The development of technology for the production of biocontrol agents against toxigenic isolates of the Aspergillus genus

Kukuruz je jedan od vodećih izvoznih proizvoda u Republici Srbiji i usev od strategijskog značaja za državnu ekonomiju. Tokom 2012. godine, Republika Srbija se našla se na listi zemalja koje su pretrpele velike ekonomske gubitke od posledica pojave aflatoksigenih gljiva na poljima kukuruza i ulaska aflatoksina u lanac ishrane. Analizom predstavnika gljiva vrste Aspergillus flavus uzorkovanih sa kukuruza uzgajanog na različitim lokalitetima u Republici Srbiji, ustanovljen je visok nivo distribucije izolata sa značajnim genetskim potencijalom za produkciju aflatoksina. Rešenje postojećeg problema, ogleda se u primeni mikrobioloških biopesticida, kao efikasne alternative sintetski dobijenim preparatima, koji i uprkos brojnim negativnim efektima, i dalje zauzimaju vodeću poziciju u trenutno aktuelnoj poljoprivrednoj praksi. Razvoj tehnologije proizvodnje agenasa biološke kontrole toksigenih izolata Aspergillus flavus iniciran je izolacijom i odabirom potentnog biokatalizatora kao nosioca bioprocesa. Među 76 novoizolovanih sojeva Bacillus spp. definisan proizvodni mikroorganizam okarakterisan je najvećim genetskim potencijalom za produkciju antimikrobnih komponenti i najizraženijim supresivnim delovanjem prema ispitivanim sojeva. Dalje faze razvoja bioprocesnog rešenja obuhvatale su optimizaciju kvalitativnog i kvantitativnog sastava hranljivog medijuma za produkciju biokontrolnih agenasa primenom odabranog proizvodnog mikroorganizma. Proizvodnja je skalirana na nivo laboratorijskog bioreaktora ukupne zapremine 16 l radi validacije definisanog optimalnog sastava kultivacionog medijuma (celuloza, 5 g/l, (NH4)2SO4 3,77 g/l, K2HPO4 0,3 g/l i MgSO4∙7H2O 0,3 g/l) i optimizacije procesnih parametara proizvodnje. Maksimizacija efikasnosti proizvedenih agenasa biološke kontrole definisana je pri uslovima koji podrazumevaju, dužinu trajanja kultivacije 96 h, brzinu mešanja 300 o/min i intenzitet aeracije 1,5 vvm. Dizajn bioprocesnog rešenja zaokružen je ispitivanjem mogućnosti formulacije krajnjeg proizvoda primenom čestica biokompatibilnog čvrstog nosača. Poslednja faza istraživanja usmerena je na sagledavanje stanja lokalnog tržišta i ispitivanje stavova poljoprivrednih proizvođača o primeni (bio)pesticida u Republici Srbiji.Maize is one of the leading export products in the Republic of Serbia and a crop of strategic importance for the country economy. In 2012. the Republic of Serbia faced an aflatoxin outbreak in maize fields, resulting in significant economic losses and aflatoxin entrance into the food chain. The analysis of the Aspergillus flavus isolates sampled from maize grown in different localities in the Republic of Serbia revealed the high distribution of strains with the genetic potential to produce aflatoxins. The solution to the existing problem was recognized in microbial biopesticides application, as effective alternative to the synthetically derived products. Besides the numerous hazardous effects, chemical pesticides still take the leading position in current agricultural practice. The development of the production technology of biocontrol agents against toxigenic Aspergillus isolates, was initiated by isolation and selection of potent biocatalyst as the central point of the bioprocess. Among 76 newly isolated strains of Bacillus spp., the producing microorganism was selected based on its highest antagonistic activity against tested phytopathogens and genetic potential for producing antimicrobial components. Further stages of the bioprocess solution development included optimizing the qualitative and quantitative composition of the cultivation medium for the production of biocontrol agents using the selected producing microorganism. Production was scaled to the level of a laboratory bioreactor with a total volume of 16 l to validate the previously defined optimal composition of the cultivation medium (cellulose, 5 g/l, (NH2)2SO4 3.77 g/l, K2HPO4 0.3 g/l and MgSO4∙7H2O 0.3 g/l) and perform optimization of process production parameters. The optimized conditions for the production of the biocontrol agents included the cultivation time of 96 h, the agitation rate of 300 rpm and the aeration intensity of 1.5 vvm. The bioprocess design was completed by investigating the possibility of the final product formulation using the biocompatible carriers. The final research step included the current state of the local market review and exploring agricultural producers’ attitudes towards the use of (bio) pesticides in the Republic of Serbia

