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Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment
Bioresource Technology 102 (2011) 8046–8051
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment Jian-Jun Qiao a,⇑, Yan-Fei Zhang a, Li-Fan Sun a, Wei-Wei Liu a, Hong-Ji Zhu a, Zhijun Zhang b,⇑ a Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin Key Laboratory of Biological and Pharmaceutical Engineering, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China b Tianjin Forestry Fruit Tree Research Institute, Tianjin 300112, PR China
a r t i c l e
i n f o
Article history: Received 3 January 2011 Received in revised form 1 May 2011 Accepted 17 May 2011 Available online 27 May 2011 Keywords: Spent mushroom substrate Dilute sulfuric acid pretreatment Lignocellulosic material Enzymatic hydrolysis Reducing sugar yield
a b s t r a c t Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (34) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120 °C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20 PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40 °C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9 1011 CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Spent mushroom substrate (SMS) is a byproduct of the mushroom production industry and an estimated 2 million tons are produced in China every year. Since SMS is not suitable for animal feed, other uses that would convert SMS into value-added products would be beneficial. As a lignocellulosic material, SMS could potentially be a source of reducing sugars for production of biofuels and other biomaterials (White et al., 2008; Kaparaju et al., 2009). Enzymatic conversion can potentially achieve nearly theoretical yields of sugars (Lee et al., 2009); however, pretreatment to improve access by enzymes to the substrate is required. Such pretreatment can be carried out using dilute sulfuric acid (Beldman et al., 1987; Sun and Cheng, 2002; Mosier et al., 2005; Bower et al., 2008; Akpinar et al., 2009). For example, Karimi et al. (2006) utilized two-stage dilute acid hydrolysis to convert rice straw into sugars. In the first stage, xylan was depolymerized to xylose with a maximum yield of 80.8% and glucan was depolymerized to glucose at a maximum of 25.8% at hydrolysis pressure of 15 bar, 10 min retention time and 0.5% acid concentration. After pretreatment, commercial enzymes ⇑ Corresponding authors. Tel.: +86 22 27405836; fax: +86 22 87402107 (J.-J. Qiao); tel.: +86 22 27798677; fax: +86 22 27798677 (Z. Zhang). E-mail addresses: [email protected] (J.-J. Qiao), [email protected] (Z. Zhang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.05.058
were used to increase the yield of reducing sugars. Cristobal et al. (2008) used cellulase and endo-glucanase to saccharify dilute acid pretreated olive tree biomass, and Yan et al. (2009) designed a cycle spray flow-through reactor to pretreat corn stover in dilute sulfuric acid which enhanced xylose sugar yields and cellulose digestibility. In the current study, dilute sulfuric acid pretreatment followed by enzymatic digestion was conducted to produce reducing sugars from SMS. The conditions for the dilute acid treatment were optimized, the effect of cellulase and xylanase was determined and the utility of SMS hydrolysates for cultivation of Lactococcus lactis was assessed.
2. Methods 2.1. Materials The SMS, the substrate after three harvesting cycles of Pleurotus ostreastus, was obtained from Tianjin, PR China. The mushroom substrate used for planting P. ostreastus is composed of wheat straw, field hay, corn cobs, cotton seed hulls. After drying SMS was ground to a particle size of 800 lm and then stored in air-tight container at 4 °C for further use. All standard chemicals, including cellulose, glucose, arabinose and xylose were purchased from Sigma Chemical Company (Shanghai, PR China).
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the first pretreatment was saved for pretreatment the new SMS at the same conditions. The reuse process was conducted six times.
