Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes

Accepted Manuscript Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes Richen Lin, Jun Cheng, Lingkan Ding, Wenlu Song, Min Liu, Junhu Zhou, Kefa Cen PII: DOI: Reference:

S0960-8524(16)30124-9 http://dx.doi.org/10.1016/j.biortech.2016.02.009 BITE 16059

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

12 December 2015 31 January 2016 1 February 2016

Please cite this article as: Lin, R., Cheng, J., Ding, L., Song, W., Liu, M., Zhou, J., Cen, K., Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.02.009

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Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes Richen Lina, Jun Chenga*, Lingkan Dinga, Wenlu Songa,b, Min Liua, Junhu Zhoua, Kefa Cena a

State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China

b

Department of Life Science and Engineering , Jining University, Jining 273155, China

Abstract Ferric oxide nanoparticles (FONPs) were used to facilitate dark hydrogen fermentation using Enterobacter aerogenes. The hydrogen yield of glucose increased from 164.5 ± 2.29 to 192.4 ± 1.14 mL/g when FONPs concentration increased from 0 to 200 mg/L. SEM images of E. aerogenes demonstrated the existence of bacterial nanowire among cells, suggesting FONPs served as electron conduits to enhance electron transfer. TEM showed cellular internalization of FONPs, indicating hydrogenase synthesis and activity was potentially promoted due to the released iron element. When further increasing FONPs concentration to 400 mg/L, the hydrogen yield of glucose decreased to 147.2 ± 2.54 mL/g. Soluble metabolic products revealed FONPs enhanced acetate pathway of hydrogen production, but weakened ethanol pathway. This shift of metabolic pathways allowed more nicotinamide adenine dinucleotide for reducing proton to hydrogen. Key words: Ferric oxide nanoparticles; Enterobacter aerogenes; Hydrogen fermentation.

*

Corresponding author: Prof. Dr. Jun Cheng, State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China. Tel.: +86 571 87952889; fax: +86 571 87951616. E-mail: [email protected]

1

1 Introduction The rapid depletion of non-renewable fossil fuels (e.g., coal, petroleum and natural gas) has led to severe energy crisis and environmental pollution, both of which emphasise the significance of renewable biofuel production. Hydrogen is a clean carbon-free fuel and an ideal secondary energy carrier with high energy density (Kothari et al., 2012; Turner, 2004). Currently hydrogen is mainly produced by steam reforming of hydrocarbons or coal gasification which entails the use of fossil fuels and emits greenhouse gas (Christopher & Dimitrios, 2012). Biohydrogen production by dark fermentation offers the advantages of energy-saving, longer-term sustainability and favourable carbon balances (Hallenbeck, 2009; Xia et al., 2015). Facultative anaerobes such as Enterobacter and strict anaerobes such as Clostridium are efficient biohydrogen producers among a large number of hydrogen-producing microorganisms. Enterobacter strains are considered to be more promising for industrial scale hydrogen production due to their rapid growth rates, ability to utilize a wide range of substrates and strong adaptability to dissolved oxygen, H2 pressure and pH (Zhang et al., 2011). To improve the hydrogen yield and production rate of Enterobacter strains, previous studies have focused on parameter optimization (e.g., pH, temperature and substrate concentration) and metabolic bioengineering (e.g., overexpression of hydrogenase) (Mishra & Das, 2014; Zhang et al., 2011). Recently, due to the unique physical and chemical properties of nanoparticles, enormous interests have been focused on their application in dark hydrogen fermentation, aiming to enhance bioactivity of hydrogen-producing microbes and further improve the yield and rate of hydrogen production. The hematite and nickel oxide nanoparticles were investigated to enhance the activity of ferredoxin oxidoreductase by increasing electron transfer rate, which in turn enhanced the 2

