Production of Cellulase and Xylanase with Selected Filamentous

Chapter 13

Production of Cellulase and Xylanase with Selected Filamentous Fungi by Solid Substrate Fermentation 1

2

G. Szakacs and R. P. Tengerdy

Downloaded by PRINCETON UNIV on September 30, 2013 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch013

1

Technical University of Budapest, Department of Agricultural Chemical Technology, HU-1521 Budapest, Gellert ter 4, Hungary Colorado State University, Department of Microbiology, Fort Collins, CO 80523-1677 2

Lignocellulolytic fungi were tested for growth and enzyme production on solid substrates such as extracted sweet sorghum pulp and wheat straw. A Gliocladium sp. was best adapted to sweet sorghum pulp with about 6.0 Filter Paper Unit (FPU)/Dry Weight (DW) cellulase activity, 14.0 IU/g DW beta glucosidase activity, 1900 IU/g DW xylanase activity and 1500 Endoglucanase Unit (EGU)/g DW endoglucanase activity. On wheat straw a Trichoderma hamatum strain produced about 7000 IU/g DW xylanase activity and a Penicillium strain 34 IU/g DW beta glucosidase activity, surpassing Trichoderma reesei Rut C30 in these categories (10.0 IU/g DW beta glucosidase and 3600 IU/g D W xylanase). All selected wild strains surpassed Trichoderma reesei Q M 6a, the parent of Rut C30 in enzyme production. The Gliocladium and Trichoderma hamatum strains grew considerably faster on both substrates than other tested fungi. Lignocellulolytic enzymes may be produced economically and efficiently by solid substrate fermentation (SSF) (1,2,3). The advantage of SSF is operational simplicity and economy in a water restricted environment, resulting in high volumetric productivity, high product concentration, and possibility for use of the product with little or no downstream processing. Crude SSF enzymes may be used directly in agrobiotechnological applications such as ensiling(40)feed additives, retting, soil additives, and in biotechnology industries such as the paper industry and the biofuel industry (3). In situ SSF enzyme production may be incorporated into such biotechnologies with great saving in process cost (5,6). The purpose of this work was finding an efficient and economical enzyme source for the bioprocessing of sweet sorghum for ethanol production. We have reported earlier the successful application of enzyme assisted ensiling (ENLAC) for increasing plant cell wall permeability, resulting in much higher yield in sugar extraction (7). Although the process is economical even with commercial enzymes, a cost reduction of about 100 0097-6156/96/0655-0175$15.00/0 © 1996 American Chemical Society

In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

176

ENZYMES FOR PULP AND PAPER PROCESSING


times may be achieved by using in situ enzymes, produced by solid substrate fermentation (SSF) on recycled extracted sweet sorghum pulp (4). The process scheme is shown in Figure 1. The advantages of the in situ process is that it uses a pretreated, near sterile, no-cost recyclable substrate, and produces enzymes that can be used without downstream processing in ENLAC. In this paper we report the cell wall degrading enzyme production of selected filamentous fungi by SSF that may be used as in situ enzyme source in sweet sorghum processing, and/or in other similar processes. The enzyme productivities of selected fungi were compared on extracted sweet sorghum pulp and wheat straw, a commonly used reference substrate in SSF. Materials and Methods Cultures. Trichoderma hamatum TUB F-105 (ATCC 62392) was isolated from decaying reed in Hungary by G. Szakacs, and identified by L. Vajna, Budapest; Trichoderma sp. TUB F-426 was isolated from soil in Queensland, Australia by G. Szakacs; Trichoderma sp. TUB F-482 was isolated from forest soil in Florida by G. Szakacs; Trichoderma sp. TUB F-486 was isolated from decaying wood, near Lake Placid, N.Y.; Gliocladium sp. TUB F-498 was isolated from soil in Germany; Pénicillium aculeatum NRRL 2129, Pencillium funiculosum NRRL 1132 and Pénicillium funiculosum NRRL 3647 strains were kindly donated from NRRL collection (Peoria, Illinois). Trichoderma reesei, Rut C30 and Q M 6a were obtained from the American Type Culture Collection (ATCC). Substrates. Extracted sweet sorghum pulp was obtained from the bioprocessing of sweet sorghum by ENLAC, followed by countercurrent diffusion for sugar extraction (4). The extracted and dried pulp contains about 25% available carbon in the form of cellulose and hemicellulose, and about 0.6% nitrogen in the form of protein. Wheat straw, supplemented with 10% wheat bran contains about 30% available carbon and 0.9% nitrogen. The dried substrates were milled to about 1 cm particle size and supplemented with the following salt solution: N H N 0 , 5.0g/l; K H P 0 , 5.0 g/1; MgS0 .7H 0, 1.0 g/1; NaCl, 1.0 g/1; MnS0 , 1.6 mg/1; ZnS0 .7H 0, 3.4 mg/1; CoCl .6H 0, 2.0 mg/1; FeS0 .7H 0, 5.0 mg/1. For 1 g dry substrate 3 ml salt solution was added to give a moisture content of 75% and approximate C:N ratio of 25 for sweet sorghum and 18 for wheat straw/bran. The initial pH before sterilization was 4.8. 4

