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Effect of ultrasonic treatment of digestion sludge on bio-hydrogen production from sucrose by anaerobic fermentation
international journal of hydrogen energy 35 (2010) 3450–3455
Available at www.sciencedirect.com
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Effect of ultrasonic treatment of digestion sludge on bio-hydrogen production from sucrose by anaerobic fermentation YiPing Guo a,b, SangHyoun Kim a, ShiHwu Sung a,*, PoHeng Lee a a
Department of Civil, Construction and Environmental Engineering, Iowa State University, 394 Town Engineering Building, Ames, IA 50011, USA b Department of Chemistry, Zhengzhou University, Zhengzhou 450052, PR China
article info
abstract
Article history:
The utilization of ultrasonic treatment on digestion sludge to enhance microbial activity
Received 18 December 2009
for bio-hydrogen production was investigated. The optimal conditions of ultrasonic time
Received in revised form
and density on digestion sludge were detected using Central Composite Experimental
21 January 2010
Design. The regression analysis showed that a significant increase of 1.34 fold in bio-
Accepted 21 January 2010
hydrogen production rate could be obtained when ultrasonic time was 10 s and ultra-
Available online 13 February 2010
sonic density around 130 W/l at digester sludge concentration of 15 g VSS/l. The analyses of biodegradation characteristics in bio-hydrogen producing process implied that ultrasound
Keywords:
did not denature the digestion sludge but just improved its biodegradation efficiency. In
Ultrasound
order to find out the mechanism of ultrasonic treatment on digestion sludge, a control
Digestion sludge
experiment was designed and COD values of digestion sludge in different treatment
Bio-hydrogen production
conditions was measured.
Mechanism
1.
Introduction
Hydrogen as an excellent alternative energy carrier candidate has been greatly promoted due to its cleanness, recycle and effectivity [1,2]. Biological hydrogen production by mixed fermentation represents an important area because it requires less input of electricity or energy and operation [3–5]. However, from current situation the improvement of biohydrogen producing efficiency is an urgent requirement for its industrialization [6–8]. The most essential solution to this problem is enhancing the hydrogen producing bacteria activity. Consequently, various studies were carried out to improve the performance of pure hydrogen producing bacteria, but the purification and maintenance of pure hydrogen producing bacteria stood out as great challenges
Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu.
[9,10]. Hence other strategies should be adopted to avoid these obstacles. Many papers focused on utilizing metal iron to enhance bio-hydrogen producing efficiency and the results were promising [11]. However, more measures are still needed to further improve the bio-hydrogen production efficiency. Ultrasound is a sound wave with frequency beyond the normal hearing range of humans (>15–20 KHz). When ultrasound wave propagates in a liquid medium, it can produce cavitation and acoustic streaming. The cavitation will generate powerful shear forces while the acoustic streaming will increase the convection of solution [12,13]. Based on these merits ultrasound has been widely utilized in many biological processes to improve the reactions. There were mainly two kinds of performances exhibited by ultrasound in these researches. One was enhancing disintegration or extraction of
* Corresponding author. Tel.: þ1 515 294 3896; fax: þ1 515 294 8216. E-mail address: [email protected] (S. Sung). 0360-3199/$ – see front matter Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu. doi:10.1016/j.ijhydene.2010.01.090
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international journal of hydrogen energy 35 (2010) 3450–3455
substrates, for example, Neis et al. improved anaerobic and aerobic degradation by ultrasonic disintegration of biomass [14], and Nitayavardhana et al. utilized ultrasound pretreatment of cassava slurry to enhance sugar release [15]. While the other function was stimulating metabolic activities of biological materials, for example, ultrasound was used to accelerate enzymatic hydrolysis of solid leather waste [16], and Runyan et al. used low-frequency ultrasound to increase outer membrane permeability of microorganism [17]. For different biological process, the significant effect could only be obtained in a special and narrow range of ultrasonic power, so investigation should be carried out to evaluate the optimal ultrasonic conditions in every biological process [18,19]. In this paper, effect of ultrasonic treatment on digestion sludge for bio-hydrogen production was studied. The objectives of this research were finding out the function of ultrasonic treatment on digestion sludge and making out ultrasonic mechanism on digestion sludge in bio-hydrogen producing process.
2.
Materials and methods
2.1.
Seed microorganisms
The seed sludge for hydrogen production experiments was collected from the anaerobic digester in a local sewage treatment works and stored at 4 C. Before inoculation the digested sludge was first filtrated through a 20-mesh sieve, and then boiled at 90 C for 15 min to harvest hydrogen-producing spore-forming anaerobes and inhibit hydrogen-consuming anaerobes.
2.2.
Ultrasonic treatment
The ultrasonic treatment was conducted using a Branson 2000 series bench-scale ultrasonic unit (Branson Ultrasonic Corporation, Danbury, CT). The ultrasonic unit had a maximum power output of 2.2 KW and operated at a constant frequency of 20 KHz. The components of the ultrasonic system included the booster (gain 1:2) and the catenoidal titanium horn (gain 1:8) with a flat 13 mm diameter face. For exposure, the horn was put into 2 cm deep into 300 ml samples. The power levels were controlled by setting the amplitude at the horn tip through pulse width modulation voltage regulation to the converter. Extrinsic parameters of amplitude (16%, 20%, 30%, 40% and 44%, corresponding ultrasonic power levels were around 100, 109, 157, 201 and 240 W/l, respectively) and time (3, 5, 10, 15 and 17 s) were chosen. The volatile suspended solids (VSS) concentration of digester sludge samples for ultrasonic exposure was kept 15 g/ L, and this value was obtained from the previous experiments in our lab [20].
2.3.
