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Solid-state fermentation plus extrusion to make biopesticide granules

D.J. Daigle1*, W.J. Connick, Jr.1, C.D. Boyette2, M.A. Jackson3 and J.W. Dorner4
1 Southern Regional Research Center, ARS, USDA, P.O. Box 19687, New Orleans, LA 70126, USA E-mail: ddaigle@nola.srrc.usda.gov 2 ARS, USDA, Stoneville, MS 38776, USA 3 NCAUR, ARS, USDA, Peoria, IL 61604, USA 4 NPRL, ARS, USDA, Dawson, GA 31742, USA

Five fungal biocontrol agents useful in agriculture were grown on rice our in plastic bags. The our, infested with Colletotrichum truncatum, an Alternaria sp., Paecilomyces fumosoroseus, or atoxigenic Aspergillus avus and A. parasiticus, was mixed with wheat our, kaolin, and water and extruded into granules. The inoculum survived extrusion and uid bed drying at 50C 392 times better than inoculum produced in liquid fermentation. Depending on the agent, the high level of our infestation permitted a 1:9 to 1:1600 dilution to yield the 1 106 cfu/g in the nal product which is usually needed for biocontrol efcacy.

Introduction Solid-state fermentation (SSF) has been used to produce fungal biocontrol agents for agricultural pests such as weeds, insects, and harmful fungi (Bartlett and Jaronski, 1988; Boyette et al. 1993; Cotty, 1994; Feng et al., 1994; Stirling et al., 1998). SSF allows a fungus to produce hardy, healthy conidia and/or mycelia (Bartlett and Jaronski, 1988; Stirling et al., 1998). Vented, autoclavable, plastic bags are convenient containers in which to conduct SSF (Goettel, 1984; Bartlett and Jaronski, 1988; Ibrahim and Low, 1993). Whole or broken cereal grains are common SSF substrates, but the use of ours also has been reported (Bartlett and Jaronski, 1988; Feng et al., 1994; McCabe et al., 1994). Some SSF systems have been developed that are economically competitive with corresponding submerged culture production (Moo-Young et al., 1983). Extrusion technology recently has been employed to make granules (Pesta) containing fungal biocontrol agent inoculum (produced in liquid culture) in a matrix containing wheat our or a mixture of wheat and rice ours (Daigle et al., 1997). The readily available food source permits rapid proliferation of the inoculum when sufcient moisture becomes available (Connick et al., 1991). Combining SSF (when the infested substrate is used directly as a formulation ingredient), extrusion, and drying processes in sequence in order to matrix-encapsulate living
1998 Chapman & Hall