2.2. Methods 2.2.1. Analysis of SMS composition The lignin content of the raw spent mushroom substrate and acid hydrolysis residues were determined according to National Renewable Energy Laboratory Procedures LAP 001, 003, and 004 (Ehrman, 1994, 1996; Templeton and Ehrman, 1994). Cellulose and hemicellulose content was determined by high performance liquid chromatography (HPLC) analysis using a Bio-Rad Aminex HPX-87P column and a refractive index detector (SHODEX). Sugars were analyzed at 65 °C using 0.00004% H2SO4 as the mobile phase (0.6 ml/min). The total amount of reducing sugars was determined using 3,5-dinitrosalicylic acid reagent (DNS method) (Miller, 1959). Before determination, the samples of raw SMS and acid treated SMS were dried to constant weight at 105 °C. The acid pretreatment liquid was neutralized with 5 mol/L ammonia and filtered through a 0.2 lm nylon filter. All analyses were performed in duplicate. 2.2.2. Dilute sulfuric acid hydrolysis The dilute sulfuric pretreatment was conducted in a 1-L Parr reactor equipped with an electric heating and magnetic stirrer system (A1120HC9) (Parr 51111, Parr Instruments) and a modular controller (Parr 4848 controller). Forty grams of dried SMS was mixed with de-ionized water to make the final solid to liquid ratio of 1:8, 1:16, 1:24 and 98% sulfuric acid was added to the total volume of the dilution water to make a final concentration of 0.5%, 1%, 2%, 4%, and 6%. The mixture was introduced into the reactor and agitated at 100 rpm at the desired temperatures. After conclusion of the dilute acid treatment, the samples were allowed to cool to room temperature and filtered through 0.2 lm nylon filter. The solids were washed with distilled water until a pH of 7 was reached, dried at 105 °C for 6 h, and subjected to composition analysis and enzymatic hydrolysis. The four factors mainly affecting dilute sulfuric acid pretreatment of SMS were temperature, the final concentration of dilute sulfuric acid, reaction time, and the final solid to liquid ratio (Noureddini and Byun, 2010). Pretreatment reactions were carried out at temperatures of 25, 50, 60, 70, 80, 90, 100, 110, and 120 °C, reaction times of 0.5, 1.0, 1.5, 2.0, and 2.5 h, and sulfuric acid concentrations of 0.5%, 1%, 2%, 4%, and 6% (v/v of 98% sulfuric acid). Based on the single factor experiments, the orthogonal experimental design was used to optimize the pretreatment process. The orthogonal table 34 was designed as shown in Table 1. The re-use of the dilute sulfuric acid was also conducted at 120 °C for 2 h with the liquid to solid ratio of 1:16. The liquid from
2.2.3. Enzymatic hydrolysis Cellulase from Trichoderma reesei (NS50013) and xylanase (NS50014) employed were purchased from Novozymes A/S (Beijing, PR China). Cellulase activity was determined as described by Ghose (1987) and expressed as FPU (filter paper unit) defined as lmol of glucose produced per minute with filter paper as the only substrate. The cellulase had an activity of 70 FPU/g. Xylanase activity was expressed as XU (xylanase unit) and estimated as reported by Bastawde et al. (1994). The xylanase employed had an activity of 600 XU/g. Cellulase loadings of 5, 10, 15, 20, 25, 30, 35, and 40 FPU/g were investigated at 40 °C for 72 h. The effect of the addition of xylanase was determined with material hydrolyzed with 20 FPU of xylanase per gram, and 50, 100, 150, and 200 units of xylanase were used. Three grams of acid pretreated samples (dried at 105 °C for 6 h) were mixed with the enzyme solutions, 60 ml of 50 mM acetate buffer (pH 4.8) was added, and the samples were incubated at 40 °C in a shaker bath at 200 rpm for 84 h. After enzyme hydrolysis, the samples were filtered through a filter paper and sugar analysis was performed on the supernatant. All the experiments were conducted in duplicate. The influence of rotation speed on cellulase and xylanase hydrolysis was determined with 20 FPU cellulase and 150 XU xylanase per gram of acid-pretreated SMS with the rotation speed of 100 and 200 rpm, and the other conditions of this process was the same as the above paragraph.
2.2.4. Culture of L. lactis subsp. lactis W28 with the SMS hydrolysates The SMS hydrolysates were subjected to vacuum filtration and the pH of the filtrate was adjusted to pH 7.2 with ammonium hydroxide sodium hydroxide, or calcium hydroxide. The fermentations were conducted in 250-ml Erlenmeyer flasks containing 100 ml hydrolysates supplemented with KH2PO3, 2 g; NaCl, 0.15 g; MgSO47H2O; 0.015 g; peptone, 1.0 g. The fermentation medium was inoculated with 5% of overnight cultures and incubated at 30 °C for 24 h without agitation. The cell density was measured using the plate count method. All the experiments were conducted in duplicate.
3. Results and discussion 3.1. Composition of SMS
Table 1 L9 (34) orthogonal experiment of H2SO4 pretreatment. Test no.
a
1 2 3 4 5 6 7 8 9
YRSAHe (g/kg SMS)
Parameters d
c
b
T
C
R
t
T1 T1 T1 T2 T2 T2 T3 T3 T3
C1 C2 C3 C2 C3 C1 C3 C1 C2
R1 R2 R3 R3 R1 R2 R2 R3 R1
t3 t1 t2 t3 t1 t2 t3 t1 t2
50.32 70.46 96.10 158.33 124.42 83.00 267.57 173.32 238.84
All data are averages of duplicates. a T: pretreatment temperature, T1, T2, T3 denote 90, 100, 120 °C, respectively. b t: pretreatment time, t1, t2, t3 denote 60, 90, 120 min, respectively. c R: liquid to solid rate, R1, R2, R3 denote 8:1, 16:1, 24:1, respectively. d C: concentration of sulfuric acid, C1, C2, C3 denote 2%, 4%, 6% (w/w), respectively. e YRSAH: yield of reducing sugar by acid hydrolysis (based on the initial SMS).
The composition of SMS is shown in Table 2. Cellulose, hemicellulose accounted for 57.1% of the total weight. The lignin content was 20.2%. The composition of SMS is in good agreement with the previous research (Kwak et al., 2008).
