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Media optimization for biohydrogen production from waste glycerol by anaerobic thermophilic mixed cultures
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 5 4 7 3 e1 5 4 8 2
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Media optimization for biohydrogen production from waste glycerol by anaerobic thermophilic mixed cultures Sureewan Sittijunda a,b, Alissara Reungsang a,c,* a
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand Center for Alternative Energy Research and Development, Khon Kaen University, Khon Kaen 40002, Thailand c Fermentation Research Center for Value-Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand b
article info
abstract
Article history:
Media compositions affecting thermophilic biohydrogen production from waste glycerol
Received 15 December 2011
were optimized using response surface methodology (RSM) with central composite design
Received in revised form
(CCD). Investigated parameters used were waste glycerol concentration, urea concentra-
26 February 2012
tion, the amount of Endo-nutrient addition and disodium hydrogen phosphate (Na2HPO4)
Accepted 28 February 2012
concentration. Waste glycerol concentration and the amount of Endo-nutrient addition
Available online 27 March 2012
had a significant individual effect on the cumulative hydrogen production (HP) ( p 0.05). The interactive effect on HP was found between waste glycerol and urea concentration as
Keywords:
well as waste glycerol concentration and the amount of Endo-nutrient addition ( p 0.05).
Waste glycerol
The optimal media compositions were 20.33 g/L of waste glycerol, 0.16 g/L of urea, 3.97 g/L
Thermophilic
of Na2HPO4 and 0.20 mL/L of the amount of Endo-nutrient addition which gave the
Response surface methodology
maximum HP of 1470.19 mL H2/L. The difference between observed HP (1502.84 mL H2/L)
Optimization
and predicted HP was 2.22%. The metabolic products from the fermentation process were 1,3-propanediol (1,3-PD), ethanol, acetic, formic, lactic, butyric, and propionic acids. Results from polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis indicated that the hydrogen producers present in the fermentation broth was Thermoanaerobacterium sp. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Hydrogen is a promising renewable energy which has received increase attention in recent years due to the high demand for sustainable energy [1]. Hydrogen has an energy yield of 122 kJ/ g, which is 2.75 times greater than hydrocarbon fuels [2]. Biologically, hydrogen is produced by photo and dark fermentation [2]. Dark fermentation is preferred over photo
fermentation process due to its cost-effective with the ability to utilize various kinds of substrates [2,3]. Biodiesel is one of alternative fuels that have been dramatically produced in recent years. The production of 10 kg of biodiesel could generate 1 kg of glycerol [4]. Therefore, it is necessary to find an alternative way to utilize this abundant waste. Glycerol is commonly used in chemicals, food, and beverages productions. It can also be used to produce 1,3-
* Corresponding author. Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand. Tel./ fax: þ66 43 362 121. E-mail address: [email protected] (A. Reungsang). 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.2012.02.185
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PD, ethanol and organic acids [4,5]. However, there is still limited information on using waste glycerol to produce hydrogen, especially under thermophilic temperature. In order to achieve efficient production of hydrogen, optimum environmental factors such as temperature, pH, inoculum size and media compositions are necessary. Temperature has a significant effect on hydrogen production. The production of hydrogen at thermophilic temperature has gained more interest due to a number of advantages over mesophilic temperature including higher hydrogen yield (HY), better pathogenic destruction, and lower risk of contamination by methanogenic archaea [1,3]. However, most of hydrogen production studies from glycerol were conducted under mesophilic conditions [4e9]. Due to the advantages of thermophilic temperature, therefore, in this study hydrogen production from waste glycerol was conducted at thermophilic condition. Microorganisms required appropriate concentrations of substrates, nitrogen, phosphate, and nutrient for metabolism during the fermentation process [10e14]. An appropriate substrate concentration could increase efficiency of hydrogen production of the hydrogen producers. Lower or higher substrate concentration than the optimum level could result in the adverse effects on hydrogen producers by causing substrate limitation and substrate inhibition, respectively [5,7,10]. Endo-nutrient is often used to enrich microorganisms capable of producing hydrogen. Important elements contained in the Endo-nutrient are Mg2þ and Fe2þ. These elements are needed for the growth of microorganisms and the hydrogenase enzyme activity [11,13]. Phosphate source such as Na2HPO4 has a buffering capacity that could reduce the pH fluctuation caused by volatile fatty acids (VFAs) accumulation during the fermentation process [12,13]. Nitrogen is required for the synthesis of nucleic acid, protein and enzyme in which an appropriate concentration of nitrogen could enhance bacterial growth and activity [13]. Optimum environmental factors affecting hydrogen production have been investigated using various kinds of carbon sources such as glucose [14], xylose [1], and lignocellulosic materials [15]. However, information on using waste glycerol to produce hydrogen under thermophilic condition is still limited. Accordingly, this study attempts to optimize media compositions used to produce hydrogen from waste glycerol under thermophilic condition employing RSM with CCD. In addition, the bacterial community presented in the fermentation broth of glycerol was analyzed by PCR-DGGE.
2.
Materials and methods
2.1.
Glycerol
Pure glycerol (analytical grade) is purchased from Ajax Finechem Pty. Ltd. Waste glycerol is obtained from biodiesel production process of the Krungtep Produce Public Company Limited, Saraburi Province, Thailand. The company produces fried chicken. Approximately 202.60 tons/month of used oil has been generated during the production process. The average 44,000 L of biodiesel as well as 4400 L of waste glycerol has been generated monthly. Waste glycerol consists of (all in
g/L) glycerol, 441.3; methanol, 230; NaCl, 10; total nitrogen, 0.5; and total phosphorus, 0.05.
2.2.
Inoculum preparation
Anaerobic thermophilic mixed cultures were enriched from the hot spring sediment of Songkla Province, Thailand. Temperature of the sampling site ranged from 53 to 68 C. The inoculum was prepared by enriching the hot spring sediment (150 mL) in the 1 L laboratory glass bottle containing 550 mL of the medium. The culture medium contained 25 g/L of pure glycerol as a carbon source in distilled water supplemented with 1 mL/L Endo-nutrient solution [11] (pH 5.5). The culture was incubated at 55 2 C in water bath. Every 4 days, 50% of culture broth was replaced with the fresh medium containing 25 g/L of pure glycerol. This process was repeated until constant hydrogen production volume was observed (4 times sub-culture). The obtained enriched culture was further used as the seed inoculums for hydrogen production. The pH and volatile suspended solid (VSS) concentration of the seed inoculum are 5.0 and 12.45 g-VSS/L, respectively.
2.3. Media preparation and biohydrogen production from pure glycerol Otherwise stated, the fermentation media contained the following constituents dissolved in distilled water: inoculum (30% (v/v)) (3.735 g-VSS/L), Endo-nutrient [11] (1 mL/L), and the different concentrations of the components that needed to be optimized i.e., peptone and urea (0.1e5 g/L), pure glycerol (20e150 g/L), and Na2HPO4 (1e7 g/L). The initial pH of the fermentation medium in this study ranged from 4 to 8 adjusted by 1 mol/L HCl or 1 mol/L NaOH. Batch fermentation of hydrogen using pure glycerol as a carbon source was conducted in 100 mL serum bottles containing 70 mL of fermentation medium. All bottles were tightly sealed with rubber septum and aluminum cap. The head space was replaced by nitrogen to create anaerobic condition. Then the serum bottles were incubated in water bath at various incubation temperatures (50e70 C). All treatments were conducted in triplicates. The volume of biogas was determined by using wetted glass syringe [16] and the biogas contents were analyzed by gas chromatography (GC).
2.4. Optimization of initial pH, incubation temperature, and determination of selected ranges of media components To obtain the optimal initial pH, incubation temperature, and to determine the ranges of media components to be used in the optimization study (Section 2.5), the hydrogen fermentation was conducted at different initial pH (4e8), nitrogen concentration (0.1e5 g/L), incubation temperature (50e70 C), pure glycerol concentration (20e150 g/L), and Na2HPO4 concentration (1e7 g/L) in a batch experiment (Table 1). First, the effects of initial pH of fermentation media and type of nitrogen source (inorganic nitrogen i.e., urea and organic nitrogen i.e., peptone) on hydrogen production were simultaneously examined at the concentrations of pure glycerol, nitrogen source, and Na2HPO4 of 25, 0.2, and 2 g/L, respectively, at the incubation temperature of 55 C (Table 1).
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Table 1 e Hydrogen production in the biohydrogen production from pure glycerol experiment. pH
Peptone conc. (g/L)
Urea conc. (g/L)
Temperature ( C)
Glycerol conc. (g/L)
Na2HPO4 conc. (g/L)
HP (mL H2/L)
4 5 5.5 6.0 6.5 8.0 5.5
0.2 0.2 0.2 0.2 0.2 0.2 e e e e e e e e e e e e e e e e e e e e
0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.5 1 3 5 0.2
55
25
2
55
25
2
25
2
0.2
50 55 60 65 70 55
2
0.2
55
20 25 50 100 150 25
440.08,a 260.50b 1009.12,a 934.69b 1087.88,a 1078.79b 590.24,a 469.08b 253.15,a 267.43b 201.22,a 132.85b 310.27b 1078.79b 676.35b 344.02b 206.84b 132.85b 449.60b 1078.79b 994.84b 330.17b 93.47b 550.86b 1078.79b 921.71b 833.00b 441.38b 421.04b 1078.79b 1051.53b 833.00b
5.5
5.5
5.5
1 2 5 7
a HP obtained by using peptone as nitrogen source. b HP obtained by using urea as nitrogen source.