Experimental design and procedure
A Box-Wilson Central Composite Design (CCD) as an effective tool in batch tests was used to optimize the two independent variables of ultrasonic time and ultrasonic power with each at five levels in this paper. The levels of independent variables
namely ultrasonic time (X1, S ) and amplitude level(X2, %) were selected based on values obtained in preliminary experiments. Ultrasonic time (X1) was varied between 3 s and 17 s while ultrasonic amplitude level (X2) between 16% and 44%. The coded values of the independent variables were a, 1, 0, þ1, þa. Experimental data from the central composite design was analyzed using response surface regression (Sigmaplot V 10.0) and fitted to a second-order polynomial model: Y ¼ b0 þ
X
bi Xi þ
X
bij Xi Xj þ
X
bii X2i
(1)
where, Y is the predicted response; b0 a constant; bi the linear coefficients; bii the squared coefficients; bij the cross-product coefficients; and Xi and Xj is variables. Furthermore, a control experiment was designed to assist in evaluating the mechanism of ultrasound on digestion sludge for biohydrogen production. In this control experiment, three different stages in hydrogen producing process were irradiated with ultrasound at the obtained optimal conditions respectively. The first stage was the boiled digestion sludge, which was exactly the same thing as mentioned above. The second stage was the substrate of sucrose, in which the sucrose concentration for ultrasonic exposure was 5 g COD/l. And the third stage was the completely solution including seed and substrate for hydrogen production. According to the result of ultrasonic effects on every stage, the function of ultrasound on digestion sludge could be distinguished. After the cumulative hydrogen production curves with respect to time were obtained from the hydrogen production experiments, a kinetic modeling of the modified Gompertz equation was applied to determine the hydrogen production potential(H), hydrogen production rate (r), and lag phase (l) [21,22] io n hr$e ðl tÞ þ 1 : H ¼ Pexp exp P
(2)
where, H is the cumulative hydrogen production (mL); l, the lag time (h), P, the hydrogen production potential (mL), r, the hydrogen production rate (mL/h), e, the constant 2.71828. The values of P, r and l were estimated using the solver function in sigmaPlot (version 10, SPSS) with a Newtonian algorithm. The results were summarized in Table 1. In order to
Table 1 – Central composite design matrix of two variables in coded and natural units along with observed responses. No. 1 2 3 4 5 6 7 8 9 10 11 12 13
X1
X2
1 1 1 1 1 1 1 1 1.4 0 0 1.4 0 1.4 1.4 0 0 0 0 0 0 0 0 0 0 0
Time(s) Amplitude level (%) r ratio 5 15 5 15 3 10 10 17 10 10 10 10 10
20 20 40 40 30 44 16 30 30 30 30 30 30
1.15 1.20 1.31 0.90 0.99 1.01 1.23 0.87 1.16 1.34 1.39 1.20 1.47
R2 0.9891 0.9872 0.9891 0.9902 0.9952 0.9955 0.9953 0.9943 0.9890 0.9938 0.9961 0.9920 0.9935
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international journal of hydrogen energy 35 (2010) 3450–3455
perceive directly the ultrasonic effect on bio-hydrogen production, r value was replaced by the ratio of r values, which meant the ratio of the r value with ultrasonic treatment digestion sludge to the one without ultrasonic treatment in the hydrogen-producing processes. And P and l values did not appear in Table 1 because they didn’t show much difference in the experimental design results. The bio-hydrogen production experiments were conducted in a series of 250-ml serum bottles. Each serum bottle had 150 ml mixture comprising the seed sludge with or without ultrasonic treatment, 5 g COD/L sucrose solution as substrate, and 5 ml of 0.72 M KHCO3 as buffer and 5 ml of nutrient solution. Each liter of nutrient solution contained 75 g of NaHCO3, 28.6 g of NH4Cl, 6.7 g of KH2PO4, 3.0 g of MgCl2$6H2O, 0.23 g of CaCl2$2H2O, 0.32 g of MnCl2$6H2O, 0.3 g of Na2MoO4$4H2O and 1.76 g of FeCl2$4H2O. Initially, the initial pH in each serum bottle was adjusted to 6.5 0.1 using potassium hydroxide or hydrochloric acid. Then the bottle was purged with nitrogen gas, sealed with butyl rubber stoppers, and incubated in a shaker at 180 rpm and 36 1 C. At each time interval, biogas sample was collected and analyzed for hydrogen content. Mixed liquor sample from each serum bottle was drawn at each time of the test, and analyzed for volatile fatty acids (VFAs) and residual carbohydrates. All the data obtained were the average of triplicate.
2.4.
Analytical methods
The biogas production was measured regularly by plunger displacement method [23]. The hydrogen content in biogas was analyzed by a gas chromatograph (Gow–Man series 350) equipped with a thermal conductivity detector and a 2.4 m 6 mm stainless column packed with Porapak Q (80/ 100 mesh). Nitrogen was used as a carrier gas at a flow rate of 30 mL/min, the temperature for the injection port, the column and the detector were set at 100, 50, and 100 C, respectively. The VFAs concentrations were analyzed by a High-
Performance Liquid chromatograph (GP40, Dionex, CA) with an absorbance detector (AD 20, Dionex) and a 300 mm 0 N 7.8 mm Metacarb 67H column (Varian, CA) using 0.05 M H2SO4 as mobile phase. COD and VSS of digestion sludge were measured according to Standard Methods [24]. Residual carbohydrates in the mixed liquor were determined following the method descried in Dubois et al. [25].
3.