The development of technology for the production of biocontrol agents against toxigenic isolates of the Aspergillus genus

Kukuruz je jedan od vodećih izvoznih proizvoda u Republici Srbiji i usev od strategijskog značaja za državnu ekonomiju. Tokom 2012. godine, Republika Srbija se našla se na listi zemalja koje su pretrpele velike ekonomske gubitke od posledica pojave aflatoksigenih gljiva na poljima kukuruza i ulaska aflatoksina u lanac ishrane. Analizom predstavnika gljiva vrste Aspergillus flavus uzorkovanih sa kukuruza uzgajanog na različitim lokalitetima u Republici Srbiji, ustanovljen je visok nivo distribucije izolata sa značajnim genetskim potencijalom za produkciju aflatoksina. Rešenje postojećeg problema, ogleda se u primeni mikrobioloških biopesticida, kao efikasne alternative sintetski dobijenim preparatima, koji i uprkos brojnim negativnim efektima, i dalje zauzimaju vodeću poziciju u trenutno aktuelnoj poljoprivrednoj praksi. Razvoj tehnologije proizvodnje agenasa biološke kontrole toksigenih izolata Aspergillus flavus iniciran je izolacijom i odabirom potentnog biokatalizatora kao nosioca bioprocesa. Među 76 novoizolovanih sojeva Bacillus spp. definisan proizvodni mikroorganizam okarakterisan je najvećim genetskim potencijalom za produkciju antimikrobnih komponenti i najizraženijim supresivnim delovanjem prema ispitivanim sojeva. Dalje faze razvoja bioprocesnog rešenja obuhvatale su optimizaciju kvalitativnog i kvantitativnog sastava hranljivog medijuma za produkciju biokontrolnih agenasa primenom odabranog proizvodnog mikroorganizma. Proizvodnja je skalirana na nivo laboratorijskog bioreaktora ukupne zapremine 16 l radi validacije definisanog optimalnog sastava kultivacionog medijuma (celuloza, 5 g/l, (NH4)2SO4 3,77 g/l, K2HPO4 0,3 g/l i MgSO4∙7H2O 0,3 g/l) i optimizacije procesnih parametara proizvodnje. Maksimizacija efikasnosti proizvedenih agenasa biološke kontrole definisana je pri uslovima koji podrazumevaju, dužinu trajanja kultivacije 96 h, brzinu mešanja 300 o/min i intenzitet aeracije 1,5 vvm. Dizajn bioprocesnog rešenja zaokružen je ispitivanjem mogućnosti formulacije krajnjeg proizvoda primenom čestica biokompatibilnog čvrstog nosača. Poslednja faza istraživanja usmerena je na sagledavanje stanja lokalnog tržišta i ispitivanje stavova poljoprivrednih proizvođača o primeni (bio)pesticida u Republici Srbiji.Maize is one of the leading export products in the Republic of Serbia and a crop of strategic importance for the country economy. In 2012. the Republic of Serbia faced an aflatoxin outbreak in maize fields, resulting in significant economic losses and aflatoxin entrance into the food chain. The analysis of the Aspergillus flavus isolates sampled from maize grown in different localities in the Republic of Serbia revealed the high distribution of strains with the genetic potential to produce aflatoxins. The solution to the existing problem was recognized in microbial biopesticides application, as effective alternative to the synthetically derived products. Besides the numerous hazardous effects, chemical pesticides still take the leading position in current agricultural practice. The development of the production technology of biocontrol agents against toxigenic Aspergillus isolates, was initiated by isolation and selection of potent biocatalyst as the central point of the bioprocess. Among 76 newly isolated strains of Bacillus spp., the producing microorganism was selected based on its highest antagonistic activity against tested phytopathogens and genetic potential for producing antimicrobial components. Further stages of the bioprocess solution development included optimizing the qualitative and quantitative composition of the cultivation medium for the production of biocontrol agents using the selected producing microorganism. Production was scaled to the level of a laboratory bioreactor with a total volume of 16 l to validate the previously defined optimal composition of the cultivation medium (cellulose, 5 g/l, (NH2)2SO4 3.77 g/l, K2HPO4 0.3 g/l and MgSO4∙7H2O 0.3 g/l) and perform optimization of process production parameters. The optimized conditions for the production of the biocontrol agents included the cultivation time of 96 h, the agitation rate of 300 rpm and the aeration intensity of 1.5 vvm. The bioprocess design was completed by investigating the possibility of the final product formulation using the biocompatible carriers. The final research step included the current state of the local market review and exploring agricultural producers’ attitudes towards the use of (bio) pesticides in the Republic of Serbia

In vitro potencijal antagonista Bacillus spp. za suzbijanje fitopatogena Xanthomonas euvesicatoria

