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Sodium inhibition of fermentative hydrogen production
international journal of hydrogen energy 34 (2009) 3295–3304
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Sodium inhibition of fermentative hydrogen production Dong-Hoon Kima, Sang-Hyoun Kimb, Hang-Sik Shina,* a Department of Civil and Environmental Engineering, Korea Advanced Institute of Science & Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea b Green Ocean Technology Center, Korea Institute of Industrial Technology, 35-3 Hongcheon-Ri, Ipchang-Myun, Seobuk-gu, Cheonan-Si, Chungnam 331-825, Republic of Korea
article info
abstract
Article history:
A continuous-stirred-tank reactor (CSTR) was fed with low-sodium influent containing
Received 27 December 2008
0.27 g of Naþ/L for 70 days (Phase I), and then subjected to higher concentrations of Naþ/L,
Received in revised form
i.e. 2.41 (Phase II), 5.36 (Phase III), and 10.14 g (Phase IV-1). At the quasi-steady state of each
22 February 2009
phase, biomass was sampled for an acute sodium toxicity assay. Unlike the control
Accepted 22 February 2009
biomass, which exhibited a monotonic decrease of specific H2 production activity (SHPA)
Available online 16 March 2009
with increasing sodium concentration from 0.27 to 21.00 g Naþ/L, the acclimated biomass maintained their activity up to 6.00 g Naþ/L. Soluble microbial product analysis revealed
Keywords:
that a sudden increase of the exterior sodium concentration changed the metabolic
Fermentative hydrogen production
pathway such that it became favorable to lactate production while depressing butyrate
Sodium inhibition
production. Meanwhile, when the biomass was allowed for sufficient time to adapt to the
Acute toxicity
chronic toxicity condition, the volumetric H2 production rate (VHPR) was maintained above
Chronic toxicity
4.05 L H2/L/d at up to Phase III. However, an irrecoverable H2 production drop was observed
Acclimation
at Phase IV-1 with a significant increase of lactate and propionate production. Although the sodium concentration decreased to 8.12 (Phase IV-2), 6.61 (Phase IV-3), and 5.36 g Naþ/L (Phase V) at further operation, the performance was never recovered. A PCR-DGGE analysis revealed that lactic acid bacteria (LAB) and propionic acid bacteria (PAB) were only detected at Phases IV and V, which are not capable of producing H2. ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The current energy system based on fossil fuels is now facing two fundamental problems in sustainability: the depletion of fossil fuel and environmental pollution. This has led to an extensive search for new alternative energy sources and carriers [1]. Among various candidates, hydrogen is regarded as the most promising energy carrier, since it produces only water when combusted and has a 2.75 times higher energy yield (122 kJ/g) than hydrocarbon fuels. In addition, as an
automotive fuel, H2 can be easily applied in proton exchange membrane fuel cell vehicles as well as conventional internal combustion engines [2]. H2 can be made via several ways, including electrolysis of water, thermocatalytic reformation of hydrogen-rich organic compounds, and biological processes. Currently, H2 is exclusively made by steam reforming of gas, requiring electricity derived from fossil fuel combustion, a process that is energy intensive and expensive. Feedstock and energy for H2 production must be renewable if its purpose is to decrease the
* Corresponding author. Tel.:þ82 42 350 3613; fax: þ82 42 350 8460. E-mail address: [email protected] (H.-S. Shin). 0360-3199/$ – see front matter ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.02.051
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current dependency on fossil fuel. Sustainable generation of H2 may be achieved by a range of technologies, including biological processes [3]. Biological H2 production can be achieved by phototrophic and non-phototrophic methods. With the help of light, autotrophs such as algae and heterotrophs such as Rhodobacter sp. produce H2 from water and organic wastewater, respectively. However, phototrophic H2 production confronts several obstacles: (1) low solar energy conversion efficiency (2) slow reaction rate, and (3) low light penetration due to biofilm formation on the reactor wall. On the other hand, non-phototrophic production, often called fermentative H2 production (FHP), offers many advantages such as fast reaction rate, degradation of solid organic wastes, technical simplicity, and no need of light. As a result, it can serve to address two critical global issues simultaneously, energy supply and environmental protection [4]. FHP is one of the ways releasing excess electrons derived from organics with the help of the ‘hydrogenase’ function in bacteria. The following equations, Eqs. (1) and (2), are the main H2 production reactions involved in FHP from glucose. Comprehensive studies have been conducted dealing with operating parameters such as pH, hydraulic retention time (HRT), carbon source, and H2 partial pressure [5]. However, little information is available about the effect of cations on FHP.
2.
Materials and methods
Glucose þ 2 H2O / 2 Acetate þ 2 CO2 þ 4 H2 þ 4 ATP
(1)
2.1.
Seed sludge and substrate
Glucose / Butyrate þ 2 CO2 þ 2 H2 þ 2 ATP
(2)
Cations play essential roles in adenosine (ATP) synthesis, nicotinamide adenine dinucleotide (NAD) oxidation/reduction, and enzyme activity, thereby accelerating microbial metabolism if maintained at proper concentrations. However, excessive cations can cause plasmolysis and loss of cell activity by creating high osmotic pressure and improper enzyme linkages. In particular, sodium, the main cation in biomass and seawater, leads to many problems in biological treatment systems of wastewater from seafood processing, the dairy industry, and chemical production. Besides, sodium concentration can be increased by buffer addition such as NaOH, Na2CO3, and NaHCO3. Therefore, research has been carried out on anaerobic digestion and biological nutrient removal processes to identify the inhibition mechanisms and alleviation of its inhibition [6,7]. For methane fermentation, it has generally been reported that sodium concentration over 2 g Naþ/L will cause a performance drop [8,9]. However, some studies have reported that continuous exposure of microorganisms to higher sodium levels increased the sodium tolerance. Seafood processing wastewater containing 5–12 g/L of sodium was successfully treated with an anaerobic filter process [10]. According to Feijoo et al. [11], sodium concentration causing 50% inhibition exceeded 10 g Naþ/L for the sludge obtained from digesters treating high saline wastewater, but it was lower than 5 g Naþ/ L when unacclimated sludge was used. Also, a stepwise increase of sodium level exhibited higher tolerance than a shock increase [12]. These findings suggest that acclimation could mitigate sodium inhibition.
There are two known bacterial strategies to adapt to and cope with high sodium concentration; salt-in and compatiblesolute strategies [13]. The main mechanism in the salt-in strategy is the extrusion of sodium ions outside cells concurrent with accumulation of potassium ions inside cells via a proton electrochemical gradient and at ATP expense. As this strategy does not reduce the osmotic pressure inside the cell, all intracellular systems should be tolerant at high osmotic pressure. On the other hand, in the compatible-solutes strategy, compatible solutes such as glycerol, ectoine, and glycine betaine are created for the proper functions of intracellular systems at high osmotic pressure. Debate continues over which strategy is more energy efficient, but it is clear that life in a salty environment is costly. Although salty organic substances, such as waste from the foodstuff industry, could be suitable sources for FHP and sodium is employed as the cation in general buffers and nutrients for FHP, research on this subject is scarce. Therefore, the present study aimed at evaluating the sodium inhibition of FHP. Chronic and acute sodium toxicity was investigated by continuous-stirred-tank reactor (CSTR) operation and batch tests at various sodium concentrations, respectively.
The seed sludge was taken from an anaerobic digester in a local wastewater treatment plant. The pH, alkalinity, and volatile suspended solids (VSS) concentration of the sludge were 7.5, 2.83 g CaCO3/L, and 5.3 g/L, respectively. The sludge was heat-treated at 90 C for 15 min to inactivate hydrogen consumers and to harvest spore-forming anaerobic bacteria such as Clostridium sp. [14]. Sucrose of 25 g COD/L was used as a substrate. Concentrations of NH4Cl, KH2PO4, and FeCl2$4H2O were added to yield a COD:N:P:Fe ratio of 100:5:1:0.33. Feed also contained the following nutrients (in mg/L): NaHCO3 1000; MgCl2$6H2O 100; CaCl2$2H2O 75; Na2MoO4$4H2O 0.01; H3BO3 0.05; MnCl2$4H2O 0.5; ZnCl2 0.05; CuCl2 0.03; NiCl2$6H2O 0.05; CoCl2$2H2O 0.5; Na2SeO3 0.05 [14].
2.2.
Continuous operation
In this study, a CSTR with a working volume of 5.0 L (325 mm high by 140 mm ID) was used. The chemical oxygen demand (COD) loading rate was maintained at 50 g/L d during the entire period of operation, which corresponded to 12 h of hydraulic retention time (HRT). The reactor was mixed by mechanical stirring at 100 rpm. The pH was maintained at 5.3 0.1 using a pH sensor, pH controller, and 3 N KOH. Biogas production was monitored by the water displacement method and then corrected to standard temperature (0 C) and pressure (760 mmHg) (STP). All experiments were conducted in a constant temperature room at 35 1 C. After being seeded with heat-treated sludge equivalent to 30% of the total effective volume, the reactor was purged with N2 gas for 5 min to provide an anaerobic condition, and then operated in batch mode. When the H2 yield reached 0.2 mol H2/mol hexoseadded in the batch operation, continuous operation started [15].
international journal of hydrogen energy 34 (2009) 3295–3304
The CSTR was fed with low-sodium influent containing 0.27 g Naþ/L by NaHCO3 (buffer solution) for 70 days; 30 days for start-up and 40 days as the control condition (Phase I). Subsequently, sodium chloride was added to increase the sodium concentration to: 2.41 (Phase II), 5.36 (Phase III), and 10.14 g Naþ/L (Phase IV-1) according to the batch test results using the control biomass. As H2 production failure was observed at Phase IV-1, sodium concentration decreased to 8.12 (Phase IV-2), 6.61 (Phase IV-3), and 5.36 g Naþ/L (Phase V) at further operation. At each phase, the reactor was operated at least 10 days, 20 times of HRT, in order to establish steadystate conditions, judging from metabolic products.
