Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet-oxidised wheat straw

Enzyme and Microbial Technology 32 (2003) 606–615

Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet-oxidised wheat straw Anders Thygesen a,b , Anne Belinda Thomsen a,∗ , Anette S. Schmidt a,1 , Henning Jørgensen b , Birgitte K. Ahring c , Lisbeth Olsson b a Risø National Laboratory, Plant Research Department, P.O. Box 49, DK-4000 Roskilde, Denmark Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Environmental Microbiology and Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark b

c

Accepted 9 January 2003

Abstract The production of cellulose and hemicellulose-degrading enzymes by cultivation of Aspergillus niger ATCC 9029, Botrytis cinerea ATCC 28466, Penicillium brasilianum IBT 20888, Schizophyllum commune ATCC 38548, and Trichoderma reesei Rut-C30 was studied. Wet-oxidised wheat straw suspension supplemented with NH4 NO3 , MgSO4 , and KH2 PO4 was used as cultivation medium aiming to obtain an enzyme mixture optimal for enzymatic hydrolysis of wet-oxidised wheat straw. The cultivations with B. cinerea and P. brasilianum gave the highest endoglucanase (EC 3.2.1.4) and ␤-glucosidase (EC 3.2.1.21) activities, in contrast to the other fungi where lower activities were found. The culture filtrates were concentrated by ammonium sulphate precipitation. After enzyme concentration, the highest enzyme activities (1.34 FPU/ml) were found in the culture broth originating from P. brasilianum. Enzymatic hydrolysis of filter cake from wet-oxidised wheat straw for 48 h with an enzyme loading of 5 FPU/g biomass resulted in glucose yields from cellulose of 58% (w/w) and 39% (w/w) using enzymes produced by P. brasilianum and a commercial enzyme mixture, respectively. At higher enzyme loading (25 FPU/g biomass) using either enzyme mixtures the glucose yield from cellulose was in the range of 77–79% (w/w). © 2003 Elsevier Science Inc. All rights reserved. Keywords: Wet oxidation; Enzymatic hydrolysis; Aspergillus niger; Botrytis cinerea; Penicillium brasilianum; Schizophyllum commune; Trichoderma reesei

1. Introduction In many countries, wheat straw is an abundant lignocellulosic by-product from farming, consisting of cellulose (35–40% w/w) and hemicellulose (25–30% w/w) in close association with lignin (10–15% w/w). The polysaccharides in lignocellulosic materials can be used for bioethanol production and efficient pre-treatment and fermentation technologies for that purpose are being developed [1]. The utilisation of both the cellulose and hemicellulose fraction is required in an economically feasible bioethanol production. The cellulose cannot be enzymatically hydrolysed to glucose without a physical and chemical pre-treatment to break down the lignin and overcome the resistance of cellulose to hydrolytic cleavage due to its partly crystalline structure [2]. ∗

Corresponding author. Tel.: +45-4677-4164; fax: +45-4677-4122. E-mail address: [email protected] (A.B. Thomsen). 1 Present address: Protein Chemistry, Novo Nordisk A/S, Hagedornsvej 1, DK-2820 Gentofte, Denmark.

The pre-treatment processes normally applied on wheat straw are acidic hydrolysis, steam explosion and wet oxidation [3]. During wet oxidation of wheat straw, a reaction involving O2 at elevated temperature and pressure, 50% (w/w) of the lignin is decomposed to low molecular weight carboxylic acids, phenolic compounds, CO2 , and H2 O [4,5]. Most of the hemicellulose (80% w/w) is dissolved and oxidised to carboxylic acids, CO2 , and H2 O leaving a solid fraction rich of cellulose [6]. Degradation products that could have inhibitory action in later fermentation steps are avoided during pre-treatment by wet oxidation [5]. The soluble sugars produced by wet oxidation can be utilised for enzyme production by cultivation of Aspergillus niger and for ethanol fermentation by Thermoanaerobacter mathranii [7] and Saccharomyces cerevisiae [8] fermenting the pentoses and hexoses, respectively. However, low contents of hemicellulose and lignin remain insoluble after wet oxidation [6] so in addition to cellulose-degrading enzymes, both hemicellulose and lignin degrading enzymes might be required for complete hydrolysis of the solid fraction.

