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Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 2 1 6 7 e1 2 1 7 4
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Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV Hongliang Han, Biqian Liu, Haijun Yang*, Jianquan Shen* Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of New Materials, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun North First Street 2, Beijing 100190, PR China
article info
abstract
Article history:
Rhodobacter sphaeroides RV was employed to produce hydrogen for the photo-fermentation
Received 24 December 2011
of sole (acetate, propionate, butyrate, lactate, malate, succinate, ethanol, glucose, citrate
Received in revised form
and sodium carbonate) and compound carbon sources (malate and succinate, lactate and
11 March 2012
succinate). The concentrations of sole carbon sources on hydrogen production were
Accepted 27 March 2012
investigated in batch assays at 0.8 g/L sodium glutamate and the maximum hydrogen yield
Available online 20 April 2012
was 424 mmol H2/mol-substrate obtained at 0.8 g/L sodium propionate. The maximum hydrogen yield reached 794 mmol H2/mol-substrate for 2.02 g lactate and 2.0 g succinate as
Keywords:
the compound carbon source. The results showed hydrogen production for the compound
Photosynthetic bacteria
carbon source was better than the sole carbon source.
Compound carbon source
Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Hydrogen production
reserved.
Rhodobacter sphaeroides RV
1.
Introduction
As an alternative to fossil fuels, hydrogen has been and remains of intense interest because of its cleanness, renewable character, and it has the highest gravimetric energy density (122 kJg1) [1,2]. As compared to the conventional chemical and electrolytical routes to hydrogen production [3,4], the biological method provides a cost-effective and an environmentally harmless alternative carried out under mild operating conditions [5,6]. The biological method mainly includes photosynthetic hydrogen production (such as green algae, cyanobacteria and photosynthetic bacteria) and fermentative hydrogen production (such as dark fermentation). Hydrogen production by fermentation has been extensively studied because of its high efficiency and its potential to use renewable sources of biomass [7,8]. Dark fermentation is a promising method considering its high hydrogen evolution rate in the absence of a light source and a great
wastewater-treated capability. However, the low theoretical conversion efficiency of heat value (4 mol H2/mol glucose) and high effluent COD restrict the industrial application [9]. Photosynthetic hydrogen production is a favorable candidate for biohydrogen production because of its high substrate conversion efficiencies and its capability of utilizing abundant substrates [10]. Photosynthetic hydrogen production is a theoretically perfect process of transforming solar energy into hydrogen by photosynthetic bacteria (12 mol H2/ mol glucose), but the actual hydrogen yields are much lower than the theoretical maximum value. Photosynthetic bacteria can produce hydrogen at the expense of solar energy and small-chain organic acids as electron donors [11]. Biohydrogen production using photosynthetic bacteria is influenced by several parameters such as carbon source, nitrogen source, C/N ratio, temperature, light source and nutrient medium [12e17]. In these factors, carbon sources are the most important which affect the electron
* Corresponding authors. Tel.: þ86 10 62620903; fax: þ86 10 62559373. E-mail address: [email protected] (J. Shen). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.03.134
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donor [10]. The conversion efficiency of light energy to hydrogen, with the supply of an appropriate carbon source, is the key factor for hydrogen production by biological systems [18]. The previous researches showed that only a portion of carbon source is suitable for hydrogen production. Some studies reported that lower hydrogen production rates occurred with acetate and butyrate than with malate and lactate [11,19,20]. Using complex substrates as carbon source for photosynthetic hydrogen production has also been explored, such as wastewater [10,15,21]. However, high conversion efficiency of substrates for photosynthetic hydrogen production has not been obtained. Rhodobacter sphaeroides is a kind of purple non-sulfur bacterium that can obtain energy through photosynthesis and has been applied into photosynthetic hydrogen production [17,21]. In the present study, photo-fermentation of sole and compound carbon sources was conducted with Rb. sphaeroides RV. The data obtained from this study are expected to provide basic and engineering data for the improvement of photosynthetic hydrogen production.
2.
Materials and methods
2.1.
Photosynthetic bacteria and growth medium
Rb. sphaeroides RV was purchased from Institute of Microbiology, Chinese Academy of Science. The strains were grown in basic medium, which was composed of a basal mineral medium (1 L of basal mineral medium was composed of K2HPO4, 600 mg; KH2PO4, 500 mg; NaCl, 400 mg; CaCl2$2H2O, 50 mg; MgCl2$6H2O, 200 mg; FeSO4$7H2O, 5 mg; ZnCl2, 7 mg; CuSO4$5H2O, 2.4 mg; H3BO3, 16 mg; MnSO4$4H2O, 10 mg; NiCl2$6H2O, 2 mg; CoCl2$6H2O, 2 mg; NaMoO4$2H2O, 4 mg; EDTA-2Na, 0.4 mg), vitamins (1.4 mg/L vitamin H, 0.5 mg/L vitamin B1, 3.0 mg/L nicotinamide), 0.3 g/L yeast extract 1.25 g/ L (NH4)2SO4, 1.0 g/L glucose and 6.84 g/L sodium succinate. The strains were cultivated anaerobically for 24 h under photosynthetic condition of 4000 lux at 32 C (light-grown cells) and used as inoculum. When required, an inoculum culture was concentrated by centrifugation at 4000 rpm for 10 min.
2.2.
Experimental method in photo-fermentation
Hydrogen-production batch experiments were performed in 120 or 320 mL glass column photobioreactor. The temperature was maintained at 32 C. Light intensity was 220 W/m2 at the outer surface of the reactor. The basic medium and additional carbon sources were used as the medium for photosynthetic hydrogen production. A required amount of sodium glutamate was added to adjust concentration to 0.8 g/L. pH was adjusted to 7.0 with 4% sodium hydroxide and hydrochloric acid. Argon gas was used to create anaerobic conditions. The gas volume was calibrated to 25 C and 760 mmHg. All the experiments were done in batch operation. Each experimental condition was carried out in triplicate. The experimental results noted were the averages (standard deviation) of the values obtained in independent experiments conducted in triplicate. All chemicals used in the experiments were of AR grade.
2.3.
Chemical analysis
The hydrogen content was determined by a gas chromatograph (Techcomp. Co., China, 7890II) equipped with a thermal conductivity detector (TCD) and a 2-m stainless steel column packed with Porapak Q (80e100 mesh). The operating temperatures of the injection port, the oven and the detector were set at 70, 50 and 70 C, respectively. Argon was used as the carrier gas at a flow rate of 30 mL/min. At each time interval, the total volume of biogas production was measured by a plunger displacement method using appropriately sized glass syringes, ranging from 10 to 100 mL [22]. The cumulative hydrogen volume was calculated by equation (1) [23]:
VH,i ¼ VH,i1 þ CH,i (VG,i VG,i1) þ VH,0 (CH,i e CH,i1)
(1)
where VH,i and VH,i1 are cumulative hydrogen volumes at the current (i) and previous (i 1) time intervals, VG,i and VG,i1 are the total biogas volumes in the current (i) and previous (i 1) time intervals, CH,i and CH,i1 are the fraction of hydrogen in the headspace of the bottle at the current (i) and previous (i 1) intervals and VH,0 is the total volume of headspace in the bottle. Detection of the alcohols and volatile fatty acids (VFAs, C2eC6) were measured by a gas chromatograph (SHIMADZU Co., Japan, GC 14B) using a flame ionization detector (FID) and a 2-m glass column packed with Unisole F-200 (30e60 mesh). The temperatures of the injection port, the oven and the detector were set at 200, 165 and 200 C, respectively. The carrier gas was argon at a flow rate of 30 mL/min. Light intensity was measured with TES-1332 luminometer (Shenzhen Langpu Electronic Tech. Co. Ltd). Cell mass concentration was determined spectrophotometrically as optical density at 660 nm (OD660).