Results and discussion
3.1. Ultrasonic effect on digestion sludge for bio-hydrogen production The optimal ultrasonic conditions of exposure time and amplitude level for maximal increase in bio-hydrogen producing efficiency were evaluated firstly. Central composite design matrix of two independent variables in coded and natural units along with the parameter of the ratio of r values was shown in Table 1. As mentioned above, here the ratio of r values referred to the ratio of the r value with ultrasonic treatment digestion sludge to the one without ultrasonic treatment in the bio-hydrogen producing processes, and P (hydrogen production potential) and l (lag time) values did not appear in Table 1 because they kept 250 20 ml/g COD and 8.0 0.5 h respectively in all the trials. Experiments of one to eight were performed at different combinations of the two independent variables while those from nine to thirteen were under the same conditions of center point. The repeated center point conditions were used to evaluate the confidence level of the method. The R2 values obtained from the modified Gompertz equation (Eq. (2)) for all experiments were lager than 0.987, indicating that experimental data fitted Eq. (2) well. Here the R2 value (0 < R2 < 1) was usually used to assess the credibility of fitted values and was different from the r (hydrogen production rate) value. And the larger the R2 values, the more credible the fitted values.
Ratio of hydrogen production rate
1.0
1.2
40
0.6 7 0.
1 1.
1.2
production rate
0.8
)
e( s
m
8
1.3
25
20
level(
%)
15
10
2
1.2
tude
20
6 4
1.1
Ampli
30
0.9
35
4
6
8
10
12
14
1.1
40
Ti
45
1.2
en Ratio of hydrog
25
1.0
0.4
1.0
0.6
1.1
18 16 14 12 10
1.3
0.8
1.1
1.0
1.3
30
1.2
1.2
35 1.2
1.4
Amplitude level(%)
0.9
1.6
16
Time(s) Fig. 1 – Effects of ultrasonic time(s) and amplitude level (%) on the ratio of hydrogen production rate based on the response plot and corresponding contour plot.
international journal of hydrogen energy 35 (2010) 3450–3455
Table 2 – Results of regression analysis of the central composite design for the ratio of hydrogen production rate. Term Intercept X1 X2 X21 X22 X1X2
Coefficient
Std. Error
T
P-value
0.3284 0.1859 0.0573 0.0065 0.0007 0.0023
0.5920 0.0528 0.0308 0.0019 0.0005 0.0012
0.5546 3.5199 1.8616 3.4732 1.4207 1.9190
0.5964 0.0097 0.1050 0.0104 0.1984 0.0965
Fig. 1 showed the response surface plot and the corresponding contour chart based on two independent variables of ultrasonic exposure time (X1/S) and ultrasonic amplitude level(X2/%), while Table 2 described the parameter estimate and the corresponding P-values obtained from multiple regression analysis on experimental data. The computed F-value of 4.19 was higher than the F (5, 7) value in F Distribution table at 5% level, which indicated the agreement between experimental data and predicted values from the mathematical model of Eq. (1) and implied the mathematical model was reliable for hydrogen production rate. The significance level of 94.76% reflected the high significance of the model. From Table 2 we can find that the liner term of ultrasonic exposure time (X1) and its square term as well as the interactive term of the two variables X1 X2 had a significant effect on the ratio of r values with the low P-values of less than 0.1, and the liner term of ultrasonic exposure time had the strongest effect on the ratio. While the liner term of ultrasonic amplitude level (X2) and its square term had less significance on correlation of coefficients because its P-value was bigger than 0.1. Following regression equation was obtained to explain the ratio of hydrogen production rate (Y ) along ultrasonic exposure time(X1) and ultrasonic amplitude level (X2): Y ¼ 0:3284 þ 0:1859X1 þ 0:0573X2 0:0065X21 0:0007X22 0:0023X1 X2
(3)
As shown in Fig. 1, the response surfaces of the ratio of r values showed a clear peak, indicating that the optimum conditions fell inside the design boundary well. By solving the regression equation of Eq. (3), the optimal values of X1 and X2 were ultrasonic amplitude level of 24% and exposure time of 10 s, respectively, while the predicted value of the ratio of hydrogen production rate obtained was 1.34. Values of optimized condition were found to be near the central level and increased or decreased amounts of the two factors would result in reductions in the ratio. The angle of inclination of the principal axis was bigger than 90 , indicating the two factors were in inverse ratio on the improvement of the ratio of r values. As can be seen from Fig. 1, increasing or decreasing of ultrasonic amplitude level and exposure time in the same pace would greatly reduce the ratio. The situation was expected because the stronger intensity of ultrasound would hurt hydrogen producing microorganism while the weaker one scarcely had effect [26,27], and this was the reason for the negative effect happened in the experiments. Herein the ratio of r values just experienced a little change when shifted the variables values from the central point to a certain range, as can be seen in Fig. 2, when the exposure time varied from 10 s to 8 s or 12 s at the amplitude level of 24%, the ratio just altered from 1.34 to 1.30, similarly, when amplitude level varied from 24% to 21% or 30% at the exposure time of 10 s, the ratio also just changed from 1.34 to around 1.30. As a result if the ultrasonic exposure time was confined in the optimal range of 8s–12 s, a significant increase of the ratio could be obtained in the range of ultrasonic amplitude level from 20% to 30%, while the corresponding ultrasonic density was from 109 W/l to 157 W/l at digester sludge concentration of 15 g VSS/l. The predicted optimal values were validated through repeated experiments and the results showed a good agreement with the predictions. Biodegradation characteristics in hydrogen production processes were also measured. Table 3 made comparisons of total volatile fatty acids (VFAs) and three main by-products as well as carbohydrates changes in bio-hydrogen production processes between the ultrasonic treatment digestion sludge and the one without ultrasonic treatment. In Table 3 the
160000 140000 120000
COD (mg/l)
3453
100000 80000 60000 40000 20000 0
Total COD
Soluble COD
Fig. 2 – CODs changes along the different treatment conditions of digestion sludge.