Bacterial pathogen Xanthomonas euvesicatoria, principal causer of bacterial spot, represents a significant problem in agricultural practice due to high yield losses in the production of pepper and tomato. The development of resistance to copper pesticides has shifted research, in the field of its suppression, towards biopesticides. In this study, several Bacillus strains were tested against Xanthomonas euvesicatoria strains, isolated from pepper leaves with symptoms of bacterial spot, to select a sufficiently effective antagonist. When it comes to the testing of cultivation broth, containing biomass of tested antagonists, the best results were achieved using isolate Bacillus sp3. On the other hand, when biomass-free supernatants, containing produced antimicrobial compounds, were tested, Bacillus sp1 and Bacillus sp2 have shown the highest antimicrobial activity. The results of this study represent a basis for further development of bioprocess solutions for the production of biopesticides based on Bacillus spp. biomass or antimicrobial compounds, showing high efficiency in suppression of pepper bacterial spot.Bakterijski patogen Xanthomonas euvesicatoria predstavlja glavnog uzročnika bakteriozne pegavosti, stoga predstavlja značajan problem u poljoprivrednoj praksi usled izazivanja velikih gubitaka prinosa u proizvodnji paprike i paradajza. Uobičajena sredstva za suzbijanje i kontrolu ovog fitopatogena predstavljaju preparati na bazi bakra. Međutim, razvoj rezistentnosti prema pesticidima na bazi bakra doveo je do zaokreta u polju suzbijanja ovih patogena ka primeni biopesticida, odnosno bioloških kontrolnih agensa. Bakterije roda Bacillus i njihovi metaboliti sa izraženom antimikrobnom aktivnošću protiv ciljanih fitopatogena predstavljaju najperspektivnije aktivne komponente biokontrolnih preparata za zaštitu bilja. U ovom istraživanju nekoliko sojeva roda Bacillus ispitano je sa ciljem odabira antagonista dovoljno efikasnih u suzbijanju sojeva Xanthomonas euvesicatoria, koji su izolovani sa listova paprike sa simptomima bakteriozne pegavosti. Prilikom testiranja antimikrobne aktivnosti uzoraka kultivacionih tečnosti, koji sadrže i biomasu testiranih antagonista, najbolji rezultati u suzbijanju testiranih fitopatogena su postignuti primenom izolata Bacillus sp3. Sa druge strane, prilikom testiranja antimikrobne aktivnosti supernatanata oslobođenih biomase antagonista, koji sadrže samo produkovana antimikrobna jedinjenja, izolati Bacillus sp1 i Bacillus sp2 su pokazali najveću antimikrobnu aktivnost protiv fitopatogena Xanthomonas euvesicatoria. Rezultati ovog istraživanja predstavljaju osnovu za dalji razvoj bioprocesnih rešenja za proizvodnju biopesticida na bazi biomase ili antimikrobnih jedinjenja koja proizvode antagonisti roda Bacillus, a koji pokazuju visoku efikasnost u suzbijanju bakteriozne pegavosti paprike

Selection of antagonists for biocontrol of xanthomonas euvesicatoria

Xanthomonas euvesicatoria is a worldwide causer of pepper bacterial spot, a bacterial plant disease responsible for massive losses of fresh pepper fruits. Considering the current problems in management of bacterial plant diseases, biological control using antagonistic microbial strains with high potential for plant pathogens suppression emerges as a possible solution. The aim of this study was to select suitable antagonists for suppression of X. euvesicatoria among the bacteria, yeast and fungi from the genera Pseudomonas, Lactobacillus, Saccharomyces and Trichoderma, based on in vitro antimicrobial activity testing using the diffusion disc method. The results of this study have revealed that cultivation broth samples of the antagonists Lactobacillus MK3 and Trichoderma reseii QM 9414, as well as supernatant samples of the antagonist Pseudomonas aeruginosa I128, have showed significant potential to be applied in biological control of X. euvesicatoria. Further research would be required to formulate suitable cultivation medium and optimize bioprocess conditions for production of the proposed pepper bacterial spot biocontrol agents

The Effect of Cultivation Conditions on Antifungal and Maize Seed Germination Activity of Bacillus-Based Biocontrol Agent

