Proceeding of the 5th International Congress on Engineering and Food, 28 May and 3 June, 1989, Cologne, Federal Republic of Germany

CAPALBO, D. M. F.

MORAES, I. O.

MORAES, R. O.

English

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SOLID AND LIQUID WASTE ÜTILIZATION IN FERMENTATION PROCESSES TO GETBACTERIAL INSECTICIDEMORAES I 0*; CAPALBO D M F**; MORAES R 0**** State University of Camplnas/UNICAMP, CP-6131 Center of Technology, 13081 Camplnas-SP, Brasil** CNPDA/EMBRAPA, CP-69 - 13820 JaguariJna-SP, Bra*ll*** CNPq (fellowBhip) - CNPDA/EHBRAPAABSTRACTEconomic reasons have delayed the commercial production of BacÂtùii thuAÎng-ie.M-Î6 insecticide, by fermentation, in Brazil, using conventional substrates. The development of this type of insecticide is very Important in our country, vhere almost AOZ of food and stored grains, "in natura" or processed, are lost by insect pests attack. Based on this fact, this vork was conducted, since 1972 at the School of Food Engineering. It was studied the feasibility of using solid or liquid wastes, as substrates, employing semi-solid or submerged fermentation to produce 6. JChatingieni-ù, endotoxins. Many agroindustrial by-products, including solid residues from paper Industry, residual fermented malt from beer Industry, wastewater from coconut processing, from alcohol fermentation (molasses), from c o m  Industry (cornsteep liquor), and a meal from cookies and biscuits residues of the bakery industry, were used to verify their abilities to support growth and sporulation of 8. X:hii'Ungieni-U, Growth and sporulation in the seml-solld process took 168 hours, and produce a spore count between 10 and 100 billions spores per gram of culture medium, but in the eubmirged fermentation, spore count reached the same range in a shorter time of A8 ~ 72 hours. The fermentative process was followed by determining the growth and sporulation kinetics, the sugar consumption, dlpicolinic acid evolution and pH behaviour. Scale up of the submerged fermentation process was done until 200 liters. In this process, aeration is very important, as well as the agitation rate, and were used 0.8 w m  and 250 rpm, respectively. The use of sôlid and liquid wastes from food Industry is advantageous because this minimized pollution problems of the environment, offering, through fermentation of B. i.huAxngie.niti, a safe product to vertebra'tes, fishes and the eco-system at leastcost. Bioassays with the product are being performed to establish rate dosages against insect-tests, and to deteralna the effective potency of the product (conclusive test), aside the »per« count, that is a presumptive test to standardize the product.INTRODUCTIONThe toxicity of chemical Insecticides to non-target species, particularly to roan, animals and beneficial Insects, In addition to the steady loss of effectiveness were some of the factors that urged the search for new means of Insect control.Pathogens, Inhibitors and competitors are generally accepted as desirable agents as alternatives for pest control. Promising candidate agents and agents already registered for use In agriculture have presented outstanding safety records [1].Biological control has been widely studied and used in many countries throughout the word and to a la'Vge extent also in Brazil. Although in a sporadic manner and in a few localities In the beginning, the study of this p«Bt control method was initiated in Brazil in 1970 [2]. Nowadays state Institutions, universities ^and the Brazilian Agricultural Research Corporation (EMBRAPA) of the Ministry of Agriculture, are carrying out •xperinents in this field [3].The National Research Center for Agriculture Defense (CNPDA) has been giving priority to blocontrol since 1985, and one of the studies that are going on, at CNPDA, is about massive production of BacÀZÎUé thiifUngiiM-U [Et) by fermentation means, that is at present, one of the moat studied and comaercially available blocontrol agent. Its use is permitted up to harvest time on several crops used for frenh consumption, and, more recently on stored grain (4),The toxic activity associated with Bi takes place primarily in the paraspotal crystal formed within the mother cell during sporulation, either ■Ln Vi\JO or in vitfio. This crystal la composed of a glycoprotein subunit that is believed to be a protoxin which is converted to a toxin by proteolytic activity. This specific protein is a feeding poison which destroy the gut eplthella of most Lepidoptera and some Diptera. Among the Lepldoptera there are numerous pest species Important In agriculture, pasture and forestry (5, 6].In addition to the crystalline inclusion, some Bt varieties release a nucleotlde-like particle, called beta exotoxin, during the vegetative growth phase. Other insecticide metabolites produced by some strains of B-i - alfa and gama exotoxlns - as well as some enzymes are of only •econdnry importance for insect control.A mixture of spores and crystals of 8-t are commercially produced and economic marketed in many countries as specific inflectlclde. However, in Brazil economic reasons have restricted the wider use of it. A s ' it is produced by fermentation means, fermentation technology in Brazil has to ■la the production of these endotoxins at reduced coats.Tl]e media for industrlai production of Bt are basically composed of complex carbon and nitrogen sources. In a rapid review one can find that starch and molasses are suitable carbon sources, and protein-rich material of plant and animal origin Such as soybean, corn steep liquor or casein hydrollsates provide good and cheap nitrogen sources for Bi growth and sporulation.Bracillan vork with Ea<Uttai thuAA.ngie.M-UStudies were initiated in 1972, at the Department of Engineering at the Faculty of Food Engineering of the State University of Campinas (UNICAMP)j to explore the feasibility of producing endotoxin preparations of Bt using subm«rged fermentation with cheap liquid by-products as components of the fermentation medium. That Department owned some submerged batch miniand pilot scale fermentors and Its facilities, so they explore the problem of the medium compoaitlon, its price and their Influence on the final cost of the product! The results obtained, generated two industrial patents, (one In 1976 and another In 1985) [7, 8), about the fermentation process by means of submerged culture using sugar cane molasse and corn steep liquor as nutrient medium.Based on the promising results obtained in laboratory and pilot seal* production of Bt, the CNPDA with the cooperation of UNICAMP, initiated in 1986 studies to employ different methods of fermentation to spare the need of large aerated fermentors.Besides these pioneer works in Braril, other institutions are developing studies on Bt genetics, Its side effects, safety, formulation techniques, Improvement of its stability In the field, and so on.THE SEMI-SOLID FERMENTATIOMSemi-solid fermentation means the growth process of microorganisms on solid materials, not in a liquid phase [9, 10]. In this type of fermentation the substrate may be put on a tray or in a flask; after the inoculation the microorganism develops on the substrate. Thé substrate can be ocasionally shaked, so that part of the bottom moves to the top providing the tieceBsary aeration for the growth of the microorganism, and promoting the uniformity of the medium.The culture mediumFirst of all, to obtain an economical production, it requires that the fermentation medium has to be as cheap as possible, as well as ablt to support conveniently the endotoxin production. Many workers have already studied the influence of the,composition media over the sponjlatlon and endotoxin formation [9, 11, 12, 13, 14, 15, 16), but did it for the submerged fermentation process. rTo Inicinte the study of the semi-solid fermentation procès« it vs* necessary to select a suitable substrate. The selection waa made based primarily on the following guidelines!agroindustrial solid by-products, available in the region where the work was going to be done; the by-product must have a stable and nearly constant composition} and, it must be really a by-product or residue of low cost.Many contacts were made with different industries to get information about their liquid and solid residues and by-products generated, their mean chemical composition, the cost and quantities available. From these data, some by-products were selected to be used In the experiments! solid residue from pulp and paper industry (A); residual fermented malt from beer Industry (B); a special kind of meal obtained from residual cookies and biscuits of bakery Industry (C)} two kinds of meal from chicken slaughter house (D and E). •The processBacittai thuAingieJiili var. was used throughout thestudy. Its culture was maintained on slants of nutrient agar'tnediua, la refrigerator. Each month it was transferred to a new slant.Erlenmeyers of 250 ml of nominal capacity, containing 10 g of each residue separately, were used for the semi-solid fermentation experiments. The solid medium stayed between 1.5 and 2.0 cm (height), to f«f1Ht»r»TABLE 1Production of BatittiU tlwAingiin6l6 spores by seml-solld fermentation processResidua used as Time of Maximum number of viablefermentation fermentation PH spores/g of fermentedaedlum (h) medium at harvest timeAsolid residue irom pulp and paper industry01697.25.5 3.0 X  10^^Bresidual fermented malt from beer Industry01442235.78.08 . 12 . 2  X  l o } ^1.0 X 10^'CB e a l  fron residual cookies / biscuits of bakery Industry01696.06.0 7*8 X  10®Dt>«al from chicken »laughter house01695.97.9 1.0 X  10̂ °Emeal from chicken ■laughter house01686.27.8 1 . 2  X  10^°C + ' A  (111)01681926.4 6.16.43.2 X  10[^ 3.0 X  10̂ ** .C + A (Itl) with Addition of mineral ■altrf01682646.96.86 . 