2.4.
Model analysis
The cumulative hydrogen production in the batch experiments followed the modified Gompertz equation [24]: Rm e H ¼ P exp exp ðl tÞ þ 1 P where H is the cumulative hydrogen production (mL), P is hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL/h), e is 2.71828, l is the lag-phase time (h), and t is the incubation time (h).The corresponding values of P, Rm and l for each batch were estimated using Origin 7.5, which is a scientific graphing and data analysis software.
3.
Results and discussion
Sodium glutamate (0.8 g/L) was used as nitrogen source and different carbon sources were tested at different initial concentrations.
3.1. Effect of sodium acetate, sodium propionate and sodium butyrate concentration on hydrogen production Acetate, propionate and butyrate were chosen as carbon sources, as they are dominant products of anaerobic
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Acetate: C2H4O2 þ 2H2O / 4H2 þ 2CO2
Propionate: C3H6O2 þ 4H2O / 7H2 þ 3CO2
Butyrate: C4H8O2 þ 6H2O / 10H2 þ 4CO2
a Volume of cumulative hydrogen (mL)
fermentation process [25e27]. The photosynthetic bacteria can further utilize these soluble metabolites to produce more hydrogen at the expense of light energy [28e30]. The theoretical equations of hydrogen production from acetate, propionate and butyrate were described as follows [11]:
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6
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1.2 g/L 2.4 g/L 3.6 g/L 4.8 g/L
2
1.8 g/L 3.0 g/L 4.2 g/L
0 0
12
24
36
48
60
72
Time (hour)
Volume of cumulative hydrogen (mL)
b
14
12
10
8
0.4 g/L 0.8 g/L 1.2 g/L 1.6 g/L 2.0 g/L 2.4 g/L 2.8 g/L
6
4
2
0 0
12
24
36
48
60
72
Time (hour)
c
16
Volume of cumulative hydrogen (mL)
The influence of sodium acetate concentration on cumulative hydrogen volume is shown in Fig. 1a. The cumulative hydrogen production was more 12 mL when sodium acetate concentration ranged 1.2e2.4 g/L. When sodium acetate concentration ranged 3.0e4.8g/L, the cumulative hydrogen dramatically decreased to about 6 mL. With the increase of sodium acetate concentration, the cumulative hydrogen gradually decreased. The maximum cumulative hydrogen was 13.7 mL obtained at 1.2 g/L sodium acetate. Table 1 shows that substrate consumption efficiency, L-sodium glutamate consumption efficiency, OD660 and hydrogen yield under different concentrations of sodium acetate. Acetate consumption efficiency, L-sodium glutamate consumption efficiency and OD660 had no obvious decreased trend along with the increase of acetate concentration. It indicated that sodium acetate as carbon source could better promote the growth of bacteria and a high acetate concentration was not toxic to photo-fermentation of hydrogen by Rb. sphaeroides RV. The result was consistent with the previous study [31]. The cumulative hydrogen production decreased with the increase of acetate concentration due to producing numerous byproducts poly-b-hydroxybutyrate (PHB). Hustede reported that acetate is favorable for PHB synthesis [32]. The byproducts competed with the hydrogen production of photosynthetic bacteria and resulted in the decrease of cumulative hydrogen production [33,34]. Therefore the optimal concentration of acetate was 1.2 g/L for the hydrogen production of photosynthetic bacteria. The maximum hydrogen yield was 165 mmol H2/mol-acetate obtained at 1.2 g/L sodium acetate. Fig. 1b illustrates the effect of the concentration of sodium propionate on hydrogen production. When sodium propionate concentration ranged in 0.8e1.6 g/L, the cumulative hydrogen volumes were about 12 mL. No PHBs were detected after the termination of hydrogen-producing reaction. It indicated propionate was not an optimal carbon source for PHB production and benefited for hydrogen production. Table 1 shows propionate consumption efficiency was lower than acetate consumption efficiency which was 70e90%. When propionate concentration ranged in 0.4e1.6 g/L, OD660 was higher than 1.1. The maximum hydrogen yield was 424 mmol H2/mol-propionate obtained at 0.4 g/L sodium propionate. The optimal concentration of propionate was ranged in 0.4e1.6 g/L for the hydrogen production of photosynthetic bacteria. The effect of butyrate concentrations on hydrogen production was similar with acetate (Fig. 1c). When butyrate concentration ranged in 1.2e1.8 g/L, the maximum
14 12
1.2 g/L 2.4 g/L 3.6 g/L 4.8 g/L
10 8
1.8 g/L 3.0 g/L 4.2 g/L
6 4 2 0 0
12
24
36
48
60
72
Time (hour)
Fig. 1 e Cumulative hydrogen volume versus corresponding fermentation time at different concentrations of sodium acetate (a), sodium propionate (b) and sodium butyrate (c).
cumulative hydrogen volume was 15.1 mL. 400 mg/L PHB was detected after the termination of hydrogen-producing reaction. It suggested that the excess of butyrate was beneficial for PHB production which was the major reason for the resulting
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3.6 65.1 88.5 1.32 28 3.0 70.9 88.3 1.38 29 2.4 73.2 88.1 1.45 28 1.8 81.6 89.6 1.29 165 1.2 80.1 82.7 1.33 242 2.8 71.6 79.6 0.83 64 2.4 70.7 75.0 0.89 70
3.2. Effect of D,L-malate, sodium lactate and sodium succinate concentration on hydrogen production
1.2 76.6 84.2 1.27 172
1.6 77.8 84.3 1.10 130
2.0 78.3 84.7 0.99 90
We choose L-lactate, D,L-malate and succinate to further the research of hydrogen production, as they are usual carbon sources for photosynthetic bacteria [35e37]. The theoretical equations of hydrogen production from malate, lactate and succinate were described as follows [11]:
Malate: C4H6O5 þ 3H2O / 6H2 þ 4CO2
0.8 75.7 84.1 1.22 276
Sodium propionate
0.4 86.4 80.3 1.17 424
Lactate: C3H6O3 þ 3H2O / 6H2 þ 3CO2
4.2 97.4 92.7 1.22 17
4.8 98.6 92.6 1.24 16
Succinate: C4H4O4 þ 4H2O / 6H2 þ 4CO2
1.8 97.7 91.1 1.18 100 1.2 99.9 90.9 1.22 165
SCE: substrate consumption efficiency (%). GCE: L-Sodium glutamate consumption efficiency (%). HY: hydrogen yield (mmol H2/mol-substrate).
3.6 98.2 92.3 1.26 27 2.4 98.0 91.0 1.19 76 Concentration (g/L) SCE GCE OD660 HY
3.0 98.7 90.7 1.08 34
Sodium acetate Carbon source
Table 1 e Effects of different concentrations of sodium acetate, sodium propionate and sodium butyrate on photo-H2 production.
Sodium butyrate
4.2 66.4 92.6 1.27 17
4.8 56.2 87.0 1.33 15
decrease of cumulative hydrogen production. The butyrate consumption efficiency and hydrogen yield decreased with the increase of sodium butyrate concentration, but OD660 and L-sodium glutamate consumption efficiency remained basically unchanged (Table 1). The maximum hydrogen yield was 242 mmol H2/mol-butyrate obtained at 1.2 g/L butyrate. Overall, the optimal butyrate concentration ranged in 1.2e1.8 g/L for photosynthetic bacteria hydrogen production. The testing results showed numerous PHB (wet cell weight 35%) and little 5-aminolevulinic acid (8e15 mg/L) were detectable for acetate and butyrate as carbon source. PHB production was adverse to the hydrogen production of photosynthetic bacteria [34]. Hustede reported that the type of carbon source, nitrogen source and the ratio of C/N can affect the production of PHB and the ratio of H2 [32]. For better hydrogen or PHB yield control, clarifying the relationship between PHB accumulation and hydrogen production is an important issue.