0141-0229/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0141-0229(03)00018-8

A. Thygesen et al. / Enzyme and Microbial Technology 32 (2003) 606–615

Cellulose-degrading enzymes are commercially available, but still too expensive for production of fuel ethanol. Another problem is that these enzymes are not developed for hydrolysis of lignocellulose [3], and usually produced using glucose as carbon source. Since wheat straw is an agricultural waste product the use of it in cultivations will lower the raw material cost and thereby the enzyme production cost. However, research and development is needed in order to make the enzyme production feasible, since cellulose is a less simple carbon source than glucose. For complete hydrolysis of cellulose, the cellulose chains are cut at random positions by endoglucanase (EC 3.2.1.4) and at the chain ends by exoglucanase (EC 3.2.1.91) producing cellobiose [9]. When commercial Celluclast is utilised for enzymatic hydrolysis of cellulose, extra ␤-glucosidase (EC 3.2.1.21) must be added for hydrolysis of the produced cellobiose. Hemicellulose is a heterogeneous branched polymer in which the composition and structure depend on the raw material source. In wheat straw, hemicellulose consists of mainly arabinoxylan (81% w/w) [2] and enzymes such as endoxylanase (EC 3.2.1.8) and ␤-xylosidase (EC 3.2.1.37) are required for hydrolysis. Many bacteria and filamentous fungi can produce cellulose-degrading enzymes. Most bacteria cannot utilise crystalline cellulose, which can be done by many filamentous fungi [3]. In this study, an enzyme mixture suitable for hydrolysis of wet-oxidised wheat straw was approached by enzyme production with four filamentous fungi, Aspergillus niger ATCC 9029, Botrytis cinerea ATCC 28466, Penicillium brasilianum IBT 20888, and Schizophyllum commune ATCC 38548. The enzyme production was compared to the frequently used Trichoderma reesei Rut-C30. Wet-oxidised wheat straw was the only carbon source and only the most essential nutrients were added. The cell mass could not be followed by optical density (OD) measurements due to interference caused by solid particles (wet-oxidised wheat straw). The cell mass was instead followed by measuring the carbon dioxide development and the composition of the cultivation solid residue. The enzyme activity of endoglucanase, ␤-glucosidase, endoxylanase, and ␤-xylosidase was determined as a function of time. Finally, a comparative study of the hydrolytic effect was made in which the produced enzymes and Celluclast were compared.

2. Materials and methods All solvents and chemicals were of analytical grade, unless otherwise stated. 2.1. Materials Wheat (Triticum aestivum L.) variety Husar was grown and harvested at Risø National Laboratory in 1997. The wheat straw was knife-milled to pass a 5 mm screen and stored in the dark at room temperature.

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2.2. Wet oxidation Wheat straw was wet-oxidised in a 2-l loop autoclave (Risø National Laboratory). Water (1 l), wheat straw (60 g dry matter (DM)), and 6.5 g Na2 CO3 were added to the autoclave followed by addition of oxygen (12 bar) to the remaining gas volume [5]. The gas–liquid mass transfer was accomplished by mixing with a pumping wheel. Thereby an excellent heat transfer was maintained which resulted in 3 min heating-up time and 0.5 min cooling-down time. The reaction temperature was kept at 195 ◦ C for a reaction time of 10 min after which the reactor was cooled and the pressure released. The product consisting of a suspension with fibrous wheat straw particles was stored at −20 ◦ C. For analysis, suspension was filtered to give a solid fibre fraction (WO filter cake) and a filtrate containing dissolved products (WO filtrate). 2.3. Cultivation and preparation of enzyme mixtures Spores from the filamentous fungi were produced by growth of A. niger ATCC 9029 on malt agar, B. cinerea ATCC 28466 on V8-juice agar, S. commune ATCC 38548 on rice agar, P. brasilianum IBT 20888 on oat bran agar, and T. reesei Rut-C30 on oat bran agar. Colonies from the agar plates were inoculated at 25 ◦ C on 57 g rice added 12 ml solution of 4 g/l yeast extract, 0.5 g/l MgSO4 ·7H2 O, 1 g/l KH2 PO4 , and 14 g/l glucose. The spores were harvested by suspending with a sterile solution of 0.1% Tween 80. All fermentors were inoculated to give 106 spores/l, except for the cultivation with B. cinerea, which was inoculated to give 104 spores/l. The batch cultivations were carried out in 1 l fermentors constructed at BioCentrum-DTU. The growth medium consisted of 900 ml wet-oxidised wheat straw supplemented with 2 g/l KH2 PO4 , 0.3 g/l MgSO4 ·7H2 O, 5.4 g/l NH4 NO3 , and 1 ml/l antifoam. The aeration rate was 0.5 l air/(l culture min), the agitation was 600 rpm, and the pH was controlled at A. niger 4.5, B. cinerea 6.5, P. brasilianum 5.0, and S. commune 6.5; by addition of HCl (2 M) and NH4 OH (2 M). The culture temperature was controlled at 24 ◦ C in the cultivation with B. cinerea and at 30 ◦ C in the other cultivations. The off-gas was passed through a condenser to avoid evaporation from the fermentors. The concentration of CO2 and O2 in the off-gas was monitored every second minute (Brüel and Kjær 1308; [10]). Samples were withdrawn from the fermentors twice a day and centrifuged. The supernatant was filtered through a 0.22 ␮m filter and stored at −20 ◦ C. The cultivations were terminated after 250 h or when the monitored CO2 level was below 0.1% (v/v). After ended cultivation, the culture broth was filtered and the cultivation solid residue was washed with water and dried at 60 ◦ C for 50 h. The cultivation filtrate was stored at −20 ◦ C. Enzymes in the cultivation filtrate were precipitated by adding 100 g (NH4 )2 SO4 slowly to 200 ml filtrate kept on ice bath, followed by stirring for 30 min. The suspension was

