EFFECT OF AGITATION RATE ON THE PRODUCTION OF …

APTEFF, 48, 1-323 (2017) UDC: 615.282:579.84:66.023.2 https://doi.org/10.2298/APT1748231M BIBLID: 1450-7188 (2017) 48, 231-244

Original scientific paper

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EFFECT OF AGITATION RATE ON THE PRODUCTION OF ANTIFUNGAL METABOLITES BY Streptomyces hygroscopicus IN A LAB-SCALE

BIOREACTOR

Ivana Ž. Mitrović1*, Jovana A. Grahovac1, Jelena M. Dodić1, Mila S. Grahovac2, Siniša N. Dodić1, Damjan G. Vučurović1, Vanja R. Vlajkov1

1 University of Novi Sad, Faculty of Technology Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

2 University of Novi Sad, Faculty of Agriculture, Trg Dositeja Obradovića 8, Novi Sad 21000, Serbia

The application of antifungal compounds produced by microorganisms in the control of plant diseases caused by phytopathogenic fungi is a promising alternative to synthetic pesticides. Among phytopathogenic fungi, Alternaria alternata and Fusarium avenaceum are significant pathogens responsible for the storage rot of apple fruits. During storage, transport and marketing A. alternata and F. avenaceum can cause significant losses of apple fruits and their control is of great importance for the producers and consumers. In the present study, the effects of agitation rate on the production of antifungal metha-bolite(s) by Streptomyces hygroscopicus in a 3-L lab-scale bioreactor (Biostat® Aplus, Sartorius AG, Germany) against two isolates of A. alternata and two isolates of F. ave-naceum were investigated. The cultivation of S. hygroscopicus was carried out at 27 °C with agitation rates of 100 rpm and 200 rpm during 7 days. The aim was to analyze the bioprocess parameters of biofungicide production in a medium containing glycerol as a carbon source, and examine the effect of agitation rate on the production of antifungal metabolite(s). The in vitro antifungal activity of the produced metabolites against fungi from the genera Alternaria and Fusarium grown on potato dextrose agar medium was determined every 24 h using wells technique. In the experiments conducted in the bioreactor at different stirring speeds, it was found that the maximum production of antifungal metabolites occurred after 96 hours of cultivation. A higher consumption of nutrients and a larger inhibition zone diameter was registered in the experiment with an agitation rate of 200 rpm. KEY WORDS. Streptomyces hygrioscopicus, biocontrol, agitation rate, Alternaria alter-

nata, Fusarium avenaceum

INTRODUCTION During marketing of apple fruits or production of apple juice, fruit may be exposed out of storage for a number of days, under conditions favorable for fungal disease de-velopment. The spoilage of apples by fungi is often accompanied by the production of

* Corresponding author: Ivana Ž. Mitrović, University of Novi Sad, Faculty of Technology Novi Sad, Bulevar

Cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]



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mycotoxins which are a potential threat to human health (1). Beside Penicillium expansum, which is one of the most important storage pathogens of apple fruit worldwide and causes blue mold, a decay that can lead to significant economic losses (2), Alternaria spp. and Fusarium spp. are also listed among the significant storage pathogens of apple fruit (3, 4). Several Alternaria species are associated with moldy core of apples while dry core rot is mostly linked to a single species, A. alternata (5). Spores infect the open calyx of young fruits, and mycelia reach the seed and carpel wall during storage. Moldy-core is characterized by the growth of the mycelia within the locules, with or without penetration into the mesoderm. The disease may become invasive and lead to a slow, dry rot confined to the flesh immediately surrounding the core. External symptoms are rare, although in-fected fruits may color and drop prematurely (6). Approximately, 30 metabolites with possible toxicity are known from various species of Alternaria. Alternariol (AOH), alte-nariol monomethyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN), and altertoxins I, II, and III are considered to be some of the important myco-toxins (7). Fusarium species are primarily known as the causal agents of plant diseases, but fungi of this genus also produce many secondary toxic metabolites that can cause acute or chronic diseases in humans and domestic animals. The most important toxins produced by F. avenaceum are moniliformin, acuminatopyrone and chrysogine, which are most commonly found in the apple products. In addition to these mycotoxins, Sorensen et al. (8) also mentioned enniatine, chlamydosporol, fusarin, and others. Fusarium rot occurrs on apples and other fruits while they are stored and shelved. It causes brown, soft, and watery circular necrosis, that gradually spreads over infected tissue, which becomes slightly sunken, sometimes with dense whitish mycelium on the surface (9). Chemical treatment of ripened fruit has many serious side-effects, especially leaving residues and sometimes causing fruit injury, in addition to the presence of offensive odors under modified storage conditions (10). The wide usage of chemical compounds to prevent plant diseases is often expensive, has adverse effects on humans, causes environmental pollution, and can also be lethal to the beneficial organisms (11). Due to adverse toxicological properties and resistance occurrence, use of chemical fungicides is being reduced, and their application after harvest is prohibited in many countries. The increasing concern for environmental protection and demand for organic farming drives research towards alternative control measures, such as the use of natural antagonists to biologically control plant pathogens (12). Various kinds of microbial antagonists have been investigated as potential antifungal biocontrol agents for plant disease management. Actinomycetes are one of the important groups of soil microorganisms. They are important producers of bioactive compounds and constitute a potential group of biocontrol agents. It is well known that actinomycetes produce 70% to 80% of bioactive secondary metabolites, where approximately 60% of antibiotics developed for agricultural use are isolated from Streptomyces spp. Growth and secondary metabolite production depends on many factors, including soil type, aeration, salinity, relative moisture content, and temperature (13). Most of active metabolites derived from actinomycetes are produced by using a submerged culture. For the production of antifungal components by microorganisms, it is



