Ind. Eng. Chem. Res. 2008, 47, 5899–5905
5899
Enhanced Anaerobic Ruminal Degradation of Bulrush through Steam Explosion Pretreatment Zheng-Bo Yue,† Rong-Hua Liu,† Han-Qing Yu,*,† Hong-Zhang Chen,‡ Bin Yu,‡ Hideki Harada,§ and Yu-You Li§ Department of Chemistry, UniVersity of Science & Technology of China, Hefei 230026, China, National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100080 China, and Department of CiVil Engineering, Tohoku UniVersity, Sendai 980-8579, Japan
Steam explosion was employed as a bulrush pretreatment process in the subsequent anaerobic fermentation by rumen microorganisms. The effects of steam pressure, moisture content, and residence time on the pretreatment efficiency were investigated by using response surface methodology and Box-Behnken design. The product formation rate (Rmax) and specific product formation potential (Ps) were respectively selected as the response. A maximum Rmax of 0.485 g chemical oxygen demand (COD)/L · d was obtained at a moisture content of 18%, a steam pressure of 1.6 MPa, and a residence time of 8 min, while a maximum Ps of 0.432 g COD/g bulrush was achieved at a moisture content of 20%, a steam pressure of 1.8 MPa and a residence time of 8 min. Batch kinetics analysis shows that increases by approximately 140% in Rmax and approximately 20% in biodegradability (Ps) were obtained, compared with the untreated bulrush under the optimum conditions. Ultraviolet (UV) analysis indicates that the soluble carbohydrate-lignin compounds or lignin fragments increased after steam explosion. Breakage of rigid lignin structure was confirmed by Fourier transform infrared spectroscopy analysis. X-ray photoelectron spectroscopy analysis shows that the ratio of oxygen to carbon atoms increased from 0.206 to 0.286, and that the relative abundance of C2 and C3 in C1s spectra increased from 27.4 to 28.7 and 8.9 to 13.8, respectively. This demonstrates the partial removal of lignin and wax from the surface of bulrush through steam explosion. Introduction Bulrush, one type of aquatic plant, is extensively used in constructed wetlands for the removal of organic pollutants, nitrogen, phosphorus, and heavy metals.1,2 However, the disposal of biomass produced in the phytoremediation processes limits its wide application in pollution abatement.3 On the other hand, since the plants are mainly composed of ligno-cellulose, they are able to be used as a renewable resource for the generation of carboxylic acids.4 Anaerobic conversion of lignocellulosic materials could be an attractive approach for waste reduction and simultaneous recovery of carboxylic acids as useful chemicals and residual solids as a green manure.4 Bioreactors seeded with rumen microorganisms have higher degradation efficiencies and rates for both ligno-cellulosic wastes and high-cell-soluble wastes, compared with the bioreactors seeded with anaerobic sludge from usual sources.5,6 Rumen microbes have been successfully applied to improve the anaerobic digestion of aquatic plants in vitro.5,6 However, the inherent nature and distribution of the lignin still restrict the microbial conversion of aquatic plants. Various studies have been carried out to improve digestion of ligno-cellosic wastes for an improved biogas yield through employing mechanical particle size reduction, alkaline hydrolysis, thermal treatment, and enzymatic degradation.7 However, so far, the modest improvements have not been proven to be commercially viable, when the additional processing costs are taken into account. Steam explosion has been recognized to have a considerable potential for the cost-effective pretreatment of ligno-cellulose.8 * To whom correspondence should be addressed. Fax: +86 551 3601592. E-mail:
[email protected]. † University of Science & Technology of China. ‡ Chinese Academy of Sciences. § Tohoku University.
Steam pressure disruption, i.e., the explosive disruptive depressurization of water-containing materials following a residence time at an elevated temperature and a pressure, has proven to be a useful adjunct treatment to enzymatic hydrolysis of cellulose materials in the case of wood fiber processing for alcohol fermentation.8 Steam pressure and residence time have been found to be the two major factors governing the pretreatment efficiency. Moisture content also plays an influential role in the severity of pretreatment, as it greatly affects the ability of heat and chemicals to penetrate wood.9 Studies have been conducted to evaluate the effects of steam pressure, residence time and moisture content on treatment efficiency of ligno-cellulose,8,9 but in an independent variable form. However, the effects of these factors, in a dependent variable form, on the microbial conversion of ligno-celluloses have not been reported yet. Furthermore, the conventional “one factor at a time” technique used for optimizing a multivariable system is time-consuming and may also result in wrong conclusions.10 Response surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models, evaluating the effects of factors, and optimizing the target function.11 It has been successfully applied in the field of biotechnology, such as optimization of enzyme synthesis,12 bacterial growth,10 and acetate production in the acidogenesis of swine wastewater.13 Therefore, individual and interactive effects of moisture content, steam pressure, and residence time on the anaerobic transformation of bulrush by rumen microorganisms were explored by using the standard response-surface technique, which involved fitting an empirical model to the experimental data and the subsequent identification of optimal condition in
10.1021/ie800202c CCC: $40.75 2008 American Chemical Society Published on Web 07/16/2008
5900 Ind. Eng. Chem. Res., Vol. 47, No. 16, 2008 Table 1. Composition of Bulrush before and after Pretreatmenta
Table 2. Experimental Design and Results
item
control
sample after pretreatment
NDF (% TS) hemicellulose (% TS) cellulose (% TS) lignin (% TS) ash (% TS) WRP (g/g TS) soluble sugars (mg/g TS) TOC (mg/g TS)
78.0 ( 0.1 21.0 ( 0.1 36.1 ( 0.8 15.0 ( 0.3 6.1 ( 0.2 5.2 ( 0.8 32.0 ( 0.4 24.1 ( 1.1
55.9 ( 0.4 2.1 ( 0.0 34.4 ( 0.9 12.7 ( 0.2 6.6 ( 0.3 7.4 ( 1.0 40.6 ( 0.5 60.3 ( 1.3
a
Standard deviations were calculated from three measurements.