87.8 X  loJJ 3.0 X  10'̂ '*C + 81‘ 0 1686.07.5 1 . 6  X  1 0 ®C + B (lil) with addition of mineral •alts01686.07.3 1 . 2  X  1 0 ^ ^S ID /C N P D Aoxlgen transfer for the growing ralcroorganlBms.Ench flnsk was previously sterilired at 1219C for 20 mlnut«s, and then humidified with 3 ml of a 22.5Z sterilized glucose solution inoculated with 7 ml of the submerged pre-fermentation of Bi (nearly 10 bacterial cells/ml, obtained in 15 h).The Incubation time ranged between 0 and 7 days. The flasks were maintained in the incubator, with only one hand-shake each day to avoid formation of clumps, and to promote a good aeration.For analysis purposes, two flasks were taken at each harvest time, and 80 ml of sterilized water was added to each one. They were agitated in a rotary-shaker for 20 minutes at 200 rpm and then the supernatant of both was used to count the viable spores and to do the pH analysis.Immediately after shaking, a sample of the liquid was transferred to a sterilized flask and heat stimulated. After this heat shock, the sample was serially diluted and the count of viable spores was done by means of the pour plate count colony method, on nutrient agar.The liquid phase previously obtained was left resting for 10 nlnute«. This pH, measured with a potenciometer, expressed the pH value of the aemi- -solid fermented sample, as suggested by A.O.A.C. (1984) methods fot meal*.The preliminary fermentation tests described, showed some Interesting results summarized on TABLE 1.The best results were attained using residues A and B or its combination with residue C. ^Many authors [17, 18) affirm that the presence of ions cslclua and magnesium Improves the aporulation of most of the spore-former bacteria.So, it was added to the culture medium a product usually reconnnended for meal animal eupplementatioa. This meal l8 rich in mineral saltft and it ii not expensive. Its addition to the residues into the media (see TABLE 1) increased the sporulation yields as expected.As the fermentation went on, it was observed that the moisture iontent of the medium decreased too much. The evaluation of its content showed nearly 30X of moisture in culture media on the 7^^ day of feraentation. Under very dry conditions, the viable cells became Unviable, and no increase in spores was attained.To overcome this problem, some experiments were done to ftttalo tha appropriated humidification needed by these growing bacteria. Periodically it was added one or two millliters of sterilized water to each {«rtaentatloQ flask. The results attained are summarized In TABLE 2.TABLE 2Influence of periodic humidification on the production of Bacittui thiVilngi.eni^ spores, by semi-solid fermentation proc««s (typical results)Harvest time (h)Moisture content (X)Maximum number of viable spores/g of fermented madiuni at harvest tim«Water added / fl^sk Water added / flask0 ml 1 ml 2 ml 0 ml 1 ml . 2 ml09619828833650.1747.5043.1640.4938.0050.1750.8552.9053.3654.1550.1751.5457.7159.9862.104.5 loj 1.4 X 1091.2 X 10^4.5 X loj 8.9 X 10|5 1.1 X 10 ' 5.3 X 10 31.8 X 10‘24.S t 10^ 9.2 * loli 1*0 it 10“ l.O * 1032 *.0 * 10^2Those data Bhowed that the addition of 2 ml of water each 48 hours was the best quantity. In the flr&t two or three days of fermentation, the erlenneyera became humid inside. Although the temperature was not measured, that humidity certainly Indicated that the temperature of the fermentation medium reached higher values than the observed outside the flasks.In a small scale work volume, the Increased temperature was not a real problem, because with only one handshake of the flask, the medium brought good mixture and the heat excess was dissipated. But it will be a real problem to be solved for the next step of scaling up the process.Th* bio*B9«yIn order to standardize the product, the spore count is not sufficient. So, it vas necessary to establish bioassays with insect-tests, pests of the agriculture, mainly Lepidoptera. Ptodia. -inteApanctitta (stored grains pest) and kiCMunonuiti oMzÂJt (foliar damage) had been chosen as insect-tests and the bioassays demonstrated the toxicity of the product obtained by •ubmerged fermentation. The products resulted from semi-solid fermentation,’ war» not bioassayed yet, but to do these assays the Laboratory of Insect- -testa/CHPDA is also doing inassive production of Attttcct'LA-ia QvmaJxJUJ> (soybaan pest insect).DISCUSSION AND CONCLUSIONfiascd on the results showed previously, it was concluded that the seml- -solid fermentation process could be used for BamUMii thu/Ungie.ni-Ci Bporea production.The fermented malt from beer industry and the solid residue from pulp and paper industry could both be used as a complete tnedlum for growth and aporulstion of 8t. Furthermore, the supllementatlon of these media witK a cheap mineral salt source could improve the production of spores.The semi-solid fermentation process showed