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necessary to provide an appropriate amount of dissolved oxygen. To achieve this, the appropriate aeration and agitation rate must be defined. When mixing rate increases and the aeration rate is constant, an increase in the amount of dissolved oxygen in the cultivating liquid is expected, but the increase in the shear force may have a negative impact on the microorganism, and further affect its productivity. The morphology of streptomycetes may have direct or indirect effects on production of target products. For example, the formation of compact pellets of S. lividans was found to be of great impor-tance for hybrid antibiotic production, while tylosin production by S. fradiae was impro-ved using a smaller pellet or clump size (14). The study of airflow rates is important in submerged bioreactors because the microorganism grows completely immersed in the culture medium without direct contact with the gas phase, which induces oxygen mass transfer limitation both inside and outside the pellets (15). In general, high agitation speeds can damage microorganisms and, as a result, the cell concentration and producti-vity do not increase (16). On the other hand, low agitation rate can lead to over-aggre-gation of cells and the formation of large pallets, which also do not represent a good form of the product. Therefore, it is very important to determine the effect of increase in agitation rate on the production of antifungal metabolites under specific conditions. In the present study, the effects of agitation rate on the production of antifungal metabolite(s) by S. hygroscopicus against two isolates of A. alternata and two isolates of F. avenaceum in a 3-L lab scale bioreactor (Biostat® Aplus, Sartorius AG, Germany) were investigated. The aim was to analyze the bioprocess parameters of biofungicide production in a medium containing glycerol as a carbon source and examine how an increase in agitation rate influences the course of cultivation under the given conditions.

EXPERIMENTAL

Fungal pathogen Isolates of A. alternata and F. avenaceum were obtained from apple fruit samples showing rot symptoms, collected during 2012, after four months storage in Ultra Low Oxygen storages in Vojvodina Province, Serbia. The pathogens were identified according to pathogenic, morphological and ecological characteristics. Two A. alternata isolates (KA10 and T1Jg3) and two F. avenaceum isolates (KA12 and KA13) were selected as representatives of the collection and used in the study. The isolates were initially grown on Potato Dextrose Agar (PDA) plates for seven days. After seven days, a small amount a mycelia plug 3 mm in diameter was taken from the margin of the colony of each isolate, and added to the flasks containing 50 ml of potato dextrose broth. The flasks were incubated for 48 hours on a rotary shaker (150 rpm) at 25°C. Before use, the culture liquid was filtered through the double layer of sterile cheesecloth.

Antifungal components production Microorganism producer, S. hygroscopicus, was isolated from the soil samples collec-ted from various locations on the territory of Novi Sad, Serbia, and stored in the Micro-



