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β-glucosidase by the mixed fungi culture Trichoderma reesei and Aspergillus phoenicis on dairy manure
Process Biochemistry 40 (2005) 3087–3094 www.elsevier.com/locate/procbio
Production of cellulase/b-glucosidase by the mixed fungi culture Trichoderma reesei and Aspergillus phoenicis on dairy manure Zhiyou Wen *, Wei Liao, Shulin Chen Department of Biological Systems Engineering, Center of Multiphase Environmental Research, Washington State University, Pullman, WA 99164-6120, USA Received 31 May 2004; accepted 21 March 2005
Abstract Trichoderma reesei was co-cultured with Aspergillus phoenicis using dairy manure as a substrate to produce cellulase with a high level of b-glucosidase. For pure cultures of T. reesei and A. phoenicis, the optimal media compositions were the same (10 g/L manure supplemented with 2 g/L KH2PO4, 2 mL/L tween-80 and 2 mg/L CoCl2), while the optimal temperature and pH were similar (25.5 8C and pH 5.76 for T. reesei; 28.2 8C and pH 5.14 for A. phoenicis). The mixed culture was therefore completed at 27 8C and pH 5.5, which is close to the optimal values for both fungi. The mixed culture resulted in a relatively high level of total cellulase and b-glucosidase. It was also found that a high manure solid concentration (>20 g/L) resulted in higher enzyme activities, probably due to an alleviation of the nutrients limitation at 10 g/L manure. The b-glucosidase activity and filter paper activity of the mixed fungi culture were 0.64 IU/mL and 1.54 FPU/mL, respectively, corresponding to a ratio of 0.41, which is an ideal ratio for hydrolyzing manure cellulose. To test the effectiveness of the enzymes produced by mixed culture, a crude enzyme broth was used for hydrolyzing the manure cellulose. The glucose concentration produced was significantly ( p < 0.01) higher than the glucose obtained when using the commercial enzyme and the enzyme broth of the pure culture T. reesei. The results indicate that the mixed culture of T. reesei and A. phoenicis is an effective approach to produce a cellulolytic enzyme system for hydrolyzing manure cellulose. # 2005 Elsevier Ltd. All rights reserved. Keywords: Cellulase; b-Glucosidase; Animal manure; Trichoderma reesei; Aspergillus phoenicis; Mixed culture
1. Introduction The environmental friendly disposal and utilization of animal manure is a significant challenge to the livestock industry. During the past decade, the U.S. animal industry has undergone a substantial structural change featured with a rapid reduction in the number of animal operations with a corresponding increase in herd size on the remaining farms. These large, concentrated animal operations have created greater environmental concern because of the amount of animal manure produced at these facilities. Currently, manure is disposed predominately through direct land application. This management practice is coming under environmental and regulatory scrutiny as the limited amount
* Corresponding author. Tel.: +1 509 335 6239; fax: +1 509 335 2722. E-mail address: [email protected] (Z. Wen). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.03.044
of land available for manure disposal may result in surface and ground water contamination. Animal manure represents a large potential bioresource for producing bio-based chemicals, materials, and energy. Manure contains a variety of nutrients, including undigested crude fibre, protein, nitrogen, phosphorus, and minerals such as K, Ca, Mg, Fe, Cu, Mn, Zn, etc. [1,2]. Although there have been some reports about manure nutrients recovery (mainly nitrogen and phosphorus) by microorganisms [3,4], limited efforts have been focused on utilizing the lignocellulosic components of manure. One possible approach to manure lignocellulose utilization is to hydrolyze the materials into fermentable saccharides, which can then be converted into value-added products or bioenergy, as in the case of dairy manure anaerobic digestion (AD). A typical AD process, however, is limited by the microbes’ inability to utilize the manure fibre as the lignocellulosic component of the fibre is very resistant to
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biodegradation [5,6]. To convert lignocellulose effectively into reducing sugars, commercial cellulase enzymes could be used [7]. The process, however, is not considered economical because the cost of commercial cellulase enzymes remains very high [8]. If cellulase could be produced directly from manure and then be applied to AD to further degrade the manure cellulose, the cost of cellulase could be significantly reduced which at the same time enhancing the methane yield and potential profitability of AD of dairy manure. Cellulolytic enzymes can be produced by a number of bacteria and fungi that can use cellulose as a primary carbon source. The cellulolytic fungus Trichoderma reesei has been widely investigated for its cellulase production from various cellulosic materials such as wood [9], wastepaper [10], corncob [11], wheat bran [12], and wheat straw [13]. A previous study showed that dairy manure was an ideal substrate for T. reesei to produce cellulase. Unfortunately, although a high total cellulase titre (1.7 FPU/mL, filter paper activity) was obtained, the activity of b-glucosidase from this culture system was very low [14]. The efficiency of cellulose hydrolysis requires the synergistic action of a cellulase system containing endocellulase (E.C. 3.2.1.4), which cleaves internal glycosidic bonds; exoglucanase (E.C. 3.2.1.91), which cuts the cellulose chain from either the reducing or non-reducing end; b-glucosidase (E.C. 3.2.1.21), which hydrolyses cellubiose to produce glucose [15,16]. A high level of bglucosidase is then necessary to avoid the accumulation of cellubiose, which is a strong inhibitor of endocellulase and exoglucanase. To overcome the deficiency of b-glucosidase in a T. reesei-derived enzyme system, T. reesei can be cocultured with another fungus: Aspergillus phoenicis, which is a good b-glucosidase producer [17–20]. The objective of the work was to develop a mixed culture of T. reesei and A. phoenicis to produce cellulase with a high b-glucosidase level so as to improve its effectiveness for enzymic hydrolysis of manure cellulose.