Fig. 2 shows the effect of D,L-malate, sodium lactate and sodium succinate concentrations on cumulative hydrogen volume and hydrogen content. Fig. 2a shows the maximum cumulative hydrogen volume and hydrogen content were 34 mL and 80.7% obtained at 6.0 g/L D,L-malate, respectively. Table 2 shows the effect of D,L-malate, sodium lactate and sodium succinate concentration on hydrogen production. L-glutamate consumption efficiencies were higher than 82% in all experiments for D,L-malate as carbon source. Malate consumption efficiency slightly decreased with the increase of D,L-malate concentration. The maximum cell density was 2.60 g/L obtained at 6.0 g/L malate. The maximum hydrogen yield was 1.34 mol H2/mol-substrate obtained at 1.0 g/L malate. Overall, the optimal concentration for D,L-malate as carbon source ranged in 1.0e6.0 g/L. Fig. 2b shows that the maximum cumulative hydrogen volume was 58.8 mL obtained at 4.0 g/L lactate. With the increase of lactate concentration, the hydrogen content firstly decreased and then increased to remain unchanged. When lactate concentration ranged in 3.0e8.0 g/L, the hydrogen content remained 50%. L-glutamate consumption efficiencies were higher than 93.2%, but lactate consumption efficiencies were very low (Table 2). The maximum lactate consumption
a
b
40
35
30
25
20
15
60 55 50 45 40 35 30 25 20 15
16
1
1
2
2
4
3
3
5
5
6
6
7
7
9
8
8
10
85 80 75 70 65 60 55 50 45 40
65 60 55 50 45 40 35 30 25
35
30
25
20
15
Hydrogen content (%)
8
Table 2 e Effects of different concentrations of D,L-malate, sodium lactate and sodium succinate on photo-H2 production.
Concentration (g/L) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 SCE 98.4 96.2 97.4 94.6 92.3 93.4 88.2 82.9 72.3 76.4 75.4 80.8 77.8 68.3 63.7 61.9 97.2 92.3 90.4 90.6 87.3 88.4 83.2 81.7 GCE 88.4 89.2 82.1 86.7 84.2 83.4 84.8 82.8 98.7 97.6 98.4 99.7 93.2 98.1 99.3 97.6 94.1 92.3 92.4 91.7 90.2 88.3 89.3 93.6 Cell density (g/L) 1.78 2.16 1.98 2.16 2.48 2.60 2.44 2.42 2.38 2.76 2.98 3.56 3.38 3.40 3.34 3.42 2.04 2.81 2.62 3.06 2.99 3.23 3.45 3.32 HY 1340 890 650 540 480 420 330 300 850 700 620 480 430 380 310 240 300 240 280 240 220 270 260 190
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4
5
Malate (g/L)
4
7
Lactate (g/L)
6
Succinate (g/L)
Hydrogen content (%)
c 14
12
10
8
6
4
2 3
Fig. 2 e Cumulative hydrogen volume versus corresponding fermentation time at different concentrations of D,L-malate (a), sodium lactate (b) and sodium succinate (c).
Hydrogen content (%)
efficiency was only 80.8% at 4.0 g/L lactate. But the maximum cell density was 3.56 g/L. With the increase of lactate concentration, the hydrogen yield gradually decreased. The maximum hydrogen yield was 850 mmol H2/mol-substrate obtained at 1.0 g/L lactate. So the optimal lactate concentration was 1.0e4.0 g/L for photosynthetic bacteria hydrogen production. Fig. 2c shows that the maximum cumulative hydrogen volume was 15.6 mL at 9.0 g/L sodium succinate. The
Sodium succinate Sodium lactate D,L-Malate
Carbon source
Volume of cumulative hydrogen (mL)
Volume of cumulative hydrogen (mL)
Volume of cumulative hydrogen (mL)
SCE: substrate consumption efficiency (%). GCE: L-Sodium glutamate consumption efficiency (%). HY: hydrogen yield (mmol H2/mol-substrate).
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Table 3 e Hydrogen production by Rb. sphaeroides RV using other carbon sources. Carbon source
Concentration (g/L)
SCE
0.2 0.4 0.5 0.8
63.2 3.2 21.1 5.1 9.4 2.6 ND
Alcohol Glucose Sodium citrate NaCO3
OD660 1.82 1.78 1.06 1.44
0.17 0.23 0.11 0.13
Cell density (g/L)
HY
8.1 0.45 11.2 0.54 ND ND
1.89 1.84 1.39 1.48
0.31 0.22 0.15 0.20
SCE: substrate consumption efficiency (%). HY: hydrogen yield (mL H2/mol-substrate). ND: no detection.
maximum hydrogen content was 35.2% at 8.0 g/L sodium succinate. L-glutamate consumption efficiencies were higher than 88%, but sodium succinate consumption efficiencies gradually decreased with the increase of sodium succinate concentration. Comparison with D,L-malate, the cell density obviously increased. The maximum cell density was 3.45 g/L obtained at 9.0 g/L sodium succinate. With the increase of sodium succinate concentration, the hydrogen yield slightly changed and remained 190e300 mmol H2/mol-substrate. So the optimal sodium succinate concentration was 3.0e9.0 g/L for photosynthetic bacteria hydrogen production.
3.3. Effect of other carbon sources on hydrogen production In this section, we studied the effects of some unusual carbon sources on hydrogen production. Table 3 shows the effects of alcohol, glucose, sodium citrate and sodium carbonate on hydrogen production. The results show the strain could utilize alcohol as carbon source for the positive growth of strain and hydrogen production. But the hydrogen yield is much lower than the foregoing carbon sources. In addition, the substrate consumption efficiency was only 63.2%. Oh suggested that minimized ethanol content could improve hydrogen production yield [38]. Glucose could provide nutrients for the growth of strain, but the cumulative hydrogen production was low. Khatipov reported that low hydrogen production rate and poor bacterial growth occurred on glucose as carbon source [34]. The strain stopped growth for sole sodium citrate as carbon source and no hydrogen was detected. Sodium carbonate could provide the nutrient for the growth of strain and no hydrogen was detected, this was because sodium
carbonate can not act as the hydrogen donor [39]. The variation of hydrogen production for various carbon sources possibly results from differences in their reduction states and patterns of metabolism [18].
3.4. The compound carbon source influenced the hydrogen production of photosynthetic bacteria Sole carbon source has the high substrate consumption efficiency and the low hydrogen yield. Sangeetha investigated the effect of mixed volatile fatty acids on the growth of Rb. Sphaeroides by response surface methodology [40]. But investigated the effect of the component of compound carbon source on hydrogen production is rare [41]. So we studied the compound carbon source that influenced the hydrogen production of photosynthetic bacteria. Fig. 3a shows malate and succinate as compound carbon source influenced the cumulative hydrogen production. The cumulative hydrogen volume for the compound carbon source was as high as over 1.4-fold than sole D,L-malate as carbon source under the fixed ratio of C/N. Table 4 shows that L-sodium glutamate consumption efficiencies were higher than 97% and no distinct differences between sole and compound carbon source. The hydrogen yield increased obviously, from 450 mmol H2/mol-substrate to 534 mmol H2/mol-substrate. The modified Gompertz model fits experimental data well with the R2 values, which are all greater than 0.98. Consequently, the modified Gompertz equation is useful and relatively accurate in predicting the cumulative hydrogen production as well as the rate of hydrogen production. The lag-phase times increased dramatically to 34.0 h, which was much longer than the 24.4 h of the sole D,L-malate. The
Table 4 e Hydrogen production and kinetic model parameters from compound carbon source. D,L-Malate
(g/L)
Sodium lactate
6.82 3.16 2.31
Sodium succinate (g/L)
1.0 2.0 4.86 3.23 2.02
1.0 2.0
GCE
98.7 97.6 98.4 98.8 98.6 98.4
GCE: L-Sodium glutamate consumption efficiency (%). HY: hydrogen yield (mmol H2/mol-substrate).