2.3.
Batch test
The inocula for batch tests were biomass collected from the continuous reactor acclimated at different sodium concentrations (Phases I, II, and III). The sludge was anaerobically centrifuged at 672g for 30 min to eliminate any salt present in the mixed liquor and avoid uncontrolled antagonistic or synergistic effects. Batch experiments were conducted using fermenters (635 mL) equipped with a pH sensor and magnetic mixing bar. Each batch fermenter contained 1g of the centrifuged sludge having 185–190 mg VSS/g, 20 mL of nutrient stock solutions (5) to yield the same final concentration as CSTR, and a predetermined amount of NaCl stock solution (40 g Naþ/L). Sodium toxicity levels were varied for each fermenter; 0.27 (control), 3.00, 6.00, 9.00, 12.00, 15.00, 18.00, and 21.00 g Naþ/L. Distilled water was added into the fermenters to adjust the final volume to 100 ml. The large headspace volume (535 mL) was to alleviate the adverse effect of high H2 partial pressure during fermentation. The initial pH was adjusted to 7.00 0.05 by 3 N KOH and HCl. The bottles were then purged with N2 gas, and placed in a water bath with a magnetic stirrer. Mixing speed and temperature were controlled at 100 rpm and 35 1 C, respectively. Biogas production and its constituents were measured periodically from each bottle to estimate H2 production. As acids are produced during fermentation, resulting in a pH drop, 3 N KOH was added to maintain the pH above 5.0 0.2. Organic acids, alcohols, and remaining substrate were measured after H2 production ceased.
2.4.
Methodology
The modified Gompertz equation was employed in this study to describe the cumulative H2 production in the batch tests [16]. 0 R $e ðl tÞ þ 1 HðtÞ ¼ P$exp exp P
(3)
where H(t) ¼ cumulative H2 production (mL) at cultivation time t (h); P ¼ H2 production potential (mL); R0 ¼ H2 production rate (mL/h); l ¼ lag period (h); and e ¼ exp(1) ¼ 2.71828. The inhibitory effect of sodium on specific H2 production activity (SHPA) was analyzed using a noncompetitive inhibition model as shown in Eq. (4) [17]. n I R ¼ R0 1 I
(4)
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where R ¼ specific H2 production rate at inhibitor concentrations of I (mL H2/g VSS/h); R0 ¼ specific hydrogen production rate without an added inhibitor (mL H2/g VSS/h); I ¼ inhibitor concentration (g/L); I* ¼ lethal inhibitor concentration beyond which the reaction cannot proceed (g/L); and n ¼ constant.
2.5.
Analytical methods
Measured biogas production was corrected to standard temperature (0 C) and pressure (760 mmHg) (STP). Hydrogen content in the biogas was determined by a gas chromatography (GC, Gow Mac series 580) using a thermal conductivity detector and a 1.8 m 3.2 mm stainless-steel column packed with molecular sieve 5A with N2 as a carrier gas. The contents of CH4, N2, and CO2 were measured using a GC of the same model noted previously with a 1.8 m 3.2 mm stainless-steel column packed with porapak Q (80/100 mesh) using helium as a carrier gas. The temperatures of the injector, detector, and column were kept at 80, 90, and 50 C, respectively, in both GCs. Volatile fatty acids (VFAs, C2–C6) and lactate were analyzed by a high performance liquid chromatograph (HPLC) (Finnigan Spectra SYSTEM LC, Thermo Electron Co.) with an ultraviolet (210 nm) detector (UV1000, Thermo Electron) and an 100 mm 7.8 mm Fast Acid Analysis column (Bio-Rad Lab.) using 0.005 M H2SO4 as a mobile phase. Aliphatic alcohol was determined using another HPLC (DX-600, Dionex) with an electrochemical detector (ED50A, Dionex) and a 250 mm 9 mm IonPac ICE-AS1 column (Dionex) using 50 mM HClO4 as a mobile phase. The liquid samples were pretreated with a 0.45 mm membrane filter prior to injection to both HPLCs. COD and VSS were measured according to Standard Methods [18]. Glucose concentration was determined by the colorimetric method [19].
2.6.
Microbial analysis
In order to analyze the microbial communities, the DNAs in the sludge sampled at each sparging condition were extracted using the Ultraclean Soil DNA Kit (Cat # 12800-50; Mo Bio Laboratory Inc., USA). The 16S rDNA fragments were amplified by polymerase chain reaction (PCR). The region corresponding to positions 357 and 518 in the 16S rDNA of Escherichia coli was PCR-amplified using the forward primer EUB357f (50 CCTACGGGAGGCAGCAG-30 ) with a GC clamp (50 -CGCCCG CCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-30 ) at the 50 end to stabilize the melting behavior of the DNA fragments and the reverse primer UNIV518r (50 -ATTACCGCGGCTGCTGG-30 ). PCR amplification was conducted in an automated thermal cycler (MWG-Bio TECH, Germany) using the protocol; that is, initial denaturation for 4 min at 94 C, annealing for 40 s at 55 C, extension for 1 min at 72 C, followed by a final extension for 8 min at 72 C. PCR mixtures had a final volume of 50 ml of 10 PCR buffer, 0.8 mM MgSO4, 0.5 mM of each primer, 0.1 mM dNTP, 25 pg template and 1 U polymerase. PCR products were electrophoresed on 2% (wt/vol) agarose gel in 1 TAE for 30 min for 50 V, and then checked with ethidium bromide staining to confirm the amplification. Denaturing gradient gel electrophoresis (DGGE) was carried out using the Dcode Universal Mutation Detection System (BioRad, USA) in accordance with the manufacturer’s instruments. PCR products were electrophoresed in 1 TAE buffer for 480 min at 70 V and
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international journal of hydrogen energy 34 (2009) 3295–3304
7.5 Control 3 g Na+/L 6 g Na+/L 9 g Na+/L 12 g Na+/L 15 g Na+/L 18 g Na+/L 21 g Na+/L
7.0
250
0.27 g Na+/L (Control) 3.00 g Na+/L 6.00 g Na+/L 9.00 g Na+/L 12.00 g Na+/L 15.00 g Na+/L 18.00 g Na+/L 21.00 g Na+/L
200
150
6.5
pH
Cumulative H2 production (ml)
300
6.0
100
5.5
50
5.0
4.5
0 0
20
40
0
60
5
10
15
20
Time (h)
Time (h) Fig. 1 – Cumulative H2 production curves for control biomass at different sodium concentrations.
Fig. 2 – pH change profiles of control biomass during fermentation.
60 C on polyacrylamide gel (7.5%) containing a linear gradient ranging from 40% to 60% denaturant. After electrophoresis, polyacrylamide gel was stained with ethidium bromide for 30 min, and then visualized on UV transilluminator. Most of the bands were excised from DGGE polyacrylamide gel for 16S rDNA sequencing. DNAs were eluted from the excised bands by immersing them in 20 ml of Tris EDTA buffer (pH 8.0) for one day, and then PCR-amplified with the forward primer EUB357f without a GC clamp and the reverse primer UNIV518r. After PCR amplification, PCR products were purified with using Multiscreen Vacuum Manifold (MILLIPORE com., USA). All the strands of the purified PCR products were sequenced with primers EUB357f by ABIPRISM Big Terminator Cycle Sequencing Kit (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Search of the GenBank database was conducted using the BLAST program.
sodium concentrations. As the H2 production was negligible at more than 15.00 g Naþ/L, Eq (3) was only applied at concentrations lower than 12.00 g Naþ/L. The lag period was also increased with sodium concentration, indicating that shock loading of sodium could result in the failure of H2 production. The pH profile shown in Fig. 2 confirms the retardation in microbial catabolism by sodium inhibition. The pH value dropped to 5.0 within 2 h up to 6.00 g Naþ/L, suggesting that the biomass could readily adapt to 6.00 g Naþ/L. However, it took more than 5 h when the sodium concentration was more than 12.00 g Naþ/L. SHPA (mL H2/g VSS/h) of the control biomass at various sodium concentrations was well fitted by Eq (4) with a high R2 value of 0.974 (graph not shown), and the sodium concentrations causing 30, 60, and 90% inhibition were found: 2.41, 5.36, and 10.14 g Naþ/L, respectively. Effects of sodium on SHPA, H2 yield (HY), and substrate removal efficiency of biomass acclimated at different sodium concentrations are illustrated in Fig. 3(a), (b), and (c), respectively. The SHPA and HY at various sodium concentrations are expressed as ‘‘the percentage (%) of the control, the results from the batch test at 0.27 g Naþ/L using each biomass’’. Acclimation substantially alleviated the acute sodium toxicity. Using the acclimated biomass the SHPA and HY were close to or even higher than those of the control at up to 6.00 g Naþ/L. The highest HY was observed at 3.00 and 6.00 g Naþ/L
3.
Results and discussion
3.1.