2. Materials and methods 2.1. Manure collection and characterization Dairy manure was collected from the Dairy Center at Washington State University in Pullman, WA and stored for later use in a freezer. The diet of the cows consisted of (DM/ animal/day): 17 lb alfalfa hay, 16 lb alfalfa haylage, 7 lb cottonseed, 7 lb wheatmill run, and 20 lb grain. The grain portion included 32% corn, 19% wheat, 17% barley, 15% peas, 4.5% soybean meal, 4.5% corn gluten, and 8% other additives such as molasses, limestone, sodium bicarbonate, and vitamins. The manure was mixed with water (ratio 2:1, w/w) and homogenized by an Osterizer1 blender. The homogenized samples were analyzed for total carbon content, total nitrogen, ammonium, phosphorus, potassium, calcium, magnesium, sodium, sulphur, and trace elements
(iron, manganese, zinc, cobalt, copper). Also, dry matter (DM) and lignocellulose content, including neutral-detergent fibre (NDF), acid-detergent fibre (ADF), and aciddetergent lignin (ADL), were analyzed. 2.2. Microorganism, medium, and culture conditions T. reesei RUT-C30 (ATCC 56765) and A. phoenicis QM 329 (ATCC 52007) were maintained (at 4 8C) in potato dextrose agar slant and malt extract agar slant, respectively. To prepare the inoculum, the spores in the slant were suspended in 2 mL medium (106–107 spores/mL) and transferred into a 250 mL Erlenmeyer flask containing 50 mL medium. The subculture medium was Mandel salt solution [21] supplemented with 2 mL/L tween-80, 1 g/L peptone, and 10 g/L glucose. Fungal cells were sub-cultured in an orbital shaker (175 rpm) at 30 8C for 1–2 generations with the mycelium being used for inoculum. For cellulase production experiments, the medium composition was manure (at different concentrations) supplemented with 2 g/L KH2PO4, 2 mL/L tween-80 and 2.0 mg/L CoCl2. Homogenized manure (as treated for characterization) was dispensed into 250 mL flasks containing 50 mL medium. The medium was autoclaved at 121 8C for 15 min. For pure culture, either T. reesei or A. phoenicis (at 10% inoculum ratio, v/v) was inoculated into each flask. For mixed culture, 10% (v/v) T. reesei and 10% (v/v) A. phoenicis inoculum was inoculated into each flask simultaneously. 2.3. Enzymic hydrolysis of manure cellulose The commercial enzyme Celluclast-1.5 L (Sigma, St. Louis, MO), crude enzyme broth from the pure culture of T. reesei and the mixed culture of T. reesei and A. phoenicis were used for hydrolyzing the manure cellulose. Before enzymic hydrolysis, manure samples were pretreated with dilute sulphuric acid to remove the protective hemicelluloselignin layer. The pretreatment procedures were the same as described previously [7]. Hydrolysis was performed in 125 mL Erlenmeyer flasks containing 50 mL of enzyme solution and 5% (w/v) substrate (pretreated manure solid). pH and temperature were adjusted to 4.8 and 50 8C, respectively. For the three types of enzyme used, the enzyme loading was adjusted to the same filter paper activity (500 FPU/L). At this FPU level, b-glucosidase activities of the commercial enzyme (Celluclast-1.5 L) and crude enzyme broth of T. reesei were less than 25 IU/L, while the mixed culture broth was around 250 IU/L. The flasks were incubated in an orbital shaker (175 rpm) and glucose concentration in the hydrolysate was determined to evaluate the hydrolysis efficiency. 2.4. Analysis Manure dry matter (DM) was determined by drying the samples at 105 8C until the weight became constant. NDF,
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ADF, and ADL were determined using the gravimetric method [22]. The apparent values of ADF and ADL were corrected by subtracting acid-detergent nitrogen, which was also determined by the gravimetric method [22]. Total carbon and total nitrogen were measured using automatic combustion (LECO CNS-2000). Ammonium was determined by titrimetric method [23]. EPA method 3050/6010 was used to analysis other elements (potassium, phosphorus, calcium, magnesium, sodium, sulphur, iron, manganese, zinc, cobalt, copper). Here, the 3050 method refers to a nitric/hydrochloric acid digest and 6010 indicates metal analysis by inductively coupled argon plasma spectroscopy (ICP). The glucose concentrations in the enzymic hydrolysate were measured using an enzyme assay kit from Sigma (Product No. GAGO-20). The activities of total cellulase (filter paper activity, FPU) and b-glucosidase were determined according to standard IUPAC procedures and expressed as an international unit [24]. One unit of FPU was defined as the amount of enzyme which releases 1 mmol of glucose equivalents from Whatman No. 1 filter paper in 1 min. One unit of b-glucosidase activity was defined as the amount of enzyme converting 1 mmol of cellubiose to produce 2 mmol of glucose in 1 min. The glucose concentrations in the cellubiose hydrolysate were also measured using the enzyme assay kit. 2.5. Experimental design and data analysis A central composite design (CC0318) [25] was used to obtain the optimal conditions (temperature, pH, and manure concentration) for b-glucosidase production by the pure culture A. phoenicis. The design matrix was a 23 factorial design combined with 4 central points and 6 axial points where one variable was set at an extreme level (1.68) while the other variable was set at its central point
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(Table 1). The coding unit of variable i was done as follows: xi ¼
ðXi Xcp Þ ; DXi
i ¼ 1; 2; 3
where xi is the coded level, Xi the true value, Xcp the true value at central point, and DXi is the step change of variable i, respectively. The true values of the variables are also given in Table 1. The activity of b-glucosidase can be written as the function of the independent variables by second-polynomial, i.e., Y ¼ a0 þ
X
aii xi þ
X
aii x2i þ
X
a i j xi x j
(1)
where Y is the predicted response (b-glucosidase activity), a the coefficients of the equation, and xi and xj are the coded levels of variables i and j, respectively. After b-glucosidase activity for each run was obtained (Table 1), the response and variables (in coded unit) were correlated by the ‘‘Response Surface Analysis’’ function of the NCSS software (NCSS Statistical Software Inc., UT, 2000) to obtain the coefficients of Eq. (1). Only the estimates of coefficients with significant levels higher than 90% (i.e., p < 0.10) were included in the final model. The correlation coefficient (R2) and significance of the model were also tested by the NCSS software. The significance of the model was evaluated by F-test. The central point (run 15) was repeated four times to obtain the standard deviation using Microsoft Excel 2000. Although the standard deviation of the central point could be used to evaluate the experimental error of the design [25], consideration that the deviation may vary under different conditions required us to perform runs 1–14 in duplicates and the deviation of each individual run was estimated.
Table 1 Central composite design of temperature (T), medium pH, and manure concentration (MC) with b-glucosidase as responsea Run
Variables
Response
Coded unit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a
True value
T
pH
MC
T (8C)
pH
MC
1 1 1 1 +1 +1 +1 +1 1.68 +1.68 0 0 0 0 0
1 1 +1 +1 1 1 +1 +1 0 0 1.68 +1.68 0 0 0
1 +1 1 +1 1 +1 1 +1 0 0 0 0 1.68 + 1.68 0
22 22 22 22 32 32 32 32 18.6 35.4 27 27 27 27 27
3.8 3.8 5.8 5.8 3.8 3.8 5.8 5.8 4.8 4.8 3.1 6.5 4.8 4.8 4.8
14 26 14 26 14 26 14 26 20 20 20 20 9.9 30 20
b-Glucosidase (IU/mL)
Deviation (%)
0.123 0.080 0.365 0.305 0.515 0.345 0.428 0.560 0.255 0.328 0.075 0.531 0.589 0.603 0.667
2.83 4.84 1.79 3.68 1.25 3.38 0.96 2.68 0.75 3.21 4.01 3.98 2.19 1.52 4.50
Runs 1–14 were performed in two duplicate, while run 15 (central point) was performed in four duplicate.