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bial Culture Collection of the Faculty of Technology in Novi Sad, Serbia. According to pathogenic, morphological and ecological characteristics, and the molecular identifica-tion, the isolate was identified as S. hygroscopicus (17). In the previous studies, the selec-ted isolate of S. hygroscopicus exhibited strong antifungal activity against different phytopathogenic fungi, causal agents of post-harvest rots (18, 19). The medium used for the growth of microorganism producer had the following composition (g/l): glucose (15.0), soybean flour (10.0), CaCO3, (3.0), NaCl, (3.0), MgSO4, (0.5), (NH4)2HPO4, (0.5), K2HPO4, (1.0). Composition of the comparative fermentation medium was as follows (g/l): glycerol (15.0), CaCO3 (3.0), NaCl (3.0), MgSO4 (0.5), (NH4)2HPO4 (0.17), K2HPO4 (0.33). The pH of the medium was adjusted to 7 ± 0.1 (Consort C863, Turnhout, Belgium) prior to autoclaving. Production of antifungal metabolites by S. hygroscopicus was carried out in a 3-L bench-scale bioreactor (Biostat Aplus) with a 2-L working volume during 7 days. The fermentation was carried out at 27 °C at the agitation rates of 100 rpm and 200 rpm. Two liters of fermentation medium was inoculated with 10% (v/v) of a preculture after the 72-h growth on a rotary shaker (IKA KS 4000i Control Incubating Shaker) at 150 rpm. The cultivation liquid was tested every 24 h. The sample of the cultivation medium was centrifuged at 10,000 g (Rotina 380 R, Hettich, Germany) for 10 min and the supernatant of cultivation medium was used for further analysis.

Analytical methods The obtained supernatants were filtered through a 0.45-μm nylon membrane (Agilent Technologies, Germany), and then analyzed by HPLC (Thermo Scientific Dionex UltiMate 3000 series) to determine residual glycerol content. The HPLC instrument was equipped with an HPG-3200SD/RS pump, autosampler WPS-3000(T) SL (10-ll injection loop), column ZORBAX NH2 (250 mm 9 4.6 mm, 5 lm), and detector Refracto- Max520. Acetonitrile (75:25, v/v) was used as eluent at a flow rate of 1.2 ml/min, and the elution time was 20 min at the column temperature of 30 °C. The residual nitrogen in the cultivation medium was determined by Total Kjeldahl Nitrogen (TKN, (EPA 60014-79-020)) method (20). A spectrophotometric method was used to determine phosphorus content (21). The biomass from the culture filtrate, separated by centrifugation, was transferred to a preweighed dry filter paper using a clean spatula and then placed in an oven at 55 °C overnight to reach a fixed weight. The growth in terms of biomass accumulation was ex-pressed as g/l culture medium. The biomass from the cultivation medium was measured every 24 h (22).

In vitro antagonistic activity assay The in vitro antagonistic activity assay was performed in 85-mm Petri plates using wells technique (23). Two layers of PDA medium were spread in the plates. The first layer consisted of 2% PDA medium. After solidification, the second layer (5 ml), composed of 1.2% PDA and filtered fungal culture liquid (35%), was added. Three wells



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per plate with a diameter of 15 mm were made and two plates represented one treatment. The antifungal activity of cell-free culture filtrate was also tested. For each treatment, 100 μl of cell-free culture filtrate was added in each well. Sterile distilled water was used as negative control treatment. Antifungal activity of the produced metabolites was tested every 24 h. The assessment of antagonistic activity was carried out by measuring diameter of inhibition zones (mm) - zones around wells with no visible mycelia growth.

Data analysis The data on mycelia growth inhibition of four fungal isolates by antifungal metabo-lites of S. hygroscopuicus during 7 days of cultivation were processed by factorial ANOVA using Software STATISTICA 12 (Statistica 2012).

RESULTS AND DISCUSSION

Effect of agitation rate on substrate consumption and cell growth during the cultivation

It is well known that the nutritional sources like carbon, nitrogen and minerals, as well and the environmental factors, have profound effect on antimicotic production by actinomycetes (24). The optimal agitation rate is very important for a maximum produc-tion of any culture in submerge cultivations and need to be carefully monitored, as it has both beneficial and deleterious effects (rupture of the cells and thus slower growth, chan-ge in cell morphology, foam production at high agitation, etc.) (25). Techapun et al. (26) in their research with Streptomyces sp. Ab 106 observed that the optimal agitation rate was between 150 and 200 rpm. However, the most favorable form of the metabolite pro-duction by streptomicetes is small pellets (27). Low stirring speed can lead to aggregation of cells and the formation of very large pallets, which are not a good form for production of targets agents. On the other hand, higher agitation speed increased the amount of dissolved oxygen and dispersion of macromolecules in the medium, and contributed to the greater growth and better production of antifungal agents, observed in this study. Figure 1 demonstrates the substrate consumption and growth of S. hygroscopicus cells in submerged cultures in the 3-L bioreactor at the agitation rates of 100 rpm and 200 rpm. The time-course of cell growth and substrate consumption during 7 days of cultivation of S. hygroscopicus in the laboratory-scale bioreactor showed that the consumption of carbon and nitrogen was in correlation with the increase in the biomass production of cells. Based on the observed consumption of nutrients during 7 days of cultivation, it can be concluded that there is no lag phase because the cells begin to consume the substrate from the start of the bioprocess. Also, the biomass of cells as an indirect indicator of the cultivation course showed that the exponential phase lasted until the third day, when the stationary phase of the bioprocess, in which the consumption of nutrients is significantly reduced, begins (Figure 1c). The results of the fermentation studies at different agitation rates suggest that the agitation rate of 200 rpm led to a higher consumption of carbon, nitrogen and phospho-



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rous sources, and consequently caused enhanced production of biomass. At the lower agitation speed (100 rpm) the cell biomass as well as substrate consumption were found to be lower. The low biomass and low activity at lower agitation speed could be attribu-ted to the dearth of oxygen being experienced by the organism due to the insufficient mixing (25).