Acute sodium toxicity
Acute sodium toxicity of control biomass (Phase I) was severe as shown in Fig. 1 and Table 1. H2 production decreased almost by half at 3.00 g Naþ/L and decreased further at higher
Table 1 – Acute sodium toxicity of FHP when using control biomass. Sodium conc. (g Naþ/L) 0.27 (Control) 3.00 6.00 9.00 12.00 15.00 18.00 21.00
H2 production (mL)
H2 yield (mol H2/mol hexoseadded)
Substrate removal (%)
P (mL)
R (mL/h)
l (h)
241 136 108 52 43 4 2 1
0.83 0.47 0.37 0.18 0.15 0.01 0.01 0.00
97.0 96.6 96.8 95.1 93.5 23.8 9.9 4.0
251 137 108 56 56 – – –
25.1 14.0 10.9 2.9 0.9 – – –
0.9 1.2 4.17 5.2 9.7 – – –
international journal of hydrogen energy 34 (2009) 3295–3304
Relative specific H2 production activity (% of control)
a
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120 100 Control Acclimated - 2.41 Acclimated - 5.36
80 60 40 20 0 0
5
10
15
20
25
Sodium conc. (g Na+/L)
Relative H2 yield (% of control)
b
140 120 100
Control Acclimated - 2.41 Acclimated - 5.36
80 60 40 20 0 0
5
10
15
20
25
20
25
Sodium conc. (g Na+/L)
Sucrose removal (%)
c
100
80
60 Control Acclimated - 2.41 Acclimated - 5.36
40
20
0 0
5
10
15
Sodium conc. (g Na+/L) Fig. 3 – Inhibitory effect of sodium on (a) relative specific H2 production activity (b) relative H2 yield, and (c) sucrose removal for biomass acclimated at different sodium concentrations.
when the biomass acclimated at 2.41 and 5.46 g Naþ/L was used, respectively. This might be attributable to the osmotic pressure formed inside the cells during the acclimation period. A drop in the sodium concentration outside of the cell would facilitate the permeation of water molecules into the cell, which would in turn adversely affect the metabolic balance. Also, acclimation showed a positive effect on substrate removal efficiency. The concentration maintaining
over 90% of substrate removal efficiency was increased to 12.00, 15.00, and 18.00 g Naþ/L when using the control and acclimated biomass at 2.41 and 5.46 g Naþ/L, respectively. At 12.00 g Naþ/L, in spite of high substrate removal, H2 production was 50% less than the control even when using acclimated biomass. This suggests that the metabolic pathway was shifted to a non-hydrogenic route by sodium inhibition under acute toxicity condition. As arranged in
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international journal of hydrogen energy 34 (2009) 3295–3304
Table 2 – Effect of sodium concentration on soluble microbial byproducts when using the acclimated biomass at different sodium concentrations (0.27 (control), 2.41, and 5.36 g NaD/L). Biomass
Sodium conc. (g Naþ/L)
Soluble microbial products, mg COD/L (% of total metabolites) Butyrate
Lactate
Acetate
Propionate
Phase I
0.27 3 6 9 12 15 18 21
7110 5000 4280 2610 530 1820 1030 710
(29.7) (22.2) (19.8) (11.5) (3.1) (26.6) (35.3) (37.2)
81,90 10,920 11,800 14,970 12,000 3950 1190 500
(34.2) (48.4) (54.6) (65.5) (69.1) (57.7) (40.8) (26.2)
3610 3400 3210 4900 3860 440 340 290
(15.1) (15.1) (14.9) (21.5) (22.2) (6.5) (11.7) (15.2)
Phase II
0.27 3 6 9 12 15 18 21
9590 9120 8850 7500 4190 3700 2500 1360
(47.0) (46.4) (39.9) (35.3) (19.7) (17.0) (17.5) (42.7)
4050 4640 5010 9150 15,000 16,200 9710 470
(19.9) (23.6) (22.6) (43) (70.5) (74.1) (67.9) (14.8)
3490 3250 6150 2700 2090 1990 2100 1000
(17.1) (16.6) (27.7) (12.7) (9.9) (9.1) (14.7) (31.4)
Phase III
0.27 3 6 9 12 15 18 21
7090 6460 10,880 6680 1660 1270 560 1850
(35.6) (32.3) (55.2) (33.6) (7.3) (6.0) (2.8) (35.7)
6530 6620 1900 9040 20,250 16,680 17,430 1120
(32.8) (33.1) (9.7) (45.5) (88.6) (77.6) (87.2) (21.6)
4180 4320 2070 810 500 3100 790 1230
(21.0) (21.6) (10.5) (4.1) (2.2) (14.5) (4.0) (23.7)
Table 2, butyrate, lactate, and acetate were the major soluble microbial products of FHP. Interestingly, metabolic shift from butyrate to lactate was concurrent with sodium inhibition. For example, when using the control biomass, the butyrate portion decreased from 29.7% to 4.7% as the sodium concentration was increased up to 12.00 g Naþ/L. Instead, lactate production was predominant, accounting for 65.6% of the total organic acids. It is known that butyrate production is accompanied by H2 production (Eq. (2)), while all lactate producing reactions are non-hydrogenic as shown in Eqs. (5) and (6) [20].
Glucose / Lactate þ Ethanol þ CO2 þ 1 ATP
Total metabolites
2850 (11.9) 1490 (6.6) 1160 (5.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
400 (1.7) 620 (2.8) 650 (3.1) 0 (0.0) 0 (0.0) 0 (0.0) 60 (2.1) 30 (1.6)
23,970 (100.0) 22,590 (100.0) 21,650 (100.0) 22,870 (100.0) 17,390 (100.0) 6850 (100.0) 2920 (100.0) 1910 (100.0)
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
3310 (16.2) 2670 (13.6) 2210 (10) 1930 (9.1) 0 (0.0) 0 (0.0) 0(0.0) 0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 360 (11.3)
20,440 (100.0) 19,680 (100.0) 22,220 (100.0) 21,280 (100.0) 21,280 (100.0) 21,890 (100.0) 14,310 (100.0) 3190 (100.0)
(4.5) (3.3) (6.0) (4.4) (2.1) (2.2) (5.7) (12.2)
1250 (6.3) 2010 (10.1) 3700 (18.8) 2500 (12.6) 0 (0.0) 0 (0.0) 100 (0.5) 0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 360 (7.0)
19,930 (100.0) 20,060 (100.0) 19,720 (100.0) 19,890 (100.0) 22,880 (100.0) 21,510 (100.0) 20,010 (100.0) 5190 (100.0)
1810 1160 550 390 1000 640 300 380
(7.6) (5.2) (2.6) (1.8) (5.8) (9.4) (10.3) (19.9)
0 0 0 0 0 0 0 0 880 650 1170 860 470 460 1130 630
(5)
(6)
Start-up Phase I
6
To the best of our knowledge, this is the first report showing a relation between lactate production with sodium
Phase III
Phase IV
Phase V 12
5
10
4
8
3
6
2
4 VHPR Sodium conc.
1
2
0 0
(7)
Phase II
50
100
150
Sodium conc. (g Na+/L)
Although it is generally accepted that both acetate and butyrate productions provide high H2 production in pure culture, previous studies in using mixed culture showed a close relationship between butyrate and H2 production rather than acetate production. It was reported that H2 yield was linearly proportional to butyrate production or B/A (butyrate/acetate) ratio and doubts were even raised regarding whether the governing acetate production pathway in mixed culture was a H2-consuming reaction as shown in the following Eq. (7) [14,21]. 4 H2 þ 2 CO2 / Acetate þ 2 H2O þ (1–2) ATP
Formate
inhibition in FHP. Previously, Xialong et al. [22] observed a gradual change of metabolic products from acetate, propionate, and butyrate to acetate with increasing sodium concentration, but did not report on lactate. The lactate production could result from either the microbial population change, which is discussed in 3.3 Microbial analysis, or the switched catabolic pathway of H2-producing bacteria. Lactate production is not commonly caused by main anaerobic H2-producing bacteria, Clostridium sp., under normal conditions. Thus far, its production is only reported when (1) H2 release is blocked; (2) hydrogenase activity is inhibited by
Volumetric H2 production rate (L/L/d)
Glucose / 2 Lactate þ 2 ATP
Ethanol
0 200
Time (d) Fig. 4 – Daily variation of volumetric H2 production rate at various sodium concentrations.
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H2 production activity (mL H2/g VSS/h)
international journal of hydrogen energy 34 (2009) 3295–3304
200 Acute toxicity Chronic toxicity
150
100 Avg. value at Phase IV
50
0 0
2
4
6
8
10
Sodium conc. (g Na+/L) Fig. 5 – Comparison of acute and chronic toxicity at different sodium concentrations.
carbon monoxide; or (3) iron or sulfur supply is limited [21]. Further study using a pure culture might reveal whether the metabolic pathway of Clostridium sp. could be altered by sodium inhibition or not.
3.2.
Chronic toxicity
Fig. 4 shows the daily variations of volumetric H2 production rate (VHPR) at various sodium concentrations. Although VHPR fluctuated at Phase III (5.36 g Naþ/L), average values at Phase II (2.41 g Naþ/L) and Phase III were higher than those at Phase I (0.27 g Naþ/L), which did not correspond with the batch test results using the control biomass. In Phase IV-1 (10.14 g Naþ/ L), a sudden deterioration of H2 production was observed, and was not recovered at further operation with lower sodium concentrations (8.12, 6.61, and 5.36 g Naþ/L). The acute sodium toxicity evaluated by a batch test using the control biomass and chronic sodium toxicity obtained in continuous operation are compared in Fig. 5. Under acute toxicity conditions, sufficient time is not provided for the biomass to adapt at the higher sodium concentration, and thus a steep decline of SHPA was shown. Meanwhile, in continuous operation, the biomass has sufficient time to adapt and retain their activity. At 5.00 g Naþ/L, the activity was decreased by 50% in the acute condition, but only 15% in the
chronic condition. This adaptation effect was also observed for substrate removal efficiency. The average continuous performances of FHP at each phase are listed in Table 3. H2 content and yield decreased with increasing sodium concentration, but the substrate removal efficiency increased due probably to the adaptation mechanism requiring more energy at a salty environment. Also, the biomass concentration increased as the substrate utilization increased. Table 4 summarizes the overall COD balance of H2, organic acids, alcohols, and biomass. The COD of biomass was calculated under the assumption that its chemical formula was C5H7O2N [24]. The COD balance was roughly 100 5% in Phases I, II, and III, but only 87% in Phases IV and V, suggesting that compatible solutes were formed for survival at the high salty condition and their leak to the broth. Several studies reported the leakage of compatible solutes such as betaines, ectoines, N-acetylated diamino acids, and Nderivatized carboxamides of glutamine as cell membranes were not perfectly impermeable to low-molecular-weight substances [13]. The butyrate portion decreased while lactate portion increased at a high sodium concentration, which agreed well with the batch test results. The portion of propionate, a by-product of the H2-consuming reaction, also increased at Phases IV and V. The reasons for the change of liquid state by-products distribution in continuous operation are discussed in 3.3 Microbial analysis.