Table 2 Composition of spent mushroom substrate.a Item
SMSb (%)
SMSc (%)
SMSd (%)
Lignin Hemicelluloses Cellulose
20.21 18.44 38.7
18.70 17.00 40.5
21.12 7.34 20.32
All data are averages of duplicates. a component percentages are based on dry-weight for all data and are averages of duplicates. b raw spent mushroom substrate in this paper. c raw spent mushroom substrate from a reference (Kwak et al., 2008). d spent mushroom substrate treated under the optimal condition.
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3.2. Effect of dilute acid sulfuric concentration, temperature and reaction time on sugar yield
Table 3 Analysis of L9 (34) experiment results. YRSAH
At a hydrolysis time of 0.5 h, acid concentration of 4%, and solid to liquid ratio of 1:8, the total amount of reducing sugars increased slowly at low temperatures, but increased significantly after the temperature reached 100 °C. At 120 °C, the maximum sugar yield was 156.88 g/kg SMS (Fig. 1a). The reducing sugar yield increased with increasing acid concentrations. When the acid concentration was increased from 1% to 4%, the reducing sugar yield increased by approximately 15%; however, it remained about the same as the acid concentration increased from 4% to 6%. As to the oligo-sugars, the glucose release increased with increasing acid concentrations. The xylose release increased as the acid concentration increased from 0.5% to 2% and reached a plateau at 2%, while arabinose release showed little change in response to increased acid concentrations (Fig. 1b). After 2 h of treatment with 4% sulfuric acid at 120 °C and a 1:8 solid to liquid ratio, the reducing sugar yield reached 260 g/kg SMS (Fig. 1c). The release of glucose and xylose increased with time and reached a plateau after 1.5 h, whereas the release of arabinose showed little change. Possibly, most of the available arabinose was released quickly early during acid hydrolysis as observed by Lee et al. (2009).
k1 k2 k3 R1 Optimal level
T
C
R
t
72.29a 121.92 226.58 154.29b T3
102.21 155.88 162.70 60.49 C3
137.86 140.34 142.58 4.72 R2
122.73 139.31 158.74 36.01 t3
YRSAH, yield of reducing sugar by acid hydrolysis. Orthogonal analysis was used to study the optimal level of each parameter for YRSAH, as shown in Table 1. According to the R values, the affecting YRSAH showed the following order, T > C > t > R. Table 2 indicated that the optimum condition for YRSAH was T3C3R2t3. All data are averages of duplicates. K Ti = the sum of YRSAH at Ti. T a ki ¼ K Ti /3. T T b R1 = max{ki } min{ki }.
The experimental conditions and results of the L9 (34) orthogonal experimental design are shown in Tables 1 and 3. According to the R values, the significance of each factor affecting the reducing sugar yield was in the order of T > C > t > R. Although the optimum condition for total reducing sugar yield of 267.57 g/kg SMS, obtained in test 7 (solid to liquid ratio of 1:16, 120 °C, 6% sulfuric acid for 120 min), was achieved with the
Fig. 1. Reducing sugar yields obtained under different conditions. (a) Yield at different temperatures and a sulfuric acid concentration 4%, solid to liquid ratio 1:8 and reaction time of 0.5 h; (b) yield after different reaction times with a sulfuric acid concentration of 4%, solid to liquid ratio of 1:8, and reaction temperature of 120 °C; (c) yield at different sulfuric acid concentrations with a solid to liquid ratio of 1:8, reaction temperature of 120 °C and reaction time of 0.5 h. All pretreatment processes were conducted in duplicate and the average data are presented.
J.-J. Qiao et al. / Bioresource Technology 102 (2011) 8046–8051
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Fig. 2. Effect of sulfuric acid concentration on reducing sugar yield (solid to liquid ratio of 1:16, 120 °C, 120 min). The average data of duplicate experiments are presented.
Fig. 3. Yield of reducing sugars after repeated use of dilute sulfuric acid. The average data of duplicate experiments are presented.
Fig. 4. Reducing sugar yield of enzymatic treatment of SMS pretreated with dilute sulfuric acid (a) effect of cellulase loading on the reducing sugar yield; (b) effect of cellulase and xylanase treatment on acid pretreated SMS; (c) effect of xylanase loading on cellulase hydrolysis. The average data of duplicate experiments are presented.
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Fig. 5. Effect of two rotation speeds on cellulase and xylanase hydrolysis. The average data of duplicate experiments are presented.
