Biohydrogen production from crude glycerol by two stage of dark and photo fermentation

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Biohydrogen production from crude glycerol by two stage of dark and photo fermentation Teera Chookaew a,b, Sompong O-Thong c, Poonsuk Prasertsan b,* a

Faculty of Agro-Industry, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla 90112, Thailand c Department of Biology, Faculty of Science, Thaksin University, Phatthalung 93110, Thailand b

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abstract

Article history:

Hydrogen production from crude glycerol by two-stage processes of dark fermentation

Received 21 August 2014

using Klebsiella sp. TR17 and photo fermentation using Rhodopseudomonas palustris TN1 (Rps.

Received in revised form

palustris TN1) was investigated in batch experiments. In dark fermentation, the cumulative

26 February 2015

hydrogen production and hydrogen yield was 64.24 mmol H2/L and 5.74 mmol H2/g COD

Accepted 27 February 2015

consumed, respectively with 80.21% of glycerol conversion rate. The dark fermentation

Available online xxx

effluent (DFE) was employed for photo fermentation. Effect of DFE concentrations (0e5

Keywords:

(0.63 g/L), and glutamate (2e8 mM) were optimized. The optimal conditions for hydrogen

Biohydrogen

production from Rps. palustris TN1 were 5 times dilution of DFE without the supplement of

Crude glycerol

yeast extract þ NaHCO3, and 2 mM glutamate. Under the optimum conditions, the cu-

Dark fermentation

mulative hydrogen production of 3.12 mmol H2/L and hydrogen yield of 0.68 mmol H2/g

Photo fermentation

COD consumed was obtained. The total hydrogen yield of two-stage processes was esti-

times dilution), with and without supplementation of yeast extract (2.3 g/L) þ NaHCO3

mated to be 6.42 mmol H2/g COD consumed which was 10.4% of the theoretical yield. Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Introduction The major global crisis is an environmental problem and energy requirement that make the increasing demand for alternative environmentally-friendly energy [1]. Hydrogen is high energy content, renewable energy carrier [2] and considered to be a clean fuel for future [3]. Among the methods for production of hydrogen, the biological process of dark and photo fermentation generates high efficiency of hydrogen [4]. Moreover, dark and photo fermentation have a

potential to use the renewable resources and wastes as substrate for production of hydrogen [5]. In dark fermentation, substrate is converted to hydrogen and organic acids, but the production of organic acids can inhibit the dark fermentative bacteria resulting in low hydrogen production [6]. In photo fermentation, the organic acids from dark fermentation effluent (DFE) can assimilate by photosynthetic bacteria [7,8]. From this point, combination of two-stage processes could achieve high hydrogen production and increase conversion efficiency [9]. Recently, waste materials such as potato starch [10], beet molasses [11], cheese whey wastewater [12], and corncob [13] can successfully be

* Corresponding author. Tel.: þ66 74 286 369; fax: þ66 74 558 866. E-mail address: [email protected] (P. Prasertsan). http://dx.doi.org/10.1016/j.ijhydene.2015.02.133 0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Chookaew T, et al., Biohydrogen production from crude glycerol by two stage of dark and photo fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.02.133

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used as substrate for two-stage processes of hydrogen production as well as waste minimization. Crude glycerol is a by-product generated from biodiesel production in the amount of 1 kg per 10 kg of biodiesel (10% w/ w) [14]. Various investigations of single stage of dark or photo fermentation for hydrogen from crude glycerol have been reported [15e17]. However, to the best of our knowledge, there was no report on two stage processes of dark and photo fermentation of crude glycerol from the biodiesel plant. So far, biohydrogen production from crude glycerol has been carried out by dark fermentation of Klebsiella sp. TR 17 [18], while the possibility of photo fermentation from the dark fermentation effluent of crude glycerol has not been explored. Since substrates to be further used should contain low concentrations of NH4þ ions and had a high C/N ratio [6]. Dark fermentation effluent of crude glycerol had a NHþ 4 ion and C/N ratio of 0.02 mg/L and 100/0.6, respectively which is suitable for photo fermentation. So, the objectives of this study were to evaluate the potential use of crude glycerol as a substrate for hydrogen production by the two-stage of dark and photo fermentation using Klebsiella sp. TR 17 and Rhodopseudomonas palustris TN1, respectively.

