum to protect biocatalysts from oxygen exposure. After 5 h of light irradiation at desired intensity the bacterial cells in the culture samples were harvested by centrifugation at 12000g for 10 min before further characterization. For the preparation of TEM (transmission electron microscope) samples to observe the TiO2 nanoparticles on the bacterial cell surface, the collected cell pellet was washed thrice with milli- Q water and freeze-dried for 48 h. After freeze drying a small piece of dried sample was dissolved in 1 mL of ethanol, and then a formvar-coated copper grid was immersed for a few seconds to collect the sample. Once the sample had been air-dried on the grid, TEM imaging was done (TECNAI 20 G2, FEI make). For SEM (scanning electron microscope) imaging, a small amount of sample from the hybrid system was dissolved in 1 mL of phosphatebuffered saline (PBS) solution, and then samples were fixed using 4% glutaraldehyde at 4 C. After 2 h of fixation, the sample was washed twice with PBS at room temperature, then the sample was serially dehydrated in 50%, 70%, 85%, 95% (twice), and 100% (thrice) with acetone, each time lasting for 10e15 min. The dehydrated sample was critical-point-dried and visualized under SEM (FEI Quanta FEG 200) at 10 kV, to observe the morphology of the E. colieTiO2 hybrid system.
The concentration of pyruvate (ab65342e abcam), lactate (ab65331 e abcam), formate (ab111748 e abcam) and NADH/ NAD (ab65348 e abcam) were determined by respective quantification assay kits. All the experiments were carried out by following the technical bulletins given in the respective assay kits.
Microscopy analysis
Assay for E. coli cell lysis To examine whether the E. coli cells in the hybrid system lyses during the photocatalytic reaction, the activity of b -galactosidase (b-gal) was used to measure the cell lysis in the supernatant of the reaction mixture of TiO2/MV2þ/E.coli hybrid system. The b -galactosidase activity can be simply monitored by 420 nm absorption by using 2-nirotophenyl b -D-galactopyranoside (ONPG) [24].
Feasibility studies A 50 mL of reaction mixture consisting of the control E. coli and the hybrid system (TiO2/MV2þ/E.coli culture) was kept under natural sunlight (10: 00 A.M to 3:00 P.M) and used for the irradiation to find the hydrogen production efficiency. For comparative analysis, TiO2/MV2þ/whole cell E. coli hybrid system under irradiation by a xenon lamp (2000 W m2 visible light) for 15 h was also performed. Hydrogen production experiments were conducted in 50 mL sealed transparent glass bottles, and the hydrogen production was monitored by GC at fixed time intervals. All the experiments were carried out in triplicate and average values were reported.
Hydrogen production under different visible light intensity The visible light (VL) intensity from the xenon (Xe) lamp was adjusted to 0, 500, 1000, 2000, 3000, and 4000 W m2 using a lux meter. The 50 mL samples of cultured hybrid system (TiO2/ MV2þ/E. coli) were illuminated under different light intensities, and E. coli cultures without TiO2/MV2þserved as the control group. The hydrogen production was monitored at fixed time intervals or at 15 h for accumulative hydrogen yield by GC (gas chromatography) analysis, (Perichrom, France) equipped with a thermal conductivity detector, a Molecular Sieve 5A 60/80 mesh column, using argon as a carrier gas.
Glucose determination Dinitrosalicylic acid (DNS) colorimetric method [23], was used to monitor the glucose utilization efficiency of the TiO2/MV2þ/ E. coli hybrid system and the control E. coli.
Results and discussion Optimization of TiO2 concentration In the aquatic ecosystem, titanium dioxide (TiO2) is highly toxic to both marine and freshwater microorganisms [25,26]. Numerous stress responses action in E. coli induced by TiO2, causes metabolic changes was reported [27]. In general, most of the polar molecules cannot cross the microbial cell membrane easily and, indeed, MV2þ (the redox mediator) have been reported as an impermeant cation for E. coli cell membrane [28]. The redox mediator (MV2þ) is known to affect the intracellular NADH level and modify the NADH related metabolic pathways [29,30]. Fig. 1 clearly demonstrated that the concentration of TiO2 higher than 0.375 mM strongly inhibited the growth of E. coli regardless of the presence or the absence of
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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large amounts of TiO2 particles were deposited on the E. coli cell surface (Fig. 2). It is well known that the particle size is a major key factor which affects the photocatalytic activity of the semiconductor materials, as it influences the particular surface area, charge carrier recombination rate and electron hole pair separation efficiency [31]. Hence, the nanosized TiO2 particles with MV2þ on the E. coli cell surface have the higher potential to display excellent photocatalytic activity. This close link and the delicate structure efficiently combined the inorganic system with the biological system. The scheme illustrating the formation of bio-hybrid system confirmed by TEM and SEM images is shown in Fig. 2.
Hydrogen production efficiency of the hybrid system Fig. 1 e Effect of TiO2 concentration on cell density (OD600) of E. coli in MOPS medium with and without 5 mM MV2þ after a culture time of 20 h. MV2þ. A slightly decreased growth of E. coli was observed at 0.375 mM TiO2, therefore this TiO2 concentration was used for further study on hydrogen production by TiO2/MV2þ/E.coli hybrid system.
