Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane bagasse

blO tSOIJ Ct IICHNOLOEI' ELSEVIER

Bioresource Technology68 (1999) 173-178

Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane bagasse Marcel Gutierrez-Correa a*, Leticia Portal a, Patricia Moreno ~, Robert P. Tengerdy u aLaboratorio de Micologia y Biotecnologia, Universidad Nacional Agraria La Molina, Apartado 456 Lima 1, Peru bDepartment of Microbiology, Colorado State University, Fort Collins, CO 80523, USA

Received 8 April 1998; revised 26 June 1998; accepted 10 July 1998

Abstract Trichoderma reesei LM-UC4, the parent strain, and its hypercellulolytic mutant LM-UC4E1 were co-cultured with Aspergillus niger ATCC 10864 in solid substrate fermentation on alkali-treated sugar cane bagasse for cellulolytic enzyme production.

Bagasse was supplemented with either soymeal or with ammonium sulfate and urea, and fermented at 80% moisture content and 30°C. Mixed culturing produced better results with the inorganic supplement. The mutant strain was more responsive to mixed culturing than the parent strain when A. niger was the cooperating partner. In a mixed culture of the mutant with inorganic N-source, 10% more biomass, but 63% more cellulase, 85% more endoglucanase and 147% more fl-glucosidase was produced than in single culture. Since co-culturing helped enzyme production more than growth, it appeared that synergistic interactions not directly related to growth were responsible for increased enzyme production. Although soymeal supplementation increased biomass production in the same mixed culture by 30%, it did not increase enzyme production. Mixed culturing is thus beneficial for the economic production of cellulases on nutritionally poor agricultural residues, without the need for supplementation with expensive organic supplements. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Cellulase; fl-glucosidase; Trichoderrna reesei; Aspergillus niger; Mixed culturing; Solid substrate fermentation

1. Introduction

Lignocellulosic biomass is the most abundant organic raw material in the world. Production of ethanol from renewable lignocellulosic resources may improve energy availability, decrease air pollution, and diminish atmospheric CO2 accumulation (Lynd et al., 1991). Reducing the cost of conversion of lignocellulosics into ethanol will stimulate new markets for the agriculture sector, thereby increasing domestic employment while reducing balance of payments deficits (Wyman, 1994; Tengerdy and Szakacs, 1998). Bioconversion of lignocellulose into ethanol is feasible, and enzyme-based technologies for cellulose hydrolysis are being developed. A critical point in these enzyme-based technologies is the production cost of an efficient enzyme system. Due to its complexity, lignocellulose bioconversion requires the action of multiple enzymes. The complete *Corresponding author. Fax: 51 1 3495670; E-mail: mgclmb@ lamolina.edu.pe

hydrolysis of cellulose requires the action of the cellulase system containing cellobiohydrolase, endoglucanase and fl-glucosidase (Kubicek, 1992). Many microorganisms, both fungi and bacteria can degrade lignocellulose. In nature, lignocellutolytic microbes interact in mixed culture to degrade lignocellulose (e.g., wood decay) (Bayer and Lamed, 1992). Lignocellulolytic enzymes are produced by both bacterial and fungal fermentation in submerged or solid substrate systems (Persson et al., 1991). Resembling the natural habitat of filamentous fungi growing on solid lignocellulosic particles, solid substrate fermentation (SSF) involves the growth of microorganisms on moist solid substrates in the absence of free water (Murthy et al., 1993; Tengerdy, 1996). Its advantages are the low capital costs for equipment, high volumetric productivity and decreased operational costs. Its disadvantages are lower overall productivity, high labor intensity and difficult operational control, but these factors are compensated in developing countries by the low cost of starting materials and the low cost of labor.