Figure 1. Effect of agitation rates on the residual glycerol content (a), residual nitrogen content (b), biomass (c), and residual phosphates (d),

during 7 days of S. hygroscopicus cultivation in a 3-L bioreactor at 100 rpm (■), and 200 rpm (▲).

At the end of the process, the residual glycerol was lower (4.06 g/l) in the bioprocess with the agitation speed of 200 rpm, than in the bioprocess with 100 rpm (6.87 g/l) (Figure 1a). Similar results were obtained for the nitrogen and phosphate sources (Figures 1b and 1d). At the end of the cultivation process at 200 rpm, the residual amounts of nitrogen and phosphorus were 0.0330 g/l and 0.1832 g/l, while in the cultivation with stirring of 100 rpm they were 0.0566 g/l and 0.3850 g/l, respectively.

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Effect of agitation rate on the production of antifungal metabolite(s) during the cultivation

The statistical analysis was performed to determine whether the duration of cultiva-tion at different agitation rates had an effect on the production of antifungal metabolites effective against four tested isolates.

Table 1. Results of the factorial analysis of variance: sources of variation of the inhibition zone diameter during 7 days of cultivation at 100 rpm

Source of variation

SS Degr. of -Freedom

MS F-value p-value

Cultivation Time

1043.67 7 149.1 318.07 0.00

Test fungi 14.08 3 4.69 10.01 0.00 Time*Fungi 83.58 21 3.98 8.49 0.00 Error 30.00 64 0.47

SS - sum of squares; MS - mean square

As expected, the cultivation time had statistically significant effect (p<0.05) on the inhibition zone diameter at both applied agitation rates (Tables 1 and 2). Also, significant differences between inhibition zones diameter were observed between the fungal isolates. Interaction between these two factors (cultivation time and test fungi) also significantly affected the inhibition zone diameter. However, regardless of the applied mixing speed, the most significant source of variation of inhibition zones diameter was the cultivation time. Since this factor influences significantly the production of antifungal components it is important to determine whether it is possible to reduce duration of the bioprocesses while maintaining or increasing the productivity of target components. Also, it is important to determine whether the optimal cultivation time depends on agitation rate, because this is of great importance for the techno-economic assessment of the justification of the increase in the agitation rate. Table 2. Results of factorial analysis of variance: sources of variation of inhibition zone

diameter during 7 days of cultivation at 200 rpm

Source of variation

SS Degr. of - Freedom

MS F-value p-value

Cultivation Time

1480.66 7 211.52 520.7 0.00

Test fungi 38.03 3 12.68 31.2 0.00 Time*Fungi 44.05 21 2.10 5.2 0.00 Error 26.00 64 0.41

SS - sum of squares; MS - mean square



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Figure 2 presents the mean values of mycelia growth inhibition zone diameter of test phytopathogenic fungi caused by supernatant of S. hygroscopicus cultivation filtrate at 100 rpm stirring speed during 7 days of cultivation. The obtained results showed that the antifungal activity was the strongest after 3 days of cultivation of S. hygroscopicus under defined conditions, with inhibition zones radius over 11 mm, indicating that the applied antifungal agent is potentially highly efficient (28). Also, the fungi from the genera Alter-naria did not show sensitivity during the first 48 hours of cultivation, while the isolates of the Fusarium genus became sensitive to the produced metabolites after 24 hours of cultivation. By inspecting Figure 2 it can be concluded that after 96 hours cultivation A. alternata KA10 was the most sensitive to the produced antifungal metabolites (average inhibition zone 25.33 mm), following by F. avenaceum KA12 (25 mm), A. alternata T1Jg3 (24.33 mm), and F. avenaceum KA13 (23.66 mm). Since the tested isolates belong to different genera their different sensitivity to antifungal components could be expected.