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international journal of hydrogen energy 35 (2010) 3450–3455
Table 3 – Changes of VFAs and carbohydrates in bio-hydrogen production processes between the ultrasonic treatment digestion sludge and the one without ultrasonic treatment. Time(h)
VFAs(gCOD/L) Total VFAs
0.00 8.00 9.00 11.00 12.00 18.00
0.19 0.90 1.24 3.96 3.99 4.56
a
0.17 0.90 1.34 4.04 4.31 5.07
Carbohydrates(mg/L)
HAc b
0.00 0.12 0.14 0.36 0.37 0.39
a
HPr 0.00 0.12 0.15 0.36 0.42 0.43
b
a
0.07 0.21 0.30 1.13 0.96 0.69
HBu 0.07 0.21 0.32 1.13 1.06 0.81
b
a
0.06 0.42 0.60 2.02 2.31 3.23
0.05b 0.41 0.65 2.10 2.47 3.57
9118.7a 3585.1 3573.6 388.2 300.7 124.0
9121.9b 3606.3 3591.4 400.9 262.5 122.7
a Control experiment without ultrasonic treatment. b Optimal experiment with ultrasonic treatment.
concentrations of total volatile fatty acids (Total VFAs) increased gradually and butyrate was the largest biodegradation product in both of the batches, which showed no distinct differences in degradation characteristics between ultrasonic treatment digestion sludge and the one without ultrasonic treatment. In addition, the carbohydrates values were also similar for both of them, so the conclusion that the utilization of ultrasound to treat digestion sludge did not denature the biodegradation paths for bio-hydrogen production could be reached.
3.2. Analyses of ultrasonic mechanism on digestion sludge for bio-hydrogen production Although the ultrasonic exposure was put on digestion sludge directly, it also needed to distinguish that the increase of hydrogen production rate came from exactly the change of substrate or digestion sludge because both of them existed in the hydrogen producing process. Therefore, a control experiment was designed to investigate the ultrasonic enhancement mechanism on digestion sludge for bio-hydrogen production. As depicted in part 2.3, three different stages in hydrogen producing process were irradiated with ultrasound at the obtained optimal conditions, respectively. The results were showed in Table 4. The R2 in Table 4 implied the experimental data fitted the Eq. (2) rather well and was believable. The highest ratio of the hydrogen production rate of 1.48 appeared when ultrasonic treatment was applied directly on both substrates and digestion sludge at the same time, and the lowest ratio of r values of 1.17 was observed when ultrasound was just executed on substrates, while the ratio of 1.30 on the digestion sludge was rather accordant with the data in Part 3.1 of this study. The interesting phenomenon observed from the results was that the sum of the ratios got when ultrasound was applied on substrates and digestion sludge respectively was exactly the ratio got when ultrasound was applied on
Table 4 – The results of the control experiment. Exposure substrates
Ratio of hydrogen production rate
R2 value
Digestion sludge Sucrose Digestion and sucrose
1.30 1.17 1.48
0.9959 0.9970 0.9967
both of them at the same time. The results implied that the change of digestion sludge played an important role in the increase of hydrogen production rate when ultrasound was utilized. And then the CODs values of different treatment conditions of digestion sludge were measured in order to find out the mechanism. As can be seen from Fig. 2, the total CODs kept the similar values from raw sludge to boiled sludge and then to both boiled and ultrasonic treated sludge, while the soluble CODs of digestion sludge increased largely from 8645 mg/L in the raw one to 12 911 mg/L when the sludge was boiled and then went to 27,667 mg/L after the boiled sludge was exposed to ultrasound. Actually, the similar results were found in the literatures by the cooperative group of Khanal et al. in which a typical soluble COD increase pattern at a longer sonication time was observed [28]. From the results we can see that both boiling and ultrasound would increase the soluble COD greatly. As is well known the boiling of digestion sludge was to harvest hydrogen-producing spore-forming anaerobes and inhibit hydrogen-consuming anaerobes [29,30]. So what was the function of ultrasound on digestion sludge? It was also in the literatures by Khanal et al. where the light microscope and scanning electron micrographs (SEM) were employed to obtain the visual observations of sludge changes before and after the ultrasonic treatment. The light-based micrograph depicted that the filaments and flocs of sludge were almost completely disintegrated and a more or less homogeneous texture was observed [31]. While the SEM figures described that the structural integrity of flocs as well as filaments was significantly disrupted without appreciable destruction of bacterial cells [32]. Thus, the function of ultrasound was assisted in decentralizing the biological floc and disrupting large organic particles into smaller-size particles but not killing the microorganisms. Meanwhile, ultrasound disassembled the flaccid surface of digestion sludge and released the intercellular materials to the aqueous phase, which could get support from several literatures, for example, Wand et al. examined that protein increased in the aqueous phase of sonicated sludge [33], while Bougrier et al. and Khanal et al. monitored the soluble organic nitrogen and ammonia-N would be released during ultrasonic disintegration of waste activated sludge, respectively [28,34]. Hence, the hydrogen producing spore-forming anaerobes in digestion sludge would be more accessible to be utilized, which resulted in the increase in the bio-hydrogen production rate.
international journal of hydrogen energy 35 (2010) 3450–3455
4.