3.3.
Microbial analysis
Fig. 6 illustrates the DGGE profiles of the 16S rDNA gene fragment amplified from the biomass sampled in Phases I–V I– V. The major bands were excised and purified to determine the sequence. Table 5 shows the results of the sequence affiliation, and isolated microorganisms and their main metabolites from the carbohydrate [23,25–32]. In total, 10 bands were detected. While similar band types were observed in Phases I, II, and III, they were changed significantly in Phases IV and V. H2-producing bacteria, Clostridium pasteurianum, and non H2-producing bacteria such as Bifidobacterium thermacidophilum and Acetobacter aceti were detected in Phases I, II, and III. Lactic acid bacteria (LAB) such as Lactococcus lactis, Lactobacillus delbrueckii, and Streptococcus bovis and propionic acid bacteria (PAB), Selenomonas sp., were only detected in Phases IV and V. As LAB and PAB were not capable of H2 production, this population shift could be linked
Table 3 – Performance of FHP at various sodium concentrations in continuous operation. Item
Unit
a
VHPR H2 content H2 yield Substrate removal Biomass
L/L/d % mol H2/mol hexoseconsumed % mg VSS/L
a VHPR ¼ Volumetric H2 production rate.
Phases I
II
III
IV
V
4.05 0.20 55.8 1.2 1.24 0.06 55.9 3.5 906 83
4.43 0.17 54.3 1.0 1.22 0.05 62.3 4.0 1040 181
4.28 0.56 51.8 1.6 0.92 0.12 79.0 6.8 1187 88
0.73 0.71 39.7 9.2 0.13 0.13 93.6 7.5 1790 241
0.16 0.11 29.8 1.0 0.03 0.02 95.7 2.7 1867 95
3302
(100.7%) (104.9%) (95.9%) (86.2%) (87.8) 25,170 26,220 23,980 21,560 21, 940 11,030 9430 5250 1600 1080 1290 1640 1790 2330 2650 1440 1580 1520 270 60
Fig. 6 – DGGE profiles of the 16S rDNA gene fragment at each phase (a [ Phase I, b [ Phase II, c [ Phase III, d [ Phase IV, e [ Phase V). 0.0 0.0 0.0 0.0 0.0 a HBu ¼ butyrate; HLa ¼ lactate; HAc ¼ acetate; HPr ¼ propionate; EtOH ¼ ethanol; HFo ¼ formate.
7.6 18.4 18.8 18.8 7.5 2.4 0.8 1.8 7.6 13.4 25.9 27.1 29.1 44.1 47.2 37.2 33.0 31.9 14.8 10.4 I II III IV V
Total (mg COD/L)
11,410 13,570 15,420 17,360 18,150
HLa (% of total)
HBu (% of total)
22.8 20.7 18.3 14.8 21.5
HPr (% of total) HAc (% of total)
a a a
Soluble microbial products
a
Phases
Table 4 – COD balance and metabolic product distribution in continuous operation.
a
EtOH (% of total)
a
HFo (% of total)
Biomass (mg COD/L) H2 (mg COD/L)
Glucose remaining (mg COD/L)
Total products (mg COD/L)
international journal of hydrogen energy 34 (2009) 3295–3304
to a decrease of H2 production with an increase of lactate and propionate productions in Phases IV and V. Sodium tolerance of LAB and PAB would be better than that of H2-producing bacteria. Recently, halotolerant L. lactis, which could be active up to 20 g Naþ/L has been isolated from the intestinal tract of coastal fish [33]. Irrecoverable H2 production at Phase V also could be explained by the abundant preexistence of non H2-producing bacteria obtained at Phase IV. Similarly, there has been one report concluding that salinity level impacted the composition of microbial communities. In an anaerobic sulfate removal reactor, the removal efficiency decreased at a sodium concentration of 5.0 g Naþ/L, which was attributed to a decrease of the sulfate reducing bacteria dominancy [34]. As the drastic H2 production drop was accompanied with a microbial community, which resulted in irrecoverable performance, special care must be taken when the sodium concentration is increased. It is recommended that the sodium concentration be increased stepwise between 5 and 10 g Naþ/L when a salty substance is fed to FHP.
4.
Conclusions
Acute sodium toxicity was evaluated by several batch tests using acclimated biomass collected from the continuous reactor operated at different sodium concentrations of 0.27 (control), 2.41, and 5.46 g Naþ/L. Unlike the control biomass, which exhibited a monotonic decrease of HPA with increasing sodium concentration from 0.27 to 21.00 g Naþ/L, the acclimated biomass maintained their activity at a concentration
international journal of hydrogen energy 34 (2009) 3295–3304
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Table 5 – Affiliation of DGGE fragments determined by the 16S rDNA and isolated microorganisms. Band
Affiliation
Similarity (%)
Samples
1 2 3 4 5 6 7 8 9 10
Lactococcus lactis Lactobacillus delbrueckii Streptococcus bovis Selenomonas ruminantium Selenomonas sp. Clostridium acidisoli Clostridium beijerinckii Clostridium pasteurianum Uncultured bacilli Bifidobacterium thermacidophilum
100 85 98 95 85 92 88 100 86 94
e d, e d d, e d, e e d, e a, b, c, d e a, b, c, d, e
Major metabolites from carbohydrates Lactate Lactate Lactate Propionate Propionate Acetate and Acetate and Acetate and – Acetate and
butyrate butyrate butyrate lactate
References [25] [26] [27] [28] [29] [30] [23] [31] – [32]
a ¼ Phase I, b ¼ Phase II, c ¼ Phase III, d ¼ Phase IV, e ¼ Phase V.
lower than 6.00 g Naþ/L. Sudden exposure of the biomass to higher sodium concentration inhibited the substrate removal efficiency and microbial catabolism. However, the H2 production drop was mainly attributed to a change of the metabolic pathway favorable to lactate production while depressing butyrate production. On the other hand, as the biomass is allowed sufficient time to adapt to the chronic toxicity condition, HPA was well maintained up to 5.36 g Naþ/ L. However, an irrecoverable H2 production drop was observed at 10.14 g Naþ/L with a significant increase of lactate and propionate production. A PCR-DGGE analysis revealed that the salty environment rendered the microbial community unfavorable to H2 production. LAB such as L. lactis, L. delbrueckii, and S. bovis and PAB, Selenomonas sp., were only detected at Phases IV and V.
[8] [9]
[10]
[11]
[12] [13] [14]
Acknowledgement This work was supported by grant No. M1-0203-00-0063 from the National Research Laboratory Program of the Korean Ministry of Science and Technology.
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thermophilic (55 C) methanol-fed sulfate reducing granular sludges. Water Sci Technol 2003;48(6):195–202. McCarty PL. Anaerobic waste treatment fundamentals III. Public Works 2004;95:91–4. Kugleman IJ, Chin KK. Toxicity, synergism, and antagonism in anaerobic waste treatment processes. Adv Chem Ser 1971; 105:55–90. Soto M, Me´ndez R, Lema JM. Sodium inhibition and sulphate reduction in the anaerobic treatment of mussel processing wastewaters. J Chem Technol Biotechnol 1993;58:1–7. Feijoo G, Soto M, Me´ndez R, Lema JM. Sodium inhibition in the anaerobic digestion process: antagonism and adaptation phenomena. Enzyme Microb Technol 1995;17:180–8. Chen WH, Han SK, Sung S. Sodium inhibition of thermophilic methanogens. J Environ Eng-ASCE 2003;129(6):506–12. Oren A. Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 1999;63(2):334–48. Kim DH, Han SK, Kim SH, Shin HS. Effect of gas sparging on continuous fermentative hydrogen production. Int J Hydrogen Energy 2006;31(15):2158–69. Kim DH, Kim SH, Ko IB, Lee CY, Shin HS. Start-up strategy for continuous fermentative hydrogen production: early switchover from batch to continuous operation. Int J Hydrogen Energy 2008;33:1532–41. Chen WH, Chen SY, Khanal S, Sung S. Kinetic study of biological hydrogen production by anaerobic fermentation. Int J Hydrogen Energy 2006;31(15):2170–8. Han K, Levenspiel O. Extended Monod kinetics for substrate product and cell inhibition. Biotechnol Bioeng 1988;32:430–7. APHA, Awwa and WEF. Standard methods for the examination of water and wastewater. 20th ed. Baltimore: American Public Health Association; 1998. 57–59. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28(3):350–6. Madigan MT, Martinko JM, Parker J. Brock biology of microorganisms. 10th ed. Pearson Education Inc; 2003. Kim SH, Han SK, Shin HS. Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge. Int J Hydrogen Energy 2004;29:1607–16. Xiaolong H, Minghua Z, Hanqing Y, Qinqin S, Lecheng L. Effect of sodium ion concentration on hydrogen production from sucrose by anaerobic hydrogen-producing granular sludge. Chin J Chem Eng 2006;14(4):511–7. Jones D, Woods D. Acetone–butanol fermentation revisited. Microbiol Rev 1986;50(4):484–524. Fang HHP, Liu H. Effect of pH on hydrogen production from glucose by a mixed culture. Bioresour Technol 2002;82:87–93. Nomura M, Kimoto H, Someya Y, Suzuki I. Novel characteristic for distinguishing Lactococcus lactis subsp. Lactis from subsp. Cremoris. Int J Syst Bacteriol 1999;49:163–6.
