Efficient hydrogen production from acetate through isolated Rhodobacter sphaeroides

Journal of Bioscience and Bioengineering VOL. 112 No. 6, 602 – 605, 2011 www.elsevier.com/locate/jbiosc

Efficient hydrogen production from acetate through isolated Rhodobacter sphaeroides Jyumpei Kobayashi, 1 Kazuaki Yoshimune, 2 Tomoe Komoriya, 3 and Hideki Kohno 2, 3,⁎ Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, 1-2-1, Izumichou, Narashino, Chiba 275–8575, Japan, 1 Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, 1-2-1, Izumichou, Narashino, Chiba 275–8575, Japan, 2 and Department of Sustainable Engineering, College of Industrial Technology, Nihon University, 1-2-1, Izumichou, Narashino, Chiba 275–8575, Japan 3 Received 8 July 2011; accepted 8 August 2011 Available online 8 September 2011

Photosynthetic bacteria produce hydrogen from lactate and acetate that are products of hydrogen producing bacteria in the dark. Thus, their coculture is a promising method for hydrogen production. However, the hydrogen production yield from acetate of Rhodobacter sphaeroides RV, which has been shown to possess the highest yield and hydrogen production rate, is low as compared to that from lactate. Photosynthetic bacteria that produce hydrogen from acetate as well as lactate were screened from lakes and swamps in the Tokyo and Chiba areas in Japan. Seventy-six strains of photosynthetic bacteria were obtained and the analysis of their 16S rRNA gene sequences revealed that they belong to R. sphaeroides. Among the isolated bacteria, R. sphaeroides HJ produced the highest amount of hydrogen from acetate and lactate. The HJ strain produced a 2300 ± 93 ml/L-broth of hydrogen from 75 mM acetate consumed during for 120 h of fermentation. The amount of hydrogen and the yield from acetate were 1.9 and 2.1 times higher, respectively, than those of R. sphaeroides RV. The amount and yield of hydrogen, produced by R. sphaeroides HJ from lactate were similar to those produced by R. sphaeroides RV. Since the amount and yield of produced hydrogen by the HJ strain were similar regardless of the substrate (acetate or lactate), its metabolic pathway could have a key to increasing hydrogen production from acetate. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Acetate; Lactate; Polyhydroxybutyrate (PHB); Photosynthetic bacteria; Hydrogen production; Rhodobacter sphaeroides]

Hydrogen is a clean and efficient energy source and has been considered an alternative to fossil fuel. Its use is increasing owing to the rapid advances in fuel cell technologies (1). Currently, most hydrogen is generated by reforming natural gas or light oil with steam at high temperatures. Compared to these methods, biological hydrogen production is an environmentally harmless and safe procedure. Moreover, biological methods for hydrogen production can achieve both energy generation and organic waste disposal. Hydrogen production by two-stage fermentation composed of dark and light phases is the most efficient method. In the first step of dark fermentation, Enterobacter aerogenes (2–4), E. cloacae (5–7), and Clostridium butyricum (8–10) are frequently used due to their efficient production of hydrogen under anaerobic conditions. These microorganisms produce lactate and acetate that are required for the phototrophic hydrogen production in the second stage. E. cloacae mainly produce acetate as byproduct during the hydrogen fermentation. In the second step of light fermentation, Rhodobacter sphaeroides RV is known for its high hydrogen productivity from various organic-acid substrates, such as lactate and acetate (11). However, the efficiency of hydrogen production from acetate by R. sphaeroides RV is lower than that from lactate. Therefore,

⁎ Corresponding author. Tel./fax: + 81 47 474 2566. E-mail address: [email protected] (H. Kohno).