activity of hydrogenase (Gadhe et al., 2015a). The resulting hydrogen yield from dairy wastewater increased 27% using mixed hydrogen-producing bacteria as inoculum. Han et al. reported the hydrogen yield from sucrose increased by 32.6% with the addition of 200 mg/L hematite nanoparticles (Han et al., 2011). The enhancement was ascribed to the iron release from hematite nanoparticles, which kept the proper iron concentrations for mixed bacteria. Beckers et al. investigated the improving effects of metal (Pd, Ag and Cu) nanoparticles and metal oxide (FexOy) nanoparticles on hydrogen fermentation using Clostridium butyricum (Beckers et al., 2013). The results suggested an improvement of electron transfer through some combinations between enzymes and nanoparticles. The related studies concluded that the application of nanoparticles was a promising approach to enhance hydrogen fermentation from various feedstock (Beckers et al., 2013; Gadhe et al., 2015b; Han et al., 2011; Mohanraj et al., 2014a; Mohanraj et al., 2014b; Mullai et al., 2013; Nasr et al., 2015; Wimonsong et al., 2013; Wimonsong et al., 2014; Zhang & Shen, 2007; Zhao et al., 2013b). Nevertheless, the researches of ferric oxide nanoparticles (FONPs) on dark hydrogen fermentation from glucose and cassava starch are still limited, especially using Enterobacter aerogenes. Furthermore, few attempts have been tried to address the morphologic response of E. aerogenes cells to nanoparticles. In the present study, the effects of FONPs on hydrogen fermentation from glucose and pre-treated cassava starch using E. aerogenes were evaluated in terms of hydrogen yield, production rate and metabolites distribution. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the morphologic changes of E. aerogenes cells in response to FONPs.

2 Materials and Methods 3

2.1 Microorganisms and materials E. aerogenes ATCC13408 was purchased from China General Microbiological Culture Collection Center. The strain was grown in Luria Bertani (LB; 5 g yeast extract, 10 g peptone, and 10 g NaCl per liter) medium in an incubator shaker at 250 rpm and 37 °C. The fermentation substrates of glucose and cassava starch were purchase from Sinopharm Chemical Reagent Co, Ltd, China and Shanghai Heyu Trade Co., Ltd., China, respectively. FONPs (γ-Fe2O3; 20 nm in diameter) were purchased from Aladdin Industrial Inc., China.

2.2 Pre-treatment of cassava starch Cassava starch was subjected to steam-heating acid pre-treatment prior to biohydrogen fermentation. Approximately 3.0 g of cassava starch and 100 mL diluted H2SO4 solution were mixed in conical flasks. The conical flasks were placed in an autoclave (Sanyo MLS-3780, Japan), and then heated by steam at 135 °C for 15 min. The pre-treated cassava starch was then used for fermentation.

2.3 Biohydrogen fermentation Experimental design on hydrogen production from glucose and pre-treated cassava starch with different concentrations of FONPs were shown in Table 1. The seed culture (1 ml) of of E. aerogenes ATCC13408 was inoculated into 100 ml of LB medium for batch cultivations. The cultivations were performed aerobically in an incubator shaker at 37 °C and 220 rpm until the cells grew to mid-log phase. After cultivation for over 8 h, the optical density at 600 nm (OD600) was determined as 0.354 after diluting the culture for 10 times. The 100 mL of E. aerogenes cultures were then added into glass bottles with 200 ml fermentative medium. The fermentative medium was sterilized at 121 °C for 30 min in an autoclave. For hydrogen fermentation of glucose, 4

the fermentative medium contained 198 mL deionized water, 3 g glucose, 0.03 g FeCl2 , 1.2 g peptone, 0.6 g yeast extract, 1 ml vitamin liquid, and 1 ml microelement liquid (Xie et al., 2008). For hydrogen fermentation of pre-treated cassava starch, the fermentative medium contained 100 mL solution of pre-treated cassava starch, 98 mL deionized water, 0.03 g FeCl2, 1.2 g peptone, 0.6 g yeast extract, 1 ml vitamin liquid, and 1 ml microelement liquid. The initial pH for fermentation of glucose and pre-treated cassava starch was both adjusted to 6.0 ± 0.1 by using 6 M HCl and 6 M NaOH solution. Then different amounts of FONPs were added to each bottle. The concentrations of FONPs were set to 0, 50, 100, 200, 300, and 400 mg/L. Subsequently, the bottles were sealed with rubber stoppers, purged with nitrogen gas for 10 min, and maintained at 37 ± 1.0 °C for fermentation. The pH value and gas compositions during fermentation were measured at an interval of 12 h as well as the analysis of gas composition. The pH value was readjusted to 6.0 every 12 h in order to prevent severe pH drop during fermentation. All the experiments were conducted in duplicate.