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[26] Dellagilo F, Felis GE, Castioni A, Torriani S, Germond JE. Lactobacillus delbrueckii subsp. Indicus subsp. nov., isolated from Indian dairy products. Int J Syst Evol Micrbiol 2005;55:401–4. [27] Poyart C, Quesne G, Trieu-Cuot P. Taxonomic dissection of the Streptococcus bovis group by analysis of manganesedependent superoxide dismutase gene (sodA) sequences: reclassification of ‘Streptococcus infantarius subsp. Coli’ as Streptococcus lutetiensis sp. nov. and of Streptococcus bovis biotype II.2 as Streptococcus pasteurianus sp. nov. Int J Syst Evol Micrbiol 2002;52:1247–55. [28] Eaton DC, Gabelman A. Fed-batch and continuous fermentation of Selenomonas ruminantium for natural propionic, acetic and succinic acids. J Ind Microbiol 1995;15: 32–8. [29] Hussy I, Hawkes FR, Dinsdale R, Hawkes DL. Continuous fermentative hydrogen production from a wheat starch coproduct by mixed microflora. Biotechnol Bioeng 2003;84(6): 619–26. } bner AM, [30] Kuhner CH, Matthies C, Acker G, Schmittroth M. Go Drake HM. Clostridium akgii sp. nov. and Clostridium acidisoli
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Available at www.sciencedirect.com
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Sodium inhibition of fermentative hydrogen production Dong-Hoon Kima, Sang-Hyoun Kimb, Hang-Sik Shina,* a Department of Civil and Environmental Engineering, Korea Advanced Institute of Science & Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea b Green Ocean Technology Center, Korea Institute of Industrial Technology, 35-3 Hongcheon-Ri, Ipchang-Myun, Seobuk-gu, Cheonan-Si, Chungnam 331-825, Republic of Korea
article info
abstract
Article history:
A continuous-stirred-tank reactor (CSTR) was fed with low-sodium influent containing
Received 27 December 2008
0.27 g of Naþ/L for 70 days (Phase I), and then subjected to higher concentrations of Naþ/L,
Received in revised form
i.e. 2.41 (Phase II), 5.36 (Phase III), and 10.14 g (Phase IV-1). At the quasi-steady state of each
22 February 2009
phase, biomass was sampled for an acute sodium toxicity assay. Unlike the control
Accepted 22 February 2009
biomass, which exhibited a monotonic decrease of specific H2 production activity (SHPA)
Available online 16 March 2009
with increasing sodium concentration from 0.27 to 21.00 g Naþ/L, the acclimated biomass maintained their activity up to 6.00 g Naþ/L. Soluble microbial product analysis revealed
Keywords:
that a sudden increase of the exterior sodium concentration changed the metabolic
Fermentative hydrogen production
pathway such that it became favorable to lactate production while depressing butyrate
Sodium inhibition
production. Meanwhile, when the biomass was allowed for sufficient time to adapt to the
Acute toxicity
chronic toxicity condition, the volumetric H2 production rate (VHPR) was maintained above
Chronic toxicity
4.05 L H2/L/d at up to Phase III. However, an irrecoverable H2 production drop was observed
Acclimation
at Phase IV-1 with a significant increase of lactate and propionate production. Although the sodium concentration decreased to 8.12 (Phase IV-2), 6.61 (Phase IV-3), and 5.36 g Naþ/L (Phase V) at further operation, the performance was never recovered. A PCR-DGGE analysis revealed that lactic acid bacteria (LAB) and propionic acid bacteria (PAB) were only detected at Phases IV and V, which are not capable of producing H2. ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The current energy system based on fossil fuels is now facing two fundamental problems in sustainability: the depletion of fossil fuel and environmental pollution. This has led to an extensive search for new alternative energy sources and carriers [1]. Among various candidates, hydrogen is regarded as the most promising energy carrier, since it produces only water when combusted and has a 2.75 times higher energy yield (122 kJ/g) than hydrocarbon fuels. In addition, as an
automotive fuel, H2 can be easily applied in proton exchange membrane fuel cell vehicles as well as conventional internal combustion engines [2]. H2 can be made via several ways, including electrolysis of water, thermocatalytic reformation of hydrogen-rich organic compounds, and biological processes. Currently, H2 is exclusively made by steam reforming of gas, requiring electricity derived from fossil fuel combustion, a process that is energy intensive and expensive. Feedstock and energy for H2 production must be renewable if its purpose is to decrease the
* Corresponding author. Tel.:þ82 42 350 3613; fax: þ82 42 350 8460. E-mail address: [email protected] (H.-S. Shin). 0360-3199/$ – see front matter ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.02.051
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international journal of hydrogen energy 34 (2009) 3295–3304
current dependency on fossil fuel. Sustainable generation of H2 may be achieved by a range of technologies, including biological processes [3]. Biological H2 production can be achieved by phototrophic and non-phototrophic methods. With the help of light, autotrophs such as algae and heterotrophs such as Rhodobacter sp. produce H2 from water and organic wastewater, respectively. However, phototrophic H2 production confronts several obstacles: (1) low solar energy conversion efficiency (2) slow reaction rate, and (3) low light penetration due to biofilm formation on the reactor wall. On the other hand, non-phototrophic production, often called fermentative H2 production (FHP), offers many advantages such as fast reaction rate, degradation of solid organic wastes, technical simplicity, and no need of light. As a result, it can serve to address two critical global issues simultaneously, energy supply and environmental protection [4]. FHP is one of the ways releasing excess electrons derived from organics with the help of the ‘hydrogenase’ function in bacteria. The following equations, Eqs. (1) and (2), are the main H2 production reactions involved in FHP from glucose. Comprehensive studies have been conducted dealing with operating parameters such as pH, hydraulic retention time (HRT), carbon source, and H2 partial pressure [5]. However, little information is available about the effect of cations on FHP.
2.
Materials and methods
Glucose þ 2 H2O / 2 Acetate þ 2 CO2 þ 4 H2 þ 4 ATP
(1)
2.1.
Seed sludge and substrate
Glucose / Butyrate þ 2 CO2 þ 2 H2 þ 2 ATP
(2)
Cations play essential roles in adenosine (ATP) synthesis, nicotinamide adenine dinucleotide (NAD) oxidation/reduction, and enzyme activity, thereby accelerating microbial metabolism if maintained at proper concentrations. However, excessive cations can cause plasmolysis and loss of cell activity by creating high osmotic pressure and improper enzyme linkages. In particular, sodium, the main cation in biomass and seawater, leads to many problems in biological treatment systems of wastewater from seafood processing, the dairy industry, and chemical production. Besides, sodium concentration can be increased by buffer addition such as NaOH, Na2CO3, and NaHCO3. Therefore, research has been carried out on anaerobic digestion and biological nutrient removal processes to identify the inhibition mechanisms and alleviation of its inhibition [6,7]. For methane fermentation, it has generally been reported that sodium concentration over 2 g Naþ/L will cause a performance drop [8,9]. However, some studies have reported that continuous exposure of microorganisms to higher sodium levels increased the sodium tolerance. Seafood processing wastewater containing 5–12 g/L of sodium was successfully treated with an anaerobic filter process [10]. According to Feijoo et al. [11], sodium concentration causing 50% inhibition exceeded 10 g Naþ/L for the sludge obtained from digesters treating high saline wastewater, but it was lower than 5 g Naþ/ L when unacclimated sludge was used. Also, a stepwise increase of sodium level exhibited higher tolerance than a shock increase [12]. These findings suggest that acclimation could mitigate sodium inhibition.
There are two known bacterial strategies to adapt to and cope with high sodium concentration; salt-in and compatiblesolute strategies [13]. The main mechanism in the salt-in strategy is the extrusion of sodium ions outside cells concurrent with accumulation of potassium ions inside cells via a proton electrochemical gradient and at ATP expense. As this strategy does not reduce the osmotic pressure inside the cell, all intracellular systems should be tolerant at high osmotic pressure. On the other hand, in the compatible-solutes strategy, compatible solutes such as glycerol, ectoine, and glycine betaine are created for the proper functions of intracellular systems at high osmotic pressure. Debate continues over which strategy is more energy efficient, but it is clear that life in a salty environment is costly. Although salty organic substances, such as waste from the foodstuff industry, could be suitable sources for FHP and sodium is employed as the cation in general buffers and nutrients for FHP, research on this subject is scarce. Therefore, the present study aimed at evaluating the sodium inhibition of FHP. Chronic and acute sodium toxicity was investigated by continuous-stirred-tank reactor (CSTR) operation and batch tests at various sodium concentrations, respectively.