Hybrid system construction and characterization From the above results, the hybrid system was constructed using 0.375 mM TiO2 and 5 mM MV2þ. The TEM images showed the strong aggregation between the nanoparticles (TiO2) and the electron mediator (MV2þ) in the reaction mixture (Fig. 2). These nanoparticles exhibited good equality and their average size was around 10e20 nm. SEM images further revealed that
The hydrogen production from the E. coli without TiO2(control) and TiO2/MV2þ/E. coli hybrid system irradiated with different visible light (VL) intensities was studied. As shown in Fig. 3a, an increase in VL intensity up to 3000 W m2 resulted in an increase of hydrogen production of the hybrid system with maximum production of 3.6 mmol/mmol glucose observed at 2000 W m2, a 2.8-fold increase than that of control. At VL intensity higher than 500 W m2, the hybrid system invariably had higher hydrogen production than the control. Too high light intensity led to a decreased hydrogen production in both the hybrid and the control systems, suggesting that under high light intensity the self defense and auto repair mechanisms of the bacterium was insufficient to protect the cell to allow for sustained production of hydrogen. As seen from material chemistry view point, these results indicate that, when an inorganic semiconductor was combined with a biological system, the efficiency of the hydrogen production can
Fig. 2 e The scheme illustrating the formation of bio-hybrid system confirmed by SEM and TEM images. Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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depletion of carbon source availability. However, the hydrogen production continued for 3 cycles up to 15 h where the hybrid system with electron mediator produced more hydrogen (3.6 mmol/mmol glucose) compared with that without electron mediator and E. coli alone (2.5 and 1.3 mmol/ mmol glucose respectively). This result clearly demonstrated that the extracellularly added MV2þ could transfer more photo-generated electrons to the intracellular bioactive compounds inside the living cells, leading to enhanced hydrogen production. The hydrogen production by different hybrid systems was compared with the results of the present study as shown in Table S1 (Supporting Information). The hybrid system using E. coli and TiO2 in the present study showed highest hydrogen yield, and TiO2 was superior to CdS as nanoparticle in the E. coli hybrid system. The results of microscopic examination and the b-galactosidase assay suggested that the cells of E. coli in the hybrid system had strong aggregation with the semiconductor and no cell lysis occurred under the conditions of photocatalytic reactions (Supporting Information, Figs. S1 and S2). This indicates the durability of TiO2/MV2þ/E.coli hybrid system for high efficiency and long term hydrogen production.
Proposed mechanism
Fig. 3 e Hydrogen accumulation under different conditions. (A) Hydrogen production of control E. coli and the hybrid system (TiO2/MV2þ/E.coli) under different visible light intensities for 15 h. (B) Hydrogen production by the hybrid system with (TiO2/MV2þ/E.coli) and without (TiO2/E.coli) electron mediator for 3 cycles of 5 h each.
be significantly improved as reported in a previous study using genetic manipulations to express exogenous [Fe -Fe]hydrogenase in E. coli cells [24]. The TiO2/MV2þ/E.coli hybrid system under 2000 W m2 irradiation in the present study could achieve a great conversion rate of glucose to hydrogen by 90% of theoretical value (100% represents 24 mmol H2/ 6 mmol glucose). Furthermore, we have done some analyses of repeated hydrogen production efficiency (with and without electron mediator (MV2þ) of TiO2/MV2þ/E.coli, TiO2/E.coli and E. coli alone under 5 h of irradiation for three cycles, where each cycle was subject to the evacuation of gas from the reaction mixture before starting the next cycle. As shown in Fig. 3b, during the first cycle hydrogen production of 2.2 mmol/mmol glucose was observed in TiO2/MV2þ/E.coli hybrid system, whereas in TiO2/E.coli hybrid system and E. coli alone produced 1.6 and 0.5 mmol H2/mmol glucose, respectively. It should be mentioned that no hydrogen production was observed in TiO2/MV2þ system lacking E. coli. After the first cycle, the hydrogen production decreased gradually in the following two cycles in the hybrid systems and the control group due to
The mechanism for the improved hydrogen production from the hybrid system is schematically illustrated in Fig. 4. When the TiO2 nanoparticles along with MV2þ (redox mediator) are irradiated by a suitable intensity of visible light, large amounts of electron-hole pairs could be generated. The electrons generated by the TiO2 nanoparticles assisted by redox
Fig. 4 e Proposed schematic mechanism of the enhanced hydrogen evolution in the hybrid system (the combination of TiO2, methyl-viologen, and the whole cell E. coli).