0.8 1.0 0.5 0.3 0.8 0.6
Cell density (g/L)
2.32 2.66 2.78 2.41 2.64 2.87
0.12 0.09 0.17 0.12 0.15 0.21
HY
450 520 534 550 690 794
Kinetic model parameters
22 15 29 19 35 39
P (mL)
Rm (mL/h)
l (h)
R2
24.2 37.2 35.1 38.4 45.9 56.5
0.85 1.07 0.95 1.24 1.19 1.98
24.4 33.8 34.0 23.7 10.7 28.3
0.986 0.994 0.994 0.995 0.992 0.991
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maximum hydrogen production rate was increased to ca. 1.0 mL/h, significantly elevated compared to the 0.85 mL/h of the sole D,L-malate. The maximum hydrogen production rate and cumulative hydrogen for compound carbon source were significantly elevated compared to the sole D,L-malate. Fig. 3b shows the cumulative hydrogen volumes gradually increased with succinate concentration from 0 to 2 g/L, from 38.4 to 56.5 mL for lactate and succinate as compound carbon source. Table 4 shows the L-glutamate consumption efficiencies hardly changed and were higher than 97%. Cell density, hydrogen yield and cumulative hydrogen were obviously increased. The maximum hydrogen yield reached 794 mmol H2/mol-substrate. Interestingly, the lag-phase time and the maximum hydrogen production rate didn’t show a regular trend. The minimum hydrogen production rate and lag-phase time were produced at 3.23 g lactate and 1.0 g succinate as compound carbon source. It indicated kinetic model parameters were remarkably affected by the component of compound carbon source. In addition, the hydrogen yield for lactate and succinate as compound carbon source was higher than malate and succinate as compound carbon
Volume of cumulative hydrogen (mL)
a
40
6.82 g/L Malate 3.16 g/L Malate+1.0 g/L Succinate 2.31 g/L Malate+2.0 g/L Succinate
35
4.
Conclusions
In the present study, hydrogen production by photosynthetic bacteria from sole and compound carbon source was investigated. The results showed hydrogen production for compound carbon sources was better than sole carbon sources. The maximum hydrogen yield reached 794 mmol H2/molsubstrate for 2.02 g lactate and 2.0 g succinate as compound carbon source. But the component of the compound carbon source needs to optimize further.
Acknowledgments 25
The authors would like to thank the Chinese Academy of Sciences for financial support (Item No. KJCX2-YW-H21).
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references
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Time ( hour )
Volume of cumulative hydrogen (mL)
source. For Rb. sphaeroides RV, different compound carbon source had different effects of hydrogen production. Overall, hydrogen production for compound carbon was better than the sole carbon source. It might be due to the various carbon sources being utilized with different efficiencies by photosynthetic bacteria [41]. The efficiency of a certain carbon source depends on factors such as the activity of tricarboxylic acid cycle, the C/N ratio, the reduction state of that material and the conversion potential of the carbon source into alternative metabolites [10]. Adding the compound carbon source into hydrogen-producing system alters the modes of metabolism, resulted in improving the hydrogen production.
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4.86 g/L Lactate 3.23 g/L Lactate+1.0 g/L Succinate 2.02 g/L Lactate+2.0 g/L Succinate
0 0
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Fig. 3 e Cumulative hydrogen volume versus corresponding fermentation time for D,L-malate and sodium succinate (a) and sodium lactate and sodium succinate (b) as compound carbon source.
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[9] Su HB, Cheng J, Zhou JH, Song WL, Cen KF. Combination of dark- and photo- fermentation to enhance hydrogen production and energy conversion efficiency. Int J Hydrogen Energy 2009;34:8846e53. lu I, Gu¨ndu¨z U, Yu¨cel M, Tu¨rker L. Aspects of the [10] Koku H, Erog metabolism of hydrogen production by Rhodobacter sphaeroides. Int J Hydrogen Energy 2002;27:1315e29. [11] Barbosa MJ, Rocha JMS, Tramper J, Wijffels RH. Acetate as a carbon source for hydrogen production by photosynthetic bacteria. J Biotechnol 2001;85:25e33. [12] Berberoglu H, Jay J, Pilon L. Effect of nutrient media on photobiological hydrogen production by Anabaena variabilis ATCC 29413. Int J Hydrogen Energy 2008;33:1172e84. [13] Allakhverdiev SI, Thavasi V, Kreslavski VD, Zharmukhamedov SK, Klimov VV, Ramakrishna S, et al. Photosynthetic hydrogen production. J Photochem Photobiol C 2010;11:101e13. [14] Dasgupta CN, Gilbert JJ, Lindblad P, Heidorn T, Borgvang SA, Skjanes K, et al. Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production. Int J Hydrogen Energy 2010;35:10218e38. [15] Ergu¨der TH, Gu¨ven E, Demirer GN. Anaerobic treatment of olive mill wastes in batch reactors. Process Biochem 2000;36: 243e8. [16] Takabatake H, Suzuki K, Ko I, Noike T. Characteristics of anaerobic ammonia removal by a mixed culture of hydrogen producing photosynthetic bacteria. Bioresour Technol 2004; 95:151e8. [17] Kondo T, Arakawa M, Hirai T, Wakayama T, Hara M, Miyake J. Enhancement of hydrogen production by a photosynthetic bacterium mutant with reduced pigment. J Biosci Bioeng 2002;93:145e50. [18] Hillmer P, Gest H. H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures. J Bacteriol 1977;129:724e31. [19] Miyake J, Mao XY, Kawamura S. Photoproduction of hydrogen from glucose by a co-culture of a photosynthetic bacterium and Clostridium butyricum. J Ferment Technol 1984; 62:531e5. [20] Kim JS, Ito K, Takahashi H. The relationship between nitrogenase activity and hydrogen evolution in Rhodopseudomonas palustris. Agric Biol Chem 1980;44:827e33. [21] Yetis M, Gu¨ndu¨z U, Eroglu I, Yu¨cel M, Tu¨rker L. Photoproduction of hydrogen from sugar refinery wastewater by Rhodobacter sphaeroides O.U. 001. Int J Hydrogen Energy 2000;25:1035e41. [22] Owen WF, Stuckey DC, Healy JB, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 1979;13:485e93. [23] Logan BE, Oh SE, Kim IS, Ginkel SV. Biological hydrogen production measured in batch anaerobic respirometers. Environ Sci Technol 2002;36:2530e5. [24] Zwietering MH, Jongenburger I, Rombouts FM, Riet KV. Modeling of the bacterial-growth curve. Appl Environ Microbiol 1990;56:1875e81. [25] Han HL, Cui MJ, Wei LL, Yang HJ, Shen JQ. Enhancement effect of hematite nanoparticles on fermentative hydrogen production. Bioresour Technol 2011;102:7903e9.