Hydrogen production in photo fermentation Dark fermentation effluent (DFE) was obtained after centrifugation (10,000 g for 10 min) and then diluted in the range 1e5 times by mixing with deionized water. The dilutions of undiluted to 5 times dilution refer to the initial COD concentration of 25,600, 22,400, 16,000, 12,800, and 9600 mg/L, respectively. Effect of nutrients supplementation with and without yeast extract (2.3 g/L) þ NaHCO3 (0.63 g/L), were performed and no any further nutrients added. The pH was adjusted to 7.0, flushed with argon to obtain an anaerobic condition and autoclaved. The inoculums of Rps. palustris TN1 (OD660 ¼ 0.5) was added in serum bottles which the initial cell concentration was 0.8e1.2 g/L. Photo fermentation was conducted in batch mode using a 60 mL of serum bottle with a 36 mL working volume. The effect of glutamate concentration on hydrogen production at 0, 2, 4, 6, and 8 mM was tested. All photo fermentation experiments were conducted at 30  C under anaerobic-light (3000 lux) condition.

Materials and methods

Analytical method

Bacterial strains and medium

Hydrogen gas was analyzed using a gas detector (Oldham MX 2100, Cambridge Sensotec Ltd., England). The organic acids in the culture broth were determined using a gas chromatography (Hewlett Packard, HP 6890) equipped with a flame ionization detector (FID) and Innowax column (dimensions 30 m  320 mm  0.25 mm). The temperature of the injection and detector were 240  C and 280  C, respectively. The chromatography was conducted using the following a program described by Yossan et al. [20]. The glycerol, succinic acid, 1,3 propanediol, and 2,3 butanediol concentration were analyzed by HPLC [18]. The liquid samples were centrifuged at 10,000 g for 10 min and then filtered through a 0.2 mm membrane before analyzed with GC and HPLC. Biomass concentration in the experiment was determined by measuring the optical density at 600 nm and dry cell weight. Total solid (TS), soluble solid (SS), pH, and ammonia nitrogen (NH3eN) were determined with the procedures described in the Standard Methods [21]. Chemical oxygen demand (COD) and total nitrogen (TN) concentrations were analyzed using commercial test kits from Spectroquant (Merck Co., Ltd., Germany). Light conversion efficiency is defined as the efficiency by which the light energy can be transformed into hydrogen [22]. The light conversion efficiency was calculated by Eq. (1).

Klebsiella sp. TR17 was isolated from crude glycerol contaminated soil [18]. The hydrogen production medium for this strain contained 11.14 g/L glycerol, 3.4 g/L K2HPO4, 2.47 g/L KH2PO4, 6.03 g/L NH4Cl, 0.2 g/L MgSO4$7H2O, 2.0 g/L yeast extract, 2.0 g/L CaCO3, 5.0 mg/L FeSO4$7H2O, 2.0 mg/L CaCl2, 2.0 mL/L and trace element solution as described by Chookaew et al. [18]. The pH was adjusted to 8.0. The crude glycerol containing 50% purity was used as a carbon source in the culture medium. Rhodopseudomonas palustris TN1 (Rps. palustris TN1) was isolated from Songkhla Lake, Thailand [19]. The medium for the inoculums preparation of Rps. palustris TN1 contained 0.5 g/L KH2PO4, 0.6 g/L K2HPO4, 0.4 g/L NaCl, 0.2 g/L MgSO4$7H2O, 0.05 g/L$CaCl22H2O, 1.0 mg/L FeSO4$7H2O, 2.0 mg/L H3BO3, 2.0 mg/L EDTA-2Na, 1.0 mg/L thiamine$HCl, 0.5 mg/L Na2Moo4$2H2O, 0.1 mg/L ZnCl2, 0.01 mg/L CoCl2$6H2O, 0.01 mg/L CuCl2, 1.5 mg/L biotin, 0.935 g/L glutamic acid, 7.076 g/L acetic acid, 0.096 g/L propionic acid, 2.753 g/L butyric acid, 2.3 g/L yeast extract, 0.63 g/L NaHCO3 [19]. The pH was adjusted to 7.0. The culture was cultivated under anaerobic-light (3000 lux) condition at 30  C.

Hydrogen production in dark fermentation Dark fermentation was performed in a 60 mL serum bottle containing 36 mL of the culture medium as described above that was flushed with nitrogen gas to create anaerobic condition, closed with rubber stoppers and aluminum cap, and then sterilized at 121  C for 20 min. Klebsiella sp. TR17 (OD660 ¼ 0.5) was inoculated in the medium and incubated at 40  C. The cumulative hydrogen production and pH were determined every 4 h.

hð%Þ ¼ ð33:6xpH2 xVH2 Þx100=ðI x A x tÞ

(1)

where pH2 is the density of hydrogen production (g/L), VH2 is the volume of hydrogen production in L, I is the light intensity (W/m2), A is the irradiated area in m2 and t is the duration of produced hydrogen (h) [22]. Hydrogen yield (mmol H2/g COD consumed) is measured as the ratio of actual mmol of hydrogen produced to the grams of COD consumed. All data were conducted based on average from triplicate experiments.