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A. Thygesen et al. / Enzyme and Microbial Technology 32 (2003) 606–615

centrifuged at 11,000 rpm and 4 ◦ C for 20 min and the pellet was dissolved in the smallest possible volume of 50 mM sodium acetate (pH 5). The solution was dialysed over a cellulose acetate membrane into 50 mM acetate buffer (pH 5) at 5 ◦ C for 15 h, filtered through a 0.45 ␮m filter, and stored at −20 ◦ C [11,12]. 2.4. Analysis The dry matter content in the WO filter cake and the cultivation solid residue was determined with a dry matter analyser HR73 (Mettler Toledeo) at 105 ◦ C. The content of dry matter in the WO filtrate was determined by drying the sample at 105 ◦ C for 20 h. The content of non-cell wall material (NCWM), hemicellulose, cellulose, and lignin was measured in the wheat straw, the WO filter cake, and the cultivation solid residue by a gravimetric detergent method [13]. Proteins, pectin, and lipids were measured as NCWM. The content of sugars in the WO filtrate was measured after acidic hydrolysis of the polysaccharides. At first 10 ml WO filtrate was mixed with 10 ml 8% (w/v) H2 SO4 followed by treatment at 121 ◦ C for 10 min. The solution was filtered and a suspension of 0.50 g Ba(OH)2 ·8H2 O and 5 ml filtrate was prepared. After 2 min reaction time, the suspension was centrifuged (4000 rpm, 5 min). A suspension of 3 ml supernatant and 0.20 g Dovex MR-3 was prepared and equilibrated for 10 min before centrifugation (4000 rpm, 5 min). A solution of 2 ml supernatant and 2 ml H2 SO4 (4 mM) was prepared. The recovery of glucose, xylose, and arabinose was determined by standard addition of sugars before addition of Ba(OH)2 ·8H2 O. The HPLC analysis was performed (Shimadzu equipment) with an Aminex HPX-87H column (Biorad) at 63 ◦ C. The eluent was 4 mM H2 SO4 , used at a flow rate of 0.6 ml/min. The components were detected refractometrically (Knauer detector). The ␤-glucosidase activity was measured using 1 mM p-nitrophenyl-␤-d-glucopyranoside (Sigma N-7006) in 50 mM citrate buffer (pH 4.8) as substrate [14]. The substrate (900 ␮l) was equilibrated at 50 ◦ C and 100 ␮l sample was added. After 10 min, 500 ␮l 1 M Na2 CO3 was added to stop the reaction. For the standards, 100 ␮l p-nitrophenol (0.05–0.5 mM) was added to 900 ␮l substrate and 500 ␮l 1 M Na2 CO3 . The absorption was measured spectroscopically at 400 nm. The ␤-xylosidase activity was measured in an analogous manner by using 1 mM p-nitrophenyl-␤-d-xylopyranoside (Sigma N-2132) as substrate at pH 4.5 and 50 ◦ C. Both activities were expressed in “nkat” (nmol converted substrate per second). The endoglucanase activity was measured using 10 g/l hydroxyethylcellulose (Fluka 54290) in 50 mM citrate buffer (pH 4.8) as substrate [14]. The substrate (900 ␮l) was equilibrated at 50 ◦ C, 100 ␮l sample was added, and after 10 min 1500 ␮l DNS reagent [15] was added. For the standards, 100 ␮l glucose (2.5–10 mM) was added to 900 ␮l substrate and 1500 ␮l DNS reagent. The samples and standards were