2.4 Microscope observation SEM micrographs of E. aerogenes in response to FONPs were obtained on a field emission scanning electronic microscope (Hitachi SU 8010, Japan) after the samples were sputtered with a thick layer of gold. The ultrastructural changes of E. aerogenes in response to FONPs were observed by TEM (Hitachi H-7650, Japan) at 120 keV electron-energy emission after staining the samples with UO2(CH3COO)2. The samples preparation for SEM and TEM imaging was detailed in a previous study (Cheng et al., 2013).

2.5 Analytical methods The optical density value of the E. aerogenes culture used for inoculation was detected by a 5

UV/VIS spectrophotometer (Unico 2600, China). The concentrations of hydrogen and carbon dioxide were analyzed on a gas chromatography system (GC; Agilent 7820A, USA) equipped with a thermal conductivity detector and a 5A column (Φ 3 mm × 3 m; Agilent, USA). The temperatures of injection port and thermal conductivity detector were 200 °C and 300 °C, respectively. The initial column temperature was set at 65 °C for 1 min, increased to 145 °C at a heating rate of 25 °C/min, and then held at 145 °C for 3 min. Argon gas was used as the carrier gas at a flow rate of 27 mL/min. Soluble metabolic products (SMPs) were analyzed on another GC system (Agilent 7820A, USA) equipped with a flame ionization detector and a DB-FFAP column (Φ 0.32 mm × 50 m; Agilent, USA). The temperatures of injection port and flame ionization detector were both 250 °C. The initial column temperature was set at 100 °C for 1 min, increased to 200 °C at a heating rate of 10 °C/min, and then held at 200 °C for 2.5 min. For the determination of specific SMPs (i.e., ethanol, acetic acid, propionic acid, iso-butyric acid, butyric acid, iso-valeric acid, valeric acid and caproic acid), the liquid samples were first centrifuged at 5000 rpm for 5 min and then adjusted with HCl to pH 2.0. The quantification of each component was determined by a standard solution, containing 0.06 v/v% of ethanol, 0.06 v/v% of acetic acid, 0.06 v/v% of propionic acid, 0.03 v/v% of iso-butyric acid, 0.06 v/v% of butyric acid, 0.03 v/v% of iso-valeric acid, 0.06 v/v% of valeric acid and 0.06 v/v% of caproic acid. Hydrogen yields were simulated by the modified Gompertz equation, and dynamic parameters (Hm, maximum hydrogen yield potential, mL/g, Rm, peak hydrogen production rate, mL/g/h; λ, lag-phase time of hydrogen production, h; and Tm, peak time, h) were calculated using Origin 8.5 software.

3 Results and discussion 6

3.1 Effects of FONPs on biohydrogen fermentation 3.1.1 Effects of FONPs on biohydrogen production from glucose and pre-treated cassava starch The effects of different concentrations (0 – 400 mg/L) of FONPs on hydrogen yield and production rate from glucose are illustrated in Fig. 1. The hydrogen yield gradually increased from 164.5 ± 2.29 to 171.8 ± 1.52, 183.3 ± 2.03, 192.4 ± 1.14 mL/g with different addition of FONPs (0 – 200 mg/L). Correspondingly, the highest peak hydrogen production rate increased to 7.2 ± 0.05 mL/g/h at fermentation time of 36 h with the addition of 200 mg/L FONPs. The addition of 200 mg/L FONPs resulted in a maximum increase of hydrogen production by 17.0% as compared to no addition of FONPs, and the corresponding peak hydrogen production rate increased by 35.8%. The enhancement of hydrogen yield and production rate in the presence of nanoparticles were possibly due to the enhanced hydrogenase activity and increased electron transfer efficiency in E. aerogenes cells. It was reported that iron was gradually released out of hematite nanoparticles in the presence of produced volatile fatty acid (Han et al., 2011). Considering that the hydrogenase enzyme is classified into [Fe-Fe] and [Ni-Fe] hydrogenase, the proper iron concentration potentially provided the essential element for hydrogenase synthesis and bacteria growth, resulting in increased hydrogen yield. Furthermore, FONPs had good conductive properties, indicating that FONPs potentially acted as electron conduits to enhance electron transfer during fermentation (Kato et al., 2012a; Kato et al., 2012b). It was reported that the nanoparticles enhance ferredoxin oxidoreductase activity by increasing electron transfer rate owing to an enhanced surface and quantum size effects, thus increase the hydrogen production yield during dark fermentation (Han et al., 2011; Mohanraj et al., 2014b). Bacterial nanowires were extracellular appendages that were 7