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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mediator could interact with bioactive components and incorporate into the hydrogen production pathway. In E. coli hydrogen production pathway, the accessible intracellular glucose acts as an electron donor and an energy source. The analysis of various intermediates as shown in Fig. 5 supports the flow of metabolites from glucose to hydrogen depicted in Fig. 4. The hybrid system enabled E. coli to increase hydrogen production with the increase in incubation time with higher production under light conditions (Fig. 5a). Glucose, an electron donor, under both dark and light conditions was rapidly decreased during the first 1 h with a continued decrease until 5 h incubation (Fig. 5b). No major difference in hydrogen production was observed under these two conditions, thus suggesting that glucose was rapidly utilized by E. coli cells and
transferred it into glycolysis pathway without any involvement of the photo-generated electrons. However, as shown in Fig. 5c, the concentration of pyruvate in light-irradiated E. coli was increased much more in the first 3 h than that of the E. coli under dark condition, suggesting that a portion of photogenerated electrons may take part in the glycolysis and accelerated the process resulting in higher pyruvate formation. On the other hand, a lower level of intracellular lactate accumulation was observed in light-irradiated E. coli compared to that under dark condition (Fig. 5d), demonstrating that the lactate fermentative pathway under light condition could be inhibited by photo-generated electrons resulting in more pyruvate conversion to formate. The results in Fig. 5e indicates that the concentration of formate in the
Fig. 5 e Hydrogen production (A) and metabolic levels of glucose (B), pyruvate(C), lactate (D), formate (E), and NADH/NAD ratio (F) of the hybrid system at various times under light (2000 W m¡2) and dark conditions. Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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dark treated E. coli decreased gradually in the first 2 h, afterward remained at nearly a constant level, whereas in the light-irradiated E. coli it decreased faster in the first 2 h, indicating the higher formate consumption. After 2 h of irradiation an increased concentration of formate was observed corresponding to the decrease of pyruvate level seen in Fig. 2c. The increased availability of formate serves as substrate for the FHL complex, which contains (NieFe) hydrogenases catalyzing the breakdown of formate into CO2 and H2 during anaerobic fermentation [32]. Hence, this superior catalytic action would lead to rapidly consumption of formate which results in increased accumulation of hydrogen. The results showed in Fig. 5f demonstrate that in the first 2 h of visible light irradiation, the NADH to NAD ratio in the TiO2/MV2þ/ E.coli hybrid system was a little higher compared to that under dark conditions. The photo-generated electrons could stimulate rapid recycling of NADH leading to high reduction potential inside the bacterial cell, which benefits the hydrogen production process. The decrease in NADH/NAD ratio suggested the increase oxidation of NADH to generate NAD in conjunction with the increased reduction of proton to generate hydrogen. Hydrogen production is usually accompanied by the production of volatile fatty acids (VFAs) such as acetic acid, butyric acid and succinic acid and solvents like ethanol. After the measurement of these metabolites, it was observed that acetic acid was found to be 0.19 mg/mL, succinic acid and ethanol were found to be 0.06 and 0.07 mg/mL respectively, whereas only trace amount of butyrate was found (data not shown). Thus, acetate was found to be the major end metabolite of this mixed acid fermentation. This hybrid system follows acetate based hydrogen production.
Feasibility study Finally, the feasibilities of applying natural sunlight (solar irradiation) for the hydrogen production using the hybrid system were thoroughly investigated as shown in Fig. 6. The hydrogen production capabilities of different combinations of natural (sunlight) and laboratory (artificial light) conditions were compared. The results illustrates that the laboratory conditions (the hybrid system using a Xe lamp for light irradiation) yielded higher hydrogen production
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than that using natural sunlight. The E. coli without the hybrid system under natural sunlight and laboratory condition had low efficiency of hydrogen production. The lower hydrogen production by the hybrid system under natural sunlight compared to that under xenon lamp due to the variation of actual intensity of sunlight with the hour of day, season, geo-graphical location and atmospheric conditions or due to the natural UV (ultraviolet) irradiation causing harmful effects and reducing the physiological action of microbes under natural conditions. Hence, more intense research work must be needed to optimize the hydrogen production and further improving the hybrid system for its application using natural resources, such as natural sunlight optimization and wastewater as culture medium.
Conclusions The possibility of combining inorganic semiconductor nanoparticles with microorganisms to enhance the renewable energy production has recently become a hot field of research. In the present study we developed a new hybrid system for the efficient photocatalytic hydrogen production. Under optimal visible light irradiation TiO2/MV2þ/E.coli hybrid system could efficiently generate 3.6 mmol H2/mmol glucose, whereas under the same condition TiO2/E.coli hybrid system and the E. coli alone produced 2.5 and 1.3 mmol/mmol glucose respectively. The results clearly demonstrate that TiO2/MV2þ/E.coli hybrid system produces a 2.8-fold increase in hydrogen production compared to the control E. coli in 15 h. The TiO2 nanoparticles with the addition of electron mediator (MV2þ) could facilitate the increase of photogenerated electrons transferred into the E. coli after visible light excitation on the extracellular side. Furthermore, the mechanistic study revealed that the increase in the rate of pyruvate formation, lactate inhibition, boost of formate concentration, and reduction of NADH/ NAD ratio were found to be the key mechanism in the hybrid system for the improved production of hydrogen. Finally, the hybrid system under natural sunlight irradiation produced 1.8 mmol H2/mmol glucose, whereas the E. coli alone under the same condition produced only 1.2 mmol H2/ mmol glucose, indicating the cost effective operation for hydrogen production. This semiconductor/intact E. coli cells hybrid system is a promising approach towards the development of a practical and clean photocatalytic hydrogen production in the future.