boiled (100 ◦ C) for 5 min and cooled on ice. The absorption was measured spectroscopically at 540 nm. The endoxylanase activity was measured in an analogous manner but with 5 min incubation time, pH 5.3, 50 ◦ C, 10 g/l xylan (Sigma X-0502) as substrate, and xylose as standard. Both activities were expressed in “nkat” (nmol converted substrate per second). The filter paper activity was measured according to IUPAC recommendations [15] using Whatman No. 1 filter paper in 50 mM citrate buffer at pH 4.8 and 50 ◦ C. The filter paper activity was expressed in FPU (␮mol/min of substrate converted). 2.5. Enzymatic hydrolysis of wet-oxidised wheat straw WO filter cake (100 mg dry matter) was milled with a knife mill (Braun 4041) and mixed with 1 ml buffer (0.2 M sodium citrate, pH 5.0). Enzyme solutions with a filter paper activity of 0.5 FPU/ml were prepared by diluting the enzyme mixtures with water. One solution contained 0.586% (v/v) Celluclast 1,5 L FG (Novozymes A/S) and 0.118% (v/v) Novozym 188 (Novozymes A/S) with the activities stated in Table 1 and based on the manufactures recommendations. The other solution contained 47.2% (v/v) P. brasilianum enzyme mixture. In the experiment with 5 FPU/g DM, 3.16 ml water and 790 ␮l enzyme solution was added to the suspended filter cake whereas in the experiment with 25 FPU/g DM, 3.95 ml enzyme solution was added to the suspended filter cake. The reaction was performed at 50 ◦ C for 24 or 48 h, respectively. The reaction was stopped by centrifugation (3000 rpm for 15 min). The concentrations of glucose, xylose, cellobiose, and arabinose were measured by HPLC (Shimadzu) in solutions of 350 ␮l supernatant and 800 ␮l 10 mM H2 SO4 . The eluent was 4 mM H2 SO4 used with a flow rate of 0.6 ml/min at 63 ◦ C. An Aminex HPX-87H column (Biorad) was used and the components were detected refractometrically (Knauer detector). 2.6. Adsorption of endoglucanase on lignocellulose Adsorption of endoglucanase to WO filter cake (32 g DM/l solution) and to filter paper (Whatman No. 42; 32 g DM/l solution) was investigated in acetate buffer (0.2 M, pH 4.8). Celluclast 1,5 L FG was added at enzyme loadings of 7.4

Table 1 Enzyme activities measured in the commercial enzyme mixtures Enzyme activity

Celluclast

Commercial mixturea

Novozym 188

Filter paper (FPU/ml) Endoglucanase (nkat/ml) ␤-Glucosidase (nkat/ml) Endoxylanase (nkat/ml) ␤-Xylosidase (nkat/ml)

67 14600 440 20600 1060

90 11500 1200 16800 950

660 4900 4000 116

a

Mixture of 83% (v/v) Celluclast and 17% (v/v) Novozym 188.

A. Thygesen et al. / Enzyme and Microbial Technology 32 (2003) 606–615

and 74 FPU/g DM, respectively, and incubated at 28 ◦ C for 100 h. 2.7. Calculations The cellulose recovery (RCellu (w/w)) during wet oxidation was calculated by Eq. (1) in which CCellu,Straw (g/kg straw) and CCellu,WOSol (g/kg straw) are contents of cellulose in straw and WO filter cake, respectively. In WO filtrate CGlucose,WOFil (g/kg straw) is the concentration of glucose: RCellu

CCellu,WOSol + CGlucose,WOFil × 162/180 = CCellu,Straw

(1)

The hemicellulose recovery (Rhemic (w/w)) during wet oxidation was calculated by Eq. (2) in which Chemic,Straw (g/kg straw) and Chemic,WOSol (g/kg straw) are the contents of hemicellulose in straw and WO filter cake, respectively. In WO filtrate CXylose,WOFil (g/kg straw) and CArabi,WOFil (g/kg straw) are the concentrations of xylose and arabinose, respectively,

Rhemic

Chemic,WOSol + (CXylose,WOFil +CArabi,WOFil ) × 132/150 = Chemic,Straw

(2)

The polymeric content of sugars after wet oxidation (CPolymSugar,WO (g/kg straw)) is calculated by Eq. (3): CPolymSugar,WO = CCellu,WOSol + Chemic,WOSol + CGlucose,WOFil × +(CXylose,WOFil + CArabi,WOFil ) ×

132 150

162 180 (3)

The specific growth rate (µ (h−1 )) in the individual growth phases was calculated by Eq. (4) in which pCO2 (g CO2 /kg straw) is the CO2 production and t (h) is the time: µ=

log(pCO2 (t2 )) − log(pCO2 (t1 )) t2 − t 1

(4)