suggested as pathways for electron transfer in microorganisms. Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1 was demonstrated (El-Naggar et al., 2010). Liu et al. reported that the addition of activated carbon, which has high electrical conductivity, facilitated electron transfer in anaerobic fermentation using co-cultures of Geobacter metallireducens and Geobacter sulfurreducens (Liu et al., 2012). By adding FONPs in fermentation, the electron transfer process was likely enhanced by FONPs due to the good conductive properties. Jiang et al. demonstrated that nanoparticles can serve as “bridges” to facilitate efficient extracellular electron transfer between interconnected microbial cells (Jiang et al., 2014). Viggi and co-workers showed that the supplementation of micrometer-size magnetite particles enhanced electron transfer efficiency and the methane production rate was increased by 33% in syntrophic microbial communities (Cruz Viggi et al., 2014). The hydrogen yield was found to be decreased with increasing the concentration above 300 mg/L. The addition 400 mg/L FONPs decreased the hydrogen yield to 147.2 ± 2.54 mL/g, representing a 10.5% decrease over the control (no addition of FONPs). An increased toxicity brought to E. aerogenes cells owing to the cell wall penetration, breakage of the cell wall and oxidative stress caused by higher concentration of nanoparticles could be a possible reason (Gadhe et al., 2015a). Therefore, it was concluded that a suitable addition of nanoparticles stimulated E. aerogenes cells and promoted hydrogen fermentation from glucose, while excess addition of nanoparticles exhibited inhibitions on E. aerogenes cells and decreased hydrogen production. Han et al. investigated the effects of haematite nanoparticles on hydrogen fermentation from sucrose using mixed bacteria, and concluded that 200 mg/L of nanoparticles was the optimal concentration (Han et al., 2011). The addition of nanoparticles above optimal level resuled in an increased oxidative stress and inhibited hydrogen fermentation. 8

Mohanraj et al. and Gadhe et al. demostrated a suitable addition of iron oxide nanoparticles enhanced hydrogen fermentation using Enterobacter cloacae (Mohanraj et al., 2014b) and mixed bacteria (Gadhe et al., 2015a), respectively. The dynamic parameters of hydrogen production from glucose fitted by the modified Gompertz equation are shown in Table 2. It confirmed that the maximum hydrogen yield potential (Hm, 205.2 mL/g) was achieved with the addition of 200 mg/L FONPs, corresponding to 18.2% higher than the control. Accordingly, the peak hydrogen production rate (Rm, 6.8 mL/g/h) was obtained with the addition of 200 mg/L FONPs, corresponding to 19.3% higher than the control. When further increasing the concentrations above 300 mg/L, the hydrogen yield potential and peak hydrogen production rate were decreased to some extent. These results suggested that the addition of nanoparticles had an evident influence on the kinetics of hydrogen production from glucose. Cassava starch is a polysaccharide consisting of a large number of glucose units joined by glycosidic bonds. Raw cassava starch is hard to be fermented by E. aerogenes. Prior to hydrogen fermentation, raw cassava starch was subjected to steam heating with dilute acid pre-treatment for saccharification. The effects of different concentrations (0 – 400 mg/L) of FONPs on hydrogen yield and production rate from hydrolysed cassava starch are illustrated in Fig. 3. The hydrogen yield gradually increased from 76.2 ± 2.29 to 77.5 ± 0.19, 90.0 ± 2.90, 124.3 ± 12.92 mL/g with the addition of FONPs (0 – 200 mg/L). Correspondingly, the peak hydrogen production rate increased to 4.5 ± 0.35 mL/g/h at fermentation time of 24 h with the addition of 200 mg/L nanoparticles. The addition 200 mg/L FONPs resulted in a maximum increase of hydrogen production by 63.1%, and the corresponding peak hydrogen production rate increased by 36.4%. It 9