[26] Yuan ZL, Yang HJ, Zhi XH, Shen JQ. Increased performance of continuous stirred tank reactor with calcium supplementation. Int J Hydrogen Energy 2010;35:2622e6. [27] Cui MJ, Yuan ZL, Zhi XH, Wei LL, Shen JQ. Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria. Int J Hydrogen Energy 2010;35:4041e7. [28] Chen CY, Yang MH, Yeh KL, Liu CH, Chang JS. Biohydrogen production using sequential two-stage dark and photo fermentation processes. Int J Hydrogen Energy 2008;33: 4755e62. ¨ zgu¨r E, Gu¨rgan M, Akko¨se S, Yu¨cel M, Gu¨ndu¨z U, [29] Afsar N, O et al. Hydrogen productivity of photosynthetic bacteria on dark fermenter effluent of potato steam peels hydrolysate. Int J Hydrogen Energy 2011;36:432e8. [30] Chader S, Haceneb H, Agathos SN. Study of hydrogen production by three strains of Chlorella isolated from the soil in the Algerian Sahara. Int J Hydrogen Energy 2009;34:941e6. [31] Oh YK, Seol EH, Kim MS, Park S. Photoproduction of hydrogen from acetate by a chemoheterotrophic bacterium Rhodopseudomonas palustris P4. Int J Hydrogen Energy 2004;29: 1115e21. [32] Hustede E, Steinbu¨chel A, Schlegel HG. Relationship between the photoproduction of hydrogen and the accumulation of PHB in non-sulphur purple bacteria. Appl Microbiol Biotechnol 1993;39:87e93. [33] Steinbu¨chel A, Fu¨chtenbusch B. Bacterial and other biological systems for polyester production. Tibtech 1998;16: 419e27. [34] Khatipove E, Miyake M, Miyake J, Asada Y. Accumulation of poly-b-hydroxybutyrate by Rhodobacter sphaeroides on various carbon and nitrogen substrates. FEMS Microbiol Lett 1998; 162:39e45. [35] Ohta Y, Frank J, Mitsui A. Hydrogen production by marine photosynthetic bacteria: effect of environmental factors and substrate specificity of the growth of a hydrogen-producing marine photosynthetic bacterium Chromatium sp. Miami PBS 1071. Int J Hydrogen Energy 1981;6:451e60. [36] Ooshima H, Takakuwa S, Katsuda T, Okuda M, Shirasawa T, Azuma M, et al. Production of hydrogen by a hydrogenasedeficient mutant of Rhodobacter capsulatus. J Ferment Bioeng 1998;85:470e5. [37] Eroglu I, Aslan K, Gunduz U, Yucel M, Turker L. Continuous hydrogen production by Rhodobacter sphaeroides O.U. 001. In: Zaborsky OR, editor. Biohydrogen. London: Plenum Press; 1998. p. 143e51. [38] Oh YK, Seol EH, Lee EY, Park S. Fermentative hydrogen production by a new chemoheterotrophic bacterium Rhodopseudomonas palustris P4. Int J Hydrogen Energy 2002;27: 1373e9. [39] Zhu HG, Shi JL. Progress of biological hydrogen production. Chin J Appl Environ Biol 2002;8:98e104. [40] Sangeetha R, Karunanithi T. Optimization of growth of Rhodobacter Sphaeroides using mixed volatile fatty acids by response surface methodology. Int J Chem Biol Eng 2010;3: 115e8. [41] Shi XY, Yu HQ. Conversion of individual and mixed volatile fatty acids to hydrogen by Rhodopseudomonas capsulate. J Chem Technol Biotechnol 2005;80:1198e203.
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Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV Hongliang Han, Biqian Liu, Haijun Yang*, Jianquan Shen* Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of New Materials, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun North First Street 2, Beijing 100190, PR China
article info
abstract
Article history:
Rhodobacter sphaeroides RV was employed to produce hydrogen for the photo-fermentation
Received 24 December 2011
of sole (acetate, propionate, butyrate, lactate, malate, succinate, ethanol, glucose, citrate
Received in revised form
and sodium carbonate) and compound carbon sources (malate and succinate, lactate and
11 March 2012
succinate). The concentrations of sole carbon sources on hydrogen production were
Accepted 27 March 2012
investigated in batch assays at 0.8 g/L sodium glutamate and the maximum hydrogen yield
Available online 20 April 2012
was 424 mmol H2/mol-substrate obtained at 0.8 g/L sodium propionate. The maximum hydrogen yield reached 794 mmol H2/mol-substrate for 2.02 g lactate and 2.0 g succinate as
Keywords:
the compound carbon source. The results showed hydrogen production for the compound
Photosynthetic bacteria
carbon source was better than the sole carbon source.
Compound carbon source
Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Hydrogen production
reserved.
Rhodobacter sphaeroides RV
1.
Introduction
As an alternative to fossil fuels, hydrogen has been and remains of intense interest because of its cleanness, renewable character, and it has the highest gravimetric energy density (122 kJg1) [1,2]. As compared to the conventional chemical and electrolytical routes to hydrogen production [3,4], the biological method provides a cost-effective and an environmentally harmless alternative carried out under mild operating conditions [5,6]. The biological method mainly includes photosynthetic hydrogen production (such as green algae, cyanobacteria and photosynthetic bacteria) and fermentative hydrogen production (such as dark fermentation). Hydrogen production by fermentation has been extensively studied because of its high efficiency and its potential to use renewable sources of biomass [7,8]. Dark fermentation is a promising method considering its high hydrogen evolution rate in the absence of a light source and a great
wastewater-treated capability. However, the low theoretical conversion efficiency of heat value (4 mol H2/mol glucose) and high effluent COD restrict the industrial application [9]. Photosynthetic hydrogen production is a favorable candidate for biohydrogen production because of its high substrate conversion efficiencies and its capability of utilizing abundant substrates [10]. Photosynthetic hydrogen production is a theoretically perfect process of transforming solar energy into hydrogen by photosynthetic bacteria (12 mol H2/ mol glucose), but the actual hydrogen yields are much lower than the theoretical maximum value. Photosynthetic bacteria can produce hydrogen at the expense of solar energy and small-chain organic acids as electron donors [11]. Biohydrogen production using photosynthetic bacteria is influenced by several parameters such as carbon source, nitrogen source, C/N ratio, temperature, light source and nutrient medium [12e17]. In these factors, carbon sources are the most important which affect the electron
* Corresponding authors. Tel.: þ86 10 62620903; fax: þ86 10 62559373. E-mail address: [email protected] (J. Shen). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.03.134
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donor [10]. The conversion efficiency of light energy to hydrogen, with the supply of an appropriate carbon source, is the key factor for hydrogen production by biological systems [18]. The previous researches showed that only a portion of carbon source is suitable for hydrogen production. Some studies reported that lower hydrogen production rates occurred with acetate and butyrate than with malate and lactate [11,19,20]. Using complex substrates as carbon source for photosynthetic hydrogen production has also been explored, such as wastewater [10,15,21]. However, high conversion efficiency of substrates for photosynthetic hydrogen production has not been obtained. Rhodobacter sphaeroides is a kind of purple non-sulfur bacterium that can obtain energy through photosynthesis and has been applied into photosynthetic hydrogen production [17,21]. In the present study, photo-fermentation of sole and compound carbon sources was conducted with Rb. sphaeroides RV. The data obtained from this study are expected to provide basic and engineering data for the improvement of photosynthetic hydrogen production.
2.
Materials and methods
2.1.