highest acid concentration tested, an acid concentration of 4% might suffice as the yield was only 3.5 g/kg SMS lower than that observed with 6% (Fig. 2). The data in Fig. 3 suggest that some reuse of the acidic solution is possible, but further studies need to be done to determine optimal conditions for reuse and to determine if unwanted by-products accumulate. 3.3. Enzymatic digestion of dilute acid pretreated SMS Treatment of SMS pretreated with 4% sulfuric acid at a solid to liquid ratio of 1:16 at 120 °C for 120 min with cellulase loading 20 FPU/g pretreated SMS resulted in 47 g/kg pretreated SMS, and enzyme loading higher than 20 FPU/g pretreated SMS showed no more effect on the reducing sugar yield (Fig. 4a). Treatment with 100 XU/g pretreated SMS of xylanase yielded 65.96 g/kg pretreated SMS when cellulase was added at 20 FPU/g pretreated SMS (Fig. 4b), and xylanase was added at 50–200 XU/g pretreated SMS, the content of reducing sugar increased from 61.97 to 80.85 g/kg SMS (Fig. 4c). Fig. 5 illustrates that increased rotation speeds improved reducing sugar yields. 3.4. Sugar analysis in pretreatment liquid HPLC analysis indicated that the reducing sugars obtained by the acid hydrolysis pretreatment process mainly consisted of xylose, glucose and arabinose. No furfural or hydroxymethylfurfual was detected. Since the total amount of reducing sugars measured with the DNS method was higher than the sum of glucose, xylose and arabinose measured with the HPLC, some oligo-sugars and/ or some non-sugar reducing compounds must have been present in the hydrolysates. 3.5. Culture of L. lactis subsp. lactis W28 with the SMS hydrolysates The reducing sugars in the SMS hydrolysates can serve as carbon source for microbes once the hydrolysates have been neutralized. Hydrolysates neutralized with calcium hydroxide provided for the highest cell density (Fig. 6), presumably because of precipitation as calcium sulfate which resulted in a lower ionic strength of the medium as compared to that obtained with sodium or ammonium hydroxide. Hydrolysates neutralized with ammonium hydroxide or sodium hydroxide had to be diluted to lower the ionic
Fig. 6. Cell density of L. lactis subsp. lactis W28 grown in undiluted and diluted SMS hydrolysates. The average data of duplicate experiments are presented.
concentration, and the highest cell densities were achieved at a twofold dilution of the hydrolysates. 4. Conclusion SMS treated with 4% sulfuric acid at 120 °C for 120 min yielded 267.57 g of reducing sugars per kg of SMS, and when this treatment was followed by hydrolysis with 20 PFU of cellulase and 200 XU xylanase at 40 °C, and additional reducing sugar release of 79.85 g/kg of pretreated SMS was obtained. The hydrolysates supported growth of L. lactis subsp. lactis W28, and the highest cell density was achieved when the hydrolysates were neutralized with calcium hydroxide. Therefore, SMS hydrolysates could be useful for the cultivation microorganisms and the production of highvalue compounds such as nisin and lactic acid. Acknowledgements This work was funded by Tianjin applied basic research projects and cutting-edge technology (Tianjin Natural Science Foundation, No. 09JCZDJC26500). We were thankful to the State Key Laboratory of Chemical Engineering of Tianjin University for making equipment and facilities vital to this research available. References Akpinar, O., Erdogan, K., Bostanci, S., 2009. Production of xylooligosaccharides by controlled acid hydrolysis of lignocellulosic materials. Carbohydr. Res. 344, 660–666. Bastawde, K.B., Puntanmbekar, U.S., Gokhale, D.V., 1994. Optimization of cellulasefree xylanase production by a novel yeast strain. J. Ind. Microbiol. 13, 220–224. Beldman, G., Hennekam, J., Voragen, A.G., 1987. Enzymatic hydrolysis of beer brewers’ spent grain and the influence of pretreatments. Biotechnol. Bioeng. 30, 668–671. Bower, S., Wickramasinghe, R., Nagle, N.J., Schell, D.J., 2008. Modeling sucrose hydrolysis in dilute sulfuric acid solutions at pretreatment conditions for lignocellulosic biomass. Bioresour. Technol. 99, 7354–7362. Cristobal, C., Encarnacion, R., Jose, M.O., Felicia, S., Eulogio, C., 2008. Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification. Bioresour. Technol. 99, 1869–1876. Ehrman, T., 1994. Method for determination of total solids in biomass. In: Laboratory Analytical Procedures No. 001. National Renewable Energy Laboratory, Golden, CO. Ehrman, T., 1996. Method for determination of acid-soluble lignin in biomass. In: Laboratory Analytical Procedures No. 004. National Renewable Energy Laboratory, Golden, CO. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. Commun. 59, 257–268.
J.-J. Qiao et al. / Bioresource Technology 102 (2011) 8046–8051 Kaparaju, P., Serrano, M., Thomsen, A.B., Kongjan, P., Angelidaki, I., 2009. Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept. Bioresour. Technol. 100, 2562–2568. Karimi, K., Kheradmandinia, S., Taherzadeh, M.J., 2006. Conversion of rice straw to sugars by dilute-acid hydrolysis. Biom. Bioenergy 30, 247–253. Kwak, W.S., Jung, S.H., Kim, Y.I., 2008. Broiler litter supplementation improves storage and feed-nutritional vale of saw dust-based spent mushroom substrate. Bioresour. Technol. 99, 2947–2955. Lee, J.M., Shi, J., Venditti, A., Jameel, R.H., 2009. Autohydrolysis pretreatment of Coastal Bermuda grass for increased enzyme hydrolysis. Bioresour. Technol. 100, 6434–6441. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. Commun. 31, 426–428. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686. Noureddini, H., Byun, J., 2010. Dilute-acid pretreatment of distillers’ grains and corn fiber. Bioresour. Technol. 101, 1060–1067.