The optimal initial pH and type of nitrogen source yielding the maximum HP were used to study the effect of nitrogen concentration on hydrogen production. The effect of nitrogen source concentration on hydrogen production was investigated at the concentrations of pure glycerol and Na2HPO4 of 25 and 2 g/L, respectively. The obtained optimal initial pH and type of nitrogen source were used in this study. The incubation temperature was 55 C. The optimal concentration of nitrogen source yielding the maximum HP value was chosen to study the effect of incubation temperature on hydrogen production. In order to study the effect of incubation temperature on hydrogen production, the experiment was conducted using pure glycerol and Na2HPO4 concentrations of 25 and 2 g/L, respectively. The optimal initial pH, type of nitrogen source as well as its optimal concentration were applied in this study. The optimal incubation temperature giving the highest HP value obtained from this study was used to study the effect of pure glycerol concentration on hydrogen production. The effect of pure glycerol concentration on hydrogen production was investigated under the optimal initial pH, type of nitrogen source and its optimum concentration, and incubation temperature. Concentration of Na2HPO4 was fixed at 2 g/L. The obtained optimal initial pH, type of nitrogen source and its optimum concentration, incubation temperature, and pure glycerol concentration were then used to examine the effect of Na2HPO4 concentration on hydrogen production.
2.5. Optimization of media components for biohydrogen production from waste glycerol 2.5.1.
Experimental design
Waste glycerol was used as the substrate for biohydrogen production. CCD was used to optimize the level of four variables i.e., waste glycerol concentration (g/L) (X1), urea concentration (g/L) (X2), the amount of Endo-nutrient addition (mL/L) (X3), and Na2HPO4 concentration (g/L) (X4). The response variable is HP (mL H2/L) (Table 2). For statistical analysis, test factors of Xi are coded as xi as appeared in the following equation: xi ¼ ðXi X0 Þ=DXi
(1)
where Xi is the actual value of the independent variable; xi is the coded value of the variable Xi; X0 is the value of Xi at the center point and DXi is the step change value. A quadratic model (Eq. (2)) is used to optimize the media compositions. X X X bi Xi þ bii X2i þ bij Xi Xj (2) Y ¼ b0 þ where Y is the predicted response (HP); b0 is a constant; bi is the linear coefficient; bii is the squared coefficient; bij is the interaction coefficient; and Xi is the variable. The response variable (HP) was fitted using a predictive polynomial quadratic equation (Eq. (2)) in order to correlate the response variable to the independent variables [17]. The statistical software Design-Expert (Demo version 7.0, Stat-Ease, Inc., Minneapolis, MN, USA) is used for regression and graphical analysis of the experimental data. The quality of fit of the
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Table 2 e Central composite experimental design matrix defining waste glycerol concentration (X1), urea concentration (X2), amount of Endo-nutrient addition (X3), Na2HPO4 concentration (X4), and result on HP. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Waste glycerol
Urea
Endo-nutrient
Na2HPO4
X1
X2
X3
X4
Code
Actual (g/L)
Code
Actual (g/L)
Code
Actual (mL/L)
Code
Actual (g/L)
Observed
Predicted
1.00 1.00 0.00 1.00 1.00 0.00 0.00 1.00 1.00 0.00 1.00 2.00 1.00 1.00 1.00 0.00 0.00 1.00 1.00 2.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00
30.88 15.35 19.19 15.15 25.58 19.19 19.19 28.88 24.62 19.19 27.77 36.81 16.73 12.27 30.58 22.54 22.69 30.31 13.77 0.23 28.50 26.58 14.77 26.88 16.62 14.88 20.42 21.69 19.19
1.00 1.00 0.00 1.00 0.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 1.00 1.00 1.00 0.00 0.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00 2.00 0.00
0.10 0.10 0.15 0.10 0.15 0.15 0.15 0.20 0.15 0.15 0.20 0.15 0.20 0.10 0.10 0.15 0.15 0.10 0.20 0.15 0.20 0.20 0.10 0.10 0.20 0.20 0.25 0.05 0.15
1.00 1.00 0.00 1.00 2.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 1.00 1.00 1.00 0.00 2.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00
0.30 0.30 0.20 0.30 0.00 0.20 0.20 0.10 0.20 0.20 0.30 0.20 0.30 0.10 0.10 0.20 0.40 0.10 0.30 0.20 0.30 0.10 0.10 0.30 0.10 0.10 0.20 0.20 0.20
1.00 1.00 0.00 1.00 0.00 0.00 0.00 1.00 2.00 0.00 1.00 0.00 1.00 1.00 1.00 2.00 0.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00
2.00 6.00 4.00 2.00 4.00 4.00 4.00 6.00 0.00 4.00 6.00 4.00 2.00 6.00 6.00 8.00 4.00 2.00 6.00 4.00 2.00 2.00 2.00 6.00 6.00 2.00 4.00 4.00 4.00
117.77 288.75 1486.07 321.08 368.44 1583.12 1383.20 161.55 175.35 1606.65 206.01 117.46 416.08 553.58 300.56 209.73 367.20 306.99 331.66 204.06 389.69 671.79 418.55 294.70 1242.07 1317.46 243.84 631.87 1183.20
311.11 365.40 1464.32 277.64 527.06 1464.32 1464.32 200.94 204.18 1464.32 172.00 180.32 555.64 659.59 214.65 192.30 219.98 202.57 361.27 154.85 218.62 582.71 615.98 487.12 1041.39 1126.08 526.97 360.14 1464.32
quadratic model is expressed by the coefficient of determination, R2, and its statistical significance is checked by the Ftest. The conditions of each trial are shown in Table 2.
2.5.2.
HP (mL H2/L)
Parameters
Biohydrogen production from waste glycerol
Biohydrogen production was conducted in 100 mL serum bottles using waste glycerol as substrate. The fermentation medium (total volume of 70 mL) contained waste glycerol, urea, Na2HPO4, Endo-nutrient [11], and 21 mL of inoculum (30% (v/v)) (3.735 g-VSS/L). The concentrations of each media component were adjusted according to the design (Table 2). Total nitrogen and phosphate concentrations contained in waste glycerol at the concentrations used in the optimization experiment (10e30 g/L of waste glycerol) were not taken into account as their concentrations are low (0.79e2.37 mg/L of total nitrogen and 0.079e0.23 mg/L of total phosphate). The initial pH of the medium was adjusted to 5.5 with 1 mol/L NaOH or 1 mol/L HCl. The serum bottles were tightly sealed with rubber stopper and aluminum cap then flushed with nitrogen gas to create the anaerobic condition. Then the serum bottles were incubated in a water bath at 55 2 C. During the incubation, the volume of biogas was measured by wetted glass syringe [16]. All treatments were conducted in triplicates. The fermentation process has been continued until biogas is no longer generated.
2.6.
Analytical methods
Biogas compositions were determined by GC (Shimadzu 2014, Japan) equipped with a thermal conductivity detector (TCD) and a 2 m stainless column packed with Unibeads C (60/80 mesh). The GC-TCD condition was set according to Saraphirom and Reungsang [18]. The volume of hydrogen in biogas was calculated by the mass balance equation [19]. Cumulative hydrogen production was calculated by the modified Gompertz equation [20]. Prior to the measurement of VFAs concentrations, one mL of liquid sample was added with 0.1 mL 34% H3PO4 to precipitate the lipid residues and acidified by 0.2 mL of 2 mol/L oxalic acid. The samples were then centrifuged at 12,000 rpm for 5 min and filtered through a 0.45 mm nylon membrane filter. The resulting filtrate was used to measure the concentrations of VFAs, acetone, alcohols, glycerol, and 1,3-PD by a high performance liquid chromatography (HPLC) equipped with the ultraviolet (UV) (210 nm) and refractive index (RI) detectors. A 7.80 300 mm Vertisep OA 8 mm column was used for HPLC analysis. The HPLC condition followed the method described by Selembo et al. [6]. The energy production (kJ/L) was calculated by multiplying the HP (mol H2/L) with molecular weight of hydrogen (2 g/mol) and the hydrogen energy yield (122 kJ/g).
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2.7.
Calculation of chemical oxygen demand (COD) balance
Glycerol consumption and concentrations of each fermentative product were converted to the COD concentration (g-COD/ L) followed the method described by Adrianus and Jeroen [21]. The fermentative products included 1,3-PD, ethanol, butanol, lactic, formic, acetic, propionic and butyric acids. The COD distribution of each fermentative product was calculated by multiplying COD concentration with 100 and then divided by COD concentration of glycerol consumed. The COD distribution for glycerol consumption was set to 100. The COD balance was then calculated as follows [22]:
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Effect of initial glycerol concentration on hydrogen production was studied at the pH of 5.5, urea concentration of 0.2 g/L, and incubation temperature of 55 C. The maximum HP of 1078.79 mL H2/L was obtained at 25 g/L of pure glycerol (Table 1). Increase in glycerol concentration from 25 to 50 g/L resulted in a slightly decrease in HP to 921.71 mL H2/L. The decrease in glycerol concentration from 25 to 20 g/L caused a sharp decrease in HP to 550.86 mL H2/L. Based on these results, the optimal concentration of pure glycerol seems to be within the range of 25e50 g/L. However, waste glycerol contains NaCl and methanol which could inhibit the growth and activity of hydrogen producers. Therefore, the selected
X COD balance % ¼ COD distribution of glycerol consumption þ ðCOD distribution of fermentative productsÞ
2.8.
PCR-DGGE
Total genomic DNA from sludge obtained from the low level (run 23), the center point (runs 6, 7, 10 and 29), the high level (run 11) and the optimum condition were extracted using phenol/chloroform method [23]. PCR-DGGE analysis of the extracted DNA and sequencing analysis were performed followed the methods described by Khamtib and Reungsang [24]. Closest matches for partial 16s rRNA gene sequences were identified by database searches in GenBank using BLAST [25].
3.