Please cite this article in press as: Chookaew T, et al., Biohydrogen production from crude glycerol by two stage of dark and photo fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.02.133

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Results and discussion

Table 1 e Characteristics of the dark fermentation effluent.

Hydrogen production in dark fermentation stage

Characteristics

The time course profiles of the cumulative hydrogen production and pH during dark fermentation of crude glycerol was illustrated in Fig. 1. Hydrogen production was generated after a lag phase of 6 h. The maximum cumulative hydrogen production (64.24 mmol H2/L), the hydrogen yield (5.74 mmol H2/g COD consumed) and hydrogen content (39%) were achieved with 80.21% of glycerol conversion rate. The initial COD of the culture medium (before the process) was 36,800 mg/L, and the dark fermentation effluent (DFE) had a COD of 25,600 mg/L, indicating a COD removal of 30.43%. The pH of the culture broth dropped from 8.0 to 5.94. The characteristics and concentration of organic acids in the DFE of crude glycerol was summarized in Table 1. The soluble metabolites were produced via the oxidative and reductive pathways for Klebsiella sp. during dark fermentation of glycerol. For oxidative pathway, glycerol is first converted to dihydroxyacetone using enzyme glycerol dehydrogenase. After that, it changes to pyruvate. Finally the pyruvate is metabolized through glycolysis and further changes to ethanol, acetate and butyrate. For reductive pathway, glycerol is finally converted to 1,3propanediol [23].

Total nitrogen (mg/L) Ammonia nitrogen (mg/L) Chemical oxygen demand (mg/L) Total solids (mg/L) Suspended solids (mg/L) Succinic acid (mM) Glycerol (mM) 1,3-Propanediol (mM) 2,3-Butanediol (mM) Ethanol (mM) Acetic acid (mM) Propionic acid (mM) Iso-butyric acid (mM) Butyric acid (mM) Iso-valeric acid (mM) n-Valeric acid (mM) Iso-caproic acid (mM) n-Caproic acid (mM) Heptanoic acid (mM)

Concentration 148 ± 11.31 0.02 25,600 ± 4525 20.99 ± 2.17 3.16 ± 0.02 4.92 ± 0.04 21.49 ± 0.24 47.06 ± 0.44 8.76 ± 0.03 47.29 ± 0.14 11.95 ± 0.29 0.40 ± 0.06 1.41 ± 0.15 0.36 ± 0.07 0.23 ± 0.01 0.26 ± 0.01 8.54 ± 0.73 0.16 ± 0.002 0.13 ± 0.006

significant factor for the hydrogen production (p < 0.05) with the optimum concentrations of 2.30 g/L and 0.63 g/L, respectively. Thus, hydrogen production on the dilution of DFE with and without a supplementation of yeast extract and NaHCO3 were monitored.

Effect of dilution and nutrients supplementation The organic acids in the DFE can be used as a substrate for production of hydrogen by the photosynthetic bacteria. However, the organic acids obtained in this study were not suitable for Rps. palustris TN1 because the concentrations may be too high that may cause a decrease in productivity and yield [8]. So, evasion from the inhibitory effect of substrate concentration could be obtained by diluting the DFE to determine the optimum substrate concentration before reutilization in photo fermentation [6]. The previous study of Rps. palustris TN1 found that yeast extract and NaHCO3 had a confidence level above 95%, indicating they are statistically

Fig. 1 e Time course of the cumulative hydrogen production and pH during dark fermentation by Klebsiella sp. TR17 under the conditions: temperature 40  C, initial pH 8.0, and 11.14 g/L crude glycerol. In each panel, symbols are A for hydrogen production, and - for pH.

Fig. 2 e Effect of dilution with supplementation yeast extract þ NaHCO3 (A), and without supplementation (B) on the cumulative hydrogen production of DFE by Rps. palustris TN1 under anaerobic-low light condition (3000 lux) at 30  C. In each panel, symbols are A for 1£, for 2£, :for 3£, C for 4£, and * for 5£; 1£ means undiluted DFE, 2£ means 50% diluted DFE.

Please cite this article in press as: Chookaew T, et al., Biohydrogen production from crude glycerol by two stage of dark and photo fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.02.133

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0.26 0.40 0.37 0.43 0.43

Without suppl. Suppl.