was noted that the increase of hydrogen yield from hydrolysed cassava starch (63.1%) was higher than that from glucose (17.0%) with the addition of 200 mg/L nanoparticles. The possible reason is that the starch after pre-treatment was further degraded with the suitable addition of nanoparticles. Nasr et al. demonstrated the addition of maghemite nanoparticles enhanced the hydrolysis of starch wastewater and promoted the anaerobic conversion of starch (Nasr et al., 2015). The hydrogen yield was found to be decreased with increasing the concentration to 300 and 400 mg/L. The addition 400 mg/L nanoparticles decreased the hydrogen yield to 72.2 ± 6.57 mL/g, representing a 5.2% decrease over the control. In conclusion, a suitable addition of nanoparticles stimulated E. aerogenes cells and promoted hydrogen fermentation from pre-treated starch, while excess addition of nanoparticles exhibited inhibitions on E. aerogenes cells and decreased hydrogen production. Table 3 shows the dynamic parameters of hydrogen production from pre-treated starch fitted by the modified Gompertz equation. The maximum hydrogen yield potential (Hm, 125.8 mL/g) was achieved with the addition of 200 mg/L FONPs, corresponding to 65.1% higher than the control. Accordingly, the peak hydrogen production rate (Rm, 5.3 mL/g/h) was obtained, corresponding to 39.5% higher than the control. When further increasing the concentrations of FONPs above 300 mg/L, the hydrogen yield potential and peak hydrogen production rate were decreased to some extent. These results suggested that the addition of nanoparticles have an evident influence on the kinetics of hydrogen production from pre-treated starch.

3.1.2 Effects of FONPs on SMPs formation after hydrogen fermentation from glucose and pre-treated cassava starch The effects of FONPs on SMPs from glucose are shown in Fig. 2. Ethanol, acetate, 10

propionate, butyrate, iso-butyrate, valerate, iso-valerate and caporate were detected as the main SMPs after hydrogen fermentation. Ethanol and acetate were the main components, accounting for 77.3-81.1% of SMPs. The main metabolic pathways of glucose by E. aerogenes are shown in Eq. (1) and (2) as follows (Azwar et al., 2014; Yuan et al., 2008): Ethanol pathway: C6 H12O6 → 2C2H6O + 2CO2

(1)

Acetic acid pathway: C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2

(2)

As shown in Fig. 2, the total amounts of SMPs (79.7 mM) in the control experiment contained ethanol (36.4 mM), acetate (25.5 mM), propionate (7.1 mM), butyrate (4.3 mM), iso-butyrate (2.7 mM), valerate (0.5 mM), iso-valerate (2.9 mM) and caporate (0.4 mM), suggesting that E. aerogenes preferred ethanol and acetate pathway. When 200 mg/L nanoparticles were added in fermentation, the total amounts of SMPs significantly increased to 107.6 mM, containing ethanol (45.0 mM), acetate (41.9 mM), propionate (8.0 mM), butyrate (8.0 mM), iso-butyrate (1.6 mM), valerate (0.6 mM), iso-valerate (2.2 mM) and caporate (0.4 mM). The metabolite distribution was similar to that of the control, viz., ethanol and acetate were the main metabolic products. However, it was observed that the proportion of ethanol decreased to 41.8% with the addition of 200 mg/L nanoparticles, compared to 45.6% in the control experiment without nanoparticles addition. In contrary, the proportion of acetate increased from 32.0% (control experiment) to 38.9% with the addition of 200 mg/L nanoparticles. As shown in Eq. (1) and (2), 1 mole glucose theoretically generates no hydrogen by ethanol pathway, while 1 mole glucose theoretically generates 4 mole hydrogen by acetic acid pathway. Therefore, the addition of nanoparticles caused the changes in metabolic pathways of E. aerogenes, resulting in increased acetate proportion and decreased ethanol proportion in SMPs. Corresponding, the hydrogen 11

