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Optimization of cultivation and separation processes for production of Bacillus sp. biomass intended for application in phytomedicine

dc.contributor.advisorGrahovac, Jovana
dc.contributor.otherDodić, Jelena
dc.contributor.otherGrahovac, Jovana
dc.contributor.otherJokić, Aleksandar
dc.contributor.otherGrahovac, Mila
dc.creatorPajčin, Ivana
dc.date.accessioned2019-11-15T10:46:07Z
dc.date.available2019-11-15T10:46:07Z
dc.date.issued2019-11-08
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/Disertacija156776580277435.pdf?controlNumber=(BISIS)111127&fileName=156776580277435.pdf&id=13357&source=NaRDuS&language=srsr
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dc.identifier.urihttp://nardus.mpn.gov.rs/123456789/11532
dc.description.abstractSve veća potreba za hranom kao posledica povećanja broja ljudi širom sveta dovela je do intenzifikacije poljoprivrednih aktivnosti usmerenih na direktnu proizvodnju hrane i hrane za životinje. U poslednjih nekoliko decenija sve više se govori o problemu prekomerne upotrebe hemijskih pesticida, što je je rezultovalo degradacijom kvaliteta zemljišta, kao i sve većim zagađenjem podzemnih i površinskih voda. Osim toga, nedovoljno naučnih podataka o uticaju hemijskih jedinjenja koja ulaze u sastav pesticida na zdravlje ljudi izaziva još veći stepen zabrinutosti. Stoga se iznalaženje novih načina za kontrolu biljnih štetočina i prevenciju i tretman bolesti biljaka postavlja kao imperativ održive poljoprivredne proizvodnje, a samim tim i održivog razvoja. Jedna od alternativa koje su pokazale najveći potencijal jeste primena biopesticida, koji po svojoj najširoj definiciji predstavljaju žive organizme ili njihove proizvode koji pokazuju sposobnost suzbijanja štetočina. Kada su u pitanju mikrobiološki biopesticidi, bakterije roda Bacillus predstavljaju najzastupljenije mikroorganizme koji se mogu sresti u komercijalno dostupnim proizvodima. Proizvodnja mikrobiolońkih biopesticida, iako u konstantnom porastu, i dalje čini samo mali deo ukupnog svetskog tržišta sredstava za zaštitu bilja, najviše zbog visokih troškova i komplikovanog procesa proizvodnje ovog tipa biokontrolnih agenasa. Stoga je kao osnovni cilj istraživanja ove doktorske disertacije postavljena optimizacija uslova umnožavanja i primarnog izdvajanja biomase soja Bacillus velezensis sa primenom za sprečavanje pojave i širenja bakterioza povrtarskih useva koje izazivaju fitopatogene vrste roda Xanthomonas, koji u Republici Srbiji izazivaju velike ekonomske gubitke u proizvodnji kupusa, paprike i paradajza. Prvi korak optimizacije bioprocesa podrazumevao je odabir optimalnog izvora ugljenika i organskog izvora azota za biotehnološku proizvodnju biokontrolnih agenasa, pri čemu su najbolji rezultati dobijeni primenom glicerola i ekstrakta kvasca, što je otvorilo mogućnost ispitivanja primene sirovog glicerola iz proizvodnje biodizela kao izvora ugljenika. Nakon toga je usledilo modelovanje sastava hranljive podloge na bazi komercijalnog i sirovog glicerola u pogledu ispitivanja uticaja komponenata podloge (glicerola, ekstrakta kvasca, (NH4)2SO4 i K2HPO4) na odabrane odzive (prečnik zona inhibicije i rezidualne koncentracije nutrijenata – glicerola, ukupnog azota i ukupnog fosfora). Dobijeni matematički modeli posmatranih odziva, za koje je dokazano da su statistički značajni na nivou značajnosti od 95%, dalje su korišćeni za optimizaciju sastava hranljivih podloga. Optimizovan sastav hranljive podloge na bazi komercijalnog glicerola je sledeći: glicerol 10 g/l, ekstrakt kvasca 2,83 g/l, (NH4)2SO4 3 g/l, K2HPO4 1,07 g/l i MgSO4∙7H2O 0,3 g/l. Sa druge strane, optimizovan sastav hranljive podloge na bazi sirovog glicerola dobija se primenom sledećih koncentracija nutrijenata: glicerol 10 g/l, K2HPO4 4,66 g/l i MgSO4∙7H2O 0,3 g/l, što ukazuje na mogućnost smanjenja troškova pripreme ove hranljive podloge zbog izostavljanja izvora azota. Validacija dobijenih matematičkih modela izvršena je eksperimentima kultivacije proizvodnog mikroorganizma u uvećanim razmerama - u Vulfovim bocama (2 l) i laboratorijskom bioreaktoru (3 l), čiji su rezultati pokazali zadovoljavajuću korelaciju sa modelima predviđenim vrednostima odziva bioprocesa, pri čemu su bolji rezultati dobijeni primenom hranljive podloge na bazi komercijalnog glicerola i kultivacijom proizvodnog mikroorganizma u laboratorijskom bioreaktoru. Unapređenje procesa unakrsne mikrofiltracije kultivacionih tečnosti dobijenih nakon kultivacije proizvodnog mikroorganizma Bacillus velezensis na hranljivoj podlozi na bazi komercijalnog glicerola primenom različitih hidrodinamiĉkih metoda pokazalo je značajan potencijal primene Kenics statičkog mešača, uduvavanja vazduha i kombinacije ovih metoda za poboljšanje fluksa permeata u stacionarnom stanju. In planta ispitivanje antimikrobne aktivnosti preparata na bazi Bacillus velezensis protiv fitopatogenih sojeva vrste Xanthomonas euvesicatoria, koji izazivaju bakterioznu pegavost paprike, pokazalo je značajan potencijal primene bikontrolnih agenasa na bazi Bacillus velezensis za suzbijanje ovog oboljenja paprike. Ispitivanje šireg spektra delovanja preparata na bazi Bacillus velezensis pokazalo je značajan potencijal za suzbijanje oboljenja izazvanih fitopatogenim sojevima roda Fusarium, koji izazivaju truljenje jabuka u toku skladištenja. Rezultati proistekli iz okvira ove doktorske disertacije predstavljaju osnovu za dalje unapređenje i povećanje razmera biotehnolońkog postupka proizvodnje biokontrolnih agenasa na bazi proizvodnog mikroorganizma Bacillus velezensis, uz predlog rešenja za iskorišćenje sirovog glicerola iz proizvodnje biodizela u biotehnolońkom postupku dobijanja proizvoda sa dodatom vrednošću.sr