Aflatoxin contamination is a global risk and a concerning problem threatening food safety. The biotechnological answer lies in the production of biocontrol agents that are effective against aflatoxins producers. In addition to their biocontrol effect, microbial-based products are recognized as efficient biosolutions for plant nutrition and growth promotion. The present study addresses the characterization of the representative of Phaseolus vulgaris rhizosphere microbiome, Bacillus sp. BioSol021, regarding plant growth promotion traits, including the activity of protease, cellulase, xylanase, and pectinase with the enzymatic activity index values 1.06, 2.04, 2.41, and 3.51, respectively. The potential for the wider commercialization of this kind of product is determined by the possibility of developing a scalable bioprocess solution suitable for technology transfer to an industrial scale. Therefore, the study addresses one of the most challenging steps in bioprocess development, including the production scale-up from the Erlenmeyer flask to the laboratory bioreactor. The results indicated the influence of the key bioprocess parameters on the dual mechanism of action of biocontrol effects against the aflatoxigenic Aspergillus flavus, as well on maize seed germination activity, pointing out the positive impact of high aeration intensity and agitation rate, resulting in inhibition zone diameters of 60 mm, a root length 96 mm, and a shoot length 27 mm

Bacillus-Loaded Biochar as Soil Amendment for Improved Germination of Maize Seeds

Biochar is considered one of the most promising long-term solutions for soil quality improvement, representing an ideal environment for microorganisms’ immobilization. Hence there is a possibility to design microbial products formulated using biochar as a solid carrier. The present study was aimed at development and characterization of Bacillus-loaded biochar to be applied as a soil amendment. The producing microorganism Bacillus sp. BioSol021 was evaluated in terms of plant growth promotion traits, indicating significant potential for production of hydrolytic enzymes, indole acetic acid (IAA) and surfactin and positive tests for ammonia and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Soybean biochar was characterised in terms of physicochemical properties to evaluate its suitability for agricultural applications. The experimental plan for Bacillus sp. BioSol021 immobilisation to biochar included variation of biochar concentration in cultivation broth and adhesion time, while the soil amendment effectiveness was evaluated during maize germination. The best results in terms of maize seed germination and seedling growth promotion were achieved by applying 5% of biochar during the 48 h immobilisation procedure. Germination percentage, root and shoot length and seed vigour index were significantly improved when using Bacillus-biochar soil amendment compared to separate treatments including biochar and Bacillus sp. BioSol021 cultivation broth. The results indicated the synergistic effect of producing microorganism and biochar on maize seed germination and seedling growth promotion, pointing out the promising potential of this proposed multi-beneficial solution for application in agricultural practices

Dairy and Wine Industry Effluents as Alternative Media for the Production of Bacillus-Based Biocontrol Agents

Food industry effluents represent one of the major concerns when it comes to environmental impact; hence, their valorization through different chemical and biological routes has been suggested as a possible solution. The vast amount of organic and inorganic nutrients present in food industry effluents makes them suitable substrates for microbial growth. This study suggests two valorization routes for whey as dairy industry effluent and flotation wastewater from the wine industry through microbial conversion to biocontrol agents as value-added products. Cultivations of the biocontrol strain Bacillus sp. BioSol021 were performed in a 16 L bioreactor to monitor the bioprocess course and investigate bioprocess kinetics in terms of microbial growth, sugar substrate consumption and surfactin synthesis, as an antimicrobial lipopeptide. The produced biocontrol agents showed high levels of biocontrol activity against mycotoxigenic strains of Aspergillus flavus, followed by a significant reduction of sugar load of the investigated effluents by the producing microorganisms. With proven high potential of whey and winery flotation wastewater to be used as substrates for microbial growth, this study provides grounds for further optimization of the suggested valorization routes, mostly in terms of bioprocess conditions to achieve maximal techno-economical feasibility, energy saving and maximal reduction of effluents’ organic and inorganic burden

Kenics Static Mixer Combined with Gas Sparging for the Improvement of Cross-Flow Microfiltration: Modeling and Optimization

The use of membrane filtration as a downstream process for microbial biomass harvesting is hampered due to the low permeate flux values achieved during the microfiltration of fermentation broths. Several hydrodynamic methods for increasing permeate flux by creating turbulent flow patterns inside the membrane module are used to overcome this problem. The main goal of this study was to investigate the combined use of a Kenics static mixer and gas sparging during cross-flow microfiltration of Bacillus velezensis IP22 cultivation broth. Optimization of the microfiltration process was performed by using the response surface methodology. It was found that the combined use of a static mixer and gas sparging leads to a considerable increase in the permeate flux, up to the optimum steady-state permeate flux value of 183.42 L·m−2·h−1 and specific energy consumption of 0.844 kW·h·m−3. The optimum steady-state permeate flux is almost four times higher, whilst, at the same time, the specific energy consumption is almost three times lower compared to the optimum results achieved using gas sparging alone. The combination of Kenics static mixer and gas sparging during cross-flow microfiltration is a promising technique for the enhancement of steady-state permeate flux with simultaneously decreasing specific energy consumption