Acknowledgements
Fig. 6 e Hydrogen production by the control E. coli and the hybrid system under sunlight and xenon lamp (2000 W m¡2).
B.R. is thankful to the Graduate School, Chulalongkorn University (CU), for post-doctoral fellowship from Ratchadaphiseksomphot Endowment Fund. A.I. acknowledges the research grants from CU on the Frontier Research Energy Cluster (CU-59-048-EN) and from Thailand Research Fund (IRG 5780008).
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2020.01.011.
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Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles Balasubramani Ramprakash a, Aran Incharoensakdi a,b,* a
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand b Academy of Science, Royal Society of Thailand, Bangkok 10300, Thailand
highlights Photogenerated electron from the NPs has ability to interact with bacterial cell. The combination of photocatalyst and biocatalyst enhances hydrogen production. TiO2/MV2þ/E. coli hybrid system produces more hydrogen compared with control. Use of natural sunlight is feasible for photocatalytic biohydrogen production.
article info
abstract
Article history:
Photocatalytic hydrogen production using an inorganic bio-hybrid system can contribute to
Received 17 October 2019
the proficient utilization of light energy, but it would necessitate the development of novel
Received in revised form
approaches for preparing a new hydrogen-producing biocatalyst. In this study, we devel-
23 December 2019
oped a hybrid system to produce hydrogen, whereby the highly efficient light-harvesting
Accepted 2 January 2020
inorganic semiconductor (TiO2) was mixed with Escherichia coli to form a biocatalyst with
Available online xxx
the addition of an electron mediator (MV2þ) under different visible light irradiation. Under
Keywords:
showing maximum production at 2000 W m2, with a 2-fold increase in the hydrogen
Escherichia coli
production compared to that without TiO2. The experiments on the continued cycle of
Hybrid system
hydrogen production revealed that the production could be continued for at least 3 cycles
Hydrogen production
of 5 h incubation for each. A possible pathway utilizing glucose for hydrogen production by
Methyl viologen
the hybrid system was proposed based on the analysis of the levels of metabolites. A
Nanoparticles
feasibility study was also conducted using natural sunlight for hydrogen production by the
this hybrid system, the hydrogen production by E. coli was light intensity-dependent
hybrid system. Overall results demonstrated that whole cells of E. coli could be employed for photocatalytic hydrogen production where the intactness of the E. coli was retained under experimental conditions. © 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. E-mail address: [email protected] (A. Incharoensakdi). https://doi.org/10.1016/j.ijhydene.2020.01.011 0360-3199/© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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Introduction The depletion of hydrocarbon fuel reserves, unstable energy prices and increased emission of environmental pollutant gases during combustion of fossil fuels have triggered a search for a new clean and sustainable energy resources [1,2]. Among the various alternative fuels, hydrogen is projected as one of the promising candidate, both from economic and environmental viewpoints, since it produces only water during combustion [3,4]. Water electrolysis, thermo-chemical method, and biological processes are the major hydrogen production methods [5]. However, still over 85% of current worldwide hydrogen production relies on the use of non-renewable fossil fuels [1]. This causes pollution and harsh conditions to the environment and also it requires high costs for the refining. Moreover, the gasification processes has created negative impact and obstructed the growth of hydrogen industry. In contrast, hydrogen production using biological methods is gaining increasing importance over challenging biofuel technologies due to its favorable characteristics such as higher energy content (120 kJ/g), that is approximately three times higher than that of fossil fuels, environmentally friendly, able to use diverse feedstocks or utilization of light/solar energy through photosynthesis. The biological hydrogen production methods include bio-photolysis, photo-fermentation and dark fermentation using obligate anaerobes, facultative anaerobes and photosynthetic bacteria [4]. Among them fermentative hydrogen production is an attractive route compared to other biological hydrogen production processes, as it can use various carbon sources, and even renewable biomass and industrial wastes as substrates [6,7]. The hydrogenase/nitrogenases present inside the microbial cells act as a catalyst and convert the electrons into hydrogen ions during transduction process [8]. Moreover, microorganisms have the selfreproducibility in nature and cost of the biotic (enzymes) catalyst is relatively low, particularly while using the whole cell microbes. Though, the commercialization of these biological methods has been hindered by its lower volumetric productivity [9]. The emergence of nano-science and nanotechnology has been gaining momentum in the past decade due to its ability to exploit diverse nano-scale materials, having sizes ranging from 1 to 100 nm [10,11]. As a consequence, nanoparticles are employed in various applications such as the agricultural, food, cosmetic, pharmaceutical, and electronic industry [12]. The advantages of nanoparticles have been recognized owing to their small size and their high surface to volume ratio which allows them to interrelate very closely with microorganisms [13]. Previous studies showed that addition of nanosized metals to dark fermentation systems could increase the efficiency of biogas production [14,15]. The use of photocatalytic semiconductor oxides emerges as a successful technology for the development of bio-hybrid materials. TiO2anatase is by far the most extensively used photocatalyst, as a wide band-gap (3.2 eV) semiconductor under UV illumination could generate energy rich electronehole pairs able to interact with the bioactive compounds inside the microbial cell. The combination of nano semiconductor materials with hydrogen forming biocatalyst has attracted more interest on clean