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3. Results and discussion 3.1. Manure characterization and cellulase production by T. reesei The chemical characteristics of dairy manure are given in Table 2. Raw manure contained 14.6% dry matter, half of which is lignocellulosics (hemicellulose, cellulose, and lignin). In terms of elements, carbon was the most abundant, followed by nitrogen, calcium, and potassium. The manure also contained phosphorus, magnesium, sodium, sulphur, iron, manganese, zinc, cobalt, and copper, which together comprised less than 1% of the dry weight. Although a small percentage of the overall dry weight, these components are notably ideal nutrients for fungi culture. A cellulase production process by T. reesei on dairy manure was developed previously [14]. Findings showed that at a 10 g/L manure level, the amounts of nitrogen, calcium, magnesium, iron, manganese, and zinc contained in the manure are sufficient to support the fungi growth, while the amounts of potassium, phosphorus, and cobalt within the manure are insufficient. As a result, the medium composition was simplified as 10 g/L (dry basis) manure supplemented with 2 g/L KH2PO4, 2 mL/L tween-80, and 2 mg/L CoCl2, with the optimal temperature and pH being held at 25.5 8C and 5.76, respectively. The reaction times of the total cellulase (filter paper activity) and b-glucosidase Table 2 Major composition of freshly collected dairy manurea Dry matter, DM (%, w/w)
14.60 0.25
Lignocellulosics (% of DM) NDF ADFb ADLc Hemicellulose (NDF–ADF) Cellulose (ADF–ADL) Lignin (ADL)
49.10 1.30 37.83 1.01 11.24 1.02 11.27 0.90 26.59 0.28 11.24 1.02
Elements (% of DM) Total carbon Total nitrogen NH4-N Potassium Phosphorus Calcium Magnesium Sodium Sulphur Iron Manganese Zinc Cobalt Copper
activities produced by T. reesei are shown in Fig. 1. Total cellulase and b-glucosidase increased in parallel with incubation time, and reached the highest levels at day 6. The highest filter paper activity and b-glucosidase activity were 1.7 FPU/mL and 0.09 IU/mL, respectively, corresponding to a ratio of b-glucosidase to total cellulase of 0.053. It has been reported that an ideal ratio of b-glucosidase activity to filter paper activity is 0.12–1.5, depending on the source of enzyme and type of substrate [16], but specifically for the hydrolysis of manure cellulose, the optimal ratio of bglucosidase activity to filter paper activity has been found to be 0.38 [7]. This suggested that the b-glucosidase contained in T. reesei-derived cellulase was insufficient to hydrolyze the cellubiose to glucose. This deficiency in b-glucosidase is common to most strains of Trichoderma and several approaches have been attempted to overcome this deficiency. For example, a temperature and pH cycling strategy was applied to the culture T. reesei RUT-C30 to increase b-glucosidase production [26]. In another study, the mutant Trichoderma E12 was grown on microcrystalline cellulose with peanut cake being used as a nitrogen source to obtain a high C/N ratio. As a result, a well balanced ratio of b-glucosidase activity to filter paper activity was observed [27]. Trichoderma could also be co-cultured with the fungi Aspergillus, which is a good producer of b-glucosidase [17,19,20,28,29]. Such a mixed culture technique was therefore, employed in this work by growing T. reesei and A. phoenicis on dairy manure to achieve the necessary high level of b-glucosidase. 3.2. b-Glucosidase production using pure culture A. phoenicis-optimization of temperature, pH, and manure concentration Before performing the mixed culture, b-glucosidase production by pure culture A. phoenicis was investigated to
50.51 1.22 3.03 0.58 0.44 0.029 1.24 0.017 0.810 0.054 2.41 0.184 0.966 0.061 0.243 0.019 0.496 0.021 0.134 0.012 0.015 0.001 0.013 0.001 0.0002 0.000 0.0046 0.000
a
Data is expressed as mean S.D. of three replicates. Data of ADF, ADL listed are the true values corrected by subtracting acid-detergent nitrogen from the apparent values [22]. c Acid-detergent nitrogen (1.57% of DM) was formed via the nonenzymatic browning reaction when nitrogen-enriched manure is heated above 50 8C [22]. The value of acid-detergent nitrogen was determined by the gravimetric method [22]. b
Fig. 1. Time course of total cellulase (filter paper activity, FPU) and bglucosidase production by the fungi T. reesei. Symbols: (*) FPU and (&) b-glucosidase activity. Data are means of three replicates and error bars show standard deviation.
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obtain the optimal conditions for temperature, pH, and manure concentration. A central composite design was used for the optimization since this statistical method has proved efficient in the optimization of various fermentation processes [25]. As shown in Table 1, b-glucosidase activity of runs 1–14 were the means of two duplicates, while the central point was run in four duplicates (run 15) and its standard deviation was 0.03 IU/mL. The results showed a high reproducibility as the deviation of each run was less than 5% (Table 1). The response and variable (in coded unit) in Table 1 were correlated as a second-order polynomial model (Eq. (1)). Table 3 lists the estimates of coefficients and the associated t-values and significant levels. It was found that the effect of manure concentration on b-glucosidase production was insignificant ( p > 0.10) within the range investigated (10–30 g/L). This suggests that the nutrients contained in manure were sufficient for A. phoenicis growth even at the lowest level (10 g/L), while there was no substrate-inhibition even at the highest level (30 g/L). In this work, only the estimates with significant levels higher than 90% ( p < 0.10) were included in the final model (Eq. (1)). Thus, the reduced model describing b-glucosidase as a function of the significant variables (T and pH) were obtained as follows: b-glucosidase ðIU=mLÞ ¼ 0:669 þ 0:080½T þ 0:100½pH 0:141½T2 0:138½pH2
(2)
The correlation coefficient (R2) of Eq. (2) was 0.91. Ftests showed that the equation had a significance of 99% ( p < 0.01). All of these indicated that the equation was reliable in reflecting the relationship between T and pH with b-glucosidase production. Eq. (2) was then used to derive the optimal values of T and pH. Using the NCSS 2000 software, the exact optimal T and pH values (in coded unit) were obtained as 0.235 and 0.336, which corresponded to true values of 28.2 8C and pH 5.14, respectively. The predicted maximum b-glucosidase activity from Eq. (2) was 0.694 IU/mL, which was less than 5% in deviation from the experimental data (0.73 IU/mL). The validity of model was thus verified. Table 3 Estimates of coefficients of the variables in second-order polynomials (Eq. (1)) and the associated statistical tests Coefficient
Variable
Estimate
t-Value
p-Level
a0 a1 a2 a3 a11 a22 a33 a12 a13 a23
Constant [T] [pH] [MC] [T]2 [pH]2 [MC]2 [T] [pH] [T] [MC] [pH] [MC]
0.669 0.080 0.100 0.009 0.142 0.138 0.034 0.042 0.008 0.036
14.56 3.230 4.005 0.347 5.470 5.312 1.305 1.304 0.249 1.094
0.000 0.012 0.004 0.738 0.001 0.001 0.228 0.229 0.810 0.305
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3.3. Enzyme production by the mixed culture T. reesei and A. phoenicis The optimal culture conditions for the pure cultures of T. reesei and A. phoenicis are summarized in Table 4. For the two pure cultures, medium composition, temperature, and pH were very similar. The response surface of b-glucosidase activity (by A. phoenicis) and filter paper activity (by T. reesei) as a function of T and pH are shown in Fig. 2. The plots are hump shaped with a clear peak within the experimental range investigated. More importantly, it was found that both filter paper activity and b-glucosidase activity did not fall steeply when the values of T and pH changed slightly from their best values (Fig. 2A and B). This is a desired property because it means that the total cellulase production by T. reesei and b-glucosidase production by A. phoenicis will remain robust even with slight fluctuations in T and pH. Such a property provides a possibility that when T and pH are set at sub-optimal values, which are very close to the optimal values, the resulting total cellulase and b-glucosidase will not decrease significantly from their maximum level. In other words, total cellulase from T. reesei and b-glucosidase from A. phoenicis could be simultaneously maintained at high levels by appropriately controlling pH and T. Based on the above analysis, 27 8C and pH 5.5 were selected as operation parameters and the enzyme production by the mixed culture were experimental determined. 3.3.1. Time course of enzyme production by the mixed culture The time course profiles of filter paper activity and b-glucosidase activity of the mixed culture and the two pure cultures are presented in Fig. 3. The trends of filter paper activity and b-glucosidase activity were in a similar pattern and increased in parallel with incubation time. T. reesei and A. phoenicis demonstrated different abilities for producing total cellulase and b-glucosidase. T. reesei produced high level of total cellulase (Fig. 3A), with very low b-glucosidase (Fig. 3B). On the contrary, the total cellulase produced by A. phoenicis was very low (Fig. 3A), while b-glucosidase activity was much higher (Fig. 3B). The mixed culture resulted in a relatively high filter paper Table 4 Summary of optimal conditions for T. reesei and A. phoenicis Parameters
Manure concentration (g/L, dry basis) Medium composition Temperature (8C) Initial medium pH Enzyme activity Reference
Fungi culture T. reesei
A. phoenicis
10
10–30
Manure + 2.0 g/L KH2PO4 + 2 mL/L Tween-80 + 2 mg/L CoCl2 25.5 28.2 5.76 5.14 1.71 FPU/mL 0.69 IU/mL [14] This work
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Fig. 3. Time course of total cellulase (filter paper activity, FPU) (A) and bglucosidase (B) production by the pure culture of T. reesei and A. phoenicis, and the mixed culture of the two fungi. Data are means of three replicates and error bars show standard deviation.