Results and discussion
3.1. Optimization of initial pH, incubation temperature, and determination of selected ranges of media components for optimization in RSM with CCD experiment Results indicated that the initial pH of 5.5 gave the highest HP values when peptone (1087.88 mL H2/L) and urea (1078.79 mL H2/L) (Table 1) were used as the nitrogen source. Since the HP obtained from urea and peptone were not significantly different, hence urea was selected as a nitrogen source, due to its low cost, throughout the experiments. The effect of urea concentration on hydrogen production was determined at the optimum pH of 5.5. HP was increased with an increase in urea concentration from 0.1 to 0.2 g/L. A further increase in urea concentration from 0.2 to 5 g/L resulted in a decrease in HP (Table 1). The highest HP of 1078.79 mL H2/L was achieved at the urea concentration of 0.2 g/L. Since the waste glycerol already contains trace amount of nitrogen, therefore the selected range of urea concentration used in the optimization experiment was chosen to be between 0.1 and 0.2 g/L. The optimum pH and urea concentration were then applied to examine the effect of incubation temperature on hydrogen production. Results indicated that incubation temperature of 55 C is the most efficient for hydrogen production from pure glycerol by thermophilic anaerobic mixed culture in which the highest HP of 1078.79 mL H2/L were achieved (Table 1). Thus, the incubation temperature of 55 C was used in the optimization experiment.
(3)
range of waste glycerol concentration for hydrogen production should be lower than the selected range of pure glycerol concentration. Hence, the tested concentration range of waste glycerol of 10e30 g/L was used in the optimization experiment. Under the optimum conditions, the effect of Na2HPO4 concentration was investigated. The highest HP of 1078.79 mL H2/L was obtained at 2 g/L of Na2HPO4. Based on this result, Na2HPO4 concentration ranged between 2 and 6 g/L was used in the optimization experiment. The results from the preliminary study indicated that the optimum initial pH and temperature for hydrogen production from pure glycerol by anaerobic thermophilic mixed cultures were 5.5 and 55 C, respectively. The selected ranges of media components to be used in the optimization study were urea concentration, waste glycerol concentration and Na2HPO4 concentration of 0.1e0.2 g/L, 10e30 g/L, and 2e6 g/L, respectively.
3.2. Optimization of media components on HP from waste glycerol by anaerobic thermophilic mixed cultures The effects of waste glycerol concentration (X1), urea concentration (X2), the amount of Endo-nutrient addition (X3), and Na2HPO4 concentration (X4) on HP were investigated. Regression analysis of the data from Table 2 resulted in the quadratic equation (Eq. (4)) as follows: HP ¼ 1458:13 107:97X1 þ 53:60X2 174:81X3 11:62X4 169:48X1 X2 þ 162:32X1 X3 31:77X1 X4 63:91X2 X3 48:91X2 X4 14:20X3 X4 388:08X21 254:69X22 229:68X23 293:50X24
(4)
The model presented a high determination coefficient (R2 ¼ 0.9249) explaining 92% of the variability in the response and a high value of the adjusted determination coefficient (adjusted R2 ¼ 0.94) suggested a high significant of the model. A very low probability ( p < 0.0001) obtained from the regression analysis of variance (ANOVA) demonstrated that the model was significant (Table 3). Linear terms of waste glycerol concentration (X1) and the amount of Endo-nutrient addition (X3) showed a significant individual effect on HP ( p 0.05).
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Table 3 e ANOVA of the fitting model for HP. HP (mL H2/L)
Source
Model X1 X2 X3 X4 X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 X21 X22 X23 X24 Residual Lack of fit Pure error Total R2
Sum of squares
Degree of freedom
Mean square
Coefficient estimate
F values
p values
6,318,677.00 166,049.30 63,659.43 606,269.91 2743.35 217,916.11 210,117.11 8035.32 64,838.45 37,990.96 3179.50 2,756,540.10 1,623,170.21 1,309,811.12 2,136,825.15 632159.42 512,978.32 119193.62 6,831,655.32 0.924912
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 10 4 28
451,334.12 166,049.31 63,659.43 606,269.92 2743.34 21,7916.11 210,117.14 8035.32 64,838.46 37,990.96 3179.50 2,756,540.12 1,623,170.21 1,409,812.31 2,136,826.22 45154.24 51,297.83 29798.41
1458.1292 107.9701 53.6046 174.8127 11.6249 169.4891 162.3230 31.7789 63.9124 48.9132 14.2011 388.0867 254.6900 229.6815 293.5074
12.3176 4.5318 1.7374 16.5460 0.0749 5.9473 5.7344 0.2193 1.7695 1.0368 0.0868 75.2303 44.3989 35.7469 58.3174
<0.0001 0.0515 0.2086 0.0012 0.7884 0.0287 0.0312 0.6468 0.2047 0.3258 0.7726 <0.0001 <0.0001 <0.0001 <0.0001
1.7215
0.3165
The quadratic model terms of all variables (X21, X22, X23 and X24) are highly significant ( p < 0.0001). A significant interaction on HP was found between waste glycerol concentration and urea concentration (X1X2) as well as waste glycerol concentration and the amount of Endo-nutrient addition (X1X3) ( p 0.05). The optimum conditions to maximize the HP were calculated using Eq. (3). The maximum HP of 1470.19 mL H2/L was estimated at a waste glycerol concentration of 20.33 g/L, urea concentration of 0.16 g/L, the amount of Endo-nutrient addition of 0.20 mL/L, and Na2HPO4 concentration of 3.97 g/L. Response surface plots based on Eq. (4) with one variable kept constant at its optimum level, and varying the other two variables within the experimental range were presented in Fig. 1AeF. Hydrogen production increased with an increase in waste glycerol concentration from 10 to 20.33 g/L but a further increase in waste glycerol concentration greater than 20.33 g/L resulted in a decrease in HP (Fig. 1AeC). A high substrate concentration, not exceed the substrate inhibition level, in the fermentation broth resulted in a high HP because substrate was used to produce hydrogen [5,14]. An increase in the concentration of waste glycerol higher than optimum level might develop osmotic pressure inside the cells which can cause cell damage [26]. This is due to the permeation of water molecules out of the cells hence a reduction of hydrogen production occurred. Waste glycerol used in this study contains high concentration of NaCl (10 g/L) and methanol (230 g/L), thus a high concentration of waste glycerol could produce high concentrations of NaCl and methanol which are toxic to the microorganisms [5,7,27,28]. Methanol is added to the used oil during the transesterification process in order to produce biodiesel. Even though the methanol is recovered after the end of transesterification process, but the methanol residue is still remained up to 200e250 g/L of waste glycerol [7]. Fig. 1A, D and E showed that an increase in urea concentration from 0.10 to 0.16 g/L resulted in an increase in HP.
However, the HP decreased when concentration of urea was greater than 0.16 g/L. Urea was used as a nitrogen source for the microorganisms in the fermentation process. Within the appropriate range, an increase in urea concentration could improve the bacterial growth and activity because nitrogen is needed for the synthesis of protein, nucleic acid, enzyme and biomass. However, a high concentration of ammonia ion released from urea might inhibit the growth of hydrogen producing bacteria. For example, Wang et al. [28] found that ammonia concentration greater than 1.2 g/L reduced the growth of Clostridium butyricum W5 and caused a reduction in hydrogen production from molasses. Fig. 1B, D, and F shows that an increase in the amount of Endo-nutrient addition from 0.10 to 0.20 mL/L resulted an increase in HP. The addition of Endo-nutrient provided the essential elements (Feþ2, Co2þ, and Mg2þ) not only for microbial growth but also for enzyme synthesis and activity. Iron is the most important element for hydrogen production because it forms ferredoxin and hydrogenase, which is directly related to the hydrogen production process [29]. Co2þ, and Mg2þ are enzyme cofactor [11,13,29,30]. NH4HCO3 in the Endo-nutrient can prevent the fluctuation of the pH during hydrogen production [12,31]. HP reduction was found when the amount of Endo-nutrient addition was greater than 0.20 mL/L. This may be caused by the dissolution of biocarbonate in NH4HCO3 which increases CO2 and reduces hydrogen content in gas phase in the fermentation system [13,31]. In addition, high ammonia concentration can also cause the adverse effect on microorganisms as described above. Moreover, Fe2þ and Cu2þ at high concentrations level than the optimum level are toxic, thus reducing the activity of hydrogen producers [13,31]. Hydrogen production increased with an increase of Na2HPO4 concentration from 2 to 3.97 g/L (Fig. 1C, E and F). An increase in phosphate concentration to the optimum level provided enough P elements which is essential for the
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Fig. 1 e Response surface plots showed the interactive effect on HP (1A the interactive effect of waste glycerol concentration and urea concentration at a fixed the amount of Endo-nutrient addition and Na2HPO4 concentration of 0.20 mL/L and 3.97 g/ L, respectively; 1B the interactive effect of waste glycerol concentration and the amount of Endo-nutrient addition at fixed urea and Na2HPO4 concentrations of 0.16 g/L and 3.97 g/L, respectively; 1C the interactive effect of waste glycerol concentration and Na2HPO4 concentration at a fixed the amount of Endo-nutrient addition and urea concentration of 0.20 mL/L and 0.16 g/L, respectively; 1D the interactive effect of the amount of Endo-nutrient addition and urea concentration at fixed waste glycerol and Na2HPO4 concentrations of 20.33 g/L and 3.97 g/L, respectively; 1E the interactive effect of Na2HPO4 and urea concentrations at fixed waste glycerol concentration and the amount of Endo-nutrient addition of 20.33 g/L and 0.20 mL/L, respectively; 1F the interactive effect of Na2HPO4 concentration and the amount of Endo-nutrient addition at fixed waste glycerol and urea concentrations of 20.33 and 0.16 g/L, respectively).
synthesis of DNA, RNA, and ATP of the hydrogen producers [13]. Moreover, Na2HPO4, as buffer in the fermentation system, prevented dramatically decrease in pH due to the accumulation of VFAs during hydrogen fermentation [12,31]. A decrease
in HP when concentration of Na2HPO4 was greater than 3.97 g/ L was observed (Fig. 1C, E, F). An increase in cytoplasmic osmotic pressure at high concentration of Na2HPO4 could damage the bacterial cells and decrease the metabolic activity
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of the microbial cells [12,31]. Consequently, the production of hydrogen decreased.
3.3.