hydrogen production in recent years in the form of inorganicebio hybrid approaches. Such systems add the great values in hydrogen production using light/solar energy. The well-known hydrogen forming biocatalyst, the hydrogenase, catalyzes the reduction of proton to form hydrogen. The addition of Cu and CuSO4 nanoparticles had a negative effect on the production of biohydrogen in Clostridium acetobutylicum NCIM 2337 and Enterobacter cloacae 811101 [16]. On the other hand, the addition of iron nanoparticles increased the biohydrogen yield by 20% in the mixed co-cultures of Rhodobacter sphaeroides NMBL02 and Escherichia coli NMBL04 [17]. In another study, TiO2 and Fe nanoparticles were shown to have a stimulatory effect on hydrogen production by Clostridium pasteurianum [18]. Recently, photocatalytic hydrogen production under visible light irradiation was achieved by dye sensitized TiO2 nanoparticles with [NieFeeSe]-hydrogenase [13], and another study with CdS nanorods and CdTe nanocrystals capped with mercaptopropionic acid combined with a recombinant Clostridial [FeeFe] hydrogenase [19]. These inorganic semiconductors and biocatalytic systems demonstrate the successful photocatalytic hydrogen production. However, the most important disadvantage of this system is that the lower growth rate of the microorganisms was observed during biocatalyst preparation. Hence, the needs of an efficient biocatalyst that can be obtain by means of simple manipulations and have a potential for high hydrogen productivity. Such a kind of biocatalyst could facilitate the construction of practical inorganic bio-hybrid system for the photocatalytic hydrogen production. The utilization of the most extensively studied bacterium E. coli, the laboratory workhorse, as biocatalyst is a promising method. Under the stimulating conditions E. coli has the capability to interact with nanoparticles on its outer cell surface [20]. Being a facultative anaerobe, its synthesis of an endogenous [NieFe]-hydrogenase is an added advantage of using this E. coli cell [21]. This endogenous [NieFe]-hydrogenase is a major component of the FHL (formate hydrogenlyase) complex, which produces hydrogen directly. There is no need for introducing exogenous hydrogenases to the E. coli cells by genetic engineering. In this study, we developed a hybrid system (TiO2/MV2þ/E. coli) and investigated the possibilities of using E. coli endogenous [NieFe] hydrogenases as biocatalyst and the photogenerated electrons from TiO2 along with electron mediator (MV2þ) on the E. coli cell surface intended for the enhancement of biohydrogen production. The basic mechanism of improved hydrogen production was also studied. Finally, the possibility of using natural sunlight (solar energy) to produce hydrogen by the hybrid system was also demonstrated.
Materials and methods TiO2 optimization The optimum TiO2 concentration was studied in MOPS (3-(Nmorpholino) propanesulfonic acid) medium [22]. E. coli K-12 strain was purchased from E. coli genetic stock centre (CGST), CT, USA. Initially E. coli cells were pre-cultured by aerobic cultivation at 37 C, 150 rpm for 6 h in 5 mL of LB Miller medium. Subsequently, the cell pellet after centrifugation of
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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200 mL of the pre-cultures at 12000 g/10 min was resuspended with 50 mL MOPS medium supplemented with 1 mM cysteine and 6 mM glucose. The final concentrations of 0.125, 0.250, 0.375, 0.500, 0.625 and 0.750 mM of TiO2 and 5 mM of MV2þ were added to parallel groups at the beginning of the inoculation. Control groups without MV2þ were also studied for comparison. All the experimental and control groups were incubated at 37 C, the cell density during the incubation period was monitored spectrophotometrically. At set time intervals, the OD at 600 nm of the culture was recorded using a UVevis spectrophotometer.
Quantification of pyruvate, lactate, formate, and NADH/ NAD
Construction and characterization of the hybrid system
For microscopic observation of E. coli whole cell, a light microscope (Seek, Melbourne, Australia) was used. The reaction mixture consists of TiO2/MV2þ/E. coli for photocatalytic hydrogen production. The samples were collected and directly subjected to microscopic observation before and after light irradiation.
In the glovebox, 50 mL of reaction mixture containing 0.375 mM of TiO2 anatase, 150 mM NaCl, 100 mM Tris-HCl (pH 7), 6 mM glucose, 100 mM ascorbic acid, 5 mM MV2þ and biocatalyst (0.1 g dry cell E. coli) was prepared in a quartz reaction bottle sealed by a rubber septum to protect biocatalysts from oxygen exposure. After 5 h of light irradiation at desired intensity the bacterial cells in the culture samples were harvested by centrifugation at 12000g for 10 min before further characterization. For the preparation of TEM (transmission electron microscope) samples to observe the TiO2 nanoparticles on the bacterial cell surface, the collected cell pellet was washed thrice with milli- Q water and freeze-dried for 48 h. After freeze drying a small piece of dried sample was dissolved in 1 mL of ethanol, and then a formvar-coated copper grid was immersed for a few seconds to collect the sample. Once the sample had been air-dried on the grid, TEM imaging was done (TECNAI 20 G2, FEI make). For SEM (scanning electron microscope) imaging, a small amount of sample from the hybrid system was dissolved in 1 mL of phosphatebuffered saline (PBS) solution, and then samples were fixed using 4% glutaraldehyde at 4 C. After 2 h of fixation, the sample was washed twice with PBS at room temperature, then the sample was serially dehydrated in 50%, 70%, 85%, 95% (twice), and 100% (thrice) with acetone, each time lasting for 10e15 min. The dehydrated sample was critical-point-dried and visualized under SEM (FEI Quanta FEG 200) at 10 kV, to observe the morphology of the E. colieTiO2 hybrid system.