Fig. 2. Three-dimension surface plot of b-glucosidase activity vs. temperature and medium pH of the pure culture A. phoenicis (A); filter paper activity (FPU) vs. temperature and medium pH of the pure culture T. reesei (B). Manure concentration was 10 g/L for both of the two pure cultures.
activity and b-glucosidase activity simultaneously, although its filter paper activity was slightly ( 15%) lower than the pure culture T. reesei (Fig. 3A), and its b-glucosidase activity was lower than ( 18%) the pure culture A. phoenicis (Fig. 3A). Compared, though, with the pure culture T. reesei, the cellulolytic potential of the mixed culture was markedly enhanced due to the increase in b-glucosidase. The mixed culture of the fungi Trichoderma and Aspergillus was studied on various substrates. Compared with the corresponding pure cultures, the enzyme levels produced by the mixed culture depended on the fungi specie
and substrate used. For example, when T. reesei LM-UC4 and A. phoenicis QM 329 were grown on bagasse, the filter paper activity and b-glucosidase activity from the mixed culture were much higher than those of the corresponding pure cultures [28]. However, b-glucosidase produced by A. niger was higher than the mixed cultures T. reesei LM-UC4 and A. niger when soymeal was added to the sugar cane bagasse [29]. Similar results were also observed in the mixed culture of T. reesei and A. terrus on bagasse [30]. When T. reesei RUT-C30 and A. phoenicis were grown on starch substrate, both filter paper activity and b-glucosidase activity were lower than those of each pure culture [17]. All of the above reports suggest that the cellulase and b-glucosidase production from mixed fungi culture are species specific and dependent on the different substrates being used. In this work, the reduced filter paper activity and b-glucosidase activity of the mixed culture may be due to the lack of synergism of the enzymes produced from the two fungi species. Another reason might be that when 10 g/L manure was used in the mixed culture, the nutrients contained in the manure were insufficient for the growth of the two fungi and as a result, a nutrients competition existed between the two fungi species. To address such an issue, the effect of manure concentration on the enzyme production by the mixed culture was investigated. 3.3.2. Effects of manure concentration The effect of manure concentration on cellulase and bglucosidase production by the mixed culture and pure cultures
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Fig. 5. Glucose concentration in the hydrolysate during enzymic hydrolysis of manure cellulose by using different enzyme sources. Data is presented as the mean of three replicates and the error bars show the standard deviation.
Fig. 4. Effects of manure concentration on total cellulase (filter paper activity, FPU) (A) and b-glucosidase (B) production by the pure culture of T. reesei and A. phoenicis, and the mixed culture of the two fungi. Data are means of three replicates and error bars show standard deviation.
are shown in Fig. 4. The pure culture of T. reesei and A. phoenicis showed almost the same level of filter paper activity (Fig. 4A) and b-glucosidase activity (Fig. 4B) at different manure concentrations. For mixed culture, filter paper activity increased from 1.38 to 1.54 FPU/mL (Fig. 4A), and b-glucosidase activity increased from 0.56 to 0.64 IU/mL (Fig. 4B) while manure concentration increased from 10 to 20 g/L. The increment for the two enzyme levels was significant ( p < 0.1), as tested by the software Statistical Analysis System (SAS 8.0). The results suggest that when a manure concentration was lower than 20 g/L the nutrients might not have been sufficient for the mixed culture, although such a level was sufficient for each of the pure cultures. However, within the range of 20–30 g/L manure concentration, enzyme production by the mixed culture leveled off, without a significant change ( p > 0.8, tested by the software SAS 8.0). This suggests that the nutrients were saturated at such manure levels. It was also noticed that the ratio of b-glucosidase activity to filter paper activity was 0.41 under 20 g/L manure level. This is an ideal ratio for effectively hydrolyzing manure cellulose (see Section 3.1) [7]. 3.4. Hydrolysis of manure cellulose by different enzymes sources To test the effectiveness of the enzymes produced from the mixed fungi culture, the hydrolysis of manure cellulose was performed by using enzyme broth from the mixed culture, the pure culture T. reesei, and commercial cellulase
(Celluclast-1.5 L). The concentration of glucose produced during hydrolysis was compared for different hydrolysis systems (Fig. 5). For each enzyme source used, the produced glucose followed a similar pattern, that is, glucose increased sharply for the first 12 h, and reached the highest levels at 96–132 h (Fig. 5). The produced glucose from mixed culture enzymes was significantly ( p < 0.01, tested by the software SAS 8.0) higher than those obtained from the other two enzyme sources. The high glucose level was due to the high b-glucosidase contained in the mixed culture broth, with the ratio of b-glucosidase activity to filter paper activity being 0.41. The result suggests that the mixed fungi culture developed in this work was an efficient method to produce cellulolytic enzymes in an effort towards manure cellulose utilization.
4. Conclusion The present work showed that the mixed culture of T. reesei and A. phoenicis could produce cellulase containing a high level of b-glucosidase from dairy manure. The medium composition for the mixed fungi culture was manure (20 g/L) supplemented with KH2PO4 (2 g/L), CoCl2 (2 mg/L) and tween-80 (2 mL/L). Under 27 8C and pH 5.5, b-glucosidase activity and filter paper activity could reach 0.64 IU/mL and 1.54 FPU/mL, respectively, with a corresponding ratio of 0.41. The hydrolysis efficiency (in terms of glucose produced) by the mixed enzymes was higher than those by commercial enzyme and enzyme from the single culture T. reesei. The work demonstrated a possible way to develop an efficient cellulolytic system for animal manure treatment.
Acknowledgments This work was supported by U.S. Department of Energy (Grant number: DE-FC3 6-01 GO 11048). The authors
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gratefully acknowledge Pacific Northwest National Laboratory (PNNL), Ms. Y. Liu, and Mr. C. Frear for their kind suggestions and technical assistance.
References [1] Fontenot JP, Smith LW, Sutton AL. Alternative utilization of animal waters. J Animal Sci 1983;(Suppl. 2):221. [2] Moller HB, Sommmer SG, Ahring BK. Separation efficiency and particle size distribution in relation to manure type and storage conditions. Biores Technol 2002;85:189–96. [3] Potter M, Oppermann-Sanio FB, Steinbuchel A. Cultivation of bacteria producing polyamino acids with liquid manure as carbon and nitrogen source. Appl Environ Microbiol 2001;67:617–22. [4] Wilkie AC, Mulbry WW. Recovery of dairy manure nutrients by benthic freshwater algae. Biores Technol 2002;84:81–91. [5] Colberg PJ. Anaerobic microbial degradation of cellulose, lignin, oligonols, and monoaromatic lignin derivatives. In: Zehnder AJ, editor. Biology of anaerobic microorganisms. New York: John Wiley & Sons; 1988. p. 333–72. [6] Nielsen HB, Mladenovska Z, Westermann P, Ahring BK. Comparison of two-stage thermophilic (68 8C/55 8C) anaerobic digestion with onestage thermophilic (55 8C) digestion of cattle manure. Biotechnol Bioeng 2004;86:291–300. [7] Wen Z, Liao W, Chen S. Hydrolysis of animal manure lignocellulosics for reducing sugar production. Biores Technol 2004;91:31–9. [8] von Sivers M, Zacchi G. A techno-economical comparison of three processes for the production of ethanol. Biores Technol 1995;51:43– 52. [9] Reczey K, Szengyel Z, Eklund R, Zacchi G. Cellulase production by T. reesei. Biores Technol 1996;57:25–30. [10] Ju LK, Afolabi OA. Wastepaper hydrolysate as soluble inducing substrate for cellulase production in continuous culture of Trichoderma reesei. Biotechnol Prog 1999;15:91–7. [11] Xia L, Shen X. High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue. Biores Technol 2004;91:259–62. [12] Smits JP, Rinzema A, Tramper J, van Sonsbeek HM, Knol W. Solidstate fermentation of wheat bran by Trichoderma reesei QM9414: substrate composition changes, C balance, enzyme production, growth and kinetics. Appl Microbol Biotechnol 1996;46:489–96. [13] Acebal C, Castillon MP, Estrada P, Mata I, Costa E, Aguado J, et al. Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw. Appl Microbiol Biotechnol 1986;24: 218–23.