The cell mass yield (Ycell mass/substrate (g/g)) was calculated by Eq. (5) in which CNCWM,CBSol (g/kg straw) and CNCWM,WOSol (g/kg straw) are the contents of non-cell wall material in the cultivation solid residue and WO filter cake, respectively. In the cultivation solid residue CCellu,CBSol (g/kg straw) and Chemic,CBSol (g/kg straw) are the contents of cellulose and hemicellulose, respectively, Ycell mass/substrate CNCWM,CBSol − CNCWM,WOSol = CPolymSugar,WO − CCellu,CBSol − Chemic,CBSol

(5)

The efficiency in concentrating enzymes in the cultivation filtrate was measured as the concentration factor (CF (nkat/nkat)). This factor was calculated by Eq. (6) in which AEnMix (nkat/ml) is the enzyme activity measured after concentrating and ACBFil (nkat/g straw) is the enzyme activity

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in the cultivation filtrate. XStraw,WO (0.060 kg straw/l) is the content of wheat straw added before wet oxidation: AEnMix CF = (6) ACBFil XStraw,WO The specific enzymatic hydrolysis rate rx (mg/(h FPU)) was calculated by Eq. (7) in which Yx,DM (g/100 g DM) is the yield of component x, tHydr (h) the hydrolysis time, and xFPU (FPU/g DM) is the enzyme loading: rx =

Yx,DM tHydr xFPU

(7)

The glucose yield relative to the cellulose content is: Yglucose,cell =

Yglucose,DM 162 · Xcellulose,DM 180

(8)

3. Results Wet oxidation of 60 g/l wheat straw resulted in a suspension of solid material (515 g DM/kg straw) in a hydrolysate containing dissolved and partially hydrolysed material (441 g DM/kg straw). The solid fraction contained remaining cellulose (356 g/kg straw) and hemicellulose (56 g/kg straw) and the hydrolysate contained dissolved and partially hydrolysed cellulose (20 g/kg straw) and arabinoxylan (119 g/kg straw) (Table 2). For the entire suspension, the recoveries of cellulose and hemicellulose were 89% (w/w) and 55% (w/w), respectively (Eqs. (1) and (2)). Since it has been shown that the carbohydrates in both fractions can supply microbial growth [5], the suspension was not separated before the cultivations. At the same wet oxidation conditions, Klinke et al. have measured low-molecular weight carboxylic acids (62 g formic acid/kg straw and 23 g acetic acid/kg straw) and phenolic compounds Table 2 Changes in chemical composition during wet oxidation of wheat straw Before wet oxidation (g/kg strawa ) Wheat straw WO filter cake NCWMb Hemicellulose Lignin Cellulose WO filtrate Glucosec Xylosec Arabinosec Formic acidd Acetic acidd Monomeric phenolsd Polymeric phenolsd a

After wet oxidation (g/kg strawa )

1000 141 320 112 421

515 60 56 29 356 441 22 101 34 62 23 4.8 35

Component produced by wet oxidation of 1 kg wheat straw. NCWM, non-cell wall material. c Monomers after acidic hydrolysis of WO filtrate. d Data from Klinke et al. [16]. b

610 A. Thygesen et al. / Enzyme and Microbial Technology 32 (2003) 606–615 Fig. 1. CO2 production, endoglucanase activity (䊐) and ␤-glucosidase activity () in the culture broth during the cultivations (—). The specific growth rate in the first (µ1 ) and the second growth phase (µ2 ) are estimated from the CO2 production.

A. Thygesen et al. / Enzyme and Microbial Technology 32 (2003) 606–615

(40 g/kg straw) as being the main degradation products (Table 2) [16]. The energy yield during the wet oxidation was 4100 kJ/kg straw, when oxidation of cellulose (11% w/w), hemicellulose (45% w/w), and lignin (74% w/w) to CO2 , formic acid, acetic acid, and phenolic compounds was taken into account (unpublished data). 3.1. Cell growth on wet-oxidised wheat straw The growth for all the filamentous fungi could be divided into two growth phases—one with a high specific growth rate followed by one with a lower specific growth rate (Fig. 1A–E). The first growth phase started after a lag phase of 10–20 h indicated by an increased CO2 production. The specific growth rate was highest for P. brasilianum (0.062 h−1 ) and lowest for B. cinerea (0.025 h−1 ) and lasted for 50 and 80 h, respectively (Eq. (4)). In the second growth phase starting after 80–100 h of cultivation, the specific growth rate was lowest for A. niger and P. brasilianum (0.001 h−1 ) and highest for B. cinerea (0.004 h−1 ). The highest CO2 production was obtained during the cultivation of P. brasilianum and B. cinerea corresponding to 780 g CO2 /kg straw and 680 g CO2 /kg straw, respectively. The chemical analysis of the cultivation solid residue showed that 76–87% (w/w) of the cellulose was degraded during cultivation of B. cinerea, P. brasilianum, and S. commune and only 30% (w/w) during the other cultivations (Fig. 2). The cultivations with high cellulose consumption resulted in a high NCWM production (64–114 g NCWM/kg straw). Therefore, an estimation of the cell mass based on the production of NCWM during the cultivations was performed. In addition to cell mass extracellular enzymes absorbed to the cultivation solid residue will also be measured as NCWM. The cell mass yield (Eq. (5)) ranged between 0.08 g/g in A. niger and 0.25 g/g in B. cinerea (Table 3).