The concentration of pyruvate (ab65342e abcam), lactate (ab65331 e abcam), formate (ab111748 e abcam) and NADH/ NAD (ab65348 e abcam) were determined by respective quantification assay kits. All the experiments were carried out by following the technical bulletins given in the respective assay kits.
Microscopy analysis
Assay for E. coli cell lysis To examine whether the E. coli cells in the hybrid system lyses during the photocatalytic reaction, the activity of b -galactosidase (b-gal) was used to measure the cell lysis in the supernatant of the reaction mixture of TiO2/MV2þ/E.coli hybrid system. The b -galactosidase activity can be simply monitored by 420 nm absorption by using 2-nirotophenyl b -D-galactopyranoside (ONPG) [24].
Feasibility studies A 50 mL of reaction mixture consisting of the control E. coli and the hybrid system (TiO2/MV2þ/E.coli culture) was kept under natural sunlight (10: 00 A.M to 3:00 P.M) and used for the irradiation to find the hydrogen production efficiency. For comparative analysis, TiO2/MV2þ/whole cell E. coli hybrid system under irradiation by a xenon lamp (2000 W m2 visible light) for 15 h was also performed. Hydrogen production experiments were conducted in 50 mL sealed transparent glass bottles, and the hydrogen production was monitored by GC at fixed time intervals. All the experiments were carried out in triplicate and average values were reported.
Hydrogen production under different visible light intensity The visible light (VL) intensity from the xenon (Xe) lamp was adjusted to 0, 500, 1000, 2000, 3000, and 4000 W m2 using a lux meter. The 50 mL samples of cultured hybrid system (TiO2/ MV2þ/E. coli) were illuminated under different light intensities, and E. coli cultures without TiO2/MV2þserved as the control group. The hydrogen production was monitored at fixed time intervals or at 15 h for accumulative hydrogen yield by GC (gas chromatography) analysis, (Perichrom, France) equipped with a thermal conductivity detector, a Molecular Sieve 5A 60/80 mesh column, using argon as a carrier gas.
Glucose determination Dinitrosalicylic acid (DNS) colorimetric method [23], was used to monitor the glucose utilization efficiency of the TiO2/MV2þ/ E. coli hybrid system and the control E. coli.
Results and discussion Optimization of TiO2 concentration In the aquatic ecosystem, titanium dioxide (TiO2) is highly toxic to both marine and freshwater microorganisms [25,26]. Numerous stress responses action in E. coli induced by TiO2, causes metabolic changes was reported [27]. In general, most of the polar molecules cannot cross the microbial cell membrane easily and, indeed, MV2þ (the redox mediator) have been reported as an impermeant cation for E. coli cell membrane [28]. The redox mediator (MV2þ) is known to affect the intracellular NADH level and modify the NADH related metabolic pathways [29,30]. Fig. 1 clearly demonstrated that the concentration of TiO2 higher than 0.375 mM strongly inhibited the growth of E. coli regardless of the presence or the absence of
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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large amounts of TiO2 particles were deposited on the E. coli cell surface (Fig. 2). It is well known that the particle size is a major key factor which affects the photocatalytic activity of the semiconductor materials, as it influences the particular surface area, charge carrier recombination rate and electron hole pair separation efficiency [31]. Hence, the nanosized TiO2 particles with MV2þ on the E. coli cell surface have the higher potential to display excellent photocatalytic activity. This close link and the delicate structure efficiently combined the inorganic system with the biological system. The scheme illustrating the formation of bio-hybrid system confirmed by TEM and SEM images is shown in Fig. 2.
Hydrogen production efficiency of the hybrid system Fig. 1 e Effect of TiO2 concentration on cell density (OD600) of E. coli in MOPS medium with and without 5 mM MV2þ after a culture time of 20 h. MV2þ. A slightly decreased growth of E. coli was observed at 0.375 mM TiO2, therefore this TiO2 concentration was used for further study on hydrogen production by TiO2/MV2þ/E.coli hybrid system.