Conclusion
The feasibility of using low energy ultrasound to enhance digestion sludge activity for bio-hydrogen production was confirmed. The optimal ultrasonic conditions were investigated using central composite design experiment. Results showed that hydrogen production rate could be increased 1.30 fold when ultrasound acted directly on digestion sludge and 1.48 fold when ultrasound applied on the solution including every composition for hydrogen production at the optimal ultrasonic time of 10 s and intensity of 130 W/l. The ultrasound made the hydrogen producing spore-forming anaerobes in digestion sludge more easily to be employed.
references
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Effect of ultrasonic treatment of digestion sludge on bio-hydrogen production from sucrose by anaerobic fermentation YiPing Guo a,b, SangHyoun Kim a, ShiHwu Sung a,*, PoHeng Lee a a
Department of Civil, Construction and Environmental Engineering, Iowa State University, 394 Town Engineering Building, Ames, IA 50011, USA b Department of Chemistry, Zhengzhou University, Zhengzhou 450052, PR China
article info
abstract
Article history:
The utilization of ultrasonic treatment on digestion sludge to enhance microbial activity
Received 18 December 2009
for bio-hydrogen production was investigated. The optimal conditions of ultrasonic time
Received in revised form
and density on digestion sludge were detected using Central Composite Experimental
21 January 2010
Design. The regression analysis showed that a significant increase of 1.34 fold in bio-
Accepted 21 January 2010
hydrogen production rate could be obtained when ultrasonic time was 10 s and ultra-
Available online 13 February 2010
sonic density around 130 W/l at digester sludge concentration of 15 g VSS/l. The analyses of biodegradation characteristics in bio-hydrogen producing process implied that ultrasound
Keywords:
did not denature the digestion sludge but just improved its biodegradation efficiency. In
Ultrasound
order to find out the mechanism of ultrasonic treatment on digestion sludge, a control
Digestion sludge
experiment was designed and COD values of digestion sludge in different treatment
Bio-hydrogen production
conditions was measured.
Mechanism
1.
Introduction
Hydrogen as an excellent alternative energy carrier candidate has been greatly promoted due to its cleanness, recycle and effectivity [1,2]. Biological hydrogen production by mixed fermentation represents an important area because it requires less input of electricity or energy and operation [3–5]. However, from current situation the improvement of biohydrogen producing efficiency is an urgent requirement for its industrialization [6–8]. The most essential solution to this problem is enhancing the hydrogen producing bacteria activity. Consequently, various studies were carried out to improve the performance of pure hydrogen producing bacteria, but the purification and maintenance of pure hydrogen producing bacteria stood out as great challenges
Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu.
[9,10]. Hence other strategies should be adopted to avoid these obstacles. Many papers focused on utilizing metal iron to enhance bio-hydrogen producing efficiency and the results were promising [11]. However, more measures are still needed to further improve the bio-hydrogen production efficiency. Ultrasound is a sound wave with frequency beyond the normal hearing range of humans (>15–20 KHz). When ultrasound wave propagates in a liquid medium, it can produce cavitation and acoustic streaming. The cavitation will generate powerful shear forces while the acoustic streaming will increase the convection of solution [12,13]. Based on these merits ultrasound has been widely utilized in many biological processes to improve the reactions. There were mainly two kinds of performances exhibited by ultrasound in these researches. One was enhancing disintegration or extraction of
* Corresponding author. Tel.: þ1 515 294 3896; fax: þ1 515 294 8216. E-mail address: [email protected] (S. Sung). 0360-3199/$ – see front matter Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu. doi:10.1016/j.ijhydene.2010.01.090
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substrates, for example, Neis et al. improved anaerobic and aerobic degradation by ultrasonic disintegration of biomass [14], and Nitayavardhana et al. utilized ultrasound pretreatment of cassava slurry to enhance sugar release [15]. While the other function was stimulating metabolic activities of biological materials, for example, ultrasound was used to accelerate enzymatic hydrolysis of solid leather waste [16], and Runyan et al. used low-frequency ultrasound to increase outer membrane permeability of microorganism [17]. For different biological process, the significant effect could only be obtained in a special and narrow range of ultrasonic power, so investigation should be carried out to evaluate the optimal ultrasonic conditions in every biological process [18,19]. In this paper, effect of ultrasonic treatment on digestion sludge for bio-hydrogen production was studied. The objectives of this research were finding out the function of ultrasonic treatment on digestion sludge and making out ultrasonic mechanism on digestion sludge in bio-hydrogen producing process.
2.
Materials and methods
2.1.
Seed microorganisms
The seed sludge for hydrogen production experiments was collected from the anaerobic digester in a local sewage treatment works and stored at 4 C. Before inoculation the digested sludge was first filtrated through a 20-mesh sieve, and then boiled at 90 C for 15 min to harvest hydrogen-producing spore-forming anaerobes and inhibit hydrogen-consuming anaerobes.
2.2.
Ultrasonic treatment
The ultrasonic treatment was conducted using a Branson 2000 series bench-scale ultrasonic unit (Branson Ultrasonic Corporation, Danbury, CT). The ultrasonic unit had a maximum power output of 2.2 KW and operated at a constant frequency of 20 KHz. The components of the ultrasonic system included the booster (gain 1:2) and the catenoidal titanium horn (gain 1:8) with a flat 13 mm diameter face. For exposure, the horn was put into 2 cm deep into 300 ml samples. The power levels were controlled by setting the amplitude at the horn tip through pulse width modulation voltage regulation to the converter. Extrinsic parameters of amplitude (16%, 20%, 30%, 40% and 44%, corresponding ultrasonic power levels were around 100, 109, 157, 201 and 240 W/l, respectively) and time (3, 5, 10, 15 and 17 s) were chosen. The volatile suspended solids (VSS) concentration of digester sludge samples for ultrasonic exposure was kept 15 g/ L, and this value was obtained from the previous experiments in our lab [20].
2.3.