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The hemicellulose degradation was 36–48% (w/w) during all the cultivations except for A. niger giving no degradation of hemicellulose (Fig. 2). 3.2. Enzyme production With exception of the cultivation with T. reesei resulting in insignificant enzyme activities (Fig. 1E), the production of endoglucanase and ␤-glucosidase was initiated at the transition between the first and second growth phase (Fig. 1A–D). The endoglucanase and ␤-glucosidase activities continued to increase throughout the cultivations. In the culture broth, the highest endoglucanase activity (1170 nkat/g straw) and ␤-glucosidase activity (3100 nkat/g straw) were obtained after the cultivation of B. cinerea (Table 3). The ratio between the endoglucanase and ␤-glucosidase activities varied from 0.2 (A. niger) to 2 (P. brasilianum) after 100 h and 0.06 (A. niger) to 1.1 (P. brasilianum) after termination of the cultivations. The highest ␤-xylosidase activity (1070 nkat/g straw) and endoxylanase activity (24,000 nkat/g straw) were obtained after the cultivation of P. brasilianum together with the second highest endoglucanase and ␤-glucosidase activity. The cultivations resulted in filter paper activities in the cultivation filtrate below the detection limit (6.2 FPU/g straw). Therefore, the enzymatic hydrolysis was performed using enzyme mixtures of concentrated cultivation filtrate. The concentration factor was highest for the enzyme mixture produced by P. brasilianum for endoglucanase (CF = 6) and endoxylanase (CF = 4.4) and lowest for the enzyme mixture produced by B. cinerea (Table 3, Eq. (6)). The enzyme mixture produced by P. brasilianum was chosen for enzymatic hydrolysis of wet-oxidised wheat straw because of high filter paper activity after concentration (1.34 FPU/ml).

Fig. 2. Content of cellulose (䊏), hemicellulose ( ), non-cell wall material ( ), and lignin (䊐) in cultivation filter cake from the terminated cultivations and wet oxidation filter cake.

Table 3 Cell mass yield and enzyme activities measured in the final cultivation filtrates and in concentrated cultivation filtrates Cell mass/enzyme activities at the end of the cultivations

A. niger

Cell massa (g/kg straw) Cell mass yieldb (g/g)

B. cinerea

20 0.08

Filter paper activityc (FPU/g straw) Endoglucanase (nkat/g strawd ) ␤-Glucosidase (nkat/g strawd ) Endoxylanase (nkat/g strawd ) ␤-Xylosidase (nkat/g strawd )

<6.2 66 1050 9300 620

Concentrated cultivation filtrate (enzyme mixtures) Filter paper activityc (FPU/ml) Endoglucanase (nkat/ml) ␤-Glucosidase (nkat/ml) Endoxylanase (nkat/ml) ␤-Xylosidase (nkat/ml)

<0.37 16 240 1300 120

P. brasilianum

114 0.25 <6.2 1170 3100 22200 340 <0.37 128 470 2600 58

S. commune

64 0.14

T. reesei

98 0.23

<6.2 890 830 24000 1070

28 0.11

<6.2 420 1160 21800 202

1.34 300 70 6300 138

<6.2 19 0 9200 17 <0.37 9 3 1840 1

0.65 190 175 4300 32

a

Calculated as produced non-cell wall material during cultivation. Produced cell mass relative to the utilised cellulose and hemicellulose (Eq. (6)). c Detection limit = 0.37 FPU/ml = 6.2 FPU/g straw. d Corresponding to the amount of straw loaded before the wet oxidation (g). b

Table 4 Released sugars after enzymatic hydrolysis of filter cake from wet-oxidised wheat straw after 24 and 48 h, respectively, at 45 ◦ C and pH 5.0 Enzyme mixture

Loading (FPU/g Dma )