0960-8524/99/$ - - see front matter © 1998 Elsevier Science Ltd. All rights reserved. PII: S0960-85 24(98)00139-4

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Mixed culture fermentations are those in which the inoculum consists of two or more organisms. Oriental food fermentations are good examples of this type of fermentation. Other examples include mushroom cultivation, composting, anaerobic digestion of organic matter, dairy fermentation and ensiling (Meers, 1974; Steinkraus, 1982; Wood, 1984; Hogan et al., 1989; Fogarty and Tuovinen, 1991). Product or processspecific mixed culture fermentations have been used for biodelignification and enzyme production (Panda et al., 1983; Duff, 1985; Duff et al., 1987; Wan Yusoff and Thayan, 1991; Castillo et al., 1994; Arora, 1995; Dueflas et al., 1995; Asiegbu et al., 1996; GutierrezCorrea and Tengerdy, 1997, 1998a, 1998b). A series of experiments with mixed culture SSF have been reported from this laboratory, indicating that strain compatibility and nutritional status are determining factors for mixed culturing (Castillo et al., 1994; Duefias et al., 1995; Gutierrez-Correa and Tengerdy, 1997, 1998a, 1998b). The most important observation was that synergistic interactions between compatible partners may overcome nutritional limitations in poor agricultural residues. In this paper earlier observations were corroborated, and strain compatibility was reexamined, using a parent-mutant pair of Trichoderma reesei strains, and a different cooperating partner than in earlier experiments.

2. Methods

2.1. Substrate treatment

Sugar cane bagasse was kindly provided by Sociedad Paramonga Ltd (Peru). This material was thoroughly washed, dried and milled to 1-2 mm particle size using a Wiley mill. The milled bagasse was mixed with 0.12 g NaOH/g dry weight (DW) and autoclaved at 121°C for 20min. After autoclaving, the treated bagasse was washed with tap water, then distilled water until neutrality, and then dried at 80°C. 2.2. Microorganisms and inoculum

The strains Trichoderma reesei LM-UC4 (Julca and Gutierrez-Correa, 1987), its mutant LM-UC4E1 (Gutierrez-Correa and Tengerdy, 1997), and Aspergillus niger ATCC 10864 (Castillo et al., 1994) were used in this work. Stock cultures were maintained on potato dextrose agar slants. Spores were washed from a 7-day agar slant culture with 10 ml sterile 0.01% Tween 80 solution, and 2 ml aliquots (10 v spores/ml) were added to each of several 250ml shake flasks containing 100 ml basal salt solution (Reese and Mandels, 1966) supplemented with 0.2% glucose, 0.2% peptone, 0.4%

treated bagasse and 0.01% Tween 80. The inoculated flasks were incubated at 30°C and 200 rpm for 48 h. 2.3. Solid substrate fermentation

Before fermentation, the C:N ratio of treated bagasse was adjusted to 10:1 by the addition of an adequate nitrogen source. Either 0.14g ammonium sulfate + 0.02 g urea, or 2.48 g soymeal was added to 3 g treated bagasse, and moistened with the basal salt solution to 80% moisture content. This substrate in a 250 ml flask was autoclaved at 121°C for 15 min. Each flask was then inoculated with 1.5% (w/w) T. reesei, either LM-UC4 or LM-UC4E1 mycelial inoculum, and incubated at 30°C in a 95% relative humidity chamber. Based on earlier determination of optimal inoculation procedure (Castillo et al., 1994; Duefias et al., 1995), A. niger (1%, w/w) mycelial inoculum was added to each flask at 36 h for soyl-neal supplemented bagasse and at 48h for ammonium sulfate and urea supplemented bagasse. Single cultures of the three strains were used as control. Triplicate flasks were set up for each experimental variation. 2. 4. Sample processing

The contents of three flasks for each time point were thoroughly blended in either 30 ml (ammonium sulfate supplement) or 50ml (soymeal supplement) distilled water by shaking for 30 min at 150 rpm, filtered and the liquid part was used for enzyme determinations. The solids were collected for dry matter and biomass determinations. Dry weight loss was determined after drying the solids in an oven at 80°C for 48 h. 2.5. Biomass determination

Fungal biomass was estimated indirectly by determining either the mycelial protein content (inorganic nitrogen) or the glucosamine content (soymeal). Mycelial protein was determined as reported by Snyder and Desborough (1978). Glucosamine was measured according to Arima and Uozumi (1967) on dried solids processed as reported by Desgranges et al. (1991). The glucosamine content was related to mycelial protein by the following correlation: y=0.13x-2.38

(r =0.96)

where y is the mycelial protein content (% DW) and x is the glucosamine content (mg/g DW) (GutierrezCorrea and Tengerdy, 1998a). Finally, fungal biomass was estimated by multiplying the mycelial protein content by 2.5 (Duefias et al., 1995).