0.17 0.18 0.19 0.39 0.41 12.5 14.2 20.0 25.0 33.3 25.0 28.5 40.0 25.0 33.3 0.56 0.66 0.61 0.72 0.72 0.56 0.60 0.62 0.64 0.69 34.30 31.35 41.84 42.92 34.40 82.83 68.92 48.72 62.43 54.05 0.03 0.49 0.07 0.04 0.07 0.28 ± 1.35 ± 1.65 ± 0.62 ± 0.60 ±

Without suppl. Suppl. Without suppl. Suppl. Without suppl. Suppl. Without suppl. Suppl.

0.90 ± 0.14 1.80 ± 0.14 1.90 ± 0.49 1.60 ± 0.28 0.73 ± 0.03 ± 0.03 ± 0.02 ± 0.01 ± 0.02 ± 0.04 7.08 7.80 8.10 8.05 7.82

Without suppl.

The cumulative hydrogen production and hydrogen yield increased with the increase of the dilution from 1 (undiluted sample) to 5 (5 times dilution) both of with and without nutrients supplementation (Fig. 2). The maximum cumulative hydrogen production and hydrogen yield of 1.37 mmol H2/L and 0.43 mmol H2/g COD consumed, respectively, were achieved at 5 times dilution of DFE without nutrients supplementation. The final pH, light conversion efficiency and COD removal efficiency both of with and without nutrients supplementation tend to increase with the increase of dilution from non-dilution to 5 times dilution (Table 2). The pH of the culture broth with and without nutrients supplementation dropped from 7.1 to 8.1 and 7.0 to 8.1, respectively. At this optimum condition, the maximum light conversion efficiency was 0.72% with the total volatile fatty acid (VFA) consumption of 43.4% and COD removal efficiency of 33.0%. The highest hydrogen production rate (0.021 mmol H2/L/h) was observed at 24 h of 5 times dilution of DFE without nutrients supplementation. While nutrients supplementation at 24 h of 5 times dilution of DFE had the hydrogen production rate of 0.014 mmol H2/L/h (data not shown). Nutrients supplementation gave higher cell concentrations than those without nutrients supplementation with the maximum cell concentration (1.90 g/L) was obtained at 3 times dilution of DFE, even though, both DFE with and without nutrients supplements achieved the same level of hydrogen production of 1.31 and 1.37 mmol H2/L, respectively, at 5 times dilution of DFE. This implied that nutrients supplementation was not significant for the hydrogen production but significant for the cell growth. It has been reported that purple non-sulfur bacteria is required NaHCO3 for supporting VFA uptake through the cell which could increase the microbial growth resulting in a significant hydrogen production [24]. As the nitrogenase enzyme has driven hydrogen producing activity of photo non-sulfur bacteria is strongly positive effect on some nitrogen source, yeast extract, which has showed to have a relatively effect on nitrogenase activity, as well as increasing in cell concentration [25]. Results of this study showed that supplementation of yeast extract and NaHCO3 could increase the cell concentration but not hydrogen production. A similar result was also reported by Xu et al. [25].

± 0.02 ± 0.02 ± 0.07 ± 0.14 ± 0.07

Suppl.

7.15 7.78 8.15 8.10 8.05 1 2 3 4 5

H2 yields (mmol H2/g COD consumed) COD removal efficiency (%) Light conversion efficiency (%) Total VFA consumption (%) Cell concentration (gram dry cell weight/L) Final pH Dilution DFE

Table 2 e Effect of dilution of the dark fermentation effluent (DFE) with and without nutrients supplementation (suppl.) by Rps. Palustris TN1 under anaerobic-low light condition (3000 lux) after 168 h of cultivation.

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Fig. 3 e Effect of glutamate concentrations on the cumulative hydrogen production of DFE by Rps. palustris TN1 under anaerobic-low light condition (3000 lux) at 30  C. In each panel, symbols are A for 0 mM (control), for 2 mM, :for 4 mM, C for 6 mM, and * for 8 mM.

Please cite this article in press as: Chookaew T, et al., Biohydrogen production from crude glycerol by two stage of dark and photo fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.02.133

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Table 3 e Effect of glutamate concentration on the hydrogen production by Rps. palustris TN1 under anaerobic-low light condition (3000 lux) after 192 h of cultivation. Glutamate concentration

Final pH

0 2 4 6 8

7.73 ± 8.15 ± 7.99 ± 8.02 ± 7.91 ±

(Control) mM mM mM mM

0.10 0.11 0.06 0.03 0.14

Cell concentration (gram dry cell weight/L) 0.80 0.88 0.63 0.73 0.67

Total VFA consumption (%)

Light conversion efficiency (%)

COD removal efficiency (%)