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NREL (National Renewable Energy Laboratories), 2000. Technical Report: Determining the Cost of Producing Ethanol from Corn Starch and Lignocellulosic Feedstocks, NREL/TP-580-28893. NREL (National Renewable Energy Laboratories), 2006. Biomass Feedstock Composition and Property Database. Available through:. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83, 1–11. Templeton, D., Ehrman, T., 1994. Determination of acid-insoluble lignin in biomass. In: Laboratory Analytical Procedures No. 003. National Renewable Energy Laboratory, Golden, CO. White, J.S., Yohannan, B.K., Walker, G.M., 2008. Bioconversion of brewer’s spent grains to bioethanol. FEMS Yeast Res. 8, 1175–1184. Yan, L., Zhang, H., Chen, J., Lin, Z., Jin, Q., Jia, H., Huang, H., 2009. Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery. Bioresour. Technol. 100, 1803–1808.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment Jian-Jun Qiao a,⇑, Yan-Fei Zhang a, Li-Fan Sun a, Wei-Wei Liu a, Hong-Ji Zhu a, Zhijun Zhang b,⇑ a Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin Key Laboratory of Biological and Pharmaceutical Engineering, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China b Tianjin Forestry Fruit Tree Research Institute, Tianjin 300112, PR China
a r t i c l e
i n f o
Article history: Received 3 January 2011 Received in revised form 1 May 2011 Accepted 17 May 2011 Available online 27 May 2011 Keywords: Spent mushroom substrate Dilute sulfuric acid pretreatment Lignocellulosic material Enzymatic hydrolysis Reducing sugar yield
a b s t r a c t Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (34) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120 °C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20 PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40 °C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9 1011 CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Spent mushroom substrate (SMS) is a byproduct of the mushroom production industry and an estimated 2 million tons are produced in China every year. Since SMS is not suitable for animal feed, other uses that would convert SMS into value-added products would be beneficial. As a lignocellulosic material, SMS could potentially be a source of reducing sugars for production of biofuels and other biomaterials (White et al., 2008; Kaparaju et al., 2009). Enzymatic conversion can potentially achieve nearly theoretical yields of sugars (Lee et al., 2009); however, pretreatment to improve access by enzymes to the substrate is required. Such pretreatment can be carried out using dilute sulfuric acid (Beldman et al., 1987; Sun and Cheng, 2002; Mosier et al., 2005; Bower et al., 2008; Akpinar et al., 2009). For example, Karimi et al. (2006) utilized two-stage dilute acid hydrolysis to convert rice straw into sugars. In the first stage, xylan was depolymerized to xylose with a maximum yield of 80.8% and glucan was depolymerized to glucose at a maximum of 25.8% at hydrolysis pressure of 15 bar, 10 min retention time and 0.5% acid concentration. After pretreatment, commercial enzymes ⇑ Corresponding authors. Tel.: +86 22 27405836; fax: +86 22 87402107 (J.-J. Qiao); tel.: +86 22 27798677; fax: +86 22 27798677 (Z. Zhang). E-mail addresses: [email protected] (J.-J. Qiao), [email protected] (Z. Zhang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.05.058
were used to increase the yield of reducing sugars. Cristobal et al. (2008) used cellulase and endo-glucanase to saccharify dilute acid pretreated olive tree biomass, and Yan et al. (2009) designed a cycle spray flow-through reactor to pretreat corn stover in dilute sulfuric acid which enhanced xylose sugar yields and cellulose digestibility. In the current study, dilute sulfuric acid pretreatment followed by enzymatic digestion was conducted to produce reducing sugars from SMS. The conditions for the dilute acid treatment were optimized, the effect of cellulase and xylanase was determined and the utility of SMS hydrolysates for cultivation of Lactococcus lactis was assessed.
2. Methods 2.1. Materials The SMS, the substrate after three harvesting cycles of Pleurotus ostreastus, was obtained from Tianjin, PR China. The mushroom substrate used for planting P. ostreastus is composed of wheat straw, field hay, corn cobs, cotton seed hulls. After drying SMS was ground to a particle size of 800 lm and then stored in air-tight container at 4 °C for further use. All standard chemicals, including cellulose, glucose, arabinose and xylose were purchased from Sigma Chemical Company (Shanghai, PR China).
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the first pretreatment was saved for pretreatment the new SMS at the same conditions. The reuse process was conducted six times.