dc.description.abstractThe raise of human population worldwide requires higher amount of available groceries, which has led to intensification of agricultural activities directed at production of food and feed. In the last few decades the problem of chemical pesticides‟ overuse has resulted in soil deterioration and contamination of surface and underground water reservoirs. Furthermore, insufficient scientific data concerning the chronic effect of chemicals used in pesticides on human health contribute to the global concern when it comes to pesticides‟ usage. Therefore finding new ways to control plant pests and to prevent or suppress plant diseases has become an imperative for sustainable agricultural production and consequently for sustainable development. One of the alternatives showing great potential is usage of biopesticides, comprising of living organisms or their products able to suppress plant pathogens. When it comes to microbial biopesticides, bacteria of the genus Bacillus are the most often used in commercial biocontrol products. Microbial biopesticides, although being a raising alternative, still represent only small share of the global pesticides‟ market, mostly due to expensive and complicated production process. Hence the main goal of the research in the framework of this PhD thesis was to optimize conditions of cultivation and separation processes for production of Bacillus velezensis biomass intended for application in prevention and suppression of plant diseases caused by phytopathogenic species of the genus Xanthomonas, which cause significant economic losses during agricultural production of cabbage, pepper and tomato in the Republic of Serbia. The first step of bioprocess optimization was to choose optimal carbon and organic nitrogen sources for biocontrol agents‟ production, where the best results have been achieved by using glycerol and yeast extract, which opened a new chapter of possibilities to utilize raw glycerol from biodiesel production as carbon source. After that, modeling of cultivation medium composition based on commercial and raw glycerol was performed in order to investigate the effect of cultivation medium components (glycerol, yeast extract, (NH4)2SO4 and K2HPO4) on the selected responses (inhibition zone diameter and residual concentration of nutrients – glycerol, total nitrogen and total phosphorus). The obtained mathematical models for the selected responses, proved to be statistically significant at the significance level of 95%, were further used for optimization of cultivation media composition. Optimized composition of the cultivation medium based on commercial glycerol was: glycerol 10 g/L, yeast extract 2.83 g/L, (NH4)2SO4 3 g/L, K2HPO4 1.07 g/L and MgSO4∙7H2O 0.3 g/L. On the other hand, optimized composition of the cultivation medium based on raw glycerol was: glycerol 10 g/L, K2HPO4 4.66 g/L and MgSO4∙7H2O 0.3 g/L, which has opened a possibility to reduce cost of cultivation medium preparation by excluding nitrogen sources. Validation of the obtained mathematical models was carried out by cultivating the producing microorganism at a larger scale – in Woulff bottles (2 L) and in a laboratory-scale bioreactor (3 L). Validation results were in good correlation with the model predicted values of the selected responses, where better results were achieved by using cultivation medium based on commercial glycerol and cultivation of the producing microorganism in the laboratory-scale bioreactor. Application of different hydrodynamic methods, such as Kenics static mixer, air sparging and their combination, has showed a significant potential for improvement of cross flow microfiltration of Bacillus velezensis cultivation broth based on commercial glycerol in terms of permeate flux enhancement. Also, significant potential of the Bаcillus velezensis cultivation broth for suppression of phytopathogenic Xanthomonas euvesicatoria strains, which cause bacterial spot of pepper, has been proven during in planta experiments. Investigation of wider spectra of antimicrobial activity expressed by preparation based on Bаcillus velezensis cultivation broth has revealed a significant potential for suppression of Fusarium phytopathogenic species responsible for apple rot during storage. The results arising from the research performed in the framework of this PhD thesis represent solid basis for further improvement and scale-up of biotechnological process for production of biocontrol agents based on Bacillus velezensis, with a proposal for utilization of raw glycerol from biodiesel production in biotechnological production of value-added products.en
dc.languagesr (latin script)
dc.publisherУниверзитет у Новом Саду, Технолошки факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Technological Development (TD or TR)/31002/RS//
dc.rightsAttribution-NonCommercial
dc.sourceУниверзитет у Новом Садуsr
dc.subjectBacillus velezensissr
dc.subjectBacillus velezensisen
dc.subjectbiological controlen
dc.subjectXanthomonasen
dc.subjectbiotechnological productionen
dc.subjectoptimizationen
dc.subjectmicrofiltrationen
dc.subjectbiološka kontrolasr
dc.subjectXanthomonassr
dc.subjectbiotehnološka proizvodnjasr
dc.subjectoptimizacijasr
dc.subjectmikrofiltracijasr
dc.titleOptimizacija uslova umnožavanja i primarnog izdvajanja biomase Bacillus sp. za primenu u fitomedicinisr
dc.title.alternativeOptimization of cultivation and separation processes for production of Bacillus sp. biomass intended for application in phytomedicineen
dc.typeDoktorska disertacijasr


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