Optimization and the confirmation experiment
The analysis of HP (Eq. (4)) model suggested that in order to obtain a maximum HP, waste glycerol concentration, urea concentration, Na2HPO4 concentration and the amount of Endo-nutrient addition should be optimized at 20.33 g/L, 0.16 g/L, 3.97 g/L, and 0.20 mL/L, respectively. The HP of 1470.19 mL H2/L was predicted under the optimum conditions. Three replications of batch fermentation experiments under the optimum condition were conducted to confirm the model validity. HP, HPR, and HY of 1502.84 mL H2/L, 17.06 mL H2/L$h, and 0.30 mol H2/mol glycerol, respectively, were obtained in the confirmation experiment with glycerol consumption of 18.99 g/L and energy production of 13.59 kJ/L. The HP results indicated that the observed HP of 1502.84 mL H2/L is in close agreement with the predicted values of HP with only 2.22% different. Results suggested that the model obtained from CCD experiment is valid. The maximum HP (1502.84 mL H2/L), obtained in this study was considered high when compared to the results of Ngo and Sim [32] (477.85 mL H2/L) whom produced hydrogen from waste glycerol by Thermotoga neapolitana. However, the initial concentration of waste glycerol used in that study was limited at 3 g/L while this study was 20.33 g/L. This indicated the advantages of using mixed culture over pure culture in terms of low operation cost (sterilization cost), easy to operate, and less sensitive to changes in environmental factors [2,13]. Additionally, the HY (0.30 mol H2/mol glycerol) obtained in this study was lower than that obtained by Ngo and Sim [32] (1.30 mol H2/mol glycerol). To improve the yield of hydrogen produced from glycerol, formic acid production has to be maximized with a reduced of 1,3-PD and lactic acid productions. The example of possible methods to improve HY were inhibition of lactic acid production by adding itaconic acid to substrate [32], and blocking the pathways of organic acid formation using the proton-suicide technique with NaBr and NaBrO3 [33]. SMPs and COD balance at the end of fermentation process in the confirmation experiment were presented in Table 4. The COD balance was only 9.72% error indicating that the measurements of SMPs were quite accurate. The yield of
hydrogen based on COD was 3.81%. The main SMPs were ethanol and 1,3-PD with the yields of 26.87 and 24.83%, respectively (based on COD). The minor metabolites, accounted for 34.78% (based on COD), were acetic, formic, butyric, lactic and propionic acids. No butanol was detected. There are two metabolic pathways of glycerol degradation i.e., oxidative and reductive pathways. In the oxidative pathway, one mole of glycerol was converted to pyruvate. The further cleavage of pyruvate to acetyl-CoA and formate yield one mole of hydrogen. Formate is easily cleaved to one mole of hydrogen and carbon dioxide while the oxidative conversion of acetylCoA to ethanol, acetate, and butyrate and oxidative conversion of pyruvate to lactic acid did not yield hydrogen [5]. In the reductive pathway, glycerol is reduced to 3hydroxypropionaldehyde and then to 1,3-PD with no generation of hydrogen. In this study, a high yield of ethanol (26.87% based on COD) was accompanied with a high yield of 1,3-PD (24.83% based on COD) which indicated that the microorganisms utilized waste glycerol through both oxidative and reductive pathways. In general, both pathways are simultaneously occurred during hydrogen fermentation from glycerol. During the growth of microorganisms, the reduced state of carbon in biomass is higher than in glycerol and the amount of NADH is generated as a result of biomass production. NADH is used in the reductive pathway to produce 1,3-PD. At the same time, microorganisms require ATP for biomass production in which ATP could be obtained from oxidative pathway [5,9]. The presence of 1,3-PD, lactic acid and propionic acid might be responsible for the low HY obtained since production of lactic acid and 1,3-PD did not yield hydrogen and production of propionic acid consume the hydrogen in the fermentation system.
3.4.
Microbial community
DGGE profile and sequence alignment results were shown in Fig. 2. Nine strongly strained distinctive bands were observed. Thermoanaerobacterium sp. (band 17), a well known thermophilic hydrogen producer [34], was mainly present in the optimum condition and the center point which coincided with the high HP obtained. Thermoanaerobacterium thermosacharolyticum (band 4) was present in all treatments. Uncultured Eubacteriaceae bacterium (band 3), Geobacillus sp. (band 6),
Table 4 e HP, metabolites production and COD balance at the end of fermentation of the optimum condition. Products Glycerol consumed Hydrogen 1,3-propanediol Ethanol Lactic acid Formic acid Acetic acid Propionic acid Butyric acid Balance
Concentration (g/L)
Concentration (g-COD/L)
COD distribution (%)
16.84 0.11 3.46 3.01 1.43 1.98 2.16 0.93 1.21
23.41 0.89 5.81 6.29 1.53 0.69 2.31 1.41 2.20
100.00 3.81 24.83 26.87 6.53 2.96 9.87 6.01 9.41 9.72
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Fig. 2 e DGGE profiles of 16s rRNA gene fragments at the end of fermentative hydrogen production at the low level (run 23), the high level (run 11), the center point (run 6, 7, 10 and 29) and the optimum conditions. Lane: O; optimum condition, M; center point, H; high level, W; low level.
Alicyclobacillus pohliae (band 11), Bacillus sp. (band 13) were present in the sample from the high level. These bacteria have not yet been reported as hydrogen producer except for Bacillus sp. which can produce hydrogen from sugar and lignocellulosic materials [35]. Dialister sp. (band 15) and Alicyclobacillus sp. (band 16) were found in the sample from the optimum condition and the low level while Acidilobus sp. (band 5) was found in the optimum condition and the center point. These bacteria are commonly found in the hot spring sediment [36,37] but have not been reported to produce hydrogen. Therefore, Thermoanaerobacterium sp. is the main hydrogen producer present in the fermentation process.
4.
Conclusions
Media compositions for thermophilic hydrogen production from waste glycerol by anaerobic thermophilic mixed cultures were optimized by RSM with CCD. The maximum HP of 1502.84 mL H2/L were obtained at 20.33 g/L waste glycerol, 0.16 g/L urea, 3.97 g/L Na2HPO4, and 0.20 mL/L the amount of Endo-nutrient addition. The hydrogen producing bacteria present in the fermentation process is Thermoanaerobacterium sp. The results obtained from this study show a potential application for converting waste glycerol into biohydrogen by thermophilic anaerobic mixed cultures.
Acknowledgments The authors appreciate the Ph.D. Scholarship to SS from Office of the Higher Education Commission, Thailand, under the Program Strategic Scholarship for Frontier Research Network for the Ph.D. Program/Thai Doctoral Degree. The authors are grateful for financial support received from the Research Group for the Development of Microbial Hydrogen Production Processes from Biomass, Office of the Higher Education Commission, the Energy Policy and Planning Office, Ministry of Energy, and the National Research University Project through Biofuel Research Cluster-Khon Kaen University, Office of the Higher Education Commission, Thailand.
references
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[3] Kongjan P, Angelidaki I. Extreme thermophilic biohydrogen production from wheat straw hydrolysate using mixed culture fermentation: effect of reactor configuration. Bioresour Technol; 2010:7780e96. [4] Temudo MF, Poldermans R, Kleerebezem R, van Loosdrecht MCM. Glycerol fermentation by open mixed cultures: a chemostat study. Biotechnol Bioeng 2008;100(6):1088e98. [5] Seifert K, Waligorska M, Wojtowski M, Laniecki M. Hydrogen generation from glycerol in batch fermentation process. Int J Hydrogen Energy 2009;34:3671e8. [6] Selembo PA, Perez JM, Lloyd WA, Logan BE. Enhanced hydrogen and 1,3-propanediol production from glycerol by fermentation using mixed cultures. Biotechol Bioeng 2009; 104(6):1098e105. [7] Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N. Hydrogen and ethanol production from glycerol-containing waste discharged after biodiesel manufacturing process. J Biosci Bioeng 2005;100(3):260e5. [8] Jitrwung R, Yargeau V. Optimization of media composition for the production of biohydrogen from waste glycerol. Int J Hydrogen Energy 2011;36:9602e11. [9] Biebl H, Menzel AP, Zeng W, Deckwer D. Microbial production of 1,3-propanediol. Appl Microbiol Biotechnol 1999;52:289e97. [10] Lin CY, Lay CH. Carbon/nitrogen-ratio effect on fermentative hydrogen production by mixed microfora. Int J Hydrogen Energy 2004;29:41e5. [11] Lin CY, Lay CH. A nutrient formulation for fermentative hydrogen production using anaerobic sewage sludge microflora. Int J Hydrogen Energy 2005;30:285e92. [12] Lin CY, Lay CH. Effects of carbonate and phosphate concentrations on hydrogen production using anaerobic sewage sludge microflora. Int J Hydrogen Energy 2004;29:275e81. [13] Wang J, Wan W. Factors influencing fermentative hydrogen production: a review. Int J Hydrogen Energy 2009;34:799e811. [14] Li Z, Wang H, Tang Z, Wang X, Bai J. Effects of pH value and substrate concentration on hydrogen production from the anaerobic fermentation of glucose. Int J Hydrogen Energy 2008;33:7413e8. [15] Pattra S, Sangyoka S, Boonmee M, Reungsang A. Biohydrogen production from the fermentation of sugarcane bagasse hydrolysate by Clostridium butyricum. Int J Hydrogen Energy 2008;33:5256e65. [16] Owen WF, Stuckey DC, Healy Jr JB, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 1979;13:485e93. [17] Lay JJ. Modeling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol Bioeng 2002;68:269e78. [18] Saraphirom P, Reungsang A. Optimization of biohydrogen production from sweet sorghum syrup using statistical methods. Int J Hydrogen Energy 2010;35(24):13435e44. [19] Zheng XJ, Yu HQ. Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic cultures. J Environ Manage 2005;74:66e70. [20] Zwietering MH, Jongenburger L, Rombouts FM, Vant RK. Modeling the bacterial growth curve. Appl Environ Microbiol 1990;56:1875e81. [21] Adrianus van H, Jeroen van der L. Handbook biological waste water treatment: measurement of organic material. Netherlands: Leidschendam; 2007. p. 8e10.