2.2. Methods 2.2.1. Analysis of SMS composition The lignin content of the raw spent mushroom substrate and acid hydrolysis residues were determined according to National Renewable Energy Laboratory Procedures LAP 001, 003, and 004 (Ehrman, 1994, 1996; Templeton and Ehrman, 1994). Cellulose and hemicellulose content was determined by high performance liquid chromatography (HPLC) analysis using a Bio-Rad Aminex HPX-87P column and a refractive index detector (SHODEX). Sugars were analyzed at 65 °C using 0.00004% H2SO4 as the mobile phase (0.6 ml/min). The total amount of reducing sugars was determined using 3,5-dinitrosalicylic acid reagent (DNS method) (Miller, 1959). Before determination, the samples of raw SMS and acid treated SMS were dried to constant weight at 105 °C. The acid pretreatment liquid was neutralized with 5 mol/L ammonia and filtered through a 0.2 lm nylon filter. All analyses were performed in duplicate. 2.2.2. Dilute sulfuric acid hydrolysis The dilute sulfuric pretreatment was conducted in a 1-L Parr reactor equipped with an electric heating and magnetic stirrer system (A1120HC9) (Parr 51111, Parr Instruments) and a modular controller (Parr 4848 controller). Forty grams of dried SMS was mixed with de-ionized water to make the final solid to liquid ratio of 1:8, 1:16, 1:24 and 98% sulfuric acid was added to the total volume of the dilution water to make a final concentration of 0.5%, 1%, 2%, 4%, and 6%. The mixture was introduced into the reactor and agitated at 100 rpm at the desired temperatures. After conclusion of the dilute acid treatment, the samples were allowed to cool to room temperature and filtered through 0.2 lm nylon filter. The solids were washed with distilled water until a pH of 7 was reached, dried at 105 °C for 6 h, and subjected to composition analysis and enzymatic hydrolysis. The four factors mainly affecting dilute sulfuric acid pretreatment of SMS were temperature, the final concentration of dilute sulfuric acid, reaction time, and the final solid to liquid ratio (Noureddini and Byun, 2010). Pretreatment reactions were carried out at temperatures of 25, 50, 60, 70, 80, 90, 100, 110, and 120 °C, reaction times of 0.5, 1.0, 1.5, 2.0, and 2.5 h, and sulfuric acid concentrations of 0.5%, 1%, 2%, 4%, and 6% (v/v of 98% sulfuric acid). Based on the single factor experiments, the orthogonal experimental design was used to optimize the pretreatment process. The orthogonal table 34 was designed as shown in Table 1. The re-use of the dilute sulfuric acid was also conducted at 120 °C for 2 h with the liquid to solid ratio of 1:16. The liquid from
2.2.3. Enzymatic hydrolysis Cellulase from Trichoderma reesei (NS50013) and xylanase (NS50014) employed were purchased from Novozymes A/S (Beijing, PR China). Cellulase activity was determined as described by Ghose (1987) and expressed as FPU (filter paper unit) defined as lmol of glucose produced per minute with filter paper as the only substrate. The cellulase had an activity of 70 FPU/g. Xylanase activity was expressed as XU (xylanase unit) and estimated as reported by Bastawde et al. (1994). The xylanase employed had an activity of 600 XU/g. Cellulase loadings of 5, 10, 15, 20, 25, 30, 35, and 40 FPU/g were investigated at 40 °C for 72 h. The effect of the addition of xylanase was determined with material hydrolyzed with 20 FPU of xylanase per gram, and 50, 100, 150, and 200 units of xylanase were used. Three grams of acid pretreated samples (dried at 105 °C for 6 h) were mixed with the enzyme solutions, 60 ml of 50 mM acetate buffer (pH 4.8) was added, and the samples were incubated at 40 °C in a shaker bath at 200 rpm for 84 h. After enzyme hydrolysis, the samples were filtered through a filter paper and sugar analysis was performed on the supernatant. All the experiments were conducted in duplicate. The influence of rotation speed on cellulase and xylanase hydrolysis was determined with 20 FPU cellulase and 150 XU xylanase per gram of acid-pretreated SMS with the rotation speed of 100 and 200 rpm, and the other conditions of this process was the same as the above paragraph.
2.2.4. Culture of L. lactis subsp. lactis W28 with the SMS hydrolysates The SMS hydrolysates were subjected to vacuum filtration and the pH of the filtrate was adjusted to pH 7.2 with ammonium hydroxide sodium hydroxide, or calcium hydroxide. The fermentations were conducted in 250-ml Erlenmeyer flasks containing 100 ml hydrolysates supplemented with KH2PO3, 2 g; NaCl, 0.15 g; MgSO47H2O; 0.015 g; peptone, 1.0 g. The fermentation medium was inoculated with 5% of overnight cultures and incubated at 30 °C for 24 h without agitation. The cell density was measured using the plate count method. All the experiments were conducted in duplicate.