Time (h)

Glucose (g/100 g DMa )

Cellobiose (g/100 g DMa )

Xylose (g/100 g DMa )

P. brasilianum

5 5 25 25

24 48 24 48

36/47∗ 45/58∗ 57/75∗ 59/77∗

1.29 0.63 0.60 0.48

10.0 12.3 16.0 16.4

Commercial enzyme mixtureb

5 5 25 25

24 48 24 48

23/30∗ 30/39∗ 49/65∗ 61/79∗

0.98 1.04 1.37 1.01

6.9 8.5 12.8 15.8

a

DM, dry matter content in the suspended filter cake with a cellulose content of 69%. Mixture of 83% (v/v) Celluclast and 17% (v/v) Novozym 188. ∗ Glucose (g/100 g cellulose) = glucose (g/100 g DM) × 515/356 × 162/180 (Eq. (8), Table 2). b

Fig. 3. Changes in endoglucanase activity vs. incubation time using two different Celluclast loadings (left axis, 50 nkat/ml; right axis, 500 nkat/ml); 50 nkat/ml (䊐), buffer (䊊), filter paper (), wet-oxidised wheat straw; 500 nkat/ml (䉱) wet-oxidised wheat straw.

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3.3. Enzymatic hydrolysis of wet-oxidised wheat straw The enzyme mixtures hydrolysed the WO filter cake to mainly glucose and xylose (Table 4). The highest specific hydrolysis rate 3.0 mg glucose/(h FPU), Eq. (7) was obtained using the enzyme mixture produced by P. brasilianum with 5 FPU/g DM for 24 h. In comparison, the specific hydrolysis rate using the mixture of Celluclast and Novozym 188 was 1.9 mg glucose/(h FPU). For both enzyme mixtures, the concentration of the intermediary hydrolysis product cellobiose was highest after 24 h. The glucose yield from cellulose was in the range of 77–79% (w/w) using both enzyme mixtures for 48 h at 25 FPU/g DM (Table 4, Eq. (8)). 3.4. Adsorption of endoglucanase on wet-oxidised wheat straw When cellulose-degrading enzymes are produced by cultivation on cellulose containing substrate there is a risk of enzyme adsorption to the solid particles. The extent of endoglucanase adsorption was investigated because it is essential when considering enzyme production in media with solid particles. Using filter paper and WO filter cake, the activity decreased by 50% after 15 min at both high (74 FPU/g DM) and low (7.4 FPU/g DM) enzyme loading (Fig. 3). At high enzyme loading using WO filter cake, the activity increased again to the initial level of activity. On the other hand, at low enzyme loading on both substrates the activity levelled off at 20% of the initial activity. This means that 80% of the initial activity was absorbed to the WO filter cake.

4. Discussion A frequent problem, when solid raw materials are used for cultivation is the lack of analytical methods suitable for estimation of produced biomass as well as for substrate utilisation. Methods relying on estimation of glucoseamin (or other cell wall components only produced by filamentous fungi) or on-line methods like IR and carbon dioxide evolution rates have been demonstrated to be useful [17,18], however, none are used extensively. In this work, it was demonstrated that off-gas analysis was valuable for estimation of the specific growth rate. With help of analysis of the composition of the solid residue before and after cultivation, it was thereby possible to compare growth and substrate utilisation by the different fungi (Figs. 1 and 2). The analysis of the cultivation solid residue showed that P. brasilianum and B. cinerea produced most NCWM and utilised most cellulose producing most CO2 . This analysis also allowed an estimation of the cell mass yield based on the cellulose and hemicellulose content in WO wheat straw. The yield of cell mass during growth on WO straw was estimated to 0.08 g/g for the cultivation of A. niger (Table 3) and the range of 0.23–0.25 g/g for the cultivations of B. cinerea and S. commune. This growth yield was lower than