Experimental design and procedure
A Box-Wilson Central Composite Design (CCD) as an effective tool in batch tests was used to optimize the two independent variables of ultrasonic time and ultrasonic power with each at five levels in this paper. The levels of independent variables
namely ultrasonic time (X1, S ) and amplitude level(X2, %) were selected based on values obtained in preliminary experiments. Ultrasonic time (X1) was varied between 3 s and 17 s while ultrasonic amplitude level (X2) between 16% and 44%. The coded values of the independent variables were a, 1, 0, þ1, þa. Experimental data from the central composite design was analyzed using response surface regression (Sigmaplot V 10.0) and fitted to a second-order polynomial model: Y ¼ b0 þ
X
bi Xi þ
X
bij Xi Xj þ
X
bii X2i
(1)
where, Y is the predicted response; b0 a constant; bi the linear coefficients; bii the squared coefficients; bij the cross-product coefficients; and Xi and Xj is variables. Furthermore, a control experiment was designed to assist in evaluating the mechanism of ultrasound on digestion sludge for biohydrogen production. In this control experiment, three different stages in hydrogen producing process were irradiated with ultrasound at the obtained optimal conditions respectively. The first stage was the boiled digestion sludge, which was exactly the same thing as mentioned above. The second stage was the substrate of sucrose, in which the sucrose concentration for ultrasonic exposure was 5 g COD/l. And the third stage was the completely solution including seed and substrate for hydrogen production. According to the result of ultrasonic effects on every stage, the function of ultrasound on digestion sludge could be distinguished. After the cumulative hydrogen production curves with respect to time were obtained from the hydrogen production experiments, a kinetic modeling of the modified Gompertz equation was applied to determine the hydrogen production potential(H), hydrogen production rate (r), and lag phase (l) [21,22] io n hr$e ðl tÞ þ 1 : H ¼ Pexp exp P
(2)
where, H is the cumulative hydrogen production (mL); l, the lag time (h), P, the hydrogen production potential (mL), r, the hydrogen production rate (mL/h), e, the constant 2.71828. The values of P, r and l were estimated using the solver function in sigmaPlot (version 10, SPSS) with a Newtonian algorithm. The results were summarized in Table 1. In order to
Table 1 – Central composite design matrix of two variables in coded and natural units along with observed responses. No. 1 2 3 4 5 6 7 8 9 10 11 12 13
X1
X2
1 1 1 1 1 1 1 1 1.4 0 0 1.4 0 1.4 1.4 0 0 0 0 0 0 0 0 0 0 0
Time(s) Amplitude level (%) r ratio 5 15 5 15 3 10 10 17 10 10 10 10 10
20 20 40 40 30 44 16 30 30 30 30 30 30
1.15 1.20 1.31 0.90 0.99 1.01 1.23 0.87 1.16 1.34 1.39 1.20 1.47
R2 0.9891 0.9872 0.9891 0.9902 0.9952 0.9955 0.9953 0.9943 0.9890 0.9938 0.9961 0.9920 0.9935
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perceive directly the ultrasonic effect on bio-hydrogen production, r value was replaced by the ratio of r values, which meant the ratio of the r value with ultrasonic treatment digestion sludge to the one without ultrasonic treatment in the hydrogen-producing processes. And P and l values did not appear in Table 1 because they didn’t show much difference in the experimental design results. The bio-hydrogen production experiments were conducted in a series of 250-ml serum bottles. Each serum bottle had 150 ml mixture comprising the seed sludge with or without ultrasonic treatment, 5 g COD/L sucrose solution as substrate, and 5 ml of 0.72 M KHCO3 as buffer and 5 ml of nutrient solution. Each liter of nutrient solution contained 75 g of NaHCO3, 28.6 g of NH4Cl, 6.7 g of KH2PO4, 3.0 g of MgCl2$6H2O, 0.23 g of CaCl2$2H2O, 0.32 g of MnCl2$6H2O, 0.3 g of Na2MoO4$4H2O and 1.76 g of FeCl2$4H2O. Initially, the initial pH in each serum bottle was adjusted to 6.5 0.1 using potassium hydroxide or hydrochloric acid. Then the bottle was purged with nitrogen gas, sealed with butyl rubber stoppers, and incubated in a shaker at 180 rpm and 36 1 C. At each time interval, biogas sample was collected and analyzed for hydrogen content. Mixed liquor sample from each serum bottle was drawn at each time of the test, and analyzed for volatile fatty acids (VFAs) and residual carbohydrates. All the data obtained were the average of triplicate.
2.4.
Analytical methods
The biogas production was measured regularly by plunger displacement method [23]. The hydrogen content in biogas was analyzed by a gas chromatograph (Gow–Man series 350) equipped with a thermal conductivity detector and a 2.4 m 6 mm stainless column packed with Porapak Q (80/ 100 mesh). Nitrogen was used as a carrier gas at a flow rate of 30 mL/min, the temperature for the injection port, the column and the detector were set at 100, 50, and 100 C, respectively. The VFAs concentrations were analyzed by a High-
Performance Liquid chromatograph (GP40, Dionex, CA) with an absorbance detector (AD 20, Dionex) and a 300 mm 0 N 7.8 mm Metacarb 67H column (Varian, CA) using 0.05 M H2SO4 as mobile phase. COD and VSS of digestion sludge were measured according to Standard Methods [24]. Residual carbohydrates in the mixed liquor were determined following the method descried in Dubois et al. [25].
3.