4

2

2

2

4

4

3

2

4

4

2

2

Solid Substrate Fermentation. SSF was performed in 250 ml double plastic cups, the inner cup having perforations for air excess, the outer one providing protection from contamination. The prepared substrate was sterilized at 121°C for 30 min, then inoculated with the spore suspension of the test fungi to a final concentration of 10 colony forming units (CFU)/g DW, and loosely packed into the inner cup (75 g/cup). The spore suspension was prepared by washing spores from the surface of 10 days old sporulating potato dextrose agar plate cultures of the respective fungi with 0.1% Tween 80 containing water. The inoculated cups were incubated at 28°C in 99% relative humidity chamber for 12 days. 7



LAB

sweet sorghum

harvested

Freshly

in situ enzyme

Silage

Animal feed

y

SSF

Pulp

COUNTER CURRENT DIFFUSER

^

Juice

Fungi

FERMENTER

Figure 1. Scheme of integrated bioprocessing of sweet sorghum for ethanol production.

A

SILO


178



Larger scale SSF with strain F-498 was performed in aluminum trays (780 χ 510 χ 80 mm), 2000 g wet substrate per tray in a layer thickness of about 5 cm. SSF was performed as above for 6 days at 28°C in a 99% relative humidity chamber. During incubation the trays were covered with plastic wrap and ventilated daily for a few minutes. Analytical Procedures. Fungal growth was determined by measuring the insoluble Ν (protein) content of the fermentum by a modified Kjeldahl procedure (8). The Ν value was multiplied by 6.25 to get the protein content, and by 2.7 to get the estimated biomass content, assuming that 37% of the fungal mycelium is protein (average value observed for 2-3 days growth of 71 reesei Rut C30). Soluble protein, indicative of secretion capability of a fungus, was measured by the bicinchoninic acid method (9). Enzyme activities were determined from the culture extract of SSF samples: 5 g DW fermented substrate was extracted with 95 ml water, containing 0.1% Tween 80, by shaking for 60 min at room temperature. From the centrifuged extract, filter paper activity (FPA) for cellulase was determined by standard IUPAC method (10). Beta-1,4endoglucanase activity (EG) was determined according to Bailey and Nevalainen (77). Xylanase activity was assayed according to Bailey et al. (72). Beta-glucosidase activity (BG) was determined following Kubicek (75). Each variable was tested in four reps (four cups), and each assay was done in duplicate. The means of these tests are shown in the results. The enzyme activities were measured daily from day 2, and the peak enzyme activities are reported on respective days. Results and Discussion The comparison of the peak lignocellulolytic enzyme activities of the selected fungi are shown in Table I. All selected strains had higher enzyme activities than the reference wild strain T. reesei Q M 6a, from which the commercially successful mutant Rut C30 has been developed. Some strains had better beta-glucosidase and xylanase activity than Rut C30. Most strains grew better and produced more enzymes on the richer wheat straw/bran medium, but one isolate, F-498, performed better on extracted sweet sorghum pulp. For industrial applications a short fermentation time is at premium, therefore, fast enzyme production is an important selection criterion. The kinetics of cellulase production on sweet sorghum pulp is shown in Figure 2. The initial rate of cellulase production of strains F-498 and F-105 was almost identical to that of Rut C30. Owing to the rapid enzyme production and the favorable cellulase-beta glucosidasexylanase ratio in the produced enzyme complex, accompanied by a rapid growth rate and lack of strong sporulation, strain F-498 was selected for in situ enzyme production in the sweet sorghum bioprocessing scheme shown in Figure 1. The enzyme production in large tray SSF was satisfactory and reproducible (Table II). The volumetric productivity in this in situ enzyme preparation was 1.35 FPU/ml for cellulase and 570 IU/ml for xylanase, surpassing the volumetric productivities achievable with this strain in submerged fermentation (SF) (1.0 FPU/ml for cellulase and 380 IU/ml for xylanase) (Szakacs, G. and Tengerdy, R.P., Techn. U . Budapest, unpublished data). The volumetric productivities were calculated on the basis of 30 g/100 ml packing density in SSF.