production was enhanced. The effects of FONPs on SMPs from pre-treated cassava starch are shown in Fig. 4. Ethanol and acetate were the main components, accounting for over 70.0% of SMPs. This result indicated that E. aerogenes preferred ethanol and acetate pathways, as shown in Eq. (1) and (2). The total amounts of SMPs (47.8 mM) in the control experiment contained ethanol (25.8 mM), acetate (12.2 mM), propionate (2.6 mM), butyrate (2.4 mM), iso-butyrate (2.3 mM), valerate (0.1 mM), iso-valerate (1.9 mM) and caporate (0.01 mM), suggesting that E. aerogenes preferred ethanol and acetate pathway. When 200 mg/L nanoparticles were added in fermentation, the total amounts of SMPs significantly increased to 64.6 mM, containing ethanol (27.9 mM), acetate (22.4 mM), propionate (3.8 mM), butyrate (5.4 mM), iso-butyrate (2.5 mM), valerate (0.2 mM), iso-valerate (2.5 mM) and caporate (0.02 mM). It was observed that the proportion of ethanol decreased to 43.2% with the addition of 200 mg/L nanoparticles, compared to 54.1% in the control experiment without nanoparticles addition. In contrary, the proportion of acetate increased from 26.6% (control experiment) to 34.6% with the addition of 200 mg/L nanoparticles. Therefore, the addition of nanoparticles caused changes in metabolic pathways of E. aerogenes, resulting in increased acetate proportion and decreased ethanol proportion in SMPs. Corresponding, the hydrogen production from hydrolysed starch was enhanced.

3.2 Effects of FONPs on morphologies of E. aerogenes cells after fermentation SEM images were obtained to understand the effects of FONPs (200 mg/L) on the surface morphologies of E. aerogenes cells after hydrogen fermentation from glucose. As shown in Fig. S1 (supplementary material), the surfaces of E. aerogenes cells treated with FONPs (Fig. S1c and d) had more aggregates than those without FONPs (Fig. S1a and b). The aggregates on the 12

surfaces of cells were likely derived from nanowire related proteins in response to the addition of FONPs, which potentially enhanced glucose transportation through cell walls. The bacterial nanowire was clearly observed among E. aerogenes cells. As extracellular appendages, bacterial nanowires played an important role in electron transfer among cells. El-Naggar et al. demonstrated electron transport along the length of bacterial nanowire in S. oneidensis MR-1 (El-Naggar et al., 2010). It appeared that the bacterial nanowire network in Fig. S1d was more complex than that in Fig. S1b. This phenomenon was possibly caused by FONPs, which had high electrical conductivity and enhanced electron transfer during fermentation. TEM analysis was used to further understand the effects of FONPs (200 mg/L) on the morphologies of E. aerogenes cells after hydrogen fermentation from glucose. As shown in Fig. S2, TEM images of E. aerogenes cells treated with FONPs (Fig. S2c and d) exhibited morphologies quite different from those of cells without FONPs (Fig. S2a and b). The cytoplasm of E. aerogenes cells treated without nanoparticles addition was relatively uniform. While a lot of dark spots (~30 nm) were observed in E. aerogenes cells with the addition of 200 mg/L nanoparticles. These specks were likely FONPs, which passed through cell walls during fermentation. This implied that there was cellular internalization and interactions between E. aerogenes cells and nanoparticles. In addition, iron was gradually released from FONPs in the presence of produced volatile fatty acid (Han et al., 2011). The proper iron concentration provided the essential element for hydrogenase synthesis and bacteria growth. Therefore, the hydrogenase synthesis and activity was potentially promoted. It was reported that small nanoparticles such as magnesium oxide (Stoimenov et al., 2002) and zinc oxide (Zhao et al., 2013a) were able to penetrate E. coli cells without leading to much disruption of cell walls. The TEM imaging of the 13

nanoparticles (i.e., TiO2 , CeO2, and ZnO) impacted cells revealed the phenomenon of intracellular nanoparticles accumulation (Yu et al., 2015).

3.3 Proposed mechanisms of enhanced hydrogen fermentation by FONPs Based on the obtained results of hydrogen production from glucose and pre-treated starch, it was concluded that the enhancement of hydrogen yield and production rate in the presence of nanoparticles (200 mg/L) were possibly due to the enhanced hydrogenase activity and electron transfer of E. aerogenes cells. The SMPs profiles suggested that the addition of nanoparticles caused changes in metabolic pathways of hydrogen production, resulting in increased acetate proportion and decreased ethanol proportion in SMPs. Previous studies (Cai et al., 2011) reported two different metabolic pathways of glucose metabolism in hydrogen fermentation as shown in Fig. S3. One is the formate hydrogen production pathway, which depends on formate decomposition by formate hydrogen lyase. The reaction pathway is shown in Eq. (3) as follows: HCOOH → H2 + CO2