Photosynthetic bacteria and growth medium
Rb. sphaeroides RV was purchased from Institute of Microbiology, Chinese Academy of Science. The strains were grown in basic medium, which was composed of a basal mineral medium (1 L of basal mineral medium was composed of K2HPO4, 600 mg; KH2PO4, 500 mg; NaCl, 400 mg; CaCl2$2H2O, 50 mg; MgCl2$6H2O, 200 mg; FeSO4$7H2O, 5 mg; ZnCl2, 7 mg; CuSO4$5H2O, 2.4 mg; H3BO3, 16 mg; MnSO4$4H2O, 10 mg; NiCl2$6H2O, 2 mg; CoCl2$6H2O, 2 mg; NaMoO4$2H2O, 4 mg; EDTA-2Na, 0.4 mg), vitamins (1.4 mg/L vitamin H, 0.5 mg/L vitamin B1, 3.0 mg/L nicotinamide), 0.3 g/L yeast extract 1.25 g/ L (NH4)2SO4, 1.0 g/L glucose and 6.84 g/L sodium succinate. The strains were cultivated anaerobically for 24 h under photosynthetic condition of 4000 lux at 32 C (light-grown cells) and used as inoculum. When required, an inoculum culture was concentrated by centrifugation at 4000 rpm for 10 min.
2.2.
Experimental method in photo-fermentation
Hydrogen-production batch experiments were performed in 120 or 320 mL glass column photobioreactor. The temperature was maintained at 32 C. Light intensity was 220 W/m2 at the outer surface of the reactor. The basic medium and additional carbon sources were used as the medium for photosynthetic hydrogen production. A required amount of sodium glutamate was added to adjust concentration to 0.8 g/L. pH was adjusted to 7.0 with 4% sodium hydroxide and hydrochloric acid. Argon gas was used to create anaerobic conditions. The gas volume was calibrated to 25 C and 760 mmHg. All the experiments were done in batch operation. Each experimental condition was carried out in triplicate. The experimental results noted were the averages (standard deviation) of the values obtained in independent experiments conducted in triplicate. All chemicals used in the experiments were of AR grade.
2.3.
Chemical analysis
The hydrogen content was determined by a gas chromatograph (Techcomp. Co., China, 7890II) equipped with a thermal conductivity detector (TCD) and a 2-m stainless steel column packed with Porapak Q (80e100 mesh). The operating temperatures of the injection port, the oven and the detector were set at 70, 50 and 70 C, respectively. Argon was used as the carrier gas at a flow rate of 30 mL/min. At each time interval, the total volume of biogas production was measured by a plunger displacement method using appropriately sized glass syringes, ranging from 10 to 100 mL [22]. The cumulative hydrogen volume was calculated by equation (1) [23]:
VH,i ¼ VH,i1 þ CH,i (VG,i VG,i1) þ VH,0 (CH,i e CH,i1)
(1)
where VH,i and VH,i1 are cumulative hydrogen volumes at the current (i) and previous (i 1) time intervals, VG,i and VG,i1 are the total biogas volumes in the current (i) and previous (i 1) time intervals, CH,i and CH,i1 are the fraction of hydrogen in the headspace of the bottle at the current (i) and previous (i 1) intervals and VH,0 is the total volume of headspace in the bottle. Detection of the alcohols and volatile fatty acids (VFAs, C2eC6) were measured by a gas chromatograph (SHIMADZU Co., Japan, GC 14B) using a flame ionization detector (FID) and a 2-m glass column packed with Unisole F-200 (30e60 mesh). The temperatures of the injection port, the oven and the detector were set at 200, 165 and 200 C, respectively. The carrier gas was argon at a flow rate of 30 mL/min. Light intensity was measured with TES-1332 luminometer (Shenzhen Langpu Electronic Tech. Co. Ltd). Cell mass concentration was determined spectrophotometrically as optical density at 660 nm (OD660).
2.4.
Model analysis
The cumulative hydrogen production in the batch experiments followed the modified Gompertz equation [24]: Rm e H ¼ P exp exp ðl tÞ þ 1 P where H is the cumulative hydrogen production (mL), P is hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL/h), e is 2.71828, l is the lag-phase time (h), and t is the incubation time (h).The corresponding values of P, Rm and l for each batch were estimated using Origin 7.5, which is a scientific graphing and data analysis software.
3.
Results and discussion
Sodium glutamate (0.8 g/L) was used as nitrogen source and different carbon sources were tested at different initial concentrations.
3.1. Effect of sodium acetate, sodium propionate and sodium butyrate concentration on hydrogen production Acetate, propionate and butyrate were chosen as carbon sources, as they are dominant products of anaerobic
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Acetate: C2H4O2 þ 2H2O / 4H2 þ 2CO2
Propionate: C3H6O2 þ 4H2O / 7H2 þ 3CO2
Butyrate: C4H8O2 þ 6H2O / 10H2 þ 4CO2
a Volume of cumulative hydrogen (mL)
fermentation process [25e27]. The photosynthetic bacteria can further utilize these soluble metabolites to produce more hydrogen at the expense of light energy [28e30]. The theoretical equations of hydrogen production from acetate, propionate and butyrate were described as follows [11]:
14
12
10
8
6
4
1.2 g/L 2.4 g/L 3.6 g/L 4.8 g/L
2
1.8 g/L 3.0 g/L 4.2 g/L
0 0
12
24
36
48
60
72
Time (hour)
Volume of cumulative hydrogen (mL)
b
14
12
10
8
0.4 g/L 0.8 g/L 1.2 g/L 1.6 g/L 2.0 g/L 2.4 g/L 2.8 g/L
6
4
2
0 0
12
24
36
48
60
72
Time (hour)
c
16
Volume of cumulative hydrogen (mL)
The influence of sodium acetate concentration on cumulative hydrogen volume is shown in Fig. 1a. The cumulative hydrogen production was more 12 mL when sodium acetate concentration ranged 1.2e2.4 g/L. When sodium acetate concentration ranged 3.0e4.8g/L, the cumulative hydrogen dramatically decreased to about 6 mL. With the increase of sodium acetate concentration, the cumulative hydrogen gradually decreased. The maximum cumulative hydrogen was 13.7 mL obtained at 1.2 g/L sodium acetate. Table 1 shows that substrate consumption efficiency, L-sodium glutamate consumption efficiency, OD660 and hydrogen yield under different concentrations of sodium acetate. Acetate consumption efficiency, L-sodium glutamate consumption efficiency and OD660 had no obvious decreased trend along with the increase of acetate concentration. It indicated that sodium acetate as carbon source could better promote the growth of bacteria and a high acetate concentration was not toxic to photo-fermentation of hydrogen by Rb. sphaeroides RV. The result was consistent with the previous study [31]. The cumulative hydrogen production decreased with the increase of acetate concentration due to producing numerous byproducts poly-b-hydroxybutyrate (PHB). Hustede reported that acetate is favorable for PHB synthesis [32]. The byproducts competed with the hydrogen production of photosynthetic bacteria and resulted in the decrease of cumulative hydrogen production [33,34]. Therefore the optimal concentration of acetate was 1.2 g/L for the hydrogen production of photosynthetic bacteria. The maximum hydrogen yield was 165 mmol H2/mol-acetate obtained at 1.2 g/L sodium acetate. Fig. 1b illustrates the effect of the concentration of sodium propionate on hydrogen production. When sodium propionate concentration ranged in 0.8e1.6 g/L, the cumulative hydrogen volumes were about 12 mL. No PHBs were detected after the termination of hydrogen-producing reaction. It indicated propionate was not an optimal carbon source for PHB production and benefited for hydrogen production. Table 1 shows propionate consumption efficiency was lower than acetate consumption efficiency which was 70e90%. When propionate concentration ranged in 0.4e1.6 g/L, OD660 was higher than 1.1. The maximum hydrogen yield was 424 mmol H2/mol-propionate obtained at 0.4 g/L sodium propionate. The optimal concentration of propionate was ranged in 0.4e1.6 g/L for the hydrogen production of photosynthetic bacteria. The effect of butyrate concentrations on hydrogen production was similar with acetate (Fig. 1c). When butyrate concentration ranged in 1.2e1.8 g/L, the maximum
14 12
1.2 g/L 2.4 g/L 3.6 g/L 4.8 g/L
10 8
1.8 g/L 3.0 g/L 4.2 g/L
6 4 2 0 0
12
24
36
48
60
72
Time (hour)
Fig. 1 e Cumulative hydrogen volume versus corresponding fermentation time at different concentrations of sodium acetate (a), sodium propionate (b) and sodium butyrate (c).