M. Gutierrez-Correa et al./Bioresource Technology 68 (1999) 173-178

2.6. Enzyme assays Endoglucanase activity (EG) and filter paper activity (FPA) for cellulase were measured according to Mandels et al. (1975). /3-glucosidase activity (BG) was measured as reported by Kubicek (1983). One international unit (IU) of enzyme activity was defined as the amount of enzyme that releases 1/zmol product per min (glucose equivalents for FPA and EG, and p-nitrophenol for BG).

175

partner). The corresponding specific enzyme activities improved even more, indicating that apparent synergistic interactions in co-culturing helped enzyme production more than growth.

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The benefit of mixed culturing was clearly manifested only with the inorganic N-source. The mutant strain was more responsive to mixed culturing than the parent strain (Fig. 2, Table 1). Although biomass production increased only by 10% in such a mixed culture, cellulase activity increased by 63%, endoglucanase activity by 85% and /3-glucosidase activity by 147% (the latter largely due to the A. niger

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Fig. 1. Growth kinetics of T. reesei LM-UC4 ([], l) or T. reesei LM-UC4E1 (A, A) with A. niger (©) in single (open symbols) and mixed (closed symbols) culture SSF on bagasse supplemented with ammoniumsulfate and urea (A) or soymeal(B).

Table 1 Maximum growth and enzyme activities in mixed culture solid substrate fermentation on sugar cane bagasse Cultures

T. reesei LM-UC4 T. reesei LM-UC4E1 A. niger T. reesei LM-UC4 andA. niger T. reesei LM-UC4E1 andA. niger

Nitrogen supplement

Inorganic Soymeal Inorganic Soymeal Inorganic Soymeal Inorganic Soymeal Inorganic Soymeal

Time (h)

120 72 120 72 120 72 120 72 120 72

Biomass (g/g DW)

0,17 0,20 0.20 0,21 0,15 0,16 0,20 0.21 0.22 0.27

Enzyme activities

Specific enzyme activities

(IU/g substrate DW)

(IU/g biomass)

FPA

EG

BG

FPA

EG

BG

6.5 10.0 9.0 13.0 5.6 6.4 8.5 10.7 14.7 15.5

57.2 58.7 70.4 61.2 65.8 51.4 89.5 63.2 129.0 73.4

7.7 19.5 8.8 25.0 13.2 27.3 17.2 18.3 21.7 23.4

38.2 50.0 45.0 61.9 37.3 40.0 42.5 50.9 66.8 57.4

336.5 293.5 352.0 291.4 438.7 321.3 447.5 300.9 586.4 271.8

55.0 97.5 44.0 119.0 88.0 170.6 86.0 87,1 98.6 86,7

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M. Gutierrez-Correa et al./Bioresource Technology 68 (1999) 173-178

The rich organic N-source, soymeal, in contrast, helped only growth but not enzyme production, with correspondingly low specific enzyme activities in mixed cultures. It appears that a nutritionally poorer substrate is actually more suitable for mixed culturing, at least for cellulase production. Soymeal, however, increased xylanase production in mixed cultures (Gutierrez-Correa and Tengerdy, 1998a). The different nutritional responses of mixed cultures to cellulase and xylanase production may be due to different enzyme regulations by nutrient components (Coughlan and Hazelwood, 1993; Haltrich and Steiner, 1994), and to complex synergistic interactions