(3)

The other is the reduced nicotinamide adenine dinucleotide (NADH)-dependent hydrogen production pathway, which depends on the activity of hydrogenase and involves re-oxidation of NAD. The reaction pathway is shown in Eq. (4) as follows: NADH + H+ → NAD+ + H2

(4)

The proposed mechanisms for enhanced hydrogen fermentation by FONPs were illustrated in Fig. S3. The mechanisms suggested that the optimum level of FONPs possibly increased the hydrogenase activity and enhanced electron transfer. In addition, the changes of metabolic pathways in the presence of FONPs resulted in the increased acetate proportion and decreased ethanol proportion in SMPs. As illustrated in Fig. S3, the generation of ethanol from 1 mol 14

glucose consumed 4 mol NADH, while the generation of acetate does not consumed NADH. Therefore, the changes in metabolic pathways allowed more NADH for converting H+ to H2, resulting in promoted hydrogen production.

4 Conclusions FONPs were used to facilitate dark hydrogen fermentation by enhancing hydrogenase activity and electron transfer of E. aerogenes. The hydrogen yields of glucose and pre-treated starch increased by 17.0% and 63.1% with 200 mg/L FONPs, respectively. TEM images of E. aerogenes showed cellular internalization of FONPs. SEM demonstrated the existence of bacterial nanowire, suggesting FONPs possibly served as electron conduits to enhance electron transfer. The hydrogen yields decreased when further increasing FONPs concentration to 400 mg/L. Metabolites analysis revealed FONPs enhanced acetate proportion in SMPs, but decreased ethanol proportion in SMPs.

Acknowledgements This study was supported by the National Natural Science Foundation – China (51476141), Zhejiang Provincial Natural Science Foundation – China (LR14E060002), National Key Technology R&D Program – China (2015BAD21B01), and Shandong Provincial Natural Science Foundation – China (ZR2014CL001).

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List of figures and tables: Fig. 1 Effects of ferric oxide nanoparticles on hydrogen production from glucose: (a) hydrogen yield, and (b) hydrogen production rate. Fig. 2 Effects of ferric oxide nanoparticles on soluble metabolic products (SMPs) in hydrogen fermentation of glucose. Fig. 3 Effects of ferric oxide nanoparticles on hydrogen production from pre-treated cassava starch: (a) hydrogen yield, and (b) hydrogen production rate. Fig. 4 Effects of ferric oxide nanoparticles on SMPs in hydrogen fermentation of pre-treated cassava starch.

Table 1 Experimental design on hydrogen production from glucose and pre-treated cassava starch with different concentrations of ferric oxide nanoparticles. Table 2 Dynamic parameters of hydrogen fermentation at various concentrations of ferric oxide nanoparticles from glucose using E. aerogenes. Table 3 Dynamic parameters of hydrogen fermentation at various concentrations of ferric oxide nanoparticles from pre-treated cassava starch using E. aerogenes.

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Hydrogen yield from glucose (mL/g)

(a) Concentration of added ferric oxide nanoparticles 0 mg/L 50 mg/L 100 mg/L 200 mg/L 300 mg/L 400 mg/L

200 160 120 80 40 0 0

12

24

36

48

60

48

60

Fermentation time (h)

Hydrogen production rate (mL/g/h)

(b) 8

Concentration of added ferric oxide nanoparticles 0 mg/L 50 mg/L 100 mg/L 200 mg/L 300 mg/L 400 mg/L

7 6 5 4 3 2 1 0 0

12

24

36

Fermentation time (h) Fig. 1 Effects of ferric oxide nanoparticles on hydrogen production from glucose: (a) hydrogen yield, and (b) hydrogen production rate.

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SMPs concentration (mM)

55 50 45 40 35

Ethanol Acetate Propionate iso-Butyrate Butyrate iso-Valerate Valerate Caproate

30 25 20 15 10 5 0

50 100 400 0 200 300 Concentration of added ferric oxide nanoparticles (mg/L) Fig. 2 Effects of ferric oxide nanoparticles on soluble metabolic products (SMPs) in hydrogen fermentation of glucose.