some advantnges over the conventional stirred or aerated liquid fermentations, and they can be described mainly because!the medium is relatively simple since only meal plus water is needed. Cheap mineral salt may be added Just to enrich it; the requireci space occupied by tha fermentation equipment is relatively small if compared to the yield of tha product! the conditions under which the microorganism grows are more like the conditions under which it grows in nature; aeration is easyly obtaine<l since there are air spaces between each particle of the substrate; Bine* the product is concentrated in the solid substrate it may be dried and Htored at less cost because less moisture must be removed.Some problems with semi-solid fermentation beccJme obvious when one worka with ittduring the fermentation of small scale batches, no problem occurs, however with large amount of material the heat becomes a problem that must be controlled; monitoring devices to determine moisture, pH and product yield, become a necessity in this type of fermentation. These parameters can not be measured in tha fermentation flask, and the removal of the samples represents a risk of contamination; the substrate treatment must ba considered; in some cases it must be cracked lightly, but the formation of flour ahould be avoided.Braiil it a big country, so as the semi-solid fermentation process dsnonatràted to be economically interesting for Et endotoxins production,it could be adopted in the future for email scale local production. Although promlaing results had been achieved, further studle* will b* required before final evaluation of the suggested approaches.REFERENCES1. Burgesi H.D, (Ed), Microbial Control of Pests and Plant Diseases, 1970- 1980. Academic Press, London, 1981.2. Moraes, I.O., EdCuZtuii thuAÀngÃ.tniÍ6 insecticide production by sumerged fermentation, M.Sc. Thesis, FEA/UNICAHP, 80pp., 1973.3. Moraea, I.O.; Capalbo, D.M.F., The use of Agricultural by product» at culture media for bioinsecticide production. In: Food Englneerins and Process Aplications, Elsevier Applied Science Publishers, 1985, pp. 377-381.4. Burguea, H.D., Strategy for the microbial control of pesta in 1980 andbeyond. In! -------- . Microbial control of pests and plant diseases1970-1980. London, Academic Press, 1985, p. 797-836.5. Andrews Jr., R.E. et alii.. Rocket imraunoelectrophoresis o*f the entomocldal parasporal crystal of BacXtioi ihuAÂngitniíi subsp. kuMtcLki. Appl. ENviron. Microbiol.. «(5) :897-900.6. Hofman, C. & Luthy, f~., Binding and activity of Bacttlui thdfUngiviiii delta-endotoxln to invertebrate cells. Arch. Microbiol., 146!7-11,1986.7. Brazil, 1976, PI 7608688. Processo de fermentação submersa para a pro- dugâo de inseticida bacteriano (BR Patent), 10 pp., 1976.8. Brazil, 1985* PI 8500663. Processo de produção de toxina termoestâval de BaciWoA thuxingitMii (BR Patent), 08 pp., 1985.9. Capalbo, D.M.F. & Moraes, 1.0., Production of proteic protoxin by EacÁZtui thuAÁngiíniii by semi-solid fermentation. Ini SIMPOSIO ANUAL DA ACADEMIA DE CIENCIA DO ESTADO DE sAo PAULO, 12., Campinas, 1988. Anais. Sào Paulo, 1988. V.2, p.46-55.10. Hesaeltine, C.W., Solid state fermentation. Part I. Process Biochea., Ui24-27, 1977.11. Dharmathiti, J.C.; Pantuwatana, S.; Bhunilratana, A., Production of BcLciWii thw’Ungie.niiô subsp. ii^aítínsli and BacJJlui ipha.vu.c.ai strain 2953 on media using a by-product from a oonosodium glutamate . factory. J. Inv. Pathol., ^ ! 231-238, 1985.12. Salama, H.S., BacÃJilui thuAÁ.ngizniii Berliner and its tolé as biological control agent in Egypt. Z. ang. Ent., £0!206-220, 1984.13.   et alii.. Novel fermentation media for production of deltaendotoxins from BCLcJJUbH, thu/úngiíníli. J. Inv. Pathol.» 41l8-19,1983.14. Salama, H.S. et alll., Utilization of fodder yeast and agro-industrial by-products in production of spores and biologically active endotoxins from BacXUui thuAingiiniU . Zbl. Microbiol., 553-563, 1983.15. Scherrer, P.j Luthy, P.; Trumpl, B., Production of delta-endotoxln by Eactttiii thukingizniii aa a function of glucose concentrations. Appl. Microbiol., 25(4):644-646, 1973.16i Smith, R.A., Effect of strain and medium variation on mosquito toxin production by BaclUai thiVdjngiiniiJ, var. ihHAilinill,. Can Microbiol., ^ ( 9 )  « 1089-1092, 1982.17. KolodiieJ, B.J. & Slepecky, R.A., Trace metal requirements for sporulation of BacJUULiU, meqeUe/Uum . J. Bacterlol., 88(4):821-830, 1964.18. Sleptcky, R. & Foster, J.W., Alteration In metal content of spores of SaciZLwi mtgatZAMiJn and the effect on some spore properties. J. Baefrlol.. 78» 117-123, 1959.

Solid and liquid waste utilization in fermentation process to get bacterial insecticide.

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Solid and liquid waste utilization in fermentation process to get bacterial insecticide.

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