agents is, we believe, a novel and practical concept for biocontrol products. One impetus for this combination of technologies is the need to increase the fungal viability in granular products above that which often results from use of liquid fermentation-produced inoculum (Daigle et al., 1997). It is not just how much you make, but how much you keep (ST Jaronski, Mycotech Corp., personal communication). Five fungi with a demonstrated utility in biocontrol were selected for this study. Colletotrichum truncatum (Schw.) Andrus and Moore, is a pathogen of hemp sesbania (Sesbania exaltata), a serious weed problem in the Southern United States (Boyette, 1991). An Alternaria pathogen of swamp dodder (Cuscuta gronovii), reduces yields of cranberry, vegetables and other eld crops (Bewick et al., 1990). Paecilomyces fumosoroseus isolate Pfr 3572 is a bioinsecticidal fungus that can infect and kill the silverleaf whitey (Bemesia argentifolii), an insect that causes great damage to numerous crops worldwide. Aspergillus avus NPL 45 and Aspergillus parasiticus CM-2 are non-aatoxinproducing strains that are used as biocompetitors to exclude aatoxin-producing strains of Aspergillus on peanuts, thereby reducing contamination by that toxic, carcinogenic, fungal metabolite (Dorner et al., 1992). The objectives of this study were to determine if: (1) fungal propagules produced by SSF survived extrusion and drying better than propagules produced in liquid culture;
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D.J. Daigle et al. (2) if the infested rice our could be used as an ingredient of an extrudable formulation; and (3) if the extent of fungal agent infestation on the our (cfu/g) would be sufcient to extend the amount of product that could be made by dilution with other formulation ingredients. Materials and methods Fungal biocontrol agents Colletotrichum truncatum was isolated from hemp sesbania seedlings. The Alternaria sp. isolated from swamp dodder was provided by T. A. Bewick, Cranberry Experiment Station, University of Massachusetts, East Wareham. Paecilomyces fumosoroseus strain (ARSEF 3572) was isolated from Bemesia argentifolii in McAllen, TX. Aspergillus avus NPL 45, isolated from peanuts in Georgia, is a naturallyoccurring strain that does not produce aatoxin. Aspergillus parasiticus CM-2 is an atoxigenic, color mutant strain that was produced by UV irradiation of A. parasiticus NRRL 6111, a toxigenic color mutant of A. parasiticus. Liquid culture fermentation Colletotrichum truncatum (mostly conidia) was grown in an aqueous cornmeal/soyour medium (Connick et al., 1996), then homogenized in a blender (6.7 106 cfu/ml). The Alternaria sp. (mycelia) was grown in a modied Richards medium for 10 days at 25C and homogenized (7.5 106 cfu/ml). Paecilomyces fumosoroseus conidia from Sabouraud dextrose maltose agar plates (7-day growth) were used to inoculate a basal salts medium (Jackson et al., 1997) with 8% (w/v) glucose and 2.5% (w/v) casamino acids. Blastospores were produced in the liquid medium for 3 days at 28C and 300 rpm in a rotary shaker incubator (5.9 108 cfu/ml). Cfu/ml were determined by serial dilution plating (spiral plater) on PDA acidied with lactic acid after incubation for 2 days at 25C (Daigle et al., 1997). Solid-state fermentation To an autoclavable polypropylene bag (36 61 cm) with a single 0.2 micron lter (7.6 25.4 cm) was added 700 g long-grain rice our. After heat sealing, a 2-cm slit was made in the bag, covered loosely with tape, and the bag was autoclaved vent-side up, for 60 min. on each of two successive days. The slit facilitated escape of steam during autoclaving. After cooling, a shaken mixture of 400 ml sterile water and 20 APDA plugs (6 mm diameter) from Petri plate cultures was added as inoculant through the slit. For P. fumosoroseus, 10 ml of liquid culture dispersed in 400 ml of sterile water was the inoculum. The slit in the bag was covered securely with tape and the bag was incubated, vent-side up, at 25C (28C for C. truncatum) with a 12-h photoperiod. Clumps of infested our were broken up by applying external pressure every few days to foster aeration, fungal dispersion, and growth during the solid-state fermentation. After the desired fermentation time (25 weeks), the infested our was homogenized in a vertical cutter mixer (Model R10, Robot-Coupe USA). The our produced about 107 cfu/g of C. truncatum, 107108 cfu/g of Alternaria sp., and 108 cfu/g of P. fumosoroseus. Three bags of rice our inoculated with each Aspergillus species were incubated for 2.6 weeks and the infested our was then transferred to a bucket with 8 l of water containing 0.01% Tween 20. The mixture was homogenized (Ultra-Turrax, Tek-Mar, Cincinnati, OH, USA), then centrifuged to concentrate the spore-our mixture for shipment to the extrusion site. The fungal viability in the our (cfu/g) was determined by weighing 2060 mg into a tube with 10 ml of 0.15% water agar and 4 glass beads (6 mm dia.). After soaking for 15 min., the sample was vortexed until dispersed. A 5-ml aliquot of the mixture was added to 95 ml of 0.15% water agar in a plastic bag and mixed for 2 min. (Stomacher Lab Blender, Seward, UK). Fungal colonies were plated (3 plates/sample, 9 plates/treatment)(spiral plater) on APDA and counted after incubation at 25C for 2 days. Preparation of granules The Pesta granules containing uniformly-incorporated fungal inoculum of C. truncatum, Alternaria sp., and P. fumosoroseus were prepared by twin-screw extrusion and uid bed drying (50C) (Daigle et al., 1997). The extruder die plate had 34 holes of 1.6-mm diameter, and extruded granules were 12 mm long. The approximate formulation in common for these agents produced by SSF was 26%(w/w) semolina, 16% kaolin (Type RC-32AF; Thiele Kaolin, Wren, GA, USA), 41% infested rice our, and 17% water (613% with the Alternaria). Variations of a few percent in the formulation ingredients were due to differing amounts of water remaining in the infested our after incubation. The formulation containing fungal agent inoculum produced in liquid culture averaged 30% semolina, 20% kaolin, 25% sterile rice our, and 25% liquid culture medium and fungal biomass. Sterile rice our was included to approximate the composition of the SSF formulation. Formulation ingredients were mixed in the vertical cutter mixer prior to extrusion in a twin-screw extruder, and the granules were dried in a uid bed drier at 50C as previously reported (Daigle et al., 1997). Samples were dried to water activity (aw) 0.10.4 (47% water). Water activity, a measure of the free water in a material,