photosynthetic bacteria possessing high hydrogen productivity from acetate were screened from the bacteria isolated from the lakes and swamps. The R. sphaeroides HJ strain was found to have high hydrogen productivity from acetate as well as lactate. The productivity from lactate by R. sphaeroides HJ was similar to that of R. sphaeroides RV. The HJ strain could be useful for efficient hydrogen production from a mixture of lactate and acetate, the by-products of dark fermentation. Furthermore, the comparison of its metabolic pathway with that of the RV strain should reveal a key to increasing the efficiency of hydrogen production from acetate. MATERIALS AND METHODS Bacteria Photosynthetic bacteria were isolated from lakes and swamps in the Tokyo and Chiba areas in Japan. The bacteria were cultivated for 2 weeks in an enrichment culture medium consisting of a basal medium (1% (v/v) inorganic solution, 1% (v/v) vitamin solution, 0.1 g/L CaCl2⋅ 2H2O, 0.2 g/L MgSO4⋅ 7H2O, 7.33 g/L K2HPO4, and 8.66 g/L KH2PO4) (12), 7.3 mM sodium acetate, and 1 wt.% yeast extract, at 30°C under illumination of 4.4 W/m2 using halogen lamps, and in anaerobic conditions in the presence of nitrogen gas. Inorganic solution contains 1.18 g/L FeSO4⋅7H2O, 0.28 g/L boric acid, 0.227 g/L MnSO4⋅ 5H2O, 0.075 g/L Na2MoO4⋅ 2H2O, 0.024 g/L ZnSO4⋅7H2O, 0.004 g/L Cu(NO3)2⋅ 3H2O, and 2.0 g/L EDTA(2Na). Vitamin solution contains 0.005 g/L biotin, 5.0 g/L thiamin hydrochloride, 0.00015 g/L p-aminobenzoic acid, 5.0 g/L nicotinic acid, and 0.00015 g/L nicotinamide. The bacteria were then cultivated on agar plate cultures of an aSy medium consisting of the basal medium, 36 mM sodium succinate, 1 wt.% yeast extract, and 9.5 mM ammonium sulphate, at 30°C under aerobic conditions, and in the dark for 2 weeks. Thereafter, a bacterial colony was picked up for

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VOL. 112, 2011

HYDROGEN PRODUCTION FROM ACETATE AND LACTATE

TABLE 1. Accumulative hydrogen produced by the five photosynthetic bacterial strains and R. sphaeroides from lactate or acetate. Substrate

Accumulative hydrogen production (ml) HJ

Lactate Acetate

360 ± 23 290 ± 9.0

HI

IH

IK

IR

RV

370 ± 26 190 ± 10

350 ± 29 200 ± 6.0

320 ± 22 210 ± 14

340 ± 14 210 ± 9.0

340 ± 17 180 ± 11

Hydrogen was produced with the hydrogen production medium containing 75 mM lactate or acetate by the fermentation at 30°C for 72 h.

isolation (13). The isolates were then cultivated in the aSy medium at 30°C under illumination of 14.6 W/m2 using halogen lamps for pre-culture. The pH of these media was adjusted to 6.8. The isolated photosynthetic bacteria were determined by the partial 16S rRNA (approximately 1.3 kbp) sequences as reported previously (14). Hydrogen production by the isolated bacteria The photosynthetic bacteria were cultured with the aSy medium at 30°C, under a light intensity of 14.6 W/m2 using halogen lamps, and under anaerobic conditions. A single colony on the aSy agar plate was subcultured in the medium in a 20-ml screw-cap test tube for 3 days. The bacteria were further cultured in the medium in a 500-ml Roux bottle for 3 days. The pre-cultured cells were centrifuged at 20,500  g for 15 min and resuspended in 20 ml of the basal medium to give an optical density (OD) at 600 nm of 3.0. A 4% agar (approximately 60°C) was added to the cell suspension and cooled and hardened in a 200-ml Roux bottle that was placed sideways. The hydrogen was produced in a hydrogen production medium (pH 7.0) consisting of a basal medium, 42 mM phosphate buffer, 75 mM organic acid (lactate or acetate), and 2 mM sodium L-glutamate at 30°C under a light intensity of 14.6 W/m2 using halogen lamps. The produced hydrogen was collected in a measuring cylinder filled with a 1 M sodium hydroxide solution to trap the by-product carbon dioxide. Experiments were conducted more than three times for each condition. Hydrogen yield The hydrogen yields were calculated by the ratio of the produced hydrogen to the consumed substrate. Although the produced gases include both hydrogen and carbon dioxide (15), carbon dioxide was trapped in a 1 M sodium hydroxide solution. The mole of the produced hydrogen was calculated using the molar volume of 22.4 L/mol. The concentrations of lactate and acetate were measured by high-performance liquid chromatography (HPLC) using a Shimadzu LC-10ATVP pump with a UV detector at a wavelength of 270 nm with an ULTRON PH-80H column (Shinwa Chemical Industries) using water-perchloric acid (1000:1.5, v/v) as the eluent at a flow rate of 1.0 ml/min. Polyhydroxybutyrate (PHB) concentration was analysed as described previously (16). The pelleted bacteria were treated with Chlorox (5.25% sodium hypochlorite) at 40°C for 2 h. PHB was pelleted by centrifugation (9000  g, 10 min) and was hydrolyzed with concentrated sulfuric acid at 90°C for 30 min to produce crotonic acid that was analysed by HPLC under the same conditions as for lactate and acetate.