H2 yields (mmol H2/g COD consumed)

37.94 41.06 34.80 32.27 34.03

0.56 1.43 0.70 0.63 0.53

24.36 41.81 24.36 24.33 12.72

0.78 0.68 0.57 0.51 0.82

± 0.14 ± 0.10 ± 0.17 ± 0.03 ± 0.03

The 5 times dilution has an initial COD of 9600 mg/L which was suitable for Rps. palustris TN1. DFE of the non-dilution to 5 times dilution had a dark yellow color and a series decreased to a light yellow color. This studied implied that the lighter color of DFE from the higher dilution resulted in higher hydrogen production. The possible reason was that light could easily pass through the medium compared to the non-dilution of DFE which had a dark yellow color. Thus, the lightest color of 5 times dilution of DFE exhibited the highest hydrogen production. These results were similar to those using an olive mill wastewater in which the lighter color from higher dilution could increase the hydrogen production [26].

Effect of glutamate concentration on hydrogen production Glutamate is one of the efficient nitrogen sources for the hydrogen production from photosynthesis bacteria [27,28]. Rps. palustris TN1 was employed to study the effect of glutamate concentrations (0e8 mM) on the hydrogen production from DFE using the optimum conditions obtained from the previous studies (5 times dilution without supplementation of yeast extract þ NaHCO3). The optimum glutamate concentration was found to be 2 mM, giving the highest hydrogen production (3.12 mmol H2/L) and cell concentration (0.87 g/L) (Fig. 3). The light conversion efficiency decreased from 1.44 to 0.53% with increasing glutamate concentrations from 2 to 8 mM. This result was similar to Shi and Yu [28], that hydrogen production was high at low concentrations of glutamate. Moreover, glutamate concentrations above 11 mM could decrease hydrogen production [29]. Higher glutamate concentrations achieved lower hydrogen production might be due to nitrogenase enzyme, as driven for the hydrogen production, was inhibited. The initial COD of the culture medium at 2 mM of glutamate was 11,000 mg/L, and the photo fermentation effluent had a COD of 6400 mg/L, indicating a COD removal of 41.82%. The final pH was 8.15 and the total VFA consumption was 41.1% (Table 3). The soluble metabolites were mainly composed of 1,3-PD (5.97e6.42 mM), 2,3-BD (0.90e1.26 mM), EtOH (8.98e10.07 mM), acetic acid (1.66e2.53 mM) and succinic acid (0.40e0.84 mM) (data not showed).

supplement of yeast extract þ NaHCO3, and 2 mM glutamate. Combined the two-stage processes obtained the hydrogen yield of 6.42 mmol H2/g COD consumed which was 10.4% of the theoretical yield. Many studies have reported on biohydrogen production by two-stage processes with the difference in various types of wastewater, photosynthetic bacterial strains, and operating conditions. Therefore, it is rather difficult to make a comparison. The total hydrogen yield obtained in this study (6.42 mmol H2/g COD consumed) was slightly lower than those from the previous reports using wastes such as ground wheat waste (6.9 mmol H2/g COD) [30], corncob (28.1 mmol H2/g COD) [13], and cheese whey wastewater (26.2 mmol H2/g COD) [12]. Possible cause for low hydrogen yield obtained in this study may be due to the interference of some organic matter such as potassium, phosphorus and sulfur in crude glycerol that might affect the hydrogen production [18].

Conclusion A two-stage of dark and photo hydrogen production could be used for conversion of crude glycerol to hydrogen. A maximum cumulative hydrogen production of 64.24 mmol H2/ L was obtained from dark fermentation. In batch experiments, the optimum condition for photo hydrogen production from DFE was 5 times dilution of DFE without supplementation of yeast extract þ NaHCO3 and 2 mM glutamate. The overall yield of 6.42 mmol H2/g COD consumed was achieved by the twostage processes.

Acknowledgment The authors thank the Royal Golden Jubilee Ph.D. Program of the Thailand Research Fund (grant No. PHD/0095/2551), the TRF Senior Research Scholar 2014 Fund (Grant No. RTA5780002), the Graduate School, the Faculty of Agro-Industry, Prince of Songkla University, Thailand. Additional acknowledge goes to Faculty of Agro-Industry, King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand

Overall yield

references In this study, the hydrogen yield obtained by dark fermentation was 5.74 mmol H2/g COD consumed. The maximum hydrogen yield of photo fermentation (0.68 mmol H2/g COD consumed) was obtained from 5 times dilution of DFE without

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Please cite this article in press as: Chookaew T, et al., Biohydrogen production from crude glycerol by two stage of dark and photo fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.02.133