3. Results and discussion 3.1. Composition of SMS
Table 1 L9 (34) orthogonal experiment of H2SO4 pretreatment. Test no.
a
1 2 3 4 5 6 7 8 9
YRSAHe (g/kg SMS)
Parameters d
c
b
T
C
R
t
T1 T1 T1 T2 T2 T2 T3 T3 T3
C1 C2 C3 C2 C3 C1 C3 C1 C2
R1 R2 R3 R3 R1 R2 R2 R3 R1
t3 t1 t2 t3 t1 t2 t3 t1 t2
50.32 70.46 96.10 158.33 124.42 83.00 267.57 173.32 238.84
All data are averages of duplicates. a T: pretreatment temperature, T1, T2, T3 denote 90, 100, 120 °C, respectively. b t: pretreatment time, t1, t2, t3 denote 60, 90, 120 min, respectively. c R: liquid to solid rate, R1, R2, R3 denote 8:1, 16:1, 24:1, respectively. d C: concentration of sulfuric acid, C1, C2, C3 denote 2%, 4%, 6% (w/w), respectively. e YRSAH: yield of reducing sugar by acid hydrolysis (based on the initial SMS).
The composition of SMS is shown in Table 2. Cellulose, hemicellulose accounted for 57.1% of the total weight. The lignin content was 20.2%. The composition of SMS is in good agreement with the previous research (Kwak et al., 2008).
Table 2 Composition of spent mushroom substrate.a Item
SMSb (%)
SMSc (%)
SMSd (%)
Lignin Hemicelluloses Cellulose
20.21 18.44 38.7
18.70 17.00 40.5
21.12 7.34 20.32
All data are averages of duplicates. a component percentages are based on dry-weight for all data and are averages of duplicates. b raw spent mushroom substrate in this paper. c raw spent mushroom substrate from a reference (Kwak et al., 2008). d spent mushroom substrate treated under the optimal condition.
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3.2. Effect of dilute acid sulfuric concentration, temperature and reaction time on sugar yield
Table 3 Analysis of L9 (34) experiment results. YRSAH
At a hydrolysis time of 0.5 h, acid concentration of 4%, and solid to liquid ratio of 1:8, the total amount of reducing sugars increased slowly at low temperatures, but increased significantly after the temperature reached 100 °C. At 120 °C, the maximum sugar yield was 156.88 g/kg SMS (Fig. 1a). The reducing sugar yield increased with increasing acid concentrations. When the acid concentration was increased from 1% to 4%, the reducing sugar yield increased by approximately 15%; however, it remained about the same as the acid concentration increased from 4% to 6%. As to the oligo-sugars, the glucose release increased with increasing acid concentrations. The xylose release increased as the acid concentration increased from 0.5% to 2% and reached a plateau at 2%, while arabinose release showed little change in response to increased acid concentrations (Fig. 1b). After 2 h of treatment with 4% sulfuric acid at 120 °C and a 1:8 solid to liquid ratio, the reducing sugar yield reached 260 g/kg SMS (Fig. 1c). The release of glucose and xylose increased with time and reached a plateau after 1.5 h, whereas the release of arabinose showed little change. Possibly, most of the available arabinose was released quickly early during acid hydrolysis as observed by Lee et al. (2009).
k1 k2 k3 R1 Optimal level
T
C
R
t
72.29a 121.92 226.58 154.29b T3
102.21 155.88 162.70 60.49 C3
137.86 140.34 142.58 4.72 R2
122.73 139.31 158.74 36.01 t3
YRSAH, yield of reducing sugar by acid hydrolysis. Orthogonal analysis was used to study the optimal level of each parameter for YRSAH, as shown in Table 1. According to the R values, the affecting YRSAH showed the following order, T > C > t > R. Table 2 indicated that the optimum condition for YRSAH was T3C3R2t3. All data are averages of duplicates. K Ti = the sum of YRSAH at Ti. T a ki ¼ K Ti /3. T T b R1 = max{ki } min{ki }.
The experimental conditions and results of the L9 (34) orthogonal experimental design are shown in Tables 1 and 3. According to the R values, the significance of each factor affecting the reducing sugar yield was in the order of T > C > t > R. Although the optimum condition for total reducing sugar yield of 267.57 g/kg SMS, obtained in test 7 (solid to liquid ratio of 1:16, 120 °C, 6% sulfuric acid for 120 min), was achieved with the
Fig. 1. Reducing sugar yields obtained under different conditions. (a) Yield at different temperatures and a sulfuric acid concentration 4%, solid to liquid ratio 1:8 and reaction time of 0.5 h; (b) yield after different reaction times with a sulfuric acid concentration of 4%, solid to liquid ratio of 1:8, and reaction temperature of 120 °C; (c) yield at different sulfuric acid concentrations with a solid to liquid ratio of 1:8, reaction temperature of 120 °C and reaction time of 0.5 h. All pretreatment processes were conducted in duplicate and the average data are presented.
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Fig. 2. Effect of sulfuric acid concentration on reducing sugar yield (solid to liquid ratio of 1:16, 120 °C, 120 min). The average data of duplicate experiments are presented.
Fig. 3. Yield of reducing sugars after repeated use of dilute sulfuric acid. The average data of duplicate experiments are presented.
Fig. 4. Reducing sugar yield of enzymatic treatment of SMS pretreated with dilute sulfuric acid (a) effect of cellulase loading on the reducing sugar yield; (b) effect of cellulase and xylanase treatment on acid pretreated SMS; (c) effect of xylanase loading on cellulase hydrolysis. The average data of duplicate experiments are presented.