RESULTS AND DISCUSSION Screening of photosynthetic bacteria Photosynthetic bacteria were screened with the aSy medium under a light, and 76 strains were isolated. All of the isolated bacteria produced hydrogen with the hydrogen production medium (200 ml). Five strains produced the highest amount of hydrogen from lactate. Table 1 shows the amount of accumulative hydrogen produced by the five isolates and

2500 2000 1500 1000 500 0 0

25

50

75

100

125

150

Initial acetate concentration (mM)

R. sphaeroides RV from lactate or acetate. The amounts of produced hydrogen from lactate were similar among the isolates. However, the HJ strain produced the highest accumulative amount of hydrogen from acetate, which was approximately 1.51-fold higher than that of the RV strain. The 16S rRNA sequence of the HJ strain showed 99.8% similarity and those of the other four strains showed the similarities of 99.4% to 99.8% with that of R. sphaeroides RV. Thus, these strains were identified as R. sphaeroides. Hydrogen production of the HJ and RV strains from acetate Hydrogen production of the isolated R. sphaeroides HJ from acetate was compared with that of R. sphaeroides RV, which is well known for efficient hydrogen production (11). Fig. 1 shows accumulative hydrogen produced by the HJ and RV strains in different acetate concentrations at 24, 72, and 120 h. The HJ strain produced more hydrogen than the RV strain in every acetate concentration (from 25 to 125 mM) and fermentation period tested. Fig. 2 shows the hydrogen yield of the HJ and RV strains from acetate. The HJ strain also exhibits a superior yield compared to the RV strain for each concentration of acetate tested. These results suggest differences between the HJ and RV strains' metabolic pathways for producing hydrogen from acetate. Further analyses of these differences should reveal key enzymes responsible for the efficient production of hydrogen. A genetic recombination study of these enzymes may increase the hydrogen productivity of R. sphaeroides. Hydrogen production of the HJ and RV strains from lactate Hydrogen production of the isolated R. sphaeroides HJ from lactate was also compared with that of R. sphaeroides RV. Fig. 3 shows accumulative hydrogen production of the HJ and RV strains in different lactate concentrations at 24, 72, and 120 h. The HJ strain produced slightly higher or similar amounts of hydrogen compared to the RV strain. On the basis of these results, the HJ strain has higher hydrogen productivity from acetate and slightly higher productivity from lactate compared to those of the RV strain. Comparison of the yields of hydrogen The hydrogen yield is the ratio of the produced hydrogen to the consumed organic-acid substrate. Fig. 2 shows the yields of hydrogen produced by R. sphaeroides HJ and R. sphaeroides RV from acetate and lactate. The HJ strain showed higher production yield from acetate than that of the RV strain, though their yields from lactate are similar. These yields are compared with the reported values of the various R. sphaeroides strains (17–29). Table 2 compares the yield and amount of hydrogen produced by the HJ strain with those by the RV strain and the other reported R. sphaeroides strains. The HJ strain produced the highest amount of hydrogen from acetate among the strains listed. Furthermore, the HJ strain showed the highest ratio (0.69) of the hydrogen

C ( 120 h) Accumulated hydrogen (ml/L-broth)

3000

Accumulated hydrogen (ml/L-broth)

B ( 72 h)

3000

Accumulated hydrogen (ml/L-broth)

A ( 24 h)

2500 2000 1500 1000 500 0 0

603

25

50

75

100

125

150

Initial acetate concentration (mM)

3000 2500 2000 1500 1000 500 0 0

25

50

75

100

125

150

Initial acetate concentration (mM)

FIG. 1. Hydrogen production of HJ and RV strains in different acetate concentrations at (A) 24, (B) 72, and (C) 120 h. The HJ (closed circles) and RV (open circles) strains were cultured at 30°C under illumination of 14.6 W/m2.