[14] Wen Z, Liao W, Chen S. Production of cellulase by Trichoderma reesei from dairy manure. Biores Technol 2005;96:491–9. [15] Bhat M, Bhat S. Cellulose degrading enzyme and their potential industrial application. Biotechnol Adv 1997;15:583–620. [16] Duff SJB. Murray WD. Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review. Biores Technol 1996;55:1– 33. [17] Duff SJB, Cooper DG, Fuller OM. Effect of medium composition and growth conditions on production of cellulase and b-glucosidase by a mixed fungi fermentation. Enzyme Micro Technol 1987;9:47–52. [18] Dduenas R, Tengerdy RP, Gutierrez-Correa M. Cellulase production by mixed fungi in solid-substrate fermentation of bagasse. World J Microbiol Biotechnol 1995;11:333–7. [19] Gutierrez-Correa M, Tengerdy RP. Production of cellulose on sugar cane bagasse by fungal mixed culture solid substrate fermentation. Biotechnol Lett 1997;19:665–7. [20] Flachner B, Brumbauer A, Reczey K. Stabilization of beta-glucosidase in Aspergillus phoenicis QM 329 pellets. Enzyme Micro Technol 1999;24:362–7. [21] Mandel M, Weber J. The production of cellulase. Adv Chem Ser 1969;95:391–414. [22] Goering HK, van Soest PJ. Forage fibre analysis (apparatus, reagent, procedures and some application). In Agricultural Handbook No. 379. Agricultural Research Service-United State Department of Agriculture, Washington D.C., 1970. 1–20. [23] Eaton AD, Clesceri LS, Greengerg AE. Standard methods for the examination of water and wastewater (19 edition). American Public Health Association, Washington D.C., 1995. [24] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59:257–68. [25] Haaland PD. Experimental Design in Biotechnology. New York: Marcel Dekker Inc., 1989. [26] Tangnu SK, Blanch HW, Wilke CR. Enhanced production of cellulase, hemicellulase and b-xylosidase by Trichoderma reesei (RUT-C30). Biotechnol Bioeng 1981;23:1837–49. [27] Cochet N. Cellulase of Trichoderma reesei: influence of culture conditions upon enzymatic profile. Enzyme Micro Technol 1991;3: 104–9. [28] Duenaa R, Tengerdy RP, Gutierrez-Correa M. Cellulase production by mixed fungi in solid-substrate fermentation of bagasse. World J Micro Biotechnol 1995;11:333–7. [29] Gutierrez-Correa M, Portal L, Moreno P, Tengerdy RP. Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane bagasse. Biores Technol 1999;68:173–8. [30] Massadeh MI, Yusoff WMW. Omar O, Kader J. Synergism of cellulase enzymes in mixed culture solid substrate fermentation. Biotechnol Lett 2001;23:1771–4.
Production of cellulase/b-glucosidase by the mixed fungi culture Trichoderma reesei and Aspergillus phoenicis on dairy manure Zhiyou Wen *, Wei Liao, Shulin Chen Department of Biological Systems Engineering, Center of Multiphase Environmental Research, Washington State University, Pullman, WA 99164-6120, USA Received 31 May 2004; accepted 21 March 2005
Abstract Trichoderma reesei was co-cultured with Aspergillus phoenicis using dairy manure as a substrate to produce cellulase with a high level of b-glucosidase. For pure cultures of T. reesei and A. phoenicis, the optimal media compositions were the same (10 g/L manure supplemented with 2 g/L KH2PO4, 2 mL/L tween-80 and 2 mg/L CoCl2), while the optimal temperature and pH were similar (25.5 8C and pH 5.76 for T. reesei; 28.2 8C and pH 5.14 for A. phoenicis). The mixed culture was therefore completed at 27 8C and pH 5.5, which is close to the optimal values for both fungi. The mixed culture resulted in a relatively high level of total cellulase and b-glucosidase. It was also found that a high manure solid concentration (>20 g/L) resulted in higher enzyme activities, probably due to an alleviation of the nutrients limitation at 10 g/L manure. The b-glucosidase activity and filter paper activity of the mixed fungi culture were 0.64 IU/mL and 1.54 FPU/mL, respectively, corresponding to a ratio of 0.41, which is an ideal ratio for hydrolyzing manure cellulose. To test the effectiveness of the enzymes produced by mixed culture, a crude enzyme broth was used for hydrolyzing the manure cellulose. The glucose concentration produced was significantly ( p < 0.01) higher than the glucose obtained when using the commercial enzyme and the enzyme broth of the pure culture T. reesei. The results indicate that the mixed culture of T. reesei and A. phoenicis is an effective approach to produce a cellulolytic enzyme system for hydrolyzing manure cellulose. # 2005 Elsevier Ltd. All rights reserved. Keywords: Cellulase; b-Glucosidase; Animal manure; Trichoderma reesei; Aspergillus phoenicis; Mixed culture
1. Introduction The environmental friendly disposal and utilization of animal manure is a significant challenge to the livestock industry. During the past decade, the U.S. animal industry has undergone a substantial structural change featured with a rapid reduction in the number of animal operations with a corresponding increase in herd size on the remaining farms. These large, concentrated animal operations have created greater environmental concern because of the amount of animal manure produced at these facilities. Currently, manure is disposed predominately through direct land application. This management practice is coming under environmental and regulatory scrutiny as the limited amount
* Corresponding author. Tel.: +1 509 335 6239; fax: +1 509 335 2722. E-mail address: [email protected] (Z. Wen). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.03.044
of land available for manure disposal may result in surface and ground water contamination. Animal manure represents a large potential bioresource for producing bio-based chemicals, materials, and energy. Manure contains a variety of nutrients, including undigested crude fibre, protein, nitrogen, phosphorus, and minerals such as K, Ca, Mg, Fe, Cu, Mn, Zn, etc. [1,2]. Although there have been some reports about manure nutrients recovery (mainly nitrogen and phosphorus) by microorganisms [3,4], limited efforts have been focused on utilizing the lignocellulosic components of manure. One possible approach to manure lignocellulose utilization is to hydrolyze the materials into fermentable saccharides, which can then be converted into value-added products or bioenergy, as in the case of dairy manure anaerobic digestion (AD). A typical AD process, however, is limited by the microbes’ inability to utilize the manure fibre as the lignocellulosic component of the fibre is very resistant to
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biodegradation [5,6]. To convert lignocellulose effectively into reducing sugars, commercial cellulase enzymes could be used [7]. The process, however, is not considered economical because the cost of commercial cellulase enzymes remains very high [8]. If cellulase could be produced directly from manure and then be applied to AD to further degrade the manure cellulose, the cost of cellulase could be significantly reduced which at the same time enhancing the methane yield and potential profitability of AD of dairy manure. Cellulolytic enzymes can be produced by a number of bacteria and fungi that can use cellulose as a primary carbon source. The cellulolytic fungus Trichoderma reesei has been widely investigated for its cellulase production from various cellulosic materials such as wood [9], wastepaper [10], corncob [11], wheat bran [12], and wheat straw [13]. A previous study showed that dairy manure was an ideal substrate for T. reesei to produce cellulase. Unfortunately, although a high total cellulase titre (1.7 FPU/mL, filter paper activity) was obtained, the activity of b-glucosidase from this culture system was very low [14]. The efficiency of cellulose hydrolysis requires the synergistic action of a cellulase system containing endocellulase (E.C. 3.2.1.4), which cleaves internal glycosidic bonds; exoglucanase (E.C. 3.2.1.91), which cuts the cellulose chain from either the reducing or non-reducing end; b-glucosidase (E.C. 3.2.1.21), which hydrolyses cellubiose to produce glucose [15,16]. A high level of bglucosidase is then necessary to avoid the accumulation of cellubiose, which is a strong inhibitor of endocellulase and exoglucanase. To overcome the deficiency of b-glucosidase in a T. reesei-derived enzyme system, T. reesei can be cocultured with another fungus: Aspergillus phoenicis, which is a good b-glucosidase producer [17–20]. The objective of the work was to develop a mixed culture of T. reesei and A. phoenicis to produce cellulase with a high b-glucosidase level so as to improve its effectiveness for enzymic hydrolysis of manure cellulose.