[22] Kongjan P, O-thong S, Angelidaki I. Biohydrogen production from desugared molasses (DM) using thermophilic mixed cultures immobilized on heat treated anaerobic sludge granules. Int J Hydrogen Energy 2011;36:14261e9. [23] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. NewYork: Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989. [24] Khamtib S, Plangklang P, Reungsang A. Optimization of fermentative hydrogen production from hydrolysate of microwave assisted sulfuric acid pretreated oil palm trunk by hot spring enriched culture. Int J Hydrogen Energy 2011; 36:14204e16. [25] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Res 1997;25: 3389e402. [26] Sreela-or C, Imai T, Plangklang P, Reungsang A. Optimization of key factors affecting hydrogen production from food waste by anaerobic mixed cultures. Int J Hydrogen Energy 2011;36:14120e33. [27] Kim DH, Kim SH, Shin HS. Sodium inhibition of fermentative hydrogen production. Int J Hydrogen Energy 2009;34: 3295e304. [28] Wang X, Jin B, Mulcahy D. Impact of carbon and nitrogen sources on hydrogen production by a newly isolated Clostridium butyricum W5. Int J Hydrogen Energy 2008;33: 4998e5005. [29] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001; 26:13e28. [30] Lay CH. Effects of nutrients on anaerobic hydrogen production. Master Degree Thesis, Feng Chia University, Taiwan; 2002. [31] Sreela-or C, Plangklang P, Imai T, Reungsang A. Co-digestion of food waste and sludge for hydrogen production by anaerobic mixed cultures: statistical key factors optimization. Int J Hydrogen Energy 2011;36:14227e37. [32] Ngo TA, Sim SJ. Dark fermentation of hydrogen from waste glycerol using hyperthermophilic eubacterium Thermotoga neapolitana. Environ Prog Sustainable Energy; 2011. doi:10.1002/ep. [33] Kumar N, Das D. Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic material as solid matrices. Enzym Microbiol Technol 2001;29:280e7. [34] Prasertsan P, O-Thong S, O-Thong NK. Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process. Int J Hydrogen Energy 2009;34:7448e59. [35] Manikkandan TR, Dhanasekar R, Thirumavalavan K. Microbial production of hydrogen from sugarcane bagasse using Bacillus Sp. Int J Chem Tech Res 2009;1(2):344e8. [36] Prokofeva MI, Miroshnichenko ML, Kostrikina NA, Chernyh NA, Kuznetsov BB, Tourova TP, et al. Acidilobus aceticus gen. nov., sp. nov., a novel anaerobic thermoacidophilic archaeon from continental hot vents in kamchatka. Int J Syst Evol Microbiol 2000;50:2001e8. [37] Ezeji TC, Bahl H. Production of raw starch hydrolyzing alpha amylase from the newlyisolatedGeobacillus thermodenitrificans HRO10. World J Microbiol Biotechnol 2007;23:1311e5.
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Media optimization for biohydrogen production from waste glycerol by anaerobic thermophilic mixed cultures Sureewan Sittijunda a,b, Alissara Reungsang a,c,* a
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand Center for Alternative Energy Research and Development, Khon Kaen University, Khon Kaen 40002, Thailand c Fermentation Research Center for Value-Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand b
article info
abstract
Article history:
Media compositions affecting thermophilic biohydrogen production from waste glycerol
Received 15 December 2011
were optimized using response surface methodology (RSM) with central composite design
Received in revised form
(CCD). Investigated parameters used were waste glycerol concentration, urea concentra-
26 February 2012
tion, the amount of Endo-nutrient addition and disodium hydrogen phosphate (Na2HPO4)
Accepted 28 February 2012
concentration. Waste glycerol concentration and the amount of Endo-nutrient addition
Available online 27 March 2012
had a significant individual effect on the cumulative hydrogen production (HP) ( p 0.05). The interactive effect on HP was found between waste glycerol and urea concentration as
Keywords:
well as waste glycerol concentration and the amount of Endo-nutrient addition ( p 0.05).
Waste glycerol
The optimal media compositions were 20.33 g/L of waste glycerol, 0.16 g/L of urea, 3.97 g/L
Thermophilic
of Na2HPO4 and 0.20 mL/L of the amount of Endo-nutrient addition which gave the
Response surface methodology
maximum HP of 1470.19 mL H2/L. The difference between observed HP (1502.84 mL H2/L)
Optimization
and predicted HP was 2.22%. The metabolic products from the fermentation process were 1,3-propanediol (1,3-PD), ethanol, acetic, formic, lactic, butyric, and propionic acids. Results from polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis indicated that the hydrogen producers present in the fermentation broth was Thermoanaerobacterium sp. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Hydrogen is a promising renewable energy which has received increase attention in recent years due to the high demand for sustainable energy [1]. Hydrogen has an energy yield of 122 kJ/ g, which is 2.75 times greater than hydrocarbon fuels [2]. Biologically, hydrogen is produced by photo and dark fermentation [2]. Dark fermentation is preferred over photo
fermentation process due to its cost-effective with the ability to utilize various kinds of substrates [2,3]. Biodiesel is one of alternative fuels that have been dramatically produced in recent years. The production of 10 kg of biodiesel could generate 1 kg of glycerol [4]. Therefore, it is necessary to find an alternative way to utilize this abundant waste. Glycerol is commonly used in chemicals, food, and beverages productions. It can also be used to produce 1,3-
* Corresponding author. Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand. Tel./ fax: þ66 43 362 121. E-mail address: [email protected] (A. Reungsang). 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.2012.02.185
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PD, ethanol and organic acids [4,5]. However, there is still limited information on using waste glycerol to produce hydrogen, especially under thermophilic temperature. In order to achieve efficient production of hydrogen, optimum environmental factors such as temperature, pH, inoculum size and media compositions are necessary. Temperature has a significant effect on hydrogen production. The production of hydrogen at thermophilic temperature has gained more interest due to a number of advantages over mesophilic temperature including higher hydrogen yield (HY), better pathogenic destruction, and lower risk of contamination by methanogenic archaea [1,3]. However, most of hydrogen production studies from glycerol were conducted under mesophilic conditions [4e9]. Due to the advantages of thermophilic temperature, therefore, in this study hydrogen production from waste glycerol was conducted at thermophilic condition. Microorganisms required appropriate concentrations of substrates, nitrogen, phosphate, and nutrient for metabolism during the fermentation process [10e14]. An appropriate substrate concentration could increase efficiency of hydrogen production of the hydrogen producers. Lower or higher substrate concentration than the optimum level could result in the adverse effects on hydrogen producers by causing substrate limitation and substrate inhibition, respectively [5,7,10]. Endo-nutrient is often used to enrich microorganisms capable of producing hydrogen. Important elements contained in the Endo-nutrient are Mg2þ and Fe2þ. These elements are needed for the growth of microorganisms and the hydrogenase enzyme activity [11,13]. Phosphate source such as Na2HPO4 has a buffering capacity that could reduce the pH fluctuation caused by volatile fatty acids (VFAs) accumulation during the fermentation process [12,13]. Nitrogen is required for the synthesis of nucleic acid, protein and enzyme in which an appropriate concentration of nitrogen could enhance bacterial growth and activity [13]. Optimum environmental factors affecting hydrogen production have been investigated using various kinds of carbon sources such as glucose [14], xylose [1], and lignocellulosic materials [15]. However, information on using waste glycerol to produce hydrogen under thermophilic condition is still limited. Accordingly, this study attempts to optimize media compositions used to produce hydrogen from waste glycerol under thermophilic condition employing RSM with CCD. In addition, the bacterial community presented in the fermentation broth of glycerol was analyzed by PCR-DGGE.
2.
Materials and methods
2.1.
Glycerol
Pure glycerol (analytical grade) is purchased from Ajax Finechem Pty. Ltd. Waste glycerol is obtained from biodiesel production process of the Krungtep Produce Public Company Limited, Saraburi Province, Thailand. The company produces fried chicken. Approximately 202.60 tons/month of used oil has been generated during the production process. The average 44,000 L of biodiesel as well as 4400 L of waste glycerol has been generated monthly. Waste glycerol consists of (all in
g/L) glycerol, 441.3; methanol, 230; NaCl, 10; total nitrogen, 0.5; and total phosphorus, 0.05.
2.2.
Inoculum preparation
Anaerobic thermophilic mixed cultures were enriched from the hot spring sediment of Songkla Province, Thailand. Temperature of the sampling site ranged from 53 to 68 C. The inoculum was prepared by enriching the hot spring sediment (150 mL) in the 1 L laboratory glass bottle containing 550 mL of the medium. The culture medium contained 25 g/L of pure glycerol as a carbon source in distilled water supplemented with 1 mL/L Endo-nutrient solution [11] (pH 5.5). The culture was incubated at 55 2 C in water bath. Every 4 days, 50% of culture broth was replaced with the fresh medium containing 25 g/L of pure glycerol. This process was repeated until constant hydrogen production volume was observed (4 times sub-culture). The obtained enriched culture was further used as the seed inoculums for hydrogen production. The pH and volatile suspended solid (VSS) concentration of the seed inoculum are 5.0 and 12.45 g-VSS/L, respectively.
2.3. Media preparation and biohydrogen production from pure glycerol Otherwise stated, the fermentation media contained the following constituents dissolved in distilled water: inoculum (30% (v/v)) (3.735 g-VSS/L), Endo-nutrient [11] (1 mL/L), and the different concentrations of the components that needed to be optimized i.e., peptone and urea (0.1e5 g/L), pure glycerol (20e150 g/L), and Na2HPO4 (1e7 g/L). The initial pH of the fermentation medium in this study ranged from 4 to 8 adjusted by 1 mol/L HCl or 1 mol/L NaOH. Batch fermentation of hydrogen using pure glycerol as a carbon source was conducted in 100 mL serum bottles containing 70 mL of fermentation medium. All bottles were tightly sealed with rubber septum and aluminum cap. The head space was replaced by nitrogen to create anaerobic condition. Then the serum bottles were incubated in water bath at various incubation temperatures (50e70 C). All treatments were conducted in triplicates. The volume of biogas was determined by using wetted glass syringe [16] and the biogas contents were analyzed by gas chromatography (GC).