Results and discussion
3.1. Ultrasonic effect on digestion sludge for bio-hydrogen production The optimal ultrasonic conditions of exposure time and amplitude level for maximal increase in bio-hydrogen producing efficiency were evaluated firstly. Central composite design matrix of two independent variables in coded and natural units along with the parameter of the ratio of r values was shown in Table 1. As mentioned above, here the ratio of r values referred to the ratio of the r value with ultrasonic treatment digestion sludge to the one without ultrasonic treatment in the bio-hydrogen producing processes, and P (hydrogen production potential) and l (lag time) values did not appear in Table 1 because they kept 250 20 ml/g COD and 8.0 0.5 h respectively in all the trials. Experiments of one to eight were performed at different combinations of the two independent variables while those from nine to thirteen were under the same conditions of center point. The repeated center point conditions were used to evaluate the confidence level of the method. The R2 values obtained from the modified Gompertz equation (Eq. (2)) for all experiments were lager than 0.987, indicating that experimental data fitted Eq. (2) well. Here the R2 value (0 < R2 < 1) was usually used to assess the credibility of fitted values and was different from the r (hydrogen production rate) value. And the larger the R2 values, the more credible the fitted values.
Ratio of hydrogen production rate
1.0
1.2
40
0.6 7 0.
1 1.
1.2
production rate
0.8
)
e( s
m
8
1.3
25
20
level(
%)
15
10
2
1.2
tude
20
6 4
1.1
Ampli
30
0.9
35
4
6
8
10
12
14
1.1
40
Ti
45
1.2
en Ratio of hydrog
25
1.0
0.4
1.0
0.6
1.1
18 16 14 12 10
1.3
0.8
1.1
1.0
1.3
30
1.2
1.2
35 1.2
1.4
Amplitude level(%)
0.9
1.6
16
Time(s) Fig. 1 – Effects of ultrasonic time(s) and amplitude level (%) on the ratio of hydrogen production rate based on the response plot and corresponding contour plot.
international journal of hydrogen energy 35 (2010) 3450–3455
Table 2 – Results of regression analysis of the central composite design for the ratio of hydrogen production rate. Term Intercept X1 X2 X21 X22 X1X2
Coefficient
Std. Error
T
P-value
0.3284 0.1859 0.0573 0.0065 0.0007 0.0023
0.5920 0.0528 0.0308 0.0019 0.0005 0.0012
0.5546 3.5199 1.8616 3.4732 1.4207 1.9190
0.5964 0.0097 0.1050 0.0104 0.1984 0.0965
Fig. 1 showed the response surface plot and the corresponding contour chart based on two independent variables of ultrasonic exposure time (X1/S) and ultrasonic amplitude level(X2/%), while Table 2 described the parameter estimate and the corresponding P-values obtained from multiple regression analysis on experimental data. The computed F-value of 4.19 was higher than the F (5, 7) value in F Distribution table at 5% level, which indicated the agreement between experimental data and predicted values from the mathematical model of Eq. (1) and implied the mathematical model was reliable for hydrogen production rate. The significance level of 94.76% reflected the high significance of the model. From Table 2 we can find that the liner term of ultrasonic exposure time (X1) and its square term as well as the interactive term of the two variables X1 X2 had a significant effect on the ratio of r values with the low P-values of less than 0.1, and the liner term of ultrasonic exposure time had the strongest effect on the ratio. While the liner term of ultrasonic amplitude level (X2) and its square term had less significance on correlation of coefficients because its P-value was bigger than 0.1. Following regression equation was obtained to explain the ratio of hydrogen production rate (Y ) along ultrasonic exposure time(X1) and ultrasonic amplitude level (X2): Y ¼ 0:3284 þ 0:1859X1 þ 0:0573X2 0:0065X21 0:0007X22 0:0023X1 X2
(3)
As shown in Fig. 1, the response surfaces of the ratio of r values showed a clear peak, indicating that the optimum conditions fell inside the design boundary well. By solving the regression equation of Eq. (3), the optimal values of X1 and X2 were ultrasonic amplitude level of 24% and exposure time of 10 s, respectively, while the predicted value of the ratio of hydrogen production rate obtained was 1.34. Values of optimized condition were found to be near the central level and increased or decreased amounts of the two factors would result in reductions in the ratio. The angle of inclination of the principal axis was bigger than 90 , indicating the two factors were in inverse ratio on the improvement of the ratio of r values. As can be seen from Fig. 1, increasing or decreasing of ultrasonic amplitude level and exposure time in the same pace would greatly reduce the ratio. The situation was expected because the stronger intensity of ultrasound would hurt hydrogen producing microorganism while the weaker one scarcely had effect [26,27], and this was the reason for the negative effect happened in the experiments. Herein the ratio of r values just experienced a little change when shifted the variables values from the central point to a certain range, as can be seen in Fig. 2, when the exposure time varied from 10 s to 8 s or 12 s at the amplitude level of 24%, the ratio just altered from 1.34 to 1.30, similarly, when amplitude level varied from 24% to 21% or 30% at the exposure time of 10 s, the ratio also just changed from 1.34 to around 1.30. As a result if the ultrasonic exposure time was confined in the optimal range of 8s–12 s, a significant increase of the ratio could be obtained in the range of ultrasonic amplitude level from 20% to 30%, while the corresponding ultrasonic density was from 109 W/l to 157 W/l at digester sludge concentration of 15 g VSS/l. The predicted optimal values were validated through repeated experiments and the results showed a good agreement with the predictions. Biodegradation characteristics in hydrogen production processes were also measured. Table 3 made comparisons of total volatile fatty acids (VFAs) and three main by-products as well as carbohydrates changes in bio-hydrogen production processes between the ultrasonic treatment digestion sludge and the one without ultrasonic treatment. In Table 3 the
160000 140000 120000
COD (mg/l)
3453
100000 80000 60000 40000 20000 0
Total COD
Soluble COD
Fig. 2 – CODs changes along the different treatment conditions of digestion sludge.