J. BIOSCI. BIOENG.,

A (Acetate)

B (Lactate) Hydrogen yield (mol-H2/mol-substrate)

KOBAYASHI ET AL.

Hydrogen yield (mol-H2/mol-substrate)

604

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

25

50

75

100

125

150

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

Initial acetate concentration (mM)

25

50

75

100

125

150

Initial lactate concentration (mM)

FIG. 2. Hydrogen yields of HJ and RV strains in different acetate and lactate concentrations at 120 h. The HJ (closed circles) and RV (open circles) strains were cultured at 30°C under illumination of 14.6 W/m2, and hydrogen was produced from acetate (A) or lactate (B).

3000

2500 2000 1500 1000 500 0 0

25

50

75

100

125

Accumulated hydrogen (ml/L-broth)

C ( 120 h)

3000

Accumulated hydrogen (ml/L-broth)

B ( 72 h)

3000

Accumulated hydrogen (ml/L-broth)

A ( 24 h)

2500 2000 1500 1000 500 0

150

Initial lactate concentration (mM)

0

25

50

75

100

125

150

Initial lactate concentration (mM)

2500 2000 1500 1000 500 0 0

25

50

75

100

125

150

Initial lactate concentration (mM)

FIG. 3. Hydrogen production of HJ and RV strains in different lactate concentrations at (A) 24, (B) 72, and (C) 120 h. The HJ (closed circles) and RV (open circles) strains were cultured at 30°C under illumination of 14.6 W/m2.

produced from acetate to that from lactate. Although R. sphaeroides O. U.001 has the highest ratio (1.5) of yield from acetate to yield from lactate, its yield from acetate (1.2) is approximately half that of the HJ strain (2.5). R. sphaeroides P1 is the PHB synthase deficient mutant of R. sphaeroides KD131 (31). The mutation is designed to improve hydrogen productivity from acetate, because PHB is often synthesised from acetate. However, the accumulative hydrogen, hydrogen yield, and the ratios are lower than those of the HJ strain. The accumulated PHB in both of the HJ and RV strains were analysed, after they were grown under the same conditions for the maximum hydrogen production in Fig. 1 (75 mM acetate, 120 h). The

PHB yields of the HJ and RV strains to the consumed acetate were 0.012 and 0.016, respectively. As compared with the hydrogen production yields that are more than 1.5 (Fig. 2), the PHB synthesis seems to be too low to influence the hydrogen production yields. A new pathway for acetate assimilation has been suggested in R. sphaeroides (32). Furthermore, the mutational analysis suggests two distinct acetate assimilation pathways that have distinct properties in hydrogen production in R. sphaeroides (33). According to these results, it is possible that the main pathway for acetate assimilation to produce hydrogen in the HJ strain is distinct from that in the RV strain. Further comparative study on the pathways of the hydrogen production of the

TABLE 2. Hydrogen amount and yield (mol-H2/mol-substrate) of R. sphaeroides strains. Organism

Substrate

Hydrogen amount (ml-H2/L-broth)

R. sphaeroides HJ R. sphaeroides RV R. sphaeroides O.U.001 R. sphaeroides KD131 R. sphaeroides P1 –, The data is unavailable.

Acetate (25 mM) Lactate (25 mM) Acetate (25 mM) Lactate (25 mM) Acetate (30 mM) Lactate (20 mM) Acetate (30 mM) Lactate (30 mM) Acetate (30 mM) Lactate (30 mM)

1200 ± 60 1700 ± 86 640 ± 64 1600 ± 130 – – 250 790 370 1100

Ratio of the amounts

Hydrogen yield

Ratio of the yields

Acetate/lactate

(mol-H2/mol-substrate)

Acetate/lactate

0.69

2.5 ± 0.13 3.7 ± 0.16 1.4 ± 0.14 3.5 ± 0.28 1.2 0.80 0.37 1.2 0.55 1.6

0.68

0.40 – 0.31 0.33

Reference

This work

0.40 1.5

30

0.31

31

0.34

VOL. 112, 2011 HJ strain and the other R. sphaeroides strains may reveal key enzymes responsible for efficient hydrogen production from acetate.

HYDROGEN PRODUCTION FROM ACETATE AND LACTATE

17.

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