2.4. Optimization of initial pH, incubation temperature, and determination of selected ranges of media components To obtain the optimal initial pH, incubation temperature, and to determine the ranges of media components to be used in the optimization study (Section 2.5), the hydrogen fermentation was conducted at different initial pH (4e8), nitrogen concentration (0.1e5 g/L), incubation temperature (50e70 C), pure glycerol concentration (20e150 g/L), and Na2HPO4 concentration (1e7 g/L) in a batch experiment (Table 1). First, the effects of initial pH of fermentation media and type of nitrogen source (inorganic nitrogen i.e., urea and organic nitrogen i.e., peptone) on hydrogen production were simultaneously examined at the concentrations of pure glycerol, nitrogen source, and Na2HPO4 of 25, 0.2, and 2 g/L, respectively, at the incubation temperature of 55 C (Table 1).
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Table 1 e Hydrogen production in the biohydrogen production from pure glycerol experiment. pH
Peptone conc. (g/L)
Urea conc. (g/L)
Temperature ( C)
Glycerol conc. (g/L)
Na2HPO4 conc. (g/L)
HP (mL H2/L)
4 5 5.5 6.0 6.5 8.0 5.5
0.2 0.2 0.2 0.2 0.2 0.2 e e e e e e e e e e e e e e e e e e e e
0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.5 1 3 5 0.2
55
25
2
55
25
2
25
2
0.2
50 55 60 65 70 55
2
0.2
55
20 25 50 100 150 25
440.08,a 260.50b 1009.12,a 934.69b 1087.88,a 1078.79b 590.24,a 469.08b 253.15,a 267.43b 201.22,a 132.85b 310.27b 1078.79b 676.35b 344.02b 206.84b 132.85b 449.60b 1078.79b 994.84b 330.17b 93.47b 550.86b 1078.79b 921.71b 833.00b 441.38b 421.04b 1078.79b 1051.53b 833.00b
5.5
5.5
5.5
1 2 5 7
a HP obtained by using peptone as nitrogen source. b HP obtained by using urea as nitrogen source.
The optimal initial pH and type of nitrogen source yielding the maximum HP were used to study the effect of nitrogen concentration on hydrogen production. The effect of nitrogen source concentration on hydrogen production was investigated at the concentrations of pure glycerol and Na2HPO4 of 25 and 2 g/L, respectively. The obtained optimal initial pH and type of nitrogen source were used in this study. The incubation temperature was 55 C. The optimal concentration of nitrogen source yielding the maximum HP value was chosen to study the effect of incubation temperature on hydrogen production. In order to study the effect of incubation temperature on hydrogen production, the experiment was conducted using pure glycerol and Na2HPO4 concentrations of 25 and 2 g/L, respectively. The optimal initial pH, type of nitrogen source as well as its optimal concentration were applied in this study. The optimal incubation temperature giving the highest HP value obtained from this study was used to study the effect of pure glycerol concentration on hydrogen production. The effect of pure glycerol concentration on hydrogen production was investigated under the optimal initial pH, type of nitrogen source and its optimum concentration, and incubation temperature. Concentration of Na2HPO4 was fixed at 2 g/L. The obtained optimal initial pH, type of nitrogen source and its optimum concentration, incubation temperature, and pure glycerol concentration were then used to examine the effect of Na2HPO4 concentration on hydrogen production.
2.5. Optimization of media components for biohydrogen production from waste glycerol 2.5.1.
Experimental design
Waste glycerol was used as the substrate for biohydrogen production. CCD was used to optimize the level of four variables i.e., waste glycerol concentration (g/L) (X1), urea concentration (g/L) (X2), the amount of Endo-nutrient addition (mL/L) (X3), and Na2HPO4 concentration (g/L) (X4). The response variable is HP (mL H2/L) (Table 2). For statistical analysis, test factors of Xi are coded as xi as appeared in the following equation: xi ¼ ðXi X0 Þ=DXi
(1)
where Xi is the actual value of the independent variable; xi is the coded value of the variable Xi; X0 is the value of Xi at the center point and DXi is the step change value. A quadratic model (Eq. (2)) is used to optimize the media compositions. X X X bi Xi þ bii X2i þ bij Xi Xj (2) Y ¼ b0 þ where Y is the predicted response (HP); b0 is a constant; bi is the linear coefficient; bii is the squared coefficient; bij is the interaction coefficient; and Xi is the variable. The response variable (HP) was fitted using a predictive polynomial quadratic equation (Eq. (2)) in order to correlate the response variable to the independent variables [17]. The statistical software Design-Expert (Demo version 7.0, Stat-Ease, Inc., Minneapolis, MN, USA) is used for regression and graphical analysis of the experimental data. The quality of fit of the
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Table 2 e Central composite experimental design matrix defining waste glycerol concentration (X1), urea concentration (X2), amount of Endo-nutrient addition (X3), Na2HPO4 concentration (X4), and result on HP. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Waste glycerol
Urea
Endo-nutrient
Na2HPO4
X1
X2
X3
X4
Code
Actual (g/L)
Code
Actual (g/L)
Code
Actual (mL/L)
Code
Actual (g/L)
Observed
Predicted
1.00 1.00 0.00 1.00 1.00 0.00 0.00 1.00 1.00 0.00 1.00 2.00 1.00 1.00 1.00 0.00 0.00 1.00 1.00 2.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00
30.88 15.35 19.19 15.15 25.58 19.19 19.19 28.88 24.62 19.19 27.77 36.81 16.73 12.27 30.58 22.54 22.69 30.31 13.77 0.23 28.50 26.58 14.77 26.88 16.62 14.88 20.42 21.69 19.19
1.00 1.00 0.00 1.00 0.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 1.00 1.00 1.00 0.00 0.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00 2.00 0.00
0.10 0.10 0.15 0.10 0.15 0.15 0.15 0.20 0.15 0.15 0.20 0.15 0.20 0.10 0.10 0.15 0.15 0.10 0.20 0.15 0.20 0.20 0.10 0.10 0.20 0.20 0.25 0.05 0.15
1.00 1.00 0.00 1.00 2.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 1.00 1.00 1.00 0.00 2.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00
0.30 0.30 0.20 0.30 0.00 0.20 0.20 0.10 0.20 0.20 0.30 0.20 0.30 0.10 0.10 0.20 0.40 0.10 0.30 0.20 0.30 0.10 0.10 0.30 0.10 0.10 0.20 0.20 0.20
1.00 1.00 0.00 1.00 0.00 0.00 0.00 1.00 2.00 0.00 1.00 0.00 1.00 1.00 1.00 2.00 0.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00
2.00 6.00 4.00 2.00 4.00 4.00 4.00 6.00 0.00 4.00 6.00 4.00 2.00 6.00 6.00 8.00 4.00 2.00 6.00 4.00 2.00 2.00 2.00 6.00 6.00 2.00 4.00 4.00 4.00
117.77 288.75 1486.07 321.08 368.44 1583.12 1383.20 161.55 175.35 1606.65 206.01 117.46 416.08 553.58 300.56 209.73 367.20 306.99 331.66 204.06 389.69 671.79 418.55 294.70 1242.07 1317.46 243.84 631.87 1183.20
311.11 365.40 1464.32 277.64 527.06 1464.32 1464.32 200.94 204.18 1464.32 172.00 180.32 555.64 659.59 214.65 192.30 219.98 202.57 361.27 154.85 218.62 582.71 615.98 487.12 1041.39 1126.08 526.97 360.14 1464.32
quadratic model is expressed by the coefficient of determination, R2, and its statistical significance is checked by the Ftest. The conditions of each trial are shown in Table 2.
2.5.2.
HP (mL H2/L)
Parameters
Biohydrogen production from waste glycerol
Biohydrogen production was conducted in 100 mL serum bottles using waste glycerol as substrate. The fermentation medium (total volume of 70 mL) contained waste glycerol, urea, Na2HPO4, Endo-nutrient [11], and 21 mL of inoculum (30% (v/v)) (3.735 g-VSS/L). The concentrations of each media component were adjusted according to the design (Table 2). Total nitrogen and phosphate concentrations contained in waste glycerol at the concentrations used in the optimization experiment (10e30 g/L of waste glycerol) were not taken into account as their concentrations are low (0.79e2.37 mg/L of total nitrogen and 0.079e0.23 mg/L of total phosphate). The initial pH of the medium was adjusted to 5.5 with 1 mol/L NaOH or 1 mol/L HCl. The serum bottles were tightly sealed with rubber stopper and aluminum cap then flushed with nitrogen gas to create the anaerobic condition. Then the serum bottles were incubated in a water bath at 55 2 C. During the incubation, the volume of biogas was measured by wetted glass syringe [16]. All treatments were conducted in triplicates. The fermentation process has been continued until biogas is no longer generated.
2.6.
Analytical methods
Biogas compositions were determined by GC (Shimadzu 2014, Japan) equipped with a thermal conductivity detector (TCD) and a 2 m stainless column packed with Unibeads C (60/80 mesh). The GC-TCD condition was set according to Saraphirom and Reungsang [18]. The volume of hydrogen in biogas was calculated by the mass balance equation [19]. Cumulative hydrogen production was calculated by the modified Gompertz equation [20]. Prior to the measurement of VFAs concentrations, one mL of liquid sample was added with 0.1 mL 34% H3PO4 to precipitate the lipid residues and acidified by 0.2 mL of 2 mol/L oxalic acid. The samples were then centrifuged at 12,000 rpm for 5 min and filtered through a 0.45 mm nylon membrane filter. The resulting filtrate was used to measure the concentrations of VFAs, acetone, alcohols, glycerol, and 1,3-PD by a high performance liquid chromatography (HPLC) equipped with the ultraviolet (UV) (210 nm) and refractive index (RI) detectors. A 7.80 300 mm Vertisep OA 8 mm column was used for HPLC analysis. The HPLC condition followed the method described by Selembo et al. [6]. The energy production (kJ/L) was calculated by multiplying the HP (mol H2/L) with molecular weight of hydrogen (2 g/mol) and the hydrogen energy yield (122 kJ/g).