2. Materials and methods 2.1. Manure collection and characterization Dairy manure was collected from the Dairy Center at Washington State University in Pullman, WA and stored for later use in a freezer. The diet of the cows consisted of (DM/ animal/day): 17 lb alfalfa hay, 16 lb alfalfa haylage, 7 lb cottonseed, 7 lb wheatmill run, and 20 lb grain. The grain portion included 32% corn, 19% wheat, 17% barley, 15% peas, 4.5% soybean meal, 4.5% corn gluten, and 8% other additives such as molasses, limestone, sodium bicarbonate, and vitamins. The manure was mixed with water (ratio 2:1, w/w) and homogenized by an Osterizer1 blender. The homogenized samples were analyzed for total carbon content, total nitrogen, ammonium, phosphorus, potassium, calcium, magnesium, sodium, sulphur, and trace elements
(iron, manganese, zinc, cobalt, copper). Also, dry matter (DM) and lignocellulose content, including neutral-detergent fibre (NDF), acid-detergent fibre (ADF), and aciddetergent lignin (ADL), were analyzed. 2.2. Microorganism, medium, and culture conditions T. reesei RUT-C30 (ATCC 56765) and A. phoenicis QM 329 (ATCC 52007) were maintained (at 4 8C) in potato dextrose agar slant and malt extract agar slant, respectively. To prepare the inoculum, the spores in the slant were suspended in 2 mL medium (106–107 spores/mL) and transferred into a 250 mL Erlenmeyer flask containing 50 mL medium. The subculture medium was Mandel salt solution [21] supplemented with 2 mL/L tween-80, 1 g/L peptone, and 10 g/L glucose. Fungal cells were sub-cultured in an orbital shaker (175 rpm) at 30 8C for 1–2 generations with the mycelium being used for inoculum. For cellulase production experiments, the medium composition was manure (at different concentrations) supplemented with 2 g/L KH2PO4, 2 mL/L tween-80 and 2.0 mg/L CoCl2. Homogenized manure (as treated for characterization) was dispensed into 250 mL flasks containing 50 mL medium. The medium was autoclaved at 121 8C for 15 min. For pure culture, either T. reesei or A. phoenicis (at 10% inoculum ratio, v/v) was inoculated into each flask. For mixed culture, 10% (v/v) T. reesei and 10% (v/v) A. phoenicis inoculum was inoculated into each flask simultaneously. 2.3. Enzymic hydrolysis of manure cellulose The commercial enzyme Celluclast-1.5 L (Sigma, St. Louis, MO), crude enzyme broth from the pure culture of T. reesei and the mixed culture of T. reesei and A. phoenicis were used for hydrolyzing the manure cellulose. Before enzymic hydrolysis, manure samples were pretreated with dilute sulphuric acid to remove the protective hemicelluloselignin layer. The pretreatment procedures were the same as described previously [7]. Hydrolysis was performed in 125 mL Erlenmeyer flasks containing 50 mL of enzyme solution and 5% (w/v) substrate (pretreated manure solid). pH and temperature were adjusted to 4.8 and 50 8C, respectively. For the three types of enzyme used, the enzyme loading was adjusted to the same filter paper activity (500 FPU/L). At this FPU level, b-glucosidase activities of the commercial enzyme (Celluclast-1.5 L) and crude enzyme broth of T. reesei were less than 25 IU/L, while the mixed culture broth was around 250 IU/L. The flasks were incubated in an orbital shaker (175 rpm) and glucose concentration in the hydrolysate was determined to evaluate the hydrolysis efficiency. 2.4. Analysis Manure dry matter (DM) was determined by drying the samples at 105 8C until the weight became constant. NDF,
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ADF, and ADL were determined using the gravimetric method [22]. The apparent values of ADF and ADL were corrected by subtracting acid-detergent nitrogen, which was also determined by the gravimetric method [22]. Total carbon and total nitrogen were measured using automatic combustion (LECO CNS-2000). Ammonium was determined by titrimetric method [23]. EPA method 3050/6010 was used to analysis other elements (potassium, phosphorus, calcium, magnesium, sodium, sulphur, iron, manganese, zinc, cobalt, copper). Here, the 3050 method refers to a nitric/hydrochloric acid digest and 6010 indicates metal analysis by inductively coupled argon plasma spectroscopy (ICP). The glucose concentrations in the enzymic hydrolysate were measured using an enzyme assay kit from Sigma (Product No. GAGO-20). The activities of total cellulase (filter paper activity, FPU) and b-glucosidase were determined according to standard IUPAC procedures and expressed as an international unit [24]. One unit of FPU was defined as the amount of enzyme which releases 1 mmol of glucose equivalents from Whatman No. 1 filter paper in 1 min. One unit of b-glucosidase activity was defined as the amount of enzyme converting 1 mmol of cellubiose to produce 2 mmol of glucose in 1 min. The glucose concentrations in the cellubiose hydrolysate were also measured using the enzyme assay kit. 2.5. Experimental design and data analysis A central composite design (CC0318) [25] was used to obtain the optimal conditions (temperature, pH, and manure concentration) for b-glucosidase production by the pure culture A. phoenicis. The design matrix was a 23 factorial design combined with 4 central points and 6 axial points where one variable was set at an extreme level (1.68) while the other variable was set at its central point
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(Table 1). The coding unit of variable i was done as follows: xi ¼
ðXi Xcp Þ ; DXi
i ¼ 1; 2; 3
where xi is the coded level, Xi the true value, Xcp the true value at central point, and DXi is the step change of variable i, respectively. The true values of the variables are also given in Table 1. The activity of b-glucosidase can be written as the function of the independent variables by second-polynomial, i.e., Y ¼ a0 þ
X
aii xi þ
X
aii x2i þ
X
a i j xi x j
(1)
where Y is the predicted response (b-glucosidase activity), a the coefficients of the equation, and xi and xj are the coded levels of variables i and j, respectively. After b-glucosidase activity for each run was obtained (Table 1), the response and variables (in coded unit) were correlated by the ‘‘Response Surface Analysis’’ function of the NCSS software (NCSS Statistical Software Inc., UT, 2000) to obtain the coefficients of Eq. (1). Only the estimates of coefficients with significant levels higher than 90% (i.e., p < 0.10) were included in the final model. The correlation coefficient (R2) and significance of the model were also tested by the NCSS software. The significance of the model was evaluated by F-test. The central point (run 15) was repeated four times to obtain the standard deviation using Microsoft Excel 2000. Although the standard deviation of the central point could be used to evaluate the experimental error of the design [25], consideration that the deviation may vary under different conditions required us to perform runs 1–14 in duplicates and the deviation of each individual run was estimated.
Table 1 Central composite design of temperature (T), medium pH, and manure concentration (MC) with b-glucosidase as responsea Run
Variables
Response
Coded unit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a
True value
T
pH
MC
T (8C)
pH
MC
1 1 1 1 +1 +1 +1 +1 1.68 +1.68 0 0 0 0 0
1 1 +1 +1 1 1 +1 +1 0 0 1.68 +1.68 0 0 0
1 +1 1 +1 1 +1 1 +1 0 0 0 0 1.68 + 1.68 0
22 22 22 22 32 32 32 32 18.6 35.4 27 27 27 27 27
3.8 3.8 5.8 5.8 3.8 3.8 5.8 5.8 4.8 4.8 3.1 6.5 4.8 4.8 4.8
14 26 14 26 14 26 14 26 20 20 20 20 9.9 30 20
b-Glucosidase (IU/mL)
Deviation (%)
0.123 0.080 0.365 0.305 0.515 0.345 0.428 0.560 0.255 0.328 0.075 0.531 0.589 0.603 0.667
2.83 4.84 1.79 3.68 1.25 3.38 0.96 2.68 0.75 3.21 4.01 3.98 2.19 1.52 4.50
Runs 1–14 were performed in two duplicate, while run 15 (central point) was performed in four duplicate.