cumulative hydrogen volume was 15.1 mL. 400 mg/L PHB was detected after the termination of hydrogen-producing reaction. It suggested that the excess of butyrate was beneficial for PHB production which was the major reason for the resulting
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3.6 65.1 88.5 1.32 28 3.0 70.9 88.3 1.38 29 2.4 73.2 88.1 1.45 28 1.8 81.6 89.6 1.29 165 1.2 80.1 82.7 1.33 242 2.8 71.6 79.6 0.83 64 2.4 70.7 75.0 0.89 70
3.2. Effect of D,L-malate, sodium lactate and sodium succinate concentration on hydrogen production
1.2 76.6 84.2 1.27 172
1.6 77.8 84.3 1.10 130
2.0 78.3 84.7 0.99 90
We choose L-lactate, D,L-malate and succinate to further the research of hydrogen production, as they are usual carbon sources for photosynthetic bacteria [35e37]. The theoretical equations of hydrogen production from malate, lactate and succinate were described as follows [11]:
Malate: C4H6O5 þ 3H2O / 6H2 þ 4CO2
0.8 75.7 84.1 1.22 276
Sodium propionate
0.4 86.4 80.3 1.17 424
Lactate: C3H6O3 þ 3H2O / 6H2 þ 3CO2
4.2 97.4 92.7 1.22 17
4.8 98.6 92.6 1.24 16
Succinate: C4H4O4 þ 4H2O / 6H2 þ 4CO2
1.8 97.7 91.1 1.18 100 1.2 99.9 90.9 1.22 165
SCE: substrate consumption efficiency (%). GCE: L-Sodium glutamate consumption efficiency (%). HY: hydrogen yield (mmol H2/mol-substrate).
3.6 98.2 92.3 1.26 27 2.4 98.0 91.0 1.19 76 Concentration (g/L) SCE GCE OD660 HY
3.0 98.7 90.7 1.08 34
Sodium acetate Carbon source
Table 1 e Effects of different concentrations of sodium acetate, sodium propionate and sodium butyrate on photo-H2 production.
Sodium butyrate
4.2 66.4 92.6 1.27 17
4.8 56.2 87.0 1.33 15
decrease of cumulative hydrogen production. The butyrate consumption efficiency and hydrogen yield decreased with the increase of sodium butyrate concentration, but OD660 and L-sodium glutamate consumption efficiency remained basically unchanged (Table 1). The maximum hydrogen yield was 242 mmol H2/mol-butyrate obtained at 1.2 g/L butyrate. Overall, the optimal butyrate concentration ranged in 1.2e1.8 g/L for photosynthetic bacteria hydrogen production. The testing results showed numerous PHB (wet cell weight 35%) and little 5-aminolevulinic acid (8e15 mg/L) were detectable for acetate and butyrate as carbon source. PHB production was adverse to the hydrogen production of photosynthetic bacteria [34]. Hustede reported that the type of carbon source, nitrogen source and the ratio of C/N can affect the production of PHB and the ratio of H2 [32]. For better hydrogen or PHB yield control, clarifying the relationship between PHB accumulation and hydrogen production is an important issue.
Fig. 2 shows the effect of D,L-malate, sodium lactate and sodium succinate concentrations on cumulative hydrogen volume and hydrogen content. Fig. 2a shows the maximum cumulative hydrogen volume and hydrogen content were 34 mL and 80.7% obtained at 6.0 g/L D,L-malate, respectively. Table 2 shows the effect of D,L-malate, sodium lactate and sodium succinate concentration on hydrogen production. L-glutamate consumption efficiencies were higher than 82% in all experiments for D,L-malate as carbon source. Malate consumption efficiency slightly decreased with the increase of D,L-malate concentration. The maximum cell density was 2.60 g/L obtained at 6.0 g/L malate. The maximum hydrogen yield was 1.34 mol H2/mol-substrate obtained at 1.0 g/L malate. Overall, the optimal concentration for D,L-malate as carbon source ranged in 1.0e6.0 g/L. Fig. 2b shows that the maximum cumulative hydrogen volume was 58.8 mL obtained at 4.0 g/L lactate. With the increase of lactate concentration, the hydrogen content firstly decreased and then increased to remain unchanged. When lactate concentration ranged in 3.0e8.0 g/L, the hydrogen content remained 50%. L-glutamate consumption efficiencies were higher than 93.2%, but lactate consumption efficiencies were very low (Table 2). The maximum lactate consumption
a
b
40
35
30
25
20
15
60 55 50 45 40 35 30 25 20 15
16
1
1
2
2
4
3
3
5
5
6
6
7
7
9
8
8
10
85 80 75 70 65 60 55 50 45 40
65 60 55 50 45 40 35 30 25
35
30
25
20
15
Hydrogen content (%)
8
Table 2 e Effects of different concentrations of D,L-malate, sodium lactate and sodium succinate on photo-H2 production.
Concentration (g/L) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 SCE 98.4 96.2 97.4 94.6 92.3 93.4 88.2 82.9 72.3 76.4 75.4 80.8 77.8 68.3 63.7 61.9 97.2 92.3 90.4 90.6 87.3 88.4 83.2 81.7 GCE 88.4 89.2 82.1 86.7 84.2 83.4 84.8 82.8 98.7 97.6 98.4 99.7 93.2 98.1 99.3 97.6 94.1 92.3 92.4 91.7 90.2 88.3 89.3 93.6 Cell density (g/L) 1.78 2.16 1.98 2.16 2.48 2.60 2.44 2.42 2.38 2.76 2.98 3.56 3.38 3.40 3.34 3.42 2.04 2.81 2.62 3.06 2.99 3.23 3.45 3.32 HY 1340 890 650 540 480 420 330 300 850 700 620 480 430 380 310 240 300 240 280 240 220 270 260 190
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 2 1 6 7 e1 2 1 7 4
4
5
Malate (g/L)
4
7
Lactate (g/L)
6
Succinate (g/L)
Hydrogen content (%)
c 14
12
10
8
6
4
2 3
Fig. 2 e Cumulative hydrogen volume versus corresponding fermentation time at different concentrations of D,L-malate (a), sodium lactate (b) and sodium succinate (c).
Hydrogen content (%)
efficiency was only 80.8% at 4.0 g/L lactate. But the maximum cell density was 3.56 g/L. With the increase of lactate concentration, the hydrogen yield gradually decreased. The maximum hydrogen yield was 850 mmol H2/mol-substrate obtained at 1.0 g/L lactate. So the optimal lactate concentration was 1.0e4.0 g/L for photosynthetic bacteria hydrogen production. Fig. 2c shows that the maximum cumulative hydrogen volume was 15.6 mL at 9.0 g/L sodium succinate. The
Sodium succinate Sodium lactate D,L-Malate
Carbon source
Volume of cumulative hydrogen (mL)
Volume of cumulative hydrogen (mL)
Volume of cumulative hydrogen (mL)
SCE: substrate consumption efficiency (%). GCE: L-Sodium glutamate consumption efficiency (%). HY: hydrogen yield (mmol H2/mol-substrate).