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Table 3 – Changes of VFAs and carbohydrates in bio-hydrogen production processes between the ultrasonic treatment digestion sludge and the one without ultrasonic treatment. Time(h)
VFAs(gCOD/L) Total VFAs
0.00 8.00 9.00 11.00 12.00 18.00
0.19 0.90 1.24 3.96 3.99 4.56
a
0.17 0.90 1.34 4.04 4.31 5.07
Carbohydrates(mg/L)
HAc b
0.00 0.12 0.14 0.36 0.37 0.39
a
HPr 0.00 0.12 0.15 0.36 0.42 0.43
b
a
0.07 0.21 0.30 1.13 0.96 0.69
HBu 0.07 0.21 0.32 1.13 1.06 0.81
b
a
0.06 0.42 0.60 2.02 2.31 3.23
0.05b 0.41 0.65 2.10 2.47 3.57
9118.7a 3585.1 3573.6 388.2 300.7 124.0
9121.9b 3606.3 3591.4 400.9 262.5 122.7
a Control experiment without ultrasonic treatment. b Optimal experiment with ultrasonic treatment.
concentrations of total volatile fatty acids (Total VFAs) increased gradually and butyrate was the largest biodegradation product in both of the batches, which showed no distinct differences in degradation characteristics between ultrasonic treatment digestion sludge and the one without ultrasonic treatment. In addition, the carbohydrates values were also similar for both of them, so the conclusion that the utilization of ultrasound to treat digestion sludge did not denature the biodegradation paths for bio-hydrogen production could be reached.
3.2. Analyses of ultrasonic mechanism on digestion sludge for bio-hydrogen production Although the ultrasonic exposure was put on digestion sludge directly, it also needed to distinguish that the increase of hydrogen production rate came from exactly the change of substrate or digestion sludge because both of them existed in the hydrogen producing process. Therefore, a control experiment was designed to investigate the ultrasonic enhancement mechanism on digestion sludge for bio-hydrogen production. As depicted in part 2.3, three different stages in hydrogen producing process were irradiated with ultrasound at the obtained optimal conditions, respectively. The results were showed in Table 4. The R2 in Table 4 implied the experimental data fitted the Eq. (2) rather well and was believable. The highest ratio of the hydrogen production rate of 1.48 appeared when ultrasonic treatment was applied directly on both substrates and digestion sludge at the same time, and the lowest ratio of r values of 1.17 was observed when ultrasound was just executed on substrates, while the ratio of 1.30 on the digestion sludge was rather accordant with the data in Part 3.1 of this study. The interesting phenomenon observed from the results was that the sum of the ratios got when ultrasound was applied on substrates and digestion sludge respectively was exactly the ratio got when ultrasound was applied on
Table 4 – The results of the control experiment. Exposure substrates
Ratio of hydrogen production rate
R2 value
Digestion sludge Sucrose Digestion and sucrose
1.30 1.17 1.48
0.9959 0.9970 0.9967
both of them at the same time. The results implied that the change of digestion sludge played an important role in the increase of hydrogen production rate when ultrasound was utilized. And then the CODs values of different treatment conditions of digestion sludge were measured in order to find out the mechanism. As can be seen from Fig. 2, the total CODs kept the similar values from raw sludge to boiled sludge and then to both boiled and ultrasonic treated sludge, while the soluble CODs of digestion sludge increased largely from 8645 mg/L in the raw one to 12 911 mg/L when the sludge was boiled and then went to 27,667 mg/L after the boiled sludge was exposed to ultrasound. Actually, the similar results were found in the literatures by the cooperative group of Khanal et al. in which a typical soluble COD increase pattern at a longer sonication time was observed [28]. From the results we can see that both boiling and ultrasound would increase the soluble COD greatly. As is well known the boiling of digestion sludge was to harvest hydrogen-producing spore-forming anaerobes and inhibit hydrogen-consuming anaerobes [29,30]. So what was the function of ultrasound on digestion sludge? It was also in the literatures by Khanal et al. where the light microscope and scanning electron micrographs (SEM) were employed to obtain the visual observations of sludge changes before and after the ultrasonic treatment. The light-based micrograph depicted that the filaments and flocs of sludge were almost completely disintegrated and a more or less homogeneous texture was observed [31]. While the SEM figures described that the structural integrity of flocs as well as filaments was significantly disrupted without appreciable destruction of bacterial cells [32]. Thus, the function of ultrasound was assisted in decentralizing the biological floc and disrupting large organic particles into smaller-size particles but not killing the microorganisms. Meanwhile, ultrasound disassembled the flaccid surface of digestion sludge and released the intercellular materials to the aqueous phase, which could get support from several literatures, for example, Wand et al. examined that protein increased in the aqueous phase of sonicated sludge [33], while Bougrier et al. and Khanal et al. monitored the soluble organic nitrogen and ammonia-N would be released during ultrasonic disintegration of waste activated sludge, respectively [28,34]. Hence, the hydrogen producing spore-forming anaerobes in digestion sludge would be more accessible to be utilized, which resulted in the increase in the bio-hydrogen production rate.
international journal of hydrogen energy 35 (2010) 3450–3455
4.
Conclusion
The feasibility of using low energy ultrasound to enhance digestion sludge activity for bio-hydrogen production was confirmed. The optimal ultrasonic conditions were investigated using central composite design experiment. Results showed that hydrogen production rate could be increased 1.30 fold when ultrasound acted directly on digestion sludge and 1.48 fold when ultrasound applied on the solution including every composition for hydrogen production at the optimal ultrasonic time of 10 s and intensity of 130 W/l. The ultrasound made the hydrogen producing spore-forming anaerobes in digestion sludge more easily to be employed.
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