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3. Results and discussion 3.1. Manure characterization and cellulase production by T. reesei The chemical characteristics of dairy manure are given in Table 2. Raw manure contained 14.6% dry matter, half of which is lignocellulosics (hemicellulose, cellulose, and lignin). In terms of elements, carbon was the most abundant, followed by nitrogen, calcium, and potassium. The manure also contained phosphorus, magnesium, sodium, sulphur, iron, manganese, zinc, cobalt, and copper, which together comprised less than 1% of the dry weight. Although a small percentage of the overall dry weight, these components are notably ideal nutrients for fungi culture. A cellulase production process by T. reesei on dairy manure was developed previously [14]. Findings showed that at a 10 g/L manure level, the amounts of nitrogen, calcium, magnesium, iron, manganese, and zinc contained in the manure are sufficient to support the fungi growth, while the amounts of potassium, phosphorus, and cobalt within the manure are insufficient. As a result, the medium composition was simplified as 10 g/L (dry basis) manure supplemented with 2 g/L KH2PO4, 2 mL/L tween-80, and 2 mg/L CoCl2, with the optimal temperature and pH being held at 25.5 8C and 5.76, respectively. The reaction times of the total cellulase (filter paper activity) and b-glucosidase Table 2 Major composition of freshly collected dairy manurea Dry matter, DM (%, w/w)
14.60 0.25
Lignocellulosics (% of DM) NDF ADFb ADLc Hemicellulose (NDF–ADF) Cellulose (ADF–ADL) Lignin (ADL)
49.10 1.30 37.83 1.01 11.24 1.02 11.27 0.90 26.59 0.28 11.24 1.02
Elements (% of DM) Total carbon Total nitrogen NH4-N Potassium Phosphorus Calcium Magnesium Sodium Sulphur Iron Manganese Zinc Cobalt Copper
activities produced by T. reesei are shown in Fig. 1. Total cellulase and b-glucosidase increased in parallel with incubation time, and reached the highest levels at day 6. The highest filter paper activity and b-glucosidase activity were 1.7 FPU/mL and 0.09 IU/mL, respectively, corresponding to a ratio of b-glucosidase to total cellulase of 0.053. It has been reported that an ideal ratio of b-glucosidase activity to filter paper activity is 0.12–1.5, depending on the source of enzyme and type of substrate [16], but specifically for the hydrolysis of manure cellulose, the optimal ratio of bglucosidase activity to filter paper activity has been found to be 0.38 [7]. This suggested that the b-glucosidase contained in T. reesei-derived cellulase was insufficient to hydrolyze the cellubiose to glucose. This deficiency in b-glucosidase is common to most strains of Trichoderma and several approaches have been attempted to overcome this deficiency. For example, a temperature and pH cycling strategy was applied to the culture T. reesei RUT-C30 to increase b-glucosidase production [26]. In another study, the mutant Trichoderma E12 was grown on microcrystalline cellulose with peanut cake being used as a nitrogen source to obtain a high C/N ratio. As a result, a well balanced ratio of b-glucosidase activity to filter paper activity was observed [27]. Trichoderma could also be co-cultured with the fungi Aspergillus, which is a good producer of b-glucosidase [17,19,20,28,29]. Such a mixed culture technique was therefore, employed in this work by growing T. reesei and A. phoenicis on dairy manure to achieve the necessary high level of b-glucosidase. 3.2. b-Glucosidase production using pure culture A. phoenicis-optimization of temperature, pH, and manure concentration Before performing the mixed culture, b-glucosidase production by pure culture A. phoenicis was investigated to
50.51 1.22 3.03 0.58 0.44 0.029 1.24 0.017 0.810 0.054 2.41 0.184 0.966 0.061 0.243 0.019 0.496 0.021 0.134 0.012 0.015 0.001 0.013 0.001 0.0002 0.000 0.0046 0.000
a
Data is expressed as mean S.D. of three replicates. Data of ADF, ADL listed are the true values corrected by subtracting acid-detergent nitrogen from the apparent values [22]. c Acid-detergent nitrogen (1.57% of DM) was formed via the nonenzymatic browning reaction when nitrogen-enriched manure is heated above 50 8C [22]. The value of acid-detergent nitrogen was determined by the gravimetric method [22]. b
Fig. 1. Time course of total cellulase (filter paper activity, FPU) and bglucosidase production by the fungi T. reesei. Symbols: (*) FPU and (&) b-glucosidase activity. Data are means of three replicates and error bars show standard deviation.
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obtain the optimal conditions for temperature, pH, and manure concentration. A central composite design was used for the optimization since this statistical method has proved efficient in the optimization of various fermentation processes [25]. As shown in Table 1, b-glucosidase activity of runs 1–14 were the means of two duplicates, while the central point was run in four duplicates (run 15) and its standard deviation was 0.03 IU/mL. The results showed a high reproducibility as the deviation of each run was less than 5% (Table 1). The response and variable (in coded unit) in Table 1 were correlated as a second-order polynomial model (Eq. (1)). Table 3 lists the estimates of coefficients and the associated t-values and significant levels. It was found that the effect of manure concentration on b-glucosidase production was insignificant ( p > 0.10) within the range investigated (10–30 g/L). This suggests that the nutrients contained in manure were sufficient for A. phoenicis growth even at the lowest level (10 g/L), while there was no substrate-inhibition even at the highest level (30 g/L). In this work, only the estimates with significant levels higher than 90% ( p < 0.10) were included in the final model (Eq. (1)). Thus, the reduced model describing b-glucosidase as a function of the significant variables (T and pH) were obtained as follows: b-glucosidase ðIU=mLÞ ¼ 0:669 þ 0:080½T þ 0:100½pH 0:141½T2 0:138½pH2
(2)
The correlation coefficient (R2) of Eq. (2) was 0.91. Ftests showed that the equation had a significance of 99% ( p < 0.01). All of these indicated that the equation was reliable in reflecting the relationship between T and pH with b-glucosidase production. Eq. (2) was then used to derive the optimal values of T and pH. Using the NCSS 2000 software, the exact optimal T and pH values (in coded unit) were obtained as 0.235 and 0.336, which corresponded to true values of 28.2 8C and pH 5.14, respectively. The predicted maximum b-glucosidase activity from Eq. (2) was 0.694 IU/mL, which was less than 5% in deviation from the experimental data (0.73 IU/mL). The validity of model was thus verified. Table 3 Estimates of coefficients of the variables in second-order polynomials (Eq. (1)) and the associated statistical tests Coefficient
Variable
Estimate
t-Value
p-Level
a0 a1 a2 a3 a11 a22 a33 a12 a13 a23
Constant [T] [pH] [MC] [T]2 [pH]2 [MC]2 [T] [pH] [T] [MC] [pH] [MC]
0.669 0.080 0.100 0.009 0.142 0.138 0.034 0.042 0.008 0.036
14.56 3.230 4.005 0.347 5.470 5.312 1.305 1.304 0.249 1.094
0.000 0.012 0.004 0.738 0.001 0.001 0.228 0.229 0.810 0.305
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3.3. Enzyme production by the mixed culture T. reesei and A. phoenicis The optimal culture conditions for the pure cultures of T. reesei and A. phoenicis are summarized in Table 4. For the two pure cultures, medium composition, temperature, and pH were very similar. The response surface of b-glucosidase activity (by A. phoenicis) and filter paper activity (by T. reesei) as a function of T and pH are shown in Fig. 2. The plots are hump shaped with a clear peak within the experimental range investigated. More importantly, it was found that both filter paper activity and b-glucosidase activity did not fall steeply when the values of T and pH changed slightly from their best values (Fig. 2A and B). This is a desired property because it means that the total cellulase production by T. reesei and b-glucosidase production by A. phoenicis will remain robust even with slight fluctuations in T and pH. Such a property provides a possibility that when T and pH are set at sub-optimal values, which are very close to the optimal values, the resulting total cellulase and b-glucosidase will not decrease significantly from their maximum level. In other words, total cellulase from T. reesei and b-glucosidase from A. phoenicis could be simultaneously maintained at high levels by appropriately controlling pH and T. Based on the above analysis, 27 8C and pH 5.5 were selected as operation parameters and the enzyme production by the mixed culture were experimental determined. 3.3.1. Time course of enzyme production by the mixed culture The time course profiles of filter paper activity and b-glucosidase activity of the mixed culture and the two pure cultures are presented in Fig. 3. The trends of filter paper activity and b-glucosidase activity were in a similar pattern and increased in parallel with incubation time. T. reesei and A. phoenicis demonstrated different abilities for producing total cellulase and b-glucosidase. T. reesei produced high level of total cellulase (Fig. 3A), with very low b-glucosidase (Fig. 3B). On the contrary, the total cellulase produced by A. phoenicis was very low (Fig. 3A), while b-glucosidase activity was much higher (Fig. 3B). The mixed culture resulted in a relatively high filter paper Table 4 Summary of optimal conditions for T. reesei and A. phoenicis Parameters
Manure concentration (g/L, dry basis) Medium composition Temperature (8C) Initial medium pH Enzyme activity Reference
Fungi culture T. reesei
A. phoenicis
10
10–30
Manure + 2.0 g/L KH2PO4 + 2 mL/L Tween-80 + 2 mg/L CoCl2 25.5 28.2 5.76 5.14 1.71 FPU/mL 0.69 IU/mL [14] This work
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Fig. 3. Time course of total cellulase (filter paper activity, FPU) (A) and bglucosidase (B) production by the pure culture of T. reesei and A. phoenicis, and the mixed culture of the two fungi. Data are means of three replicates and error bars show standard deviation.