Hybrid system construction and characterization From the above results, the hybrid system was constructed using 0.375 mM TiO2 and 5 mM MV2þ. The TEM images showed the strong aggregation between the nanoparticles (TiO2) and the electron mediator (MV2þ) in the reaction mixture (Fig. 2). These nanoparticles exhibited good equality and their average size was around 10e20 nm. SEM images further revealed that
The hydrogen production from the E. coli without TiO2(control) and TiO2/MV2þ/E. coli hybrid system irradiated with different visible light (VL) intensities was studied. As shown in Fig. 3a, an increase in VL intensity up to 3000 W m2 resulted in an increase of hydrogen production of the hybrid system with maximum production of 3.6 mmol/mmol glucose observed at 2000 W m2, a 2.8-fold increase than that of control. At VL intensity higher than 500 W m2, the hybrid system invariably had higher hydrogen production than the control. Too high light intensity led to a decreased hydrogen production in both the hybrid and the control systems, suggesting that under high light intensity the self defense and auto repair mechanisms of the bacterium was insufficient to protect the cell to allow for sustained production of hydrogen. As seen from material chemistry view point, these results indicate that, when an inorganic semiconductor was combined with a biological system, the efficiency of the hydrogen production can
Fig. 2 e The scheme illustrating the formation of bio-hybrid system confirmed by SEM and TEM images. Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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depletion of carbon source availability. However, the hydrogen production continued for 3 cycles up to 15 h where the hybrid system with electron mediator produced more hydrogen (3.6 mmol/mmol glucose) compared with that without electron mediator and E. coli alone (2.5 and 1.3 mmol/ mmol glucose respectively). This result clearly demonstrated that the extracellularly added MV2þ could transfer more photo-generated electrons to the intracellular bioactive compounds inside the living cells, leading to enhanced hydrogen production. The hydrogen production by different hybrid systems was compared with the results of the present study as shown in Table S1 (Supporting Information). The hybrid system using E. coli and TiO2 in the present study showed highest hydrogen yield, and TiO2 was superior to CdS as nanoparticle in the E. coli hybrid system. The results of microscopic examination and the b-galactosidase assay suggested that the cells of E. coli in the hybrid system had strong aggregation with the semiconductor and no cell lysis occurred under the conditions of photocatalytic reactions (Supporting Information, Figs. S1 and S2). This indicates the durability of TiO2/MV2þ/E.coli hybrid system for high efficiency and long term hydrogen production.
Proposed mechanism
Fig. 3 e Hydrogen accumulation under different conditions. (A) Hydrogen production of control E. coli and the hybrid system (TiO2/MV2þ/E.coli) under different visible light intensities for 15 h. (B) Hydrogen production by the hybrid system with (TiO2/MV2þ/E.coli) and without (TiO2/E.coli) electron mediator for 3 cycles of 5 h each.
be significantly improved as reported in a previous study using genetic manipulations to express exogenous [Fe -Fe]hydrogenase in E. coli cells [24]. The TiO2/MV2þ/E.coli hybrid system under 2000 W m2 irradiation in the present study could achieve a great conversion rate of glucose to hydrogen by 90% of theoretical value (100% represents 24 mmol H2/ 6 mmol glucose). Furthermore, we have done some analyses of repeated hydrogen production efficiency (with and without electron mediator (MV2þ) of TiO2/MV2þ/E.coli, TiO2/E.coli and E. coli alone under 5 h of irradiation for three cycles, where each cycle was subject to the evacuation of gas from the reaction mixture before starting the next cycle. As shown in Fig. 3b, during the first cycle hydrogen production of 2.2 mmol/mmol glucose was observed in TiO2/MV2þ/E.coli hybrid system, whereas in TiO2/E.coli hybrid system and E. coli alone produced 1.6 and 0.5 mmol H2/mmol glucose, respectively. It should be mentioned that no hydrogen production was observed in TiO2/MV2þ system lacking E. coli. After the first cycle, the hydrogen production decreased gradually in the following two cycles in the hybrid systems and the control group due to
The mechanism for the improved hydrogen production from the hybrid system is schematically illustrated in Fig. 4. When the TiO2 nanoparticles along with MV2þ (redox mediator) are irradiated by a suitable intensity of visible light, large amounts of electron-hole pairs could be generated. The electrons generated by the TiO2 nanoparticles assisted by redox
Fig. 4 e Proposed schematic mechanism of the enhanced hydrogen evolution in the hybrid system (the combination of TiO2, methyl-viologen, and the whole cell E. coli).