the pretreatment stage. Furthermore, the reasons behind the enhancement by steam explosion were also elucidated. Materials and Methods Seed Culture and Substrate. The mixed rumen microorganisms were obtained from a fistulated goat fed twice every day with 4 kg of corn stalks as previously reported.5 Bulrush was collected from the Jishui River in Jiyuan, China. The sun-dried bulrush was used as the resources for steam explosion treatment. As shown in Table 1, bulrush contained 78.0% of the neutral detergent fiber (NDF) on the basis of total solid (TS). Cellulose, hemicellulose, lignin, and ash respectively accounted for 36.1, 21.0, 15.0, and 6.1% of TS. Chopped bulrush (3-4 cm in length) was put into a streamexploded vessel of 1 m3. After that, a valve on the vessel was suddenly opened to expose the vessel to atmospheric pressure. Experimental Conditions. Batch fermentation experiments were conducted in 250-mL serum vials with 150 mL of working volume. The strained rumen fluid of 30 mL was used as the inoculums. Nutrition medium of 120 mL was added and the initial pH was adjusted to 7.0 ( 0.1. The initial bulrush concentration was 5.0 g TS/L. After the substrate and rumen microbe were added as designed, the vials were purged with N2 and CO2 (40:60) for 30 s and sealed with aluminum rubbers. Each trial was replicated three times and the averaged results are reported in this paper. All vials were placed in an air-bath shaker at 120 rpm and 40 ( 1 °C. Fermentation products, mainly composed of volatile fatty acid (VFA) and methane, were determined and expressed as chemical oxygen demand (COD). Experimental Design and Data Analysis. A modified Gompertz equation was employed to model the formation of fermentation products: 14
{ [
P ) Pmax exp -exp
]}
Rmaxe (λ - t) + 1 Pmax
(1)
where P (g COD/L) is the products formed per liter of reactor volume at fermentation time t; Pmax (g COD/L) is the maximum product formed per liter of reactor volume; Rmax (g COD/L · d) is the maximum formation rate of product; λ (d) is the lag time to exponential formation of product. Once cumulative product formation curves were obtained over the course of an entire batch experiment, a curve was drawn using the modified Gompertz equation and the values of Pmax and Rmax were estimated. The specific product formation potential (Ps) is defined as Pmax/[S] (g COD/g), in which [S] is bulrush concentration (g/L). In order to describe the nature of the response surface in the optimum region, a Box-Behnken design with three coded levels was provided. The range and levels of the variables investigated are given in Table 2. Moisture, steam pressure, and residence time were selected as independent variables. The central values chosen for experimental design were: 20% for moisture, 1.5
run 1 2 3 4 5 6 7 8 9 10 11 12 13
moisture steam pressure time Rmax Ps (%) (MPa) (min) (g COD/L · d) (g COD/g) λ (d) 10 30 10 30 10 30 10 30 20 20 20 20 20
1.0 1.0 2.0 2.0 1.5 1.5 1.5 1.5 1.0 2.0 1.0 2.0 1.5
8 8 8 8 6 6 10 10 6 6 10 10 8
0.300 0.298 0.366 0.345 0.335 0.264 0.343 0.301 0.207 0.241 0.238 0.296 0.490
0.370 0.375 0.400 0.396 0.392 0.388 0.385 0.380 0.379 0.428 0.385 0.410 0.429
0.097 0.123 0.142 0.213 0.142 0.123 0.135 0.241 0.134 0.142 0.147 0.214 0.121
R2 0.912 0.930 0.937 0.970 0.934 0.949 0.920 0.936 0.940 0.909 0.945 0.974 0.912
MPa for steam pressure, and 8 min for residence time. To predict the optimal point, a second order polynomial function was employed as: Y ) b0 + b1x1 + b2x2 + b3x3 + b11x12 + b22x22 + b33x32 + b12x1x2 + b13x1x3 + b23x2x3 (2) where Y is the predicted response, stands for Rmax and Ps, respectively, b0, b1, b2, b11, b22, and b12 are regression coefficients, and x1, x2, and x3 represent moisture, steam pressure, and residence time in the pretreatment phase, respectively. This is a square regression model in terms of real values. After eq 2 is determined, it can be used to locate the optimum for the set of independent variables by the partial derivatives of the model response with respect to the individual independent variables is equal to zero. Minitab, Version 14 (Minitab Inc., USA), was used for experimental design and regression analysis of the experimental data. Analytical Methods. The VFA concentration and biogas content were determined as reported previously.5 TS and volatile solid (VS) were analyzed according to the standard methods.15 The water retention power (WRP) was determined following the method reported by Ribitsch et al.16 Water- or dioxanesoluble compounds were obtained with the bulrush being soaked for 2 h at a dry matter content of 20% (w/w) with deionized water or dioxane. The ultraviolet (UV) spectra of the extractions were recorded with a spectrophotometer (UV-2450, Shimadzu Co., Japan). The pretreated and control samples were dried at vacuum, coated with Au and were then imaged using KYKY-1000 scanning electric microscope (SEM) (Keyi Co., China). X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) analyses were performed as described previously.17 Results Effects of Steam Explosion on Components and Accessibility of Bulrush. Table 1 shows the variations of the bulrush components before and after steam explosion. Compared with the untreated bulrush, the treated contained a lower lignin fraction and the hemicellulose decreased by about 90%. Soluble sugar fraction of bulrush increased by about 25% and soluble total organic carbon (TOC) increased by 150% (Table 1). The NDF fraction also decreased significantly after steam explosion. Cellulose accessibility reflects the level of cellulose hydroxyl being contacted by reagents, which depends on the structure of cellulose materials, micropore diameters and their distributions. The gross cellulose accessibility could be represented by WRP. WRP of the bulrush pretreated under optimal conditions increased by 42%, compared with the controls (Table 1).
Ind. Eng. Chem. Res., Vol. 47, No. 16, 2008 5901 Table 3. Estimated Regression Coefficients for Equations 3 and 4 SE-Coea
p valueb
term
coefficient
t-value
Rmax
b0 b1 b2 b3 b11 b22 b33
Ps
b0 b1 b2 b3 b11 b22 b33
-2.92287 0.172061 -16.987 0.01773 0.003632 4.883 1.41725 0.108105 13.110 0.52952 0.035935 14.736 -0.00049 0.00009 -5.427 -0.45533 0.035806 -12.717 -0.03258 0.002238 -14.560 R2 ) 98.0% R2(adj) ) 96.5% -0.06663 0.075432 -0.883 0.01143 0.001592 7.181 0.20625 0.047394 4.352 0.05265 0.015754 3.342 -0.00029 0.000039 -7.347 -0.05833 0.015698 -3.716 -0.00340 0.000981 -3.461 2 2 R ) 93.1% R (adj) ) 87.9%
a “SE-Coe” means standard error of coefficient. significant.
b
0.000** 0.001** 0.000** 0.000** 0.001** 0.000** 0.000** 0.403 0.000** 0.002** 0.010** 0.000** 0.006** 0.009**
** means highly
Table 4. ANOVA Analysis of Equations 3 and 4 Figure 1. Anaerobic fermentation of bulrush: (a) without pretreatment and (b) with pretreatment. Rmax
Product Formation from Bulrush Fermentation. Product formation from the anaerobic digestion of untreated bulrush with rumen microbes is illustrated in Figure 1a. VFA and methane were the major fermentation products. Figure 1 shows the cumulative production measured and simulated by eq 1, using the untreated bulrush and the bulrush pretreated at a moisture content of 20%, a steam pressure of 1.6 MPa, and a residence time of 8 min. With the modified Gompertz equation, Ps and Rmax of each batch test were calculated and are summarized in Table 2. Values are the means of results of each run in duplicate. The regression coefficients values were greater than 0.90 for all trials (data not shown), indicating that the parameters were statistically significant. However, the productions of VFA and methane from the two types of bulrush were different with each other. This might be attributed to the changed structures or components of bulrush induced by the steam explosion. Optimization of Steam Explosion Pretreatment for Rmax and Ps. A preliminary study on the effect of moisture, steam pressure and residence time on Rmax in the anaerobic conversion of the steam explosion-pretreated bulrush by rumen microbes was carried out, in order to determine the most critical factors and their region of interest. The experimental data shown in Table 2 were further subjected to regression analysis, generating the following quadratic regression equation including the coefficients, which shows the statistical significance with an R value of 0.05: Y ) -2.9287 + 0.01773x1 + 1.41725x2 + 0.52952x3 0.00049x12 - 0.45533x22 - 0.03258x32
(3)
From eq 3, the optimization conditions of steam explosion were obtained as follows: x1 ) 18 (%), x2 ) 1.6 (MPa), and x3 ) 8.0 (min), where Rmax was 0.485 g COD/L · d. A high correlation coefficient (R2) of 0.980 indicates that this model fitted to the experimental results well (Table 3). ANOVA analysis was carried out to evaluate the regression equation. A low value of p (