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2.7.
Calculation of chemical oxygen demand (COD) balance
Glycerol consumption and concentrations of each fermentative product were converted to the COD concentration (g-COD/ L) followed the method described by Adrianus and Jeroen [21]. The fermentative products included 1,3-PD, ethanol, butanol, lactic, formic, acetic, propionic and butyric acids. The COD distribution of each fermentative product was calculated by multiplying COD concentration with 100 and then divided by COD concentration of glycerol consumed. The COD distribution for glycerol consumption was set to 100. The COD balance was then calculated as follows [22]:
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Effect of initial glycerol concentration on hydrogen production was studied at the pH of 5.5, urea concentration of 0.2 g/L, and incubation temperature of 55 C. The maximum HP of 1078.79 mL H2/L was obtained at 25 g/L of pure glycerol (Table 1). Increase in glycerol concentration from 25 to 50 g/L resulted in a slightly decrease in HP to 921.71 mL H2/L. The decrease in glycerol concentration from 25 to 20 g/L caused a sharp decrease in HP to 550.86 mL H2/L. Based on these results, the optimal concentration of pure glycerol seems to be within the range of 25e50 g/L. However, waste glycerol contains NaCl and methanol which could inhibit the growth and activity of hydrogen producers. Therefore, the selected
X COD balance % ¼ COD distribution of glycerol consumption þ ðCOD distribution of fermentative productsÞ
2.8.
PCR-DGGE
Total genomic DNA from sludge obtained from the low level (run 23), the center point (runs 6, 7, 10 and 29), the high level (run 11) and the optimum condition were extracted using phenol/chloroform method [23]. PCR-DGGE analysis of the extracted DNA and sequencing analysis were performed followed the methods described by Khamtib and Reungsang [24]. Closest matches for partial 16s rRNA gene sequences were identified by database searches in GenBank using BLAST [25].
3.
Results and discussion
3.1. Optimization of initial pH, incubation temperature, and determination of selected ranges of media components for optimization in RSM with CCD experiment Results indicated that the initial pH of 5.5 gave the highest HP values when peptone (1087.88 mL H2/L) and urea (1078.79 mL H2/L) (Table 1) were used as the nitrogen source. Since the HP obtained from urea and peptone were not significantly different, hence urea was selected as a nitrogen source, due to its low cost, throughout the experiments. The effect of urea concentration on hydrogen production was determined at the optimum pH of 5.5. HP was increased with an increase in urea concentration from 0.1 to 0.2 g/L. A further increase in urea concentration from 0.2 to 5 g/L resulted in a decrease in HP (Table 1). The highest HP of 1078.79 mL H2/L was achieved at the urea concentration of 0.2 g/L. Since the waste glycerol already contains trace amount of nitrogen, therefore the selected range of urea concentration used in the optimization experiment was chosen to be between 0.1 and 0.2 g/L. The optimum pH and urea concentration were then applied to examine the effect of incubation temperature on hydrogen production. Results indicated that incubation temperature of 55 C is the most efficient for hydrogen production from pure glycerol by thermophilic anaerobic mixed culture in which the highest HP of 1078.79 mL H2/L were achieved (Table 1). Thus, the incubation temperature of 55 C was used in the optimization experiment.
(3)
range of waste glycerol concentration for hydrogen production should be lower than the selected range of pure glycerol concentration. Hence, the tested concentration range of waste glycerol of 10e30 g/L was used in the optimization experiment. Under the optimum conditions, the effect of Na2HPO4 concentration was investigated. The highest HP of 1078.79 mL H2/L was obtained at 2 g/L of Na2HPO4. Based on this result, Na2HPO4 concentration ranged between 2 and 6 g/L was used in the optimization experiment. The results from the preliminary study indicated that the optimum initial pH and temperature for hydrogen production from pure glycerol by anaerobic thermophilic mixed cultures were 5.5 and 55 C, respectively. The selected ranges of media components to be used in the optimization study were urea concentration, waste glycerol concentration and Na2HPO4 concentration of 0.1e0.2 g/L, 10e30 g/L, and 2e6 g/L, respectively.
3.2. Optimization of media components on HP from waste glycerol by anaerobic thermophilic mixed cultures The effects of waste glycerol concentration (X1), urea concentration (X2), the amount of Endo-nutrient addition (X3), and Na2HPO4 concentration (X4) on HP were investigated. Regression analysis of the data from Table 2 resulted in the quadratic equation (Eq. (4)) as follows: HP ¼ 1458:13 107:97X1 þ 53:60X2 174:81X3 11:62X4 169:48X1 X2 þ 162:32X1 X3 31:77X1 X4 63:91X2 X3 48:91X2 X4 14:20X3 X4 388:08X21 254:69X22 229:68X23 293:50X24
(4)
The model presented a high determination coefficient (R2 ¼ 0.9249) explaining 92% of the variability in the response and a high value of the adjusted determination coefficient (adjusted R2 ¼ 0.94) suggested a high significant of the model. A very low probability ( p < 0.0001) obtained from the regression analysis of variance (ANOVA) demonstrated that the model was significant (Table 3). Linear terms of waste glycerol concentration (X1) and the amount of Endo-nutrient addition (X3) showed a significant individual effect on HP ( p 0.05).
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Table 3 e ANOVA of the fitting model for HP. HP (mL H2/L)
Source
Model X1 X2 X3 X4 X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 X21 X22 X23 X24 Residual Lack of fit Pure error Total R2
Sum of squares
Degree of freedom
Mean square
Coefficient estimate
F values
p values
6,318,677.00 166,049.30 63,659.43 606,269.91 2743.35 217,916.11 210,117.11 8035.32 64,838.45 37,990.96 3179.50 2,756,540.10 1,623,170.21 1,309,811.12 2,136,825.15 632159.42 512,978.32 119193.62 6,831,655.32 0.924912
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 10 4 28
451,334.12 166,049.31 63,659.43 606,269.92 2743.34 21,7916.11 210,117.14 8035.32 64,838.46 37,990.96 3179.50 2,756,540.12 1,623,170.21 1,409,812.31 2,136,826.22 45154.24 51,297.83 29798.41
1458.1292 107.9701 53.6046 174.8127 11.6249 169.4891 162.3230 31.7789 63.9124 48.9132 14.2011 388.0867 254.6900 229.6815 293.5074
12.3176 4.5318 1.7374 16.5460 0.0749 5.9473 5.7344 0.2193 1.7695 1.0368 0.0868 75.2303 44.3989 35.7469 58.3174
<0.0001 0.0515 0.2086 0.0012 0.7884 0.0287 0.0312 0.6468 0.2047 0.3258 0.7726 <0.0001 <0.0001 <0.0001 <0.0001
1.7215
0.3165
The quadratic model terms of all variables (X21, X22, X23 and X24) are highly significant ( p < 0.0001). A significant interaction on HP was found between waste glycerol concentration and urea concentration (X1X2) as well as waste glycerol concentration and the amount of Endo-nutrient addition (X1X3) ( p 0.05). The optimum conditions to maximize the HP were calculated using Eq. (3). The maximum HP of 1470.19 mL H2/L was estimated at a waste glycerol concentration of 20.33 g/L, urea concentration of 0.16 g/L, the amount of Endo-nutrient addition of 0.20 mL/L, and Na2HPO4 concentration of 3.97 g/L. Response surface plots based on Eq. (4) with one variable kept constant at its optimum level, and varying the other two variables within the experimental range were presented in Fig. 1AeF. Hydrogen production increased with an increase in waste glycerol concentration from 10 to 20.33 g/L but a further increase in waste glycerol concentration greater than 20.33 g/L resulted in a decrease in HP (Fig. 1AeC). A high substrate concentration, not exceed the substrate inhibition level, in the fermentation broth resulted in a high HP because substrate was used to produce hydrogen [5,14]. An increase in the concentration of waste glycerol higher than optimum level might develop osmotic pressure inside the cells which can cause cell damage [26]. This is due to the permeation of water molecules out of the cells hence a reduction of hydrogen production occurred. Waste glycerol used in this study contains high concentration of NaCl (10 g/L) and methanol (230 g/L), thus a high concentration of waste glycerol could produce high concentrations of NaCl and methanol which are toxic to the microorganisms [5,7,27,28]. Methanol is added to the used oil during the transesterification process in order to produce biodiesel. Even though the methanol is recovered after the end of transesterification process, but the methanol residue is still remained up to 200e250 g/L of waste glycerol [7]. Fig. 1A, D and E showed that an increase in urea concentration from 0.10 to 0.16 g/L resulted in an increase in HP.