The seed sludge was taken from an anaerobic digester in a local wastewater treatment plant. The pH, alkalinity, and volatile suspended solids (VSS) concentration of the sludge were 7.5, 2.83 g CaCO3/L, and 5.3 g/L, respectively. The sludge was heat-treated at 90 C for 15 min to inactivate hydrogen consumers and to harvest spore-forming anaerobic bacteria such as Clostridium sp. [14]. Sucrose of 25 g COD/L was used as a substrate. Concentrations of NH4Cl, KH2PO4, and FeCl2$4H2O were added to yield a COD:N:P:Fe ratio of 100:5:1:0.33. Feed also contained the following nutrients (in mg/L): NaHCO3 1000; MgCl2$6H2O 100; CaCl2$2H2O 75; Na2MoO4$4H2O 0.01; H3BO3 0.05; MnCl2$4H2O 0.5; ZnCl2 0.05; CuCl2 0.03; NiCl2$6H2O 0.05; CoCl2$2H2O 0.5; Na2SeO3 0.05 [14].
2.2.
Continuous operation
In this study, a CSTR with a working volume of 5.0 L (325 mm high by 140 mm ID) was used. The chemical oxygen demand (COD) loading rate was maintained at 50 g/L d during the entire period of operation, which corresponded to 12 h of hydraulic retention time (HRT). The reactor was mixed by mechanical stirring at 100 rpm. The pH was maintained at 5.3 0.1 using a pH sensor, pH controller, and 3 N KOH. Biogas production was monitored by the water displacement method and then corrected to standard temperature (0 C) and pressure (760 mmHg) (STP). All experiments were conducted in a constant temperature room at 35 1 C. After being seeded with heat-treated sludge equivalent to 30% of the total effective volume, the reactor was purged with N2 gas for 5 min to provide an anaerobic condition, and then operated in batch mode. When the H2 yield reached 0.2 mol H2/mol hexoseadded in the batch operation, continuous operation started [15].
international journal of hydrogen energy 34 (2009) 3295–3304
The CSTR was fed with low-sodium influent containing 0.27 g Naþ/L by NaHCO3 (buffer solution) for 70 days; 30 days for start-up and 40 days as the control condition (Phase I). Subsequently, sodium chloride was added to increase the sodium concentration to: 2.41 (Phase II), 5.36 (Phase III), and 10.14 g Naþ/L (Phase IV-1) according to the batch test results using the control biomass. As H2 production failure was observed at Phase IV-1, sodium concentration decreased to 8.12 (Phase IV-2), 6.61 (Phase IV-3), and 5.36 g Naþ/L (Phase V) at further operation. At each phase, the reactor was operated at least 10 days, 20 times of HRT, in order to establish steadystate conditions, judging from metabolic products.
2.3.
Batch test
The inocula for batch tests were biomass collected from the continuous reactor acclimated at different sodium concentrations (Phases I, II, and III). The sludge was anaerobically centrifuged at 672g for 30 min to eliminate any salt present in the mixed liquor and avoid uncontrolled antagonistic or synergistic effects. Batch experiments were conducted using fermenters (635 mL) equipped with a pH sensor and magnetic mixing bar. Each batch fermenter contained 1g of the centrifuged sludge having 185–190 mg VSS/g, 20 mL of nutrient stock solutions (5) to yield the same final concentration as CSTR, and a predetermined amount of NaCl stock solution (40 g Naþ/L). Sodium toxicity levels were varied for each fermenter; 0.27 (control), 3.00, 6.00, 9.00, 12.00, 15.00, 18.00, and 21.00 g Naþ/L. Distilled water was added into the fermenters to adjust the final volume to 100 ml. The large headspace volume (535 mL) was to alleviate the adverse effect of high H2 partial pressure during fermentation. The initial pH was adjusted to 7.00 0.05 by 3 N KOH and HCl. The bottles were then purged with N2 gas, and placed in a water bath with a magnetic stirrer. Mixing speed and temperature were controlled at 100 rpm and 35 1 C, respectively. Biogas production and its constituents were measured periodically from each bottle to estimate H2 production. As acids are produced during fermentation, resulting in a pH drop, 3 N KOH was added to maintain the pH above 5.0 0.2. Organic acids, alcohols, and remaining substrate were measured after H2 production ceased.
2.4.
Methodology
The modified Gompertz equation was employed in this study to describe the cumulative H2 production in the batch tests [16]. 0 R $e ðl tÞ þ 1 HðtÞ ¼ P$exp exp P
(3)
where H(t) ¼ cumulative H2 production (mL) at cultivation time t (h); P ¼ H2 production potential (mL); R0 ¼ H2 production rate (mL/h); l ¼ lag period (h); and e ¼ exp(1) ¼ 2.71828. The inhibitory effect of sodium on specific H2 production activity (SHPA) was analyzed using a noncompetitive inhibition model as shown in Eq. (4) [17]. n I R ¼ R0 1 I
(4)
3297
where R ¼ specific H2 production rate at inhibitor concentrations of I (mL H2/g VSS/h); R0 ¼ specific hydrogen production rate without an added inhibitor (mL H2/g VSS/h); I ¼ inhibitor concentration (g/L); I* ¼ lethal inhibitor concentration beyond which the reaction cannot proceed (g/L); and n ¼ constant.
2.5.
Analytical methods
Measured biogas production was corrected to standard temperature (0 C) and pressure (760 mmHg) (STP). Hydrogen content in the biogas was determined by a gas chromatography (GC, Gow Mac series 580) using a thermal conductivity detector and a 1.8 m 3.2 mm stainless-steel column packed with molecular sieve 5A with N2 as a carrier gas. The contents of CH4, N2, and CO2 were measured using a GC of the same model noted previously with a 1.8 m 3.2 mm stainless-steel column packed with porapak Q (80/100 mesh) using helium as a carrier gas. The temperatures of the injector, detector, and column were kept at 80, 90, and 50 C, respectively, in both GCs. Volatile fatty acids (VFAs, C2–C6) and lactate were analyzed by a high performance liquid chromatograph (HPLC) (Finnigan Spectra SYSTEM LC, Thermo Electron Co.) with an ultraviolet (210 nm) detector (UV1000, Thermo Electron) and an 100 mm 7.8 mm Fast Acid Analysis column (Bio-Rad Lab.) using 0.005 M H2SO4 as a mobile phase. Aliphatic alcohol was determined using another HPLC (DX-600, Dionex) with an electrochemical detector (ED50A, Dionex) and a 250 mm 9 mm IonPac ICE-AS1 column (Dionex) using 50 mM HClO4 as a mobile phase. The liquid samples were pretreated with a 0.45 mm membrane filter prior to injection to both HPLCs. COD and VSS were measured according to Standard Methods [18]. Glucose concentration was determined by the colorimetric method [19].
2.6.
Microbial analysis
In order to analyze the microbial communities, the DNAs in the sludge sampled at each sparging condition were extracted using the Ultraclean Soil DNA Kit (Cat # 12800-50; Mo Bio Laboratory Inc., USA). The 16S rDNA fragments were amplified by polymerase chain reaction (PCR). The region corresponding to positions 357 and 518 in the 16S rDNA of Escherichia coli was PCR-amplified using the forward primer EUB357f (50 CCTACGGGAGGCAGCAG-30 ) with a GC clamp (50 -CGCCCG CCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-30 ) at the 50 end to stabilize the melting behavior of the DNA fragments and the reverse primer UNIV518r (50 -ATTACCGCGGCTGCTGG-30 ). PCR amplification was conducted in an automated thermal cycler (MWG-Bio TECH, Germany) using the protocol; that is, initial denaturation for 4 min at 94 C, annealing for 40 s at 55 C, extension for 1 min at 72 C, followed by a final extension for 8 min at 72 C. PCR mixtures had a final volume of 50 ml of 10 PCR buffer, 0.8 mM MgSO4, 0.5 mM of each primer, 0.1 mM dNTP, 25 pg template and 1 U polymerase. PCR products were electrophoresed on 2% (wt/vol) agarose gel in 1 TAE for 30 min for 50 V, and then checked with ethidium bromide staining to confirm the amplification. Denaturing gradient gel electrophoresis (DGGE) was carried out using the Dcode Universal Mutation Detection System (BioRad, USA) in accordance with the manufacturer’s instruments. PCR products were electrophoresed in 1 TAE buffer for 480 min at 70 V and
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7.5 Control 3 g Na+/L 6 g Na+/L 9 g Na+/L 12 g Na+/L 15 g Na+/L 18 g Na+/L 21 g Na+/L
7.0
250
0.27 g Na+/L (Control) 3.00 g Na+/L 6.00 g Na+/L 9.00 g Na+/L 12.00 g Na+/L 15.00 g Na+/L 18.00 g Na+/L 21.00 g Na+/L
200
150
6.5
pH
Cumulative H2 production (ml)
300
6.0
100
5.5
50
5.0
4.5
0 0
20
40
0
60
5
10
15
20
Time (h)
Time (h) Fig. 1 – Cumulative H2 production curves for control biomass at different sodium concentrations.
Fig. 2 – pH change profiles of control biomass during fermentation.