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the one found in cultivation of A. niger on the easy accessible substrate glucose (0.46 g cell mass/g glucose) [19]. The resulting filter paper activities in the culture broths were low (<6.2 FPU/g straw) (Table 3), compared with previous studies (18–200 FPU/g raw material) using cellulose or lignocellulosic materials as substrates [20–24]. In these studies, complex nutrients like yeast extract and peptone have been used, in contrast to this study, where only NH4 NO3 , MgSO4 , and KH2 PO4 were added to the pre-treated wheat straw. The investigated filamentous fungi grew well initially (Fig. 1A–E), after which the growth levelled off. After termination of cultivation, A. niger and T. reesei had only used 30% (w/w) of the available cellulose (Fig. 2), indicating either that nutrient limitation resulted in decreased cell growth or that the level of endoglucanase produced were too low to support further degradation of the substrate. In many cases, the nutrient content in the cultivation medium has shown to be very important for the enzyme production. In a cultivation of Penicillium occitanis, the resulting filter paper activity increased five-fold when the composition and content of the nitrogen source were optimised [24]. In cultivations with Trichoderma viride, the resulting filter paper activity has been found to depend on the nutrient additions. Up to a two-fold increase of the filter paper activity could be achieved with 0.5 g/l additions of peptone, casein hydrolysate, cottonseed flour or yeast extract to cellulose containing medium [25]. During wet oxidation, not only carbohydrates (hemicellulose) are released from the solid substrate, but also other components, such as low molecular carboxylic acids and phenolic compounds [16]. These components may also influence the growth of the filamentous fungi and may explain the comparatively slow growth for T. reesei. Previous studies cultivating T. reesei in WO filtrate, showed also poor growth and enzyme production compared with two Aspergillus strains [26]. According to the experiments on the enzyme adsorption, endoglucanase was adsorbed to both filter paper and the WO solid residue (Fig. 3). Adsorption of cellulose degrading enzymes to solid substrates is well known and has been described both as a reversible [27] and an irreversible process [28,29]. The low level of endoglucanase measured in the broths resulting from the cultivations of T. reesei and A. niger could be due to irreversible adsorption of the enzymes, as indicated in the sorption studies (Fig. 3). However, further studies that are focusing on adsorption and action of the individual cellulose and hemicellulose degrading enzymes needs to be carried out to confirm this assumption. Enzymatic hydrolysis with the mixture of Celluclast and Novozym 188 (25 FPU/g DM for 24 h) resulted in a low yield of glucose from cellulose (65% w/w, Table 4). Accordingly, Szczodrak [30] has reported the yield to 65% (w/w) when hydrolysing alkaline treated wheat straw with enzymes produced by T. reesei (25 FPU/g DM for 24 h). Differences in the chemical composition and fraction of crystalline cellulose in the different materials has been shown to influence their hydrolysis potentials [9], which

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might explain the slightly different hydrolysis results obtained on WO solid residue here compared to what earlier has been found in alkali treated wheat straw [30]. Using the enzyme mixture produced by P. brasilianum resulted in a yield of glucose from cellulose at 75% (w/w) (Table 4). The superior efficiency of the P. brasilianum enzymes might be due to a higher ␤-glucosidase activity relative to the filter paper activity, since ␤-glucosidase has been found rate limiting during enzymatic hydrolysis of cellulose (Tables 1 and 3) [30]. The possible difference in adsorption of the different enzymes on the substrate might be yet another factor of substantial importance for the hydrolysis result.

5. Conclusion In this study it was found that: • For hydrolysis of wet oxidised wheat straw, the enzyme mixture produced by cultivation of P. brasilianum on wet oxidised wheat straw resulted in a higher glucose yield than the mixture of Celluclast and Novozym 188. Using the high enzyme activity, the P. brasilianum enzyme mixture showed the highest initial hydrolysis rate. • The highest utilisation of cellulose and the highest endoglucanase activity was obtained by the cultivations of B. cinerea and P. brasilianum.

Acknowledgments Prof. Jens Christian Frisvad from the Technical University of Denmark is gratefully acknowledged for donation of P. brasilianum IBT 20888 from his strain collection as a potentially interesting enzyme producing fungi. Mr. Tomas Fernqvist, Mrs. Ingelis Larsen and Dr. Helene Benstrup Klinke from Risø National Laboratory are acknowledged for their technical assistance. Dr. Lisbeth Olsson and Ph.D. student Henning Jørgensen acknowledge the financial support from the Danish Technical Research Council on their research on enzyme production in filamentous fungi. The authors would also like to thank the Danish Ministry of Environment and Energy (1383/97-0009) for financial support. References [1] Zaldivar J, Nielsen J, Olsson L. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 2001;56:17–34. [2] Puls J, Schuseil J. Chemistry of hemicelluloses: relationship between hemicellulose structure and enzymes required for hydrolysis. In: Coughlan MP, Hazlewood GP, editors. Hemicellulose and hemicellulases. Portland Press Research Monograph; 1993. p. 1–27. [3] Cen P, Xia L. Production of cellulase by solid-state fermentation. Adv Biochem Eng/Biotechnol 1999;65:69–92. [4] McGinnis GD, Wilson WW, Mullen CE. Biomass pretreatment with water and high-pressure oxygen. The wet-oxidation process. Ind Eng Chem Prod Res Dev 1983;22:352–7.

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