13. SZAKACS & TENGERDY

179

Production of Cellulase & Xylanase

Table I. Lignocellulolytic fermentation (SSF)

enzyme

production

in solid

substrate

Enzyme activities Strain


Trichoderma reesei Q M 6a Trichoderma reesei Rut C30 Trichoderma hamatum TUB F-105 Gliocladium sp. TUB F-498 Trichoderma sp TUB F-426 Pénicillium aculeatum N R R L 2129 Pénicillium funiculosum N R R L 1132 Trichoderma sp. TUB F-482 Trichoderma sp TUB F-486

Substrate

FPA

BG

Xylanase

EG

S

2.68 (8)

1.43 (8)

1500 (8)

420 (8)

W

2.92 (8)

1.82 (8)

2090 (6)

580 (8)

S

9.87(10)

8.70(10)

2020(10)

2410(12)

W

10.60(10)

10.30(10)

3580(10)

4120(10)

S

4.87 (6)

6.07 (8)

3470 (4)

960 (4)

w

6.12(4)

11.41 (6)

7070 (4)

1580 (4)

S

5.67 (6)

14.00(10)

1890 (4)

1510(6)

w

1.53 (6)

10.21 (6)

830 (6)

130(6)

S

5.02 (8)

6.06 (8)

3050 (8)

1180 (8)

S

3.87(12)

18.02(12)

2480 (12)

1170 (8)

w

5.92(10)

33.75 (10)

3060 (8)

1530(10)

S

4.02 (8)

22.83 (8)

2480 (12)

890 (8)

w

2.10(10)

15.30(10)

260(10)

330 (10)

S

3.71 (12)

12.72(12)

2000 (12)

730(12)

w

3.80(10)

14.52(10)

1170(10)

1150(10)

S

3,82(10)

5.01 (10)

1430(10)

610(10)

w

4.60(10)

10.43 (10)

1670(10)

840(10)

w

Conditions: Fermentation in perforated plastic cups, 75 g inoculated substrate/250 ml cup; inoculum 10 spores/g DW; moisture content 75%, t=28°C. The peak enzyme activities are shown on days in parenthesis on two substrates: S = extracted sweet sorghum pulp; W: wheat straw + wheat bran (9+1); FPA = filter paper activity, FPU/g DW; B G = beta glucosidase activity, IU/g DW; xylanase activity, IU/g DW; E G = 1,4-beta-endoglucanase activity, EGU/g DW. The data shown are averages from four parallel cups. 7


180



DAYS Figure 2. The kinetics of cellulase production on sweet sorghum pulp in solid substrate fermentation. A T. reesei Q M 6a; • T. reesei Rut C30; X Gliocladium sp. F-498; • T. hamatum F-105. Table IL Enzyme production by Gliocladium sp. F-498 in large tray SSF Enzyme activities Run No.