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Fig. 5. Effect of two rotation speeds on cellulase and xylanase hydrolysis. The average data of duplicate experiments are presented.
highest acid concentration tested, an acid concentration of 4% might suffice as the yield was only 3.5 g/kg SMS lower than that observed with 6% (Fig. 2). The data in Fig. 3 suggest that some reuse of the acidic solution is possible, but further studies need to be done to determine optimal conditions for reuse and to determine if unwanted by-products accumulate. 3.3. Enzymatic digestion of dilute acid pretreated SMS Treatment of SMS pretreated with 4% sulfuric acid at a solid to liquid ratio of 1:16 at 120 °C for 120 min with cellulase loading 20 FPU/g pretreated SMS resulted in 47 g/kg pretreated SMS, and enzyme loading higher than 20 FPU/g pretreated SMS showed no more effect on the reducing sugar yield (Fig. 4a). Treatment with 100 XU/g pretreated SMS of xylanase yielded 65.96 g/kg pretreated SMS when cellulase was added at 20 FPU/g pretreated SMS (Fig. 4b), and xylanase was added at 50–200 XU/g pretreated SMS, the content of reducing sugar increased from 61.97 to 80.85 g/kg SMS (Fig. 4c). Fig. 5 illustrates that increased rotation speeds improved reducing sugar yields. 3.4. Sugar analysis in pretreatment liquid HPLC analysis indicated that the reducing sugars obtained by the acid hydrolysis pretreatment process mainly consisted of xylose, glucose and arabinose. No furfural or hydroxymethylfurfual was detected. Since the total amount of reducing sugars measured with the DNS method was higher than the sum of glucose, xylose and arabinose measured with the HPLC, some oligo-sugars and/ or some non-sugar reducing compounds must have been present in the hydrolysates. 3.5. Culture of L. lactis subsp. lactis W28 with the SMS hydrolysates The reducing sugars in the SMS hydrolysates can serve as carbon source for microbes once the hydrolysates have been neutralized. Hydrolysates neutralized with calcium hydroxide provided for the highest cell density (Fig. 6), presumably because of precipitation as calcium sulfate which resulted in a lower ionic strength of the medium as compared to that obtained with sodium or ammonium hydroxide. Hydrolysates neutralized with ammonium hydroxide or sodium hydroxide had to be diluted to lower the ionic
Fig. 6. Cell density of L. lactis subsp. lactis W28 grown in undiluted and diluted SMS hydrolysates. The average data of duplicate experiments are presented.
concentration, and the highest cell densities were achieved at a twofold dilution of the hydrolysates. 4. Conclusion SMS treated with 4% sulfuric acid at 120 °C for 120 min yielded 267.57 g of reducing sugars per kg of SMS, and when this treatment was followed by hydrolysis with 20 PFU of cellulase and 200 XU xylanase at 40 °C, and additional reducing sugar release of 79.85 g/kg of pretreated SMS was obtained. The hydrolysates supported growth of L. lactis subsp. lactis W28, and the highest cell density was achieved when the hydrolysates were neutralized with calcium hydroxide. Therefore, SMS hydrolysates could be useful for the cultivation microorganisms and the production of highvalue compounds such as nisin and lactic acid. Acknowledgements This work was funded by Tianjin applied basic research projects and cutting-edge technology (Tianjin Natural Science Foundation, No. 09JCZDJC26500). We were thankful to the State Key Laboratory of Chemical Engineering of Tianjin University for making equipment and facilities vital to this research available. References Akpinar, O., Erdogan, K., Bostanci, S., 2009. Production of xylooligosaccharides by controlled acid hydrolysis of lignocellulosic materials. Carbohydr. Res. 344, 660–666. Bastawde, K.B., Puntanmbekar, U.S., Gokhale, D.V., 1994. Optimization of cellulasefree xylanase production by a novel yeast strain. J. Ind. Microbiol. 13, 220–224. Beldman, G., Hennekam, J., Voragen, A.G., 1987. Enzymatic hydrolysis of beer brewers’ spent grain and the influence of pretreatments. Biotechnol. Bioeng. 30, 668–671. Bower, S., Wickramasinghe, R., Nagle, N.J., Schell, D.J., 2008. Modeling sucrose hydrolysis in dilute sulfuric acid solutions at pretreatment conditions for lignocellulosic biomass. Bioresour. Technol. 99, 7354–7362. Cristobal, C., Encarnacion, R., Jose, M.O., Felicia, S., Eulogio, C., 2008. Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification. Bioresour. Technol. 99, 1869–1876. Ehrman, T., 1994. Method for determination of total solids in biomass. In: Laboratory Analytical Procedures No. 001. National Renewable Energy Laboratory, Golden, CO. Ehrman, T., 1996. Method for determination of acid-soluble lignin in biomass. In: Laboratory Analytical Procedures No. 004. National Renewable Energy Laboratory, Golden, CO. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. Commun. 59, 257–268.
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