60 C on polyacrylamide gel (7.5%) containing a linear gradient ranging from 40% to 60% denaturant. After electrophoresis, polyacrylamide gel was stained with ethidium bromide for 30 min, and then visualized on UV transilluminator. Most of the bands were excised from DGGE polyacrylamide gel for 16S rDNA sequencing. DNAs were eluted from the excised bands by immersing them in 20 ml of Tris EDTA buffer (pH 8.0) for one day, and then PCR-amplified with the forward primer EUB357f without a GC clamp and the reverse primer UNIV518r. After PCR amplification, PCR products were purified with using Multiscreen Vacuum Manifold (MILLIPORE com., USA). All the strands of the purified PCR products were sequenced with primers EUB357f by ABIPRISM Big Terminator Cycle Sequencing Kit (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Search of the GenBank database was conducted using the BLAST program.
sodium concentrations. As the H2 production was negligible at more than 15.00 g Naþ/L, Eq (3) was only applied at concentrations lower than 12.00 g Naþ/L. The lag period was also increased with sodium concentration, indicating that shock loading of sodium could result in the failure of H2 production. The pH profile shown in Fig. 2 confirms the retardation in microbial catabolism by sodium inhibition. The pH value dropped to 5.0 within 2 h up to 6.00 g Naþ/L, suggesting that the biomass could readily adapt to 6.00 g Naþ/L. However, it took more than 5 h when the sodium concentration was more than 12.00 g Naþ/L. SHPA (mL H2/g VSS/h) of the control biomass at various sodium concentrations was well fitted by Eq (4) with a high R2 value of 0.974 (graph not shown), and the sodium concentrations causing 30, 60, and 90% inhibition were found: 2.41, 5.36, and 10.14 g Naþ/L, respectively. Effects of sodium on SHPA, H2 yield (HY), and substrate removal efficiency of biomass acclimated at different sodium concentrations are illustrated in Fig. 3(a), (b), and (c), respectively. The SHPA and HY at various sodium concentrations are expressed as ‘‘the percentage (%) of the control, the results from the batch test at 0.27 g Naþ/L using each biomass’’. Acclimation substantially alleviated the acute sodium toxicity. Using the acclimated biomass the SHPA and HY were close to or even higher than those of the control at up to 6.00 g Naþ/L. The highest HY was observed at 3.00 and 6.00 g Naþ/L
3.
Results and discussion
3.1.
Acute sodium toxicity
Acute sodium toxicity of control biomass (Phase I) was severe as shown in Fig. 1 and Table 1. H2 production decreased almost by half at 3.00 g Naþ/L and decreased further at higher
Table 1 – Acute sodium toxicity of FHP when using control biomass. Sodium conc. (g Naþ/L) 0.27 (Control) 3.00 6.00 9.00 12.00 15.00 18.00 21.00
H2 production (mL)
H2 yield (mol H2/mol hexoseadded)
Substrate removal (%)
P (mL)
R (mL/h)
l (h)
241 136 108 52 43 4 2 1
0.83 0.47 0.37 0.18 0.15 0.01 0.01 0.00
97.0 96.6 96.8 95.1 93.5 23.8 9.9 4.0
251 137 108 56 56 – – –
25.1 14.0 10.9 2.9 0.9 – – –
0.9 1.2 4.17 5.2 9.7 – – –
international journal of hydrogen energy 34 (2009) 3295–3304
Relative specific H2 production activity (% of control)
a
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120 100 Control Acclimated - 2.41 Acclimated - 5.36
80 60 40 20 0 0
5
10
15
20
25
Sodium conc. (g Na+/L)
Relative H2 yield (% of control)
b
140 120 100
Control Acclimated - 2.41 Acclimated - 5.36
80 60 40 20 0 0
5
10
15
20
25
20
25
Sodium conc. (g Na+/L)
Sucrose removal (%)
c
100
80
60 Control Acclimated - 2.41 Acclimated - 5.36
40
20
0 0
5
10
15
Sodium conc. (g Na+/L) Fig. 3 – Inhibitory effect of sodium on (a) relative specific H2 production activity (b) relative H2 yield, and (c) sucrose removal for biomass acclimated at different sodium concentrations.
when the biomass acclimated at 2.41 and 5.46 g Naþ/L was used, respectively. This might be attributable to the osmotic pressure formed inside the cells during the acclimation period. A drop in the sodium concentration outside of the cell would facilitate the permeation of water molecules into the cell, which would in turn adversely affect the metabolic balance. Also, acclimation showed a positive effect on substrate removal efficiency. The concentration maintaining
over 90% of substrate removal efficiency was increased to 12.00, 15.00, and 18.00 g Naþ/L when using the control and acclimated biomass at 2.41 and 5.46 g Naþ/L, respectively. At 12.00 g Naþ/L, in spite of high substrate removal, H2 production was 50% less than the control even when using acclimated biomass. This suggests that the metabolic pathway was shifted to a non-hydrogenic route by sodium inhibition under acute toxicity condition. As arranged in
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Table 2 – Effect of sodium concentration on soluble microbial byproducts when using the acclimated biomass at different sodium concentrations (0.27 (control), 2.41, and 5.36 g NaD/L). Biomass
Sodium conc. (g Naþ/L)
Soluble microbial products, mg COD/L (% of total metabolites) Butyrate
Lactate
Acetate
Propionate
Phase I
0.27 3 6 9 12 15 18 21
7110 5000 4280 2610 530 1820 1030 710
(29.7) (22.2) (19.8) (11.5) (3.1) (26.6) (35.3) (37.2)
81,90 10,920 11,800 14,970 12,000 3950 1190 500
(34.2) (48.4) (54.6) (65.5) (69.1) (57.7) (40.8) (26.2)
3610 3400 3210 4900 3860 440 340 290
(15.1) (15.1) (14.9) (21.5) (22.2) (6.5) (11.7) (15.2)
Phase II
0.27 3 6 9 12 15 18 21
9590 9120 8850 7500 4190 3700 2500 1360
(47.0) (46.4) (39.9) (35.3) (19.7) (17.0) (17.5) (42.7)
4050 4640 5010 9150 15,000 16,200 9710 470
(19.9) (23.6) (22.6) (43) (70.5) (74.1) (67.9) (14.8)
3490 3250 6150 2700 2090 1990 2100 1000
(17.1) (16.6) (27.7) (12.7) (9.9) (9.1) (14.7) (31.4)
Phase III
0.27 3 6 9 12 15 18 21
7090 6460 10,880 6680 1660 1270 560 1850
(35.6) (32.3) (55.2) (33.6) (7.3) (6.0) (2.8) (35.7)
6530 6620 1900 9040 20,250 16,680 17,430 1120
(32.8) (33.1) (9.7) (45.5) (88.6) (77.6) (87.2) (21.6)
4180 4320 2070 810 500 3100 790 1230
(21.0) (21.6) (10.5) (4.1) (2.2) (14.5) (4.0) (23.7)
Table 2, butyrate, lactate, and acetate were the major soluble microbial products of FHP. Interestingly, metabolic shift from butyrate to lactate was concurrent with sodium inhibition. For example, when using the control biomass, the butyrate portion decreased from 29.7% to 4.7% as the sodium concentration was increased up to 12.00 g Naþ/L. Instead, lactate production was predominant, accounting for 65.6% of the total organic acids. It is known that butyrate production is accompanied by H2 production (Eq. (2)), while all lactate producing reactions are non-hydrogenic as shown in Eqs. (5) and (6) [20].
Glucose / Lactate þ Ethanol þ CO2 þ 1 ATP
Total metabolites
2850 (11.9) 1490 (6.6) 1160 (5.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
400 (1.7) 620 (2.8) 650 (3.1) 0 (0.0) 0 (0.0) 0 (0.0) 60 (2.1) 30 (1.6)
23,970 (100.0) 22,590 (100.0) 21,650 (100.0) 22,870 (100.0) 17,390 (100.0) 6850 (100.0) 2920 (100.0) 1910 (100.0)
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
3310 (16.2) 2670 (13.6) 2210 (10) 1930 (9.1) 0 (0.0) 0 (0.0) 0(0.0) 0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 360 (11.3)
20,440 (100.0) 19,680 (100.0) 22,220 (100.0) 21,280 (100.0) 21,280 (100.0) 21,890 (100.0) 14,310 (100.0) 3190 (100.0)
(4.5) (3.3) (6.0) (4.4) (2.1) (2.2) (5.7) (12.2)
1250 (6.3) 2010 (10.1) 3700 (18.8) 2500 (12.6) 0 (0.0) 0 (0.0) 100 (0.5) 0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 360 (7.0)
19,930 (100.0) 20,060 (100.0) 19,720 (100.0) 19,890 (100.0) 22,880 (100.0) 21,510 (100.0) 20,010 (100.0) 5190 (100.0)
1810 1160 550 390 1000 640 300 380
(7.6) (5.2) (2.6) (1.8) (5.8) (9.4) (10.3) (19.9)
0 0 0 0 0 0 0 0 880 650 1170 860 470 460 1130 630
(5)
(6)
Start-up Phase I
6
To the best of our knowledge, this is the first report showing a relation between lactate production with sodium
Phase III
Phase IV
Phase V 12
5
10
4
8
3
6
2
4 VHPR Sodium conc.
1
2
0 0
(7)
Phase II
50
100
150
Sodium conc. (g Na+/L)
Although it is generally accepted that both acetate and butyrate productions provide high H2 production in pure culture, previous studies in using mixed culture showed a close relationship between butyrate and H2 production rather than acetate production. It was reported that H2 yield was linearly proportional to butyrate production or B/A (butyrate/acetate) ratio and doubts were even raised regarding whether the governing acetate production pathway in mixed culture was a H2-consuming reaction as shown in the following Eq. (7) [14,21]. 4 H2 þ 2 CO2 / Acetate þ 2 H2O þ (1–2) ATP
Formate
inhibition in FHP. Previously, Xialong et al. [22] observed a gradual change of metabolic products from acetate, propionate, and butyrate to acetate with increasing sodium concentration, but did not report on lactate. The lactate production could result from either the microbial population change, which is discussed in 3.3 Microbial analysis, or the switched catabolic pathway of H2-producing bacteria. Lactate production is not commonly caused by main anaerobic H2-producing bacteria, Clostridium sp., under normal conditions. Thus far, its production is only reported when (1) H2 release is blocked; (2) hydrogenase activity is inhibited by
Volumetric H2 production rate (L/L/d)
Glucose / 2 Lactate þ 2 ATP
Ethanol
0 200
Time (d) Fig. 4 – Daily variation of volumetric H2 production rate at various sodium concentrations.