Final pH

FPA

BG

Xylanase

EG

1

6.0

4.2

13.4

2370

1570

2

5.9

4.1

7.1

1680

1510

3

6.0

4.0

7.5

2560

1150

4

6.1

4.7

11.7

3090

1530

Conditions: 2000g inoculated extracted sweet sorghum pulp in 780x510x80 mm aluminum tray, 5 cm layer thickness, inoculum 10 spores/g DW, moisture content 75%, t=28°C., fermentation time 6 days, FPA = filter paper activity, FPU/g DW; B G = beta glucosidase activity, IU/g DW; xylanase activity, IU/g DW; E G = 1,4-betaendoglucanase activity, EGU/g DW.



13.

SZAKACS & TENGERDY

181

Production of Cellulase & Xylanase

The conditions of SSF particularly enhanced the production of xylanase compared with SF: for Rut C30 in SF the volumetric productivity was 200 IU/ml, in SSF 1075 IU/ml (Szakacs, G. and Tengerdy, R.P., Techn. U . Budapest, unpublished data). The most rapid and best xylanase producer was strain F-105 as shown in Figure 3. The peak production, 7070 IU/g DW, corresponds to a volumetric productivity of 2140 IU/ml. In conclusion, in situ enzymes produced by SSF are in sufficiently concentrated form to be applied economically in biotechnology processes. These results show that one of the most important critérium for strain selection in SSF is the substrate specificity under SSF conditions. The wild strains selected under these conditions all produced higher levels of enzymes than T. reesei Q M 6a, the parent of the highly successful Rut C30 mutant selected under SF conditions. These strains probably have a greater potential for genetic improvement aimed specifically for enhancing enzyme production by SSF. —ι

8000 η

DAYS

Figure 3. The kinetics of xylanase production on wheat straw in solid substrate fermentation. A T. reesei Q M 6a; • T. reesei Rut C30; • T. hamatum¥-\05.


182


Acknowledgement This research was supported by a USA-Hungary Science Cooperation Grant NSF INT 8722686, by a USA-Hungary Joint Board Grant No. 307/92, and by a grant from the Hungarian Academy of Sciences, OTKA T-017201. Literature Cited


1. 2. 3. 4. 5. 6. 7.

8. 9.

10. 11. 12. 13.

Lonsane, B.K.; Ghildyal, N.P. In "SolidState Cultivation", H.W. Doelle, D.A. Mitchell, C E . Rolz, Eds.; Elsevier, N.Y. 1992, pp. 191-209. Persson, I.; Tjerneld, F.; Hahn-Hagerdal, B. Proc. Biochem. 1991, 26, 65-74. Tengerdy, R.P.; Journal of Scientific and Industrial Research, 1996, 55, 313-316. Tengerdy, R.P.; Szakacs, G.; Sipocz, J. Appl. Biochem. Biotechn. 1996, 57/58, 563-569. Pandey, A. Proc. Biochem. 1992, 27, 109-117. Tengerdy, R.P. In "Solid State Cultivation", H.W. Doelle, D.A. Mitchell, C E . Rolz, Eds.; Elsevier,N.Y. 1992, pp. 269-282. Tengerdy, R.P.; Schmidt, J.; Nyeste, L.; Castillo, M.R.; Szakacs, G.; Pecs, M . ; Sipocz, J. In "Biomass for Energy, Environment, Agriculture and Industry", P. Chattier, A. Beenackers, G. Grossi, Eds.; Pergamon Press, Oxford, U.K. 1995, Vol. 2, pp. 1455-1459. Hach Inc. Manual. In "Procedures Manual: Systems for Food, Feed and Beverage Analysis", Hach Inc., Loveland, CO, 1990, pp. 15-21. Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gardner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N . M . ; Olson, B.J.; Klenk, D . C Anal. Biochem. 1985,150, 76-85. Ghose, T.K. Pure and Appl. Chem. 1987, 59, 257-268. Bailey, M.J.; Nevalainen, K.M.H. Enzyme Microb. TechnoL 1981, 3, 153-157. Bailey, M.J.; Biely, P.; Poutanen, K. J. Biotechnol. 1992, 23, 257-270. Kubicek, C P . Arch. Microbiol. 1982,132, 349-354.


Production of Cellulase and Xylanase with Selected Filamentous

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