22

(a) Hydrogen yield from hydrolyzed cassava starch (mg/L)

150

Concentration of added ferric oxide nanoparticles 0 mg/L 50 mg/L 100 mg/L 200 mg/L 300 mg/L 400 mg/L

120 90 60 30 0 0

12

24

36

48

60

Fermentation time (h)

Hydrogen production rate (mL/g/h)

(b)

Concentration of added ferric oxide nanoparticles

5

0 mg/L 50 mg/L 100 mg/L 200 mg/L 300 mg/L 400 mg/L

4 3 2 1 0 0

12

24

36

48

60

Fermentation time (h) Fig. 3 Effects of ferric oxide nanoparticles on hydrogen production from pre-treated cassava starch: (a) hydrogen yield, and (b) hydrogen production rate.

23

45

SMPs concentration (mM)

40 35 30

Ethanol Acetate Propionate iso-Butyrate Butyrate iso-Valerate Valerate Caproate

25 20 15 10 5 0

0 50 100 400 200 300 Concentration of added ferric oxide nanoparticles (mg/L) Fig. 4 Effects of ferric oxide nanoparticles on SMPs in hydrogen fermentation of pre-treated cassava starch.

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Table 1 Experimental design on hydrogen production from glucose and pre-treated cassava starch with different concentrations of ferric oxide nanoparticles. Feedstock (10 g/L)

Inoculum (100 mL)

Glucose

Enterobacter aerogenes

0

50

100

200

300

400

Pre-treated cassava starch Enterobacter aerogenes

0

50

100

200

300

400

FONPs: ferric oxide (γ-Fe2O3) nanoparticles.

25

FONPs concentration(mg/L)

Table 2 Dynamic parameters of hydrogen fermentation at various concentrations of ferric oxide nanoparticles from glucose using E. aerogenes.

Concentration of

H2 yield

H2 production rate

Kinetic model parameters

nanoparticles (mL/g)

(mL/g)

(mL/g/h)

Hm (mL/g)

Rm (mL/g/h)

λ (h)

Tm (h)

R2

0

164.5 ± 2.29

5.3 ± 0.14

173.6

5.7

7.3

18.5

0.987

50

171.8 ± 1.52

5.3 ± 0.01

181.8

5.8

6.8

18.3

0.988

100

183.3 ± 2.03

5.8 ± 0.07

194.2

6.1

5.5

17.2

0.983

200

192.4 ± 1.14

7.2 ± 0.05

205.2

6.8

8.4

19.5

0.976

300

178.8 ± 1.96

5.6 ± 0.02

189.2

6.1

6.4

17.8

0.986

400

147.2 ± 2.54

4.5 ± 0.02

152.9

5.6

4.8

14.8

0.994

Feedstock

Glucose

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Table 3 Dynamic parameters of hydrogen fermentation at various concentrations of ferric oxide nanoparticles from pre-treated cassava starch using E. aerogenes.

Concentration of

H2 yield

H2 production rate

Kinetic model parameters

nanoparticles (mL/g)

(mL/g)

(mL/g/h)

Hm (mL/g)

Rm (mL/g/h)

λ (h)

Tm (h)

R2

0

76.2 ± 3.67

3.3 ± 0.22

76.2

3.8

7.2

14.6

0.999

50

77.5 ± 0.19

4.0 ± 0.04

77.5

5.9

8.4

13.2

0.999

Pre-treated

100

90.0 ± 2.90

4.5 ± 0.06

89.1

5.2

8.7

15.0

0.997

cassava starch

200

124.3 ± 12.92

4.5 ± 0.35

125.8

5.3

5.0

13.7

0.999

300

86.4 ± 2.64

4.3 ± 0.04

85.7

5.5

8.0

13.7

0.999

400

72.2 ± 6.57

3.2 ± 0.15

72.2

5.3

6.8

11.8

0.999

Feedstock

27

>Effect of γ-Fe2O3 nanoparticles on H2 fermentation using E. aerogenes was studied. >Addition of 200 mg/L nanoparticles enhanced H2 production by 17.0% from glucose. >Hydrogenase activity and electron transfer were enhanced by 200 mg/L nanoparticles. >SEM and TEM were used to investigate bacteria changes in response to nanoparticles.

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