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Table 3 e Hydrogen production by Rb. sphaeroides RV using other carbon sources. Carbon source
Concentration (g/L)
SCE
0.2 0.4 0.5 0.8
63.2 3.2 21.1 5.1 9.4 2.6 ND
Alcohol Glucose Sodium citrate NaCO3
OD660 1.82 1.78 1.06 1.44
0.17 0.23 0.11 0.13
Cell density (g/L)
HY
8.1 0.45 11.2 0.54 ND ND
1.89 1.84 1.39 1.48
0.31 0.22 0.15 0.20
SCE: substrate consumption efficiency (%). HY: hydrogen yield (mL H2/mol-substrate). ND: no detection.
maximum hydrogen content was 35.2% at 8.0 g/L sodium succinate. L-glutamate consumption efficiencies were higher than 88%, but sodium succinate consumption efficiencies gradually decreased with the increase of sodium succinate concentration. Comparison with D,L-malate, the cell density obviously increased. The maximum cell density was 3.45 g/L obtained at 9.0 g/L sodium succinate. With the increase of sodium succinate concentration, the hydrogen yield slightly changed and remained 190e300 mmol H2/mol-substrate. So the optimal sodium succinate concentration was 3.0e9.0 g/L for photosynthetic bacteria hydrogen production.
3.3. Effect of other carbon sources on hydrogen production In this section, we studied the effects of some unusual carbon sources on hydrogen production. Table 3 shows the effects of alcohol, glucose, sodium citrate and sodium carbonate on hydrogen production. The results show the strain could utilize alcohol as carbon source for the positive growth of strain and hydrogen production. But the hydrogen yield is much lower than the foregoing carbon sources. In addition, the substrate consumption efficiency was only 63.2%. Oh suggested that minimized ethanol content could improve hydrogen production yield [38]. Glucose could provide nutrients for the growth of strain, but the cumulative hydrogen production was low. Khatipov reported that low hydrogen production rate and poor bacterial growth occurred on glucose as carbon source [34]. The strain stopped growth for sole sodium citrate as carbon source and no hydrogen was detected. Sodium carbonate could provide the nutrient for the growth of strain and no hydrogen was detected, this was because sodium
carbonate can not act as the hydrogen donor [39]. The variation of hydrogen production for various carbon sources possibly results from differences in their reduction states and patterns of metabolism [18].
3.4. The compound carbon source influenced the hydrogen production of photosynthetic bacteria Sole carbon source has the high substrate consumption efficiency and the low hydrogen yield. Sangeetha investigated the effect of mixed volatile fatty acids on the growth of Rb. Sphaeroides by response surface methodology [40]. But investigated the effect of the component of compound carbon source on hydrogen production is rare [41]. So we studied the compound carbon source that influenced the hydrogen production of photosynthetic bacteria. Fig. 3a shows malate and succinate as compound carbon source influenced the cumulative hydrogen production. The cumulative hydrogen volume for the compound carbon source was as high as over 1.4-fold than sole D,L-malate as carbon source under the fixed ratio of C/N. Table 4 shows that L-sodium glutamate consumption efficiencies were higher than 97% and no distinct differences between sole and compound carbon source. The hydrogen yield increased obviously, from 450 mmol H2/mol-substrate to 534 mmol H2/mol-substrate. The modified Gompertz model fits experimental data well with the R2 values, which are all greater than 0.98. Consequently, the modified Gompertz equation is useful and relatively accurate in predicting the cumulative hydrogen production as well as the rate of hydrogen production. The lag-phase times increased dramatically to 34.0 h, which was much longer than the 24.4 h of the sole D,L-malate. The
Table 4 e Hydrogen production and kinetic model parameters from compound carbon source. D,L-Malate
(g/L)
Sodium lactate
6.82 3.16 2.31
Sodium succinate (g/L)
1.0 2.0 4.86 3.23 2.02
1.0 2.0
GCE
98.7 97.6 98.4 98.8 98.6 98.4
GCE: L-Sodium glutamate consumption efficiency (%). HY: hydrogen yield (mmol H2/mol-substrate).
0.8 1.0 0.5 0.3 0.8 0.6
Cell density (g/L)
2.32 2.66 2.78 2.41 2.64 2.87
0.12 0.09 0.17 0.12 0.15 0.21
HY
450 520 534 550 690 794
Kinetic model parameters
22 15 29 19 35 39
P (mL)
Rm (mL/h)
l (h)
R2
24.2 37.2 35.1 38.4 45.9 56.5
0.85 1.07 0.95 1.24 1.19 1.98
24.4 33.8 34.0 23.7 10.7 28.3
0.986 0.994 0.994 0.995 0.992 0.991
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 2 1 6 7 e1 2 1 7 4
maximum hydrogen production rate was increased to ca. 1.0 mL/h, significantly elevated compared to the 0.85 mL/h of the sole D,L-malate. The maximum hydrogen production rate and cumulative hydrogen for compound carbon source were significantly elevated compared to the sole D,L-malate. Fig. 3b shows the cumulative hydrogen volumes gradually increased with succinate concentration from 0 to 2 g/L, from 38.4 to 56.5 mL for lactate and succinate as compound carbon source. Table 4 shows the L-glutamate consumption efficiencies hardly changed and were higher than 97%. Cell density, hydrogen yield and cumulative hydrogen were obviously increased. The maximum hydrogen yield reached 794 mmol H2/mol-substrate. Interestingly, the lag-phase time and the maximum hydrogen production rate didn’t show a regular trend. The minimum hydrogen production rate and lag-phase time were produced at 3.23 g lactate and 1.0 g succinate as compound carbon source. It indicated kinetic model parameters were remarkably affected by the component of compound carbon source. In addition, the hydrogen yield for lactate and succinate as compound carbon source was higher than malate and succinate as compound carbon
Volume of cumulative hydrogen (mL)
a
40
6.82 g/L Malate 3.16 g/L Malate+1.0 g/L Succinate 2.31 g/L Malate+2.0 g/L Succinate
35
4.
Conclusions
In the present study, hydrogen production by photosynthetic bacteria from sole and compound carbon source was investigated. The results showed hydrogen production for compound carbon sources was better than sole carbon sources. The maximum hydrogen yield reached 794 mmol H2/molsubstrate for 2.02 g lactate and 2.0 g succinate as compound carbon source. But the component of the compound carbon source needs to optimize further.
Acknowledgments 25
The authors would like to thank the Chinese Academy of Sciences for financial support (Item No. KJCX2-YW-H21).
20 15
references
10 5
0
20
40
60
80
100
Time ( hour )
Volume of cumulative hydrogen (mL)
source. For Rb. sphaeroides RV, different compound carbon source had different effects of hydrogen production. Overall, hydrogen production for compound carbon was better than the sole carbon source. It might be due to the various carbon sources being utilized with different efficiencies by photosynthetic bacteria [41]. The efficiency of a certain carbon source depends on factors such as the activity of tricarboxylic acid cycle, the C/N ratio, the reduction state of that material and the conversion potential of the carbon source into alternative metabolites [10]. Adding the compound carbon source into hydrogen-producing system alters the modes of metabolism, resulted in improving the hydrogen production.
30
0
b
12173
60
50
40
30
20
10
4.86 g/L Lactate 3.23 g/L Lactate+1.0 g/L Succinate 2.02 g/L Lactate+2.0 g/L Succinate
0 0
20
40
60
80
Time ( hour )
Fig. 3 e Cumulative hydrogen volume versus corresponding fermentation time for D,L-malate and sodium succinate (a) and sodium lactate and sodium succinate (b) as compound carbon source.
100
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