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Biopesticide granule manufacture has been shown to be an important factor in biocontrol products (Connick et al., 1996). Each batch weighed about 600 g on a dry weight basis. Fungal agentviability in granules with C. truncatum was 103106 cfu/g, 104107 cfu/g with the Alternaria, and 106108 cfu/g with P. fumosoroseus (the lower values for each fungus resulted from use of inoculum from liquid fermentation). The data reported are averages from 24 repeated experiments. Granules containing the Aspergillus spp. were prepared on a 170-kg scale using a twin-screw extruder with a preconditioner (Model TX-52, Wenger, Sabetha, KS, USA). A mixture of 136 kg semolina and 34 kg kaolin was preconditioned with 36.3 kg of water. This mixture was amended further by continuous addition (peristaltic pump) of 27.2 kg of spore suspension (homogenized spore-our concentrate plus water) containing 39 g xanthan gum (Kelzan, Kelco, San Diego, CA, USA) and 5 g Tween 20. After extruding through a die plate with 1.6-mm holes and cutting to 12 mm lengths, the granules were dried at 50C in a continuous bed dryer (Model 4800, Wenger) to aw 0.40.5. Inoculum viability was 107 cfu/g. Data are from one preparation of granules for each Aspergillus species. The fungal viability in the product granules was determined by the method, described above, for infested our. The percent inoculum survival after processing was calculated by dividing the actual cfu/g of the product granules by the theoretical cfu/g (which assumes no cfu losses in processing) times 100. The theoretical cfu/g is the quotient of the weight of rice ourinoculum divided by the total weight of dry solids in the formulation multiplied by the actual cfu/g in the our. The extension factor (EF) is an estimate of the extent to which one unit (by weight) of infested our can be diluted, or extended, to produce X units of product at 1 106 cfu/g. The EF factor was calculated by dividing the total weight of the dry solids (DS) in the formulation by the weight of dry infested rice our (DIRF) times the cfu/g in the product divided by 1 106 cfu/g: EF DS DIRF cfu/g 106

The weight of dry infested our is its weight in the formulation minus the water content determined instrumentally. The value, 1 106 cfu/g, was selected as an often-efcacious concentration based on our experiences with a variety of biocontrol formulations (Connick et al., 1998). Water activity was determined with a CX-2 wateractivity system (Decagon Devices, Pullman, WA, USA) at 2024C, and water content was determined with a moisture determination balance (Model MB 200, Ohaus, Florham Park, NJ, USA). Results and discussion Fungal inoculum produced by SSF survived processing up to 88 times better than liquid fermentation inoculum of C. truncatum, 92 times better for the Alternaria, and 5.5 times better for P. fumosoroseus (Table 1). From the standpoint of

Table 1 Percent inoculum survival and extension factors for fungal biocontrol agents incorporated in Pesta from inoculum produced by liquid or solid-state fermentation (on rice our).
Ferment. Time, (wks) 1.5 2 3 4 5 1.4 2 3 4 0.4 2 2.6 2.6 Ferment. Medium liquid rice our rice our rice our rice our liquid rice our rice our rice our liquid rice our rice our rice our Inoculum Survivala, (%) 0.1 0.3 8.8 3.8 5.3 0.9 82.0 83.0 67.7 16.7 91.5 86.0c 100.0c Extension Factorb 0.2 4.0 0.7 2.7 78 59 79 336 1500 1600

Fungal Agent Colletotrichum truncatum

Alternaria sp.

Paecilomyces fumosoroseus Aspergillus avus Aspergillus parasiticus

a b

Percentage of fungal inoculum in the formulation that survived extrusion and drying (50C). Estimated extent to which one unit by weight of infested our can be diluted (extended) to produce X units of product at 1 106 cfu/g. c Daigle et al., 1997.