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H2 production activity (mL H2/g VSS/h)
international journal of hydrogen energy 34 (2009) 3295–3304
200 Acute toxicity Chronic toxicity
150
100 Avg. value at Phase IV
50
0 0
2
4
6
8
10
Sodium conc. (g Na+/L) Fig. 5 – Comparison of acute and chronic toxicity at different sodium concentrations.
carbon monoxide; or (3) iron or sulfur supply is limited [21]. Further study using a pure culture might reveal whether the metabolic pathway of Clostridium sp. could be altered by sodium inhibition or not.
3.2.
Chronic toxicity
Fig. 4 shows the daily variations of volumetric H2 production rate (VHPR) at various sodium concentrations. Although VHPR fluctuated at Phase III (5.36 g Naþ/L), average values at Phase II (2.41 g Naþ/L) and Phase III were higher than those at Phase I (0.27 g Naþ/L), which did not correspond with the batch test results using the control biomass. In Phase IV-1 (10.14 g Naþ/ L), a sudden deterioration of H2 production was observed, and was not recovered at further operation with lower sodium concentrations (8.12, 6.61, and 5.36 g Naþ/L). The acute sodium toxicity evaluated by a batch test using the control biomass and chronic sodium toxicity obtained in continuous operation are compared in Fig. 5. Under acute toxicity conditions, sufficient time is not provided for the biomass to adapt at the higher sodium concentration, and thus a steep decline of SHPA was shown. Meanwhile, in continuous operation, the biomass has sufficient time to adapt and retain their activity. At 5.00 g Naþ/L, the activity was decreased by 50% in the acute condition, but only 15% in the
chronic condition. This adaptation effect was also observed for substrate removal efficiency. The average continuous performances of FHP at each phase are listed in Table 3. H2 content and yield decreased with increasing sodium concentration, but the substrate removal efficiency increased due probably to the adaptation mechanism requiring more energy at a salty environment. Also, the biomass concentration increased as the substrate utilization increased. Table 4 summarizes the overall COD balance of H2, organic acids, alcohols, and biomass. The COD of biomass was calculated under the assumption that its chemical formula was C5H7O2N [24]. The COD balance was roughly 100 5% in Phases I, II, and III, but only 87% in Phases IV and V, suggesting that compatible solutes were formed for survival at the high salty condition and their leak to the broth. Several studies reported the leakage of compatible solutes such as betaines, ectoines, N-acetylated diamino acids, and Nderivatized carboxamides of glutamine as cell membranes were not perfectly impermeable to low-molecular-weight substances [13]. The butyrate portion decreased while lactate portion increased at a high sodium concentration, which agreed well with the batch test results. The portion of propionate, a by-product of the H2-consuming reaction, also increased at Phases IV and V. The reasons for the change of liquid state by-products distribution in continuous operation are discussed in 3.3 Microbial analysis.
3.3.
Microbial analysis
Fig. 6 illustrates the DGGE profiles of the 16S rDNA gene fragment amplified from the biomass sampled in Phases I–V I– V. The major bands were excised and purified to determine the sequence. Table 5 shows the results of the sequence affiliation, and isolated microorganisms and their main metabolites from the carbohydrate [23,25–32]. In total, 10 bands were detected. While similar band types were observed in Phases I, II, and III, they were changed significantly in Phases IV and V. H2-producing bacteria, Clostridium pasteurianum, and non H2-producing bacteria such as Bifidobacterium thermacidophilum and Acetobacter aceti were detected in Phases I, II, and III. Lactic acid bacteria (LAB) such as Lactococcus lactis, Lactobacillus delbrueckii, and Streptococcus bovis and propionic acid bacteria (PAB), Selenomonas sp., were only detected in Phases IV and V. As LAB and PAB were not capable of H2 production, this population shift could be linked
Table 3 – Performance of FHP at various sodium concentrations in continuous operation. Item
Unit
a
VHPR H2 content H2 yield Substrate removal Biomass
L/L/d % mol H2/mol hexoseconsumed % mg VSS/L
a VHPR ¼ Volumetric H2 production rate.
Phases I
II
III
IV
V
4.05 0.20 55.8 1.2 1.24 0.06 55.9 3.5 906 83
4.43 0.17 54.3 1.0 1.22 0.05 62.3 4.0 1040 181
4.28 0.56 51.8 1.6 0.92 0.12 79.0 6.8 1187 88
0.73 0.71 39.7 9.2 0.13 0.13 93.6 7.5 1790 241
0.16 0.11 29.8 1.0 0.03 0.02 95.7 2.7 1867 95
3302
(100.7%) (104.9%) (95.9%) (86.2%) (87.8) 25,170 26,220 23,980 21,560 21, 940 11,030 9430 5250 1600 1080 1290 1640 1790 2330 2650 1440 1580 1520 270 60
Fig. 6 – DGGE profiles of the 16S rDNA gene fragment at each phase (a [ Phase I, b [ Phase II, c [ Phase III, d [ Phase IV, e [ Phase V). 0.0 0.0 0.0 0.0 0.0 a HBu ¼ butyrate; HLa ¼ lactate; HAc ¼ acetate; HPr ¼ propionate; EtOH ¼ ethanol; HFo ¼ formate.
7.6 18.4 18.8 18.8 7.5 2.4 0.8 1.8 7.6 13.4 25.9 27.1 29.1 44.1 47.2 37.2 33.0 31.9 14.8 10.4 I II III IV V
Total (mg COD/L)
11,410 13,570 15,420 17,360 18,150
HLa (% of total)
HBu (% of total)
22.8 20.7 18.3 14.8 21.5
HPr (% of total) HAc (% of total)
a a a
Soluble microbial products
a
Phases
Table 4 – COD balance and metabolic product distribution in continuous operation.
a
EtOH (% of total)
a
HFo (% of total)
Biomass (mg COD/L) H2 (mg COD/L)
Glucose remaining (mg COD/L)
Total products (mg COD/L)
international journal of hydrogen energy 34 (2009) 3295–3304
to a decrease of H2 production with an increase of lactate and propionate productions in Phases IV and V. Sodium tolerance of LAB and PAB would be better than that of H2-producing bacteria. Recently, halotolerant L. lactis, which could be active up to 20 g Naþ/L has been isolated from the intestinal tract of coastal fish [33]. Irrecoverable H2 production at Phase V also could be explained by the abundant preexistence of non H2-producing bacteria obtained at Phase IV. Similarly, there has been one report concluding that salinity level impacted the composition of microbial communities. In an anaerobic sulfate removal reactor, the removal efficiency decreased at a sodium concentration of 5.0 g Naþ/L, which was attributed to a decrease of the sulfate reducing bacteria dominancy [34]. As the drastic H2 production drop was accompanied with a microbial community, which resulted in irrecoverable performance, special care must be taken when the sodium concentration is increased. It is recommended that the sodium concentration be increased stepwise between 5 and 10 g Naþ/L when a salty substance is fed to FHP.
4.
Conclusions
Acute sodium toxicity was evaluated by several batch tests using acclimated biomass collected from the continuous reactor operated at different sodium concentrations of 0.27 (control), 2.41, and 5.46 g Naþ/L. Unlike the control biomass, which exhibited a monotonic decrease of HPA with increasing sodium concentration from 0.27 to 21.00 g Naþ/L, the acclimated biomass maintained their activity at a concentration
international journal of hydrogen energy 34 (2009) 3295–3304
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Table 5 – Affiliation of DGGE fragments determined by the 16S rDNA and isolated microorganisms. Band
Affiliation
Similarity (%)
Samples
1 2 3 4 5 6 7 8 9 10
Lactococcus lactis Lactobacillus delbrueckii Streptococcus bovis Selenomonas ruminantium Selenomonas sp. Clostridium acidisoli Clostridium beijerinckii Clostridium pasteurianum Uncultured bacilli Bifidobacterium thermacidophilum
100 85 98 95 85 92 88 100 86 94
e d, e d d, e d, e e d, e a, b, c, d e a, b, c, d, e
Major metabolites from carbohydrates Lactate Lactate Lactate Propionate Propionate Acetate and Acetate and Acetate and – Acetate and
butyrate butyrate butyrate lactate
References [25] [26] [27] [28] [29] [30] [23] [31] – [32]
a ¼ Phase I, b ¼ Phase II, c ¼ Phase III, d ¼ Phase IV, e ¼ Phase V.
lower than 6.00 g Naþ/L. Sudden exposure of the biomass to higher sodium concentration inhibited the substrate removal efficiency and microbial catabolism. However, the H2 production drop was mainly attributed to a change of the metabolic pathway favorable to lactate production while depressing butyrate production. On the other hand, as the biomass is allowed sufficient time to adapt to the chronic toxicity condition, HPA was well maintained up to 5.36 g Naþ/ L. However, an irrecoverable H2 production drop was observed at 10.14 g Naþ/L with a significant increase of lactate and propionate production. A PCR-DGGE analysis revealed that the salty environment rendered the microbial community unfavorable to H2 production. LAB such as L. lactis, L. delbrueckii, and S. bovis and PAB, Selenomonas sp., were only detected at Phases IV and V.
[8] [9]
[10]
[11]
[12] [13] [14]
Acknowledgement This work was supported by grant No. M1-0203-00-0063 from the National Research Laboratory Program of the Korean Ministry of Science and Technology.
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