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in co-culturing (Stahl and Christensen, 1992; White and Boddy, 1992). The somewhat conflicting results in the literature on the effect of nutrients on mixed culturing indicate the need for a careful scrutiny of nutrients, fermentation conditions and microbial interactions on a case-by-case basis (Wan Yusoff and Thayan, 1991; Madamwar and Patel, 1992; Castillo et al., 1994). A nutrient sparing effect is evident particularly for the case of the T. reesei LM-UC4E1 and A. niger interaction in cellulase production. The reason may be symbiotic associations in mixed cultures that not only overcome but overcompensate for nutrient limitations

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Fig. 2. Time course profiles of enzyme production by single and mixed culture SSF on bagasse supplemented with ammonium sulfate and urea (A) or soymeal (B). Symbolsas in Fig. 1.

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Table 2 Comparison of maximum growth and enzyme activities in mixed culture solid substrate fermentation on sugar cane bagasse Cultures

T. reesei LM-UC4 and A. niger T. reesei LM-UC4E1 and A. niger T. reesei LM-UC4 and A. phoenicis T. reesei LM-UC4E1 and A. phoenicis T. reesei LM-UC4 and A. phoenicis

Nitrogen supplement

Inorganic Soymeal Inorganic Soymeal Inorganic Inorganic Inorganic Soymeal

Biomass (g/g DW)

0.20 0.21 0.22 0.27 0.35 0.29 0.20 0.23

Reference

Enzyme activities (IU/g substrate DW) FPA

EG

BG

8.5 10.7 14.7 15.5 13.4 9.7 11.0 9.4

89.5 63.2 129.0 73.4 73.8 20.0 113.2 48.3

17.2 18.3 21.7 23.4 18.1 8.5 18.0 17.9

(1) (1) (2) (2) (3)

(1) This work; (2) Gutierrez-Correa and Tengerdy (1997); (3) Gutierrez-Correa and Tengerdy (1998b).

in t h e substrate. This n u t r i e n t s p a r i n g effect gives a solid e c o n o m i c a d v a n t a g e to m i x e d culturing over single culturing for e n z y m e p r o d u c t i o n on n u t r i e n t limited agricultural waste p r o d u c t s , saving the cost of expensive o r g a n i c s u p p l e m e n t s . The maximum biomass production and enzyme activities o b s e r v e d in p r e v i o u s a n d c u r r e n t e x p e r i m e n t s are c o m p a r e d in T a b l e 2. T h e n u t r i e n t s p a r i n g effect is c o m p a r a b l e with e i t h e r A . niger o r A . p h o e n i c i s as c o o p e r a t i n g p a r t n e r s . Strain compatibility, however, is an i m p o r t a n t m o d i f y i n g factor. F o r the p a r e n t strain, A . p h o e n i c i s was a b e t t e r p a r t n e r for the m u t a n t A. niger. Strain c o m p a t i b i l i t y is a critical f a c t o r in m i x e d culturing, a n d has to b e e s t a b l i s h e d case by case for e a c h application.

3.3. Conclusion

It has b e e n e s t a b l i s h e d in this r e s e a r c h that strain c o m p a t i b i l i t y a n d n u t r i t i o n a l status o f the s u b s t r a t e a r e d e t e r m i n i n g factors for successful m i x e d c u l t u r e f e r m e n t a t i o n . T h e m o s t i m p o r t a n t conclusion f r o m these results is that m i x e d culturing is a d v a n t a g e o u s in n u t r i e n t - l i m i t e d conditions, w h e r e symbiotic associations m a y o v e r c o m e such limitations. In p r a c t i c a l t e r m s it m e a n s that c h e a p e r m e d i a m a y b e u s e d for e n z y m e p r o d u c t i o n by m i x e d cultures t h a n by single cultures, w i t h o u t sacrificing e n z y m e yields.

Acknowledgements This r e s e a r c h was s u p p o r t e d by grants f r o m the Multinational Project of Biotechnology of the Organization o f A m e r i c a n States a n d by N S F g r a n t INT-9214903. T h e a u t h o r s wish to t h a n k M r M a r i o G a r c i a for his technical assistance.

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