However, the HP decreased when concentration of urea was greater than 0.16 g/L. Urea was used as a nitrogen source for the microorganisms in the fermentation process. Within the appropriate range, an increase in urea concentration could improve the bacterial growth and activity because nitrogen is needed for the synthesis of protein, nucleic acid, enzyme and biomass. However, a high concentration of ammonia ion released from urea might inhibit the growth of hydrogen producing bacteria. For example, Wang et al. [28] found that ammonia concentration greater than 1.2 g/L reduced the growth of Clostridium butyricum W5 and caused a reduction in hydrogen production from molasses. Fig. 1B, D, and F shows that an increase in the amount of Endo-nutrient addition from 0.10 to 0.20 mL/L resulted an increase in HP. The addition of Endo-nutrient provided the essential elements (Feþ2, Co2þ, and Mg2þ) not only for microbial growth but also for enzyme synthesis and activity. Iron is the most important element for hydrogen production because it forms ferredoxin and hydrogenase, which is directly related to the hydrogen production process [29]. Co2þ, and Mg2þ are enzyme cofactor [11,13,29,30]. NH4HCO3 in the Endo-nutrient can prevent the fluctuation of the pH during hydrogen production [12,31]. HP reduction was found when the amount of Endo-nutrient addition was greater than 0.20 mL/L. This may be caused by the dissolution of biocarbonate in NH4HCO3 which increases CO2 and reduces hydrogen content in gas phase in the fermentation system [13,31]. In addition, high ammonia concentration can also cause the adverse effect on microorganisms as described above. Moreover, Fe2þ and Cu2þ at high concentrations level than the optimum level are toxic, thus reducing the activity of hydrogen producers [13,31]. Hydrogen production increased with an increase of Na2HPO4 concentration from 2 to 3.97 g/L (Fig. 1C, E and F). An increase in phosphate concentration to the optimum level provided enough P elements which is essential for the
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Fig. 1 e Response surface plots showed the interactive effect on HP (1A the interactive effect of waste glycerol concentration and urea concentration at a fixed the amount of Endo-nutrient addition and Na2HPO4 concentration of 0.20 mL/L and 3.97 g/ L, respectively; 1B the interactive effect of waste glycerol concentration and the amount of Endo-nutrient addition at fixed urea and Na2HPO4 concentrations of 0.16 g/L and 3.97 g/L, respectively; 1C the interactive effect of waste glycerol concentration and Na2HPO4 concentration at a fixed the amount of Endo-nutrient addition and urea concentration of 0.20 mL/L and 0.16 g/L, respectively; 1D the interactive effect of the amount of Endo-nutrient addition and urea concentration at fixed waste glycerol and Na2HPO4 concentrations of 20.33 g/L and 3.97 g/L, respectively; 1E the interactive effect of Na2HPO4 and urea concentrations at fixed waste glycerol concentration and the amount of Endo-nutrient addition of 20.33 g/L and 0.20 mL/L, respectively; 1F the interactive effect of Na2HPO4 concentration and the amount of Endo-nutrient addition at fixed waste glycerol and urea concentrations of 20.33 and 0.16 g/L, respectively).
synthesis of DNA, RNA, and ATP of the hydrogen producers [13]. Moreover, Na2HPO4, as buffer in the fermentation system, prevented dramatically decrease in pH due to the accumulation of VFAs during hydrogen fermentation [12,31]. A decrease
in HP when concentration of Na2HPO4 was greater than 3.97 g/ L was observed (Fig. 1C, E, F). An increase in cytoplasmic osmotic pressure at high concentration of Na2HPO4 could damage the bacterial cells and decrease the metabolic activity
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of the microbial cells [12,31]. Consequently, the production of hydrogen decreased.
3.3.
Optimization and the confirmation experiment
The analysis of HP (Eq. (4)) model suggested that in order to obtain a maximum HP, waste glycerol concentration, urea concentration, Na2HPO4 concentration and the amount of Endo-nutrient addition should be optimized at 20.33 g/L, 0.16 g/L, 3.97 g/L, and 0.20 mL/L, respectively. The HP of 1470.19 mL H2/L was predicted under the optimum conditions. Three replications of batch fermentation experiments under the optimum condition were conducted to confirm the model validity. HP, HPR, and HY of 1502.84 mL H2/L, 17.06 mL H2/L$h, and 0.30 mol H2/mol glycerol, respectively, were obtained in the confirmation experiment with glycerol consumption of 18.99 g/L and energy production of 13.59 kJ/L. The HP results indicated that the observed HP of 1502.84 mL H2/L is in close agreement with the predicted values of HP with only 2.22% different. Results suggested that the model obtained from CCD experiment is valid. The maximum HP (1502.84 mL H2/L), obtained in this study was considered high when compared to the results of Ngo and Sim [32] (477.85 mL H2/L) whom produced hydrogen from waste glycerol by Thermotoga neapolitana. However, the initial concentration of waste glycerol used in that study was limited at 3 g/L while this study was 20.33 g/L. This indicated the advantages of using mixed culture over pure culture in terms of low operation cost (sterilization cost), easy to operate, and less sensitive to changes in environmental factors [2,13]. Additionally, the HY (0.30 mol H2/mol glycerol) obtained in this study was lower than that obtained by Ngo and Sim [32] (1.30 mol H2/mol glycerol). To improve the yield of hydrogen produced from glycerol, formic acid production has to be maximized with a reduced of 1,3-PD and lactic acid productions. The example of possible methods to improve HY were inhibition of lactic acid production by adding itaconic acid to substrate [32], and blocking the pathways of organic acid formation using the proton-suicide technique with NaBr and NaBrO3 [33]. SMPs and COD balance at the end of fermentation process in the confirmation experiment were presented in Table 4. The COD balance was only 9.72% error indicating that the measurements of SMPs were quite accurate. The yield of
hydrogen based on COD was 3.81%. The main SMPs were ethanol and 1,3-PD with the yields of 26.87 and 24.83%, respectively (based on COD). The minor metabolites, accounted for 34.78% (based on COD), were acetic, formic, butyric, lactic and propionic acids. No butanol was detected. There are two metabolic pathways of glycerol degradation i.e., oxidative and reductive pathways. In the oxidative pathway, one mole of glycerol was converted to pyruvate. The further cleavage of pyruvate to acetyl-CoA and formate yield one mole of hydrogen. Formate is easily cleaved to one mole of hydrogen and carbon dioxide while the oxidative conversion of acetylCoA to ethanol, acetate, and butyrate and oxidative conversion of pyruvate to lactic acid did not yield hydrogen [5]. In the reductive pathway, glycerol is reduced to 3hydroxypropionaldehyde and then to 1,3-PD with no generation of hydrogen. In this study, a high yield of ethanol (26.87% based on COD) was accompanied with a high yield of 1,3-PD (24.83% based on COD) which indicated that the microorganisms utilized waste glycerol through both oxidative and reductive pathways. In general, both pathways are simultaneously occurred during hydrogen fermentation from glycerol. During the growth of microorganisms, the reduced state of carbon in biomass is higher than in glycerol and the amount of NADH is generated as a result of biomass production. NADH is used in the reductive pathway to produce 1,3-PD. At the same time, microorganisms require ATP for biomass production in which ATP could be obtained from oxidative pathway [5,9]. The presence of 1,3-PD, lactic acid and propionic acid might be responsible for the low HY obtained since production of lactic acid and 1,3-PD did not yield hydrogen and production of propionic acid consume the hydrogen in the fermentation system.
3.4.
Microbial community
DGGE profile and sequence alignment results were shown in Fig. 2. Nine strongly strained distinctive bands were observed. Thermoanaerobacterium sp. (band 17), a well known thermophilic hydrogen producer [34], was mainly present in the optimum condition and the center point which coincided with the high HP obtained. Thermoanaerobacterium thermosacharolyticum (band 4) was present in all treatments. Uncultured Eubacteriaceae bacterium (band 3), Geobacillus sp. (band 6),
Table 4 e HP, metabolites production and COD balance at the end of fermentation of the optimum condition. Products Glycerol consumed Hydrogen 1,3-propanediol Ethanol Lactic acid Formic acid Acetic acid Propionic acid Butyric acid Balance
Concentration (g/L)
Concentration (g-COD/L)
COD distribution (%)
16.84 0.11 3.46 3.01 1.43 1.98 2.16 0.93 1.21
23.41 0.89 5.81 6.29 1.53 0.69 2.31 1.41 2.20
100.00 3.81 24.83 26.87 6.53 2.96 9.87 6.01 9.41 9.72
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Fig. 2 e DGGE profiles of 16s rRNA gene fragments at the end of fermentative hydrogen production at the low level (run 23), the high level (run 11), the center point (run 6, 7, 10 and 29) and the optimum conditions. Lane: O; optimum condition, M; center point, H; high level, W; low level.
Alicyclobacillus pohliae (band 11), Bacillus sp. (band 13) were present in the sample from the high level. These bacteria have not yet been reported as hydrogen producer except for Bacillus sp. which can produce hydrogen from sugar and lignocellulosic materials [35]. Dialister sp. (band 15) and Alicyclobacillus sp. (band 16) were found in the sample from the optimum condition and the low level while Acidilobus sp. (band 5) was found in the optimum condition and the center point. These bacteria are commonly found in the hot spring sediment [36,37] but have not been reported to produce hydrogen. Therefore, Thermoanaerobacterium sp. is the main hydrogen producer present in the fermentation process.
4.
Conclusions
Media compositions for thermophilic hydrogen production from waste glycerol by anaerobic thermophilic mixed cultures were optimized by RSM with CCD. The maximum HP of 1502.84 mL H2/L were obtained at 20.33 g/L waste glycerol, 0.16 g/L urea, 3.97 g/L Na2HPO4, and 0.20 mL/L the amount of Endo-nutrient addition. The hydrogen producing bacteria present in the fermentation process is Thermoanaerobacterium sp. The results obtained from this study show a potential application for converting waste glycerol into biohydrogen by thermophilic anaerobic mixed cultures.
Acknowledgments The authors appreciate the Ph.D. Scholarship to SS from Office of the Higher Education Commission, Thailand, under the Program Strategic Scholarship for Frontier Research Network for the Ph.D. Program/Thai Doctoral Degree. The authors are grateful for financial support received from the Research Group for the Development of Microbial Hydrogen Production Processes from Biomass, Office of the Higher Education Commission, the Energy Policy and Planning Office, Ministry of Energy, and the National Research University Project through Biofuel Research Cluster-Khon Kaen University, Office of the Higher Education Commission, Thailand.
references
[1] Kongjan P, Min B, Angelidaki I. Biohydrogen production from xylose at extreme thermophilic temperatures (70 C) by mixed culture fermentation. Water Res 2009;43:1414e24. [2] Hawkes FR, Hussy I, Kyazze G, Dinsdale R, Hawkes DL. Continuous dark fermentative hydrogen production by mesophilic microflora: principles and progress. Int J Hydrogen Energy 2007;32:172e84.
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