Fig. 2. Three-dimension surface plot of b-glucosidase activity vs. temperature and medium pH of the pure culture A. phoenicis (A); filter paper activity (FPU) vs. temperature and medium pH of the pure culture T. reesei (B). Manure concentration was 10 g/L for both of the two pure cultures.
activity and b-glucosidase activity simultaneously, although its filter paper activity was slightly ( 15%) lower than the pure culture T. reesei (Fig. 3A), and its b-glucosidase activity was lower than ( 18%) the pure culture A. phoenicis (Fig. 3A). Compared, though, with the pure culture T. reesei, the cellulolytic potential of the mixed culture was markedly enhanced due to the increase in b-glucosidase. The mixed culture of the fungi Trichoderma and Aspergillus was studied on various substrates. Compared with the corresponding pure cultures, the enzyme levels produced by the mixed culture depended on the fungi specie
and substrate used. For example, when T. reesei LM-UC4 and A. phoenicis QM 329 were grown on bagasse, the filter paper activity and b-glucosidase activity from the mixed culture were much higher than those of the corresponding pure cultures [28]. However, b-glucosidase produced by A. niger was higher than the mixed cultures T. reesei LM-UC4 and A. niger when soymeal was added to the sugar cane bagasse [29]. Similar results were also observed in the mixed culture of T. reesei and A. terrus on bagasse [30]. When T. reesei RUT-C30 and A. phoenicis were grown on starch substrate, both filter paper activity and b-glucosidase activity were lower than those of each pure culture [17]. All of the above reports suggest that the cellulase and b-glucosidase production from mixed fungi culture are species specific and dependent on the different substrates being used. In this work, the reduced filter paper activity and b-glucosidase activity of the mixed culture may be due to the lack of synergism of the enzymes produced from the two fungi species. Another reason might be that when 10 g/L manure was used in the mixed culture, the nutrients contained in the manure were insufficient for the growth of the two fungi and as a result, a nutrients competition existed between the two fungi species. To address such an issue, the effect of manure concentration on the enzyme production by the mixed culture was investigated. 3.3.2. Effects of manure concentration The effect of manure concentration on cellulase and bglucosidase production by the mixed culture and pure cultures
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Fig. 5. Glucose concentration in the hydrolysate during enzymic hydrolysis of manure cellulose by using different enzyme sources. Data is presented as the mean of three replicates and the error bars show the standard deviation.
Fig. 4. Effects of manure concentration on total cellulase (filter paper activity, FPU) (A) and b-glucosidase (B) production by the pure culture of T. reesei and A. phoenicis, and the mixed culture of the two fungi. Data are means of three replicates and error bars show standard deviation.
are shown in Fig. 4. The pure culture of T. reesei and A. phoenicis showed almost the same level of filter paper activity (Fig. 4A) and b-glucosidase activity (Fig. 4B) at different manure concentrations. For mixed culture, filter paper activity increased from 1.38 to 1.54 FPU/mL (Fig. 4A), and b-glucosidase activity increased from 0.56 to 0.64 IU/mL (Fig. 4B) while manure concentration increased from 10 to 20 g/L. The increment for the two enzyme levels was significant ( p < 0.1), as tested by the software Statistical Analysis System (SAS 8.0). The results suggest that when a manure concentration was lower than 20 g/L the nutrients might not have been sufficient for the mixed culture, although such a level was sufficient for each of the pure cultures. However, within the range of 20–30 g/L manure concentration, enzyme production by the mixed culture leveled off, without a significant change ( p > 0.8, tested by the software SAS 8.0). This suggests that the nutrients were saturated at such manure levels. It was also noticed that the ratio of b-glucosidase activity to filter paper activity was 0.41 under 20 g/L manure level. This is an ideal ratio for effectively hydrolyzing manure cellulose (see Section 3.1) [7]. 3.4. Hydrolysis of manure cellulose by different enzymes sources To test the effectiveness of the enzymes produced from the mixed fungi culture, the hydrolysis of manure cellulose was performed by using enzyme broth from the mixed culture, the pure culture T. reesei, and commercial cellulase
(Celluclast-1.5 L). The concentration of glucose produced during hydrolysis was compared for different hydrolysis systems (Fig. 5). For each enzyme source used, the produced glucose followed a similar pattern, that is, glucose increased sharply for the first 12 h, and reached the highest levels at 96–132 h (Fig. 5). The produced glucose from mixed culture enzymes was significantly ( p < 0.01, tested by the software SAS 8.0) higher than those obtained from the other two enzyme sources. The high glucose level was due to the high b-glucosidase contained in the mixed culture broth, with the ratio of b-glucosidase activity to filter paper activity being 0.41. The result suggests that the mixed fungi culture developed in this work was an efficient method to produce cellulolytic enzymes in an effort towards manure cellulose utilization.
4. Conclusion The present work showed that the mixed culture of T. reesei and A. phoenicis could produce cellulase containing a high level of b-glucosidase from dairy manure. The medium composition for the mixed fungi culture was manure (20 g/L) supplemented with KH2PO4 (2 g/L), CoCl2 (2 mg/L) and tween-80 (2 mL/L). Under 27 8C and pH 5.5, b-glucosidase activity and filter paper activity could reach 0.64 IU/mL and 1.54 FPU/mL, respectively, with a corresponding ratio of 0.41. The hydrolysis efficiency (in terms of glucose produced) by the mixed enzymes was higher than those by commercial enzyme and enzyme from the single culture T. reesei. The work demonstrated a possible way to develop an efficient cellulolytic system for animal manure treatment.
Acknowledgments This work was supported by U.S. Department of Energy (Grant number: DE-FC3 6-01 GO 11048). The authors
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gratefully acknowledge Pacific Northwest National Laboratory (PNNL), Ms. Y. Liu, and Mr. C. Frear for their kind suggestions and technical assistance.
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