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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mediator could interact with bioactive components and incorporate into the hydrogen production pathway. In E. coli hydrogen production pathway, the accessible intracellular glucose acts as an electron donor and an energy source. The analysis of various intermediates as shown in Fig. 5 supports the flow of metabolites from glucose to hydrogen depicted in Fig. 4. The hybrid system enabled E. coli to increase hydrogen production with the increase in incubation time with higher production under light conditions (Fig. 5a). Glucose, an electron donor, under both dark and light conditions was rapidly decreased during the first 1 h with a continued decrease until 5 h incubation (Fig. 5b). No major difference in hydrogen production was observed under these two conditions, thus suggesting that glucose was rapidly utilized by E. coli cells and
transferred it into glycolysis pathway without any involvement of the photo-generated electrons. However, as shown in Fig. 5c, the concentration of pyruvate in light-irradiated E. coli was increased much more in the first 3 h than that of the E. coli under dark condition, suggesting that a portion of photogenerated electrons may take part in the glycolysis and accelerated the process resulting in higher pyruvate formation. On the other hand, a lower level of intracellular lactate accumulation was observed in light-irradiated E. coli compared to that under dark condition (Fig. 5d), demonstrating that the lactate fermentative pathway under light condition could be inhibited by photo-generated electrons resulting in more pyruvate conversion to formate. The results in Fig. 5e indicates that the concentration of formate in the
Fig. 5 e Hydrogen production (A) and metabolic levels of glucose (B), pyruvate(C), lactate (D), formate (E), and NADH/NAD ratio (F) of the hybrid system at various times under light (2000 W m¡2) and dark conditions. Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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dark treated E. coli decreased gradually in the first 2 h, afterward remained at nearly a constant level, whereas in the light-irradiated E. coli it decreased faster in the first 2 h, indicating the higher formate consumption. After 2 h of irradiation an increased concentration of formate was observed corresponding to the decrease of pyruvate level seen in Fig. 2c. The increased availability of formate serves as substrate for the FHL complex, which contains (NieFe) hydrogenases catalyzing the breakdown of formate into CO2 and H2 during anaerobic fermentation [32]. Hence, this superior catalytic action would lead to rapidly consumption of formate which results in increased accumulation of hydrogen. The results showed in Fig. 5f demonstrate that in the first 2 h of visible light irradiation, the NADH to NAD ratio in the TiO2/MV2þ/ E.coli hybrid system was a little higher compared to that under dark conditions. The photo-generated electrons could stimulate rapid recycling of NADH leading to high reduction potential inside the bacterial cell, which benefits the hydrogen production process. The decrease in NADH/NAD ratio suggested the increase oxidation of NADH to generate NAD in conjunction with the increased reduction of proton to generate hydrogen. Hydrogen production is usually accompanied by the production of volatile fatty acids (VFAs) such as acetic acid, butyric acid and succinic acid and solvents like ethanol. After the measurement of these metabolites, it was observed that acetic acid was found to be 0.19 mg/mL, succinic acid and ethanol were found to be 0.06 and 0.07 mg/mL respectively, whereas only trace amount of butyrate was found (data not shown). Thus, acetate was found to be the major end metabolite of this mixed acid fermentation. This hybrid system follows acetate based hydrogen production.
Feasibility study Finally, the feasibilities of applying natural sunlight (solar irradiation) for the hydrogen production using the hybrid system were thoroughly investigated as shown in Fig. 6. The hydrogen production capabilities of different combinations of natural (sunlight) and laboratory (artificial light) conditions were compared. The results illustrates that the laboratory conditions (the hybrid system using a Xe lamp for light irradiation) yielded higher hydrogen production
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than that using natural sunlight. The E. coli without the hybrid system under natural sunlight and laboratory condition had low efficiency of hydrogen production. The lower hydrogen production by the hybrid system under natural sunlight compared to that under xenon lamp due to the variation of actual intensity of sunlight with the hour of day, season, geo-graphical location and atmospheric conditions or due to the natural UV (ultraviolet) irradiation causing harmful effects and reducing the physiological action of microbes under natural conditions. Hence, more intense research work must be needed to optimize the hydrogen production and further improving the hybrid system for its application using natural resources, such as natural sunlight optimization and wastewater as culture medium.
Conclusions The possibility of combining inorganic semiconductor nanoparticles with microorganisms to enhance the renewable energy production has recently become a hot field of research. In the present study we developed a new hybrid system for the efficient photocatalytic hydrogen production. Under optimal visible light irradiation TiO2/MV2þ/E.coli hybrid system could efficiently generate 3.6 mmol H2/mmol glucose, whereas under the same condition TiO2/E.coli hybrid system and the E. coli alone produced 2.5 and 1.3 mmol/mmol glucose respectively. The results clearly demonstrate that TiO2/MV2þ/E.coli hybrid system produces a 2.8-fold increase in hydrogen production compared to the control E. coli in 15 h. The TiO2 nanoparticles with the addition of electron mediator (MV2þ) could facilitate the increase of photogenerated electrons transferred into the E. coli after visible light excitation on the extracellular side. Furthermore, the mechanistic study revealed that the increase in the rate of pyruvate formation, lactate inhibition, boost of formate concentration, and reduction of NADH/ NAD ratio were found to be the key mechanism in the hybrid system for the improved production of hydrogen. Finally, the hybrid system under natural sunlight irradiation produced 1.8 mmol H2/mmol glucose, whereas the E. coli alone under the same condition produced only 1.2 mmol H2/ mmol glucose, indicating the cost effective operation for hydrogen production. This semiconductor/intact E. coli cells hybrid system is a promising approach towards the development of a practical and clean photocatalytic hydrogen production in the future.
Acknowledgements
Fig. 6 e Hydrogen production by the control E. coli and the hybrid system under sunlight and xenon lamp (2000 W m¡2).
B.R. is thankful to the Graduate School, Chulalongkorn University (CU), for post-doctoral fellowship from Ratchadaphiseksomphot Endowment Fund. A.I. acknowledges the research grants from CU on the Frontier Research Energy Cluster (CU-59-048-EN) and from Thailand Research Fund (IRG 5780008).
Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2020.01.011.
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Please cite this article as: Ramprakash B, Incharoensakdi A, Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles, International Journal of Hydrogen Energy, https://doi.org/10.1016/j.ijhydene.2020.01.011