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D.J. Daigle et al. inoculumsurvival, a SSF fermentation of 3 weeks was best for C. truncatum, and 23 weeks were best for the Alternaria. A time study was not conducted for P. fumosoroseus, but survival of inoculum was greater than 91% at 2 weeks. Aspergillus spp. inoculum produced by SSF gave 86100% survival in previously reported work (Daigle et al., 1997). Each fungus has an optimal incubation time and temperature for a particular substrate, and will eventually reach a limit of population and become dormant (McCabe et al., 1994). Like survival, the extension factor varied widely among the fungi tested (Table 1). The least extension was obtained with C. truncatum where 1 kg infested rice could, for example, be made into 4 kg product at 1 106 cfu/g (EF 4). Higher extension factors were obtained with the Alternaria (EF 78) and P. fumosoroseus (EF 336). The highest EF, 1600, was obtained with Aspergillus parasiticus. Depending on the fungal agent, it is possible to make a relatively large amount of product from a relatively small amount of infested rice our, thereby minimizing bulk storage and contamination problems. In this study, microbial contamination of the rice our occurred in about 20% of the bags. However, it may be preferable to produce inoculum in several bags so that an entire production run is not jeopardized by contamination as it would be in the case of one large fermentation batch. Drying at 50C is severe for living fungal agents, but this temperature was chosen because it is the minimum that can be controlled in some industrial dryers. The survivability and EF may be greater for some fungi if dried at a lower temperature. Optimizing water activity, mixing, and aeration (vent size andporosity) during the SSF would be of critical importance in a commercial application (MooYoung et al., 1983). Conclusions SSF plus extrusion is an effective combination of commercially-available technologies that encapsulates hardy fungal propagules in granular products for biocontrol of agricultural pests. Rice our and/or other cereal grain ours infested with propagules of a fungal biocontrol agent can be incorporated directly as a formulation ingredient. Even infested whole or broken grains may be suitable in place of our if the material is soft enough to extrude through holes in the die plate. No separate step is needed to harvest the conidia or other propagules. Higher equipment costs and longer fermentation times may be offset by increased inoculum survival, extension of SSFproduced inoculum, greater exibility in choice of ingredients, and control over size, shape, and activity of the product. Field test comparisons are needed to help determine if Pesta granules made by this SSF-extrusion method are more commercially viable than fermented whole grain products. Acknowledgements The authors are grateful to Mary Lovisa, Sarah Joyce, Kelley Williams, Michael Watson, Angela Payne, Valerie Vanderpool, and Jimmy McAlpine (ARS, USDA), as well as B. S. Strahm (Wenger) and personnel at the Extrusion Laboratory, Kansas State University,Manhattan, for technical assistance. We also thank R. C. Ostrowski (United Agri Products), G. Weaver (ConAgra), E. T. Champagne (SRRC), T. A. Bewick (Cranberry Experiment Station, UMass), G. Walker and J. Cascino (Sylvan Bioproducts), and P. Vegas (Comet Rice Ingredients Co.) for helpful discussions and formulation ingredients. References
Bartlett, MC and Jaronski, ST (1988). Mass production of entomogenous fungi for biological control of insects. In: Fungi in Biological Control Systems, MN Burge, ed pp 6185, Manchester: Manchester University Press. Bewick, TA, Stewart, JS, Binning, LK and Stevenson, WR (1990). US Patent 4,915,726. Boyette, CD (1991). Plant Dis 75:62-64. Boyette, CD, Abbas, HK and Connick, WJ, Jr. (1993). Weed Sci 41:678681. Connick, WJ, Jr., Boyette, CD and McAlpine, JR (1991). Biol Control 1:281287. Connick, WJ, Jr., Daigle, DJ, Boyette, CD, Williams, KS, Vinyard, BT and Quimby, PC, Jr. (1996). Biocontrol Sci Technol 6:277284. Connick, WJ, Jr., Daigle, DJ, Pepperman, AB, Hebbar, KP, Lumsden, RD, Anderson, TW and Sands, DC. (1998). Biol Control, in press. Cotty, PJ (1994). Phytopathology 84:12701277. Daigle, DJ, Connick, WJ, Jr., Boyette, CD, Lovisa, MP, Williams, KS and Watson, M (1997). World J Microbiol Biotechnol 6:277284. Dorner, JW, Cole, RJ and Blankenship, PD (1992). J Food Prot 55:888892. Feng, MG, Poprawski, TJ and Khachatourians, GG (1994). Biocontrol Sci Technol 4:334. Goettel, MS (1984). J Microbiol Methods 3:1520. Ibrahim, YB and Low, W (1993). Int J Pest Manage 39:288292. Jackson, MA, McGuire, MR, Lacey, LA and Wraight, SP (1997). Mycol Res 101:3541. McCabe, DE, Martinell, BJ, Paau, A and Graham-Weiss, LL (1994). US Patent 5,288,296. Moo-Young, M, Moreira, AR and Tengerdy, RP (1983). Principles of solid-state substrate fermentation. In: The Filamentous

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Received: 26 May Revisions requested: 17 June Revisions received: 4 August Accepted: 6 August

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