Time*Fungi; LS Means

Current effect: F(21, 64)=8.4910, p=.00000

Effective hypothesis decomposition

T1Jg3 KA10 KA13 KA12

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Figure 2. Mean values of mycelia growth inhibition zone diameter (mm) of test

phytopathogenic fungi caused by supernatant of the S. hygroscopicus filtrate during cultivation at 100 rpm for 7 days.

The experimental results obtained at a stirring speed of 200 rpm are presented in Figure 3. It can be seen that the largest inhibition zone diameter for all test phytopatho-genic fungi occurred in 96 h of cultivation, in the stationary phase of the bioprocess. In view of the fact that the stationary phase of the bioprocess begins between the third and fourth day of cultivation and that maximum amount of antifungal metabolites was formed after the fourth day of cultivation, it can be concluded that the synthesized antifungal metabolites are secondary metabolites.



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Time*Fungi; LS Means

Current effect: F(21, 64)=5.1636, p=.00000

Effective hy pothesis decomposition

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Figure 3. Mean values of mycelia growth inhibition zone diameter (mm) of test phytopathogenic fungi caused by supernatant of the S. hygroscopicus filtrate during

cultivation at 200 rpm for 7 days.

During the first 24 hours of cultivation, no antifungal activity against the isolates was observed, while significant growth inhibition appeared after 72 hours of cultivation of S. hygroscopicus (Figure 3). The largest zone of mycelia growth inhibition was noticed in the phytopathogenic fungi A. alternata KA10 (average inhibition zone diameter 29 mm) and A. alternata T1Jg3 (27.33 mm) in 96 h of cultivation (Figures 3 and 4). Slightly smaller inhibition zones gave phythopatogenic fungi from the Fusarium genus.

Figure 4. Inhibition zones caused by 100 μl of S. hygroscopicus cultivation filtrate

at 200 rpm in 96 h of cultivation: a) A. alternata KA10, b) A. alternata T1Jg3, c) F. avenaceum KA12, d) F. avenaceum KA13



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Since the inhibition zones caused by S. hygroscopicus cultivation filtrate at 200 rpm are larger than in the case of 100 rpm, it can be assumed that a higher mixing rate sig-nificantly affects the morphology of the cells and, consequently, the larger production of antifungal metabolites. Leda et al. (15), in their study using S. viridosporus as micro-organism producer concluded that the increase in agitation rate from 200 to 500 rpm resulted in a consistent decrease in the cell growth and production as well. It is possible that an increase in the mixing rate resulted in high shearing effects that were detrimental to mycelium integrity. Besides, the streptomycin isolates acted differently at different mixing rates.

CONCLUSION The work was concerned with the influence of different agitation rate on the substrate consumption, biomass growth, and the biosynthesis of antifungal metabolite(s) during cultivation of S. hygroscopicus. Observing the time-course of substrate consumption and the cells growth in both bioprocesses it can be concluded that there is no lag phase and the exponential phase lasts until the third day when the stationary phase begins. The re-sults of this study showed that cultivation time has a statistically significant effect on the antifungal activity of S. hygroscopicus. Since the maximum productivity was observed after 96 h of cultivation in both agitation rates, it can be noticed that the produced active components are secondary metabolite(s). In accordance with this fact, it can be concluded that at both agitation rates (100 rpm and 200 rpm), the bioprocess could be stopped after 96 h of cultivation, which is a significant fact from an economical aspect. A larger inhibition zone diameter of the test phytopathogenic fungi observed at the higher mixing rate (200 rpm) after 96 h of cultivation indicates an enhanced oxygen transfer to the cells of S. hygroscopicus and a favorable effect on the cells morphology, making them more productive. Moreover, the results of the study indicate that the isolate of S. hygroscopicus has potential as an antagonist of tested postharvest apple pathogens from the genera Alterna-ria and Fusarium. Hence, after additional studies regarding its activity in vivo under realistic production conditions, it could be used for protection of apple fruits from storage pathogens, while simultaneously helping to solve the problem of using chemical pesticides, which present a great environmental problem.

Acknowledgement

The authors gratefully acknowledge the support of the Ministry of Education, Science and Technological Development of the Republic of Serbia, Project Number: TR- 31002.

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УТИЦАЈ БРЗИНЕ МЕШАЊА НА ПРОДУКЦИЈУ АНТИФУНГАЛНИХ МЕТАБОЛИТА Streptomyces hygroscopicus У ЛАБОРАТОРИЈСКОМ

БИОРЕАКТОРУ

Ивана Ж. Митровић1*, Јована А. Граховац1, Јелена М. Додић1, Мила С. Граховац2, Синиша Н. Додић1, Дамјан Г. Вучуровић1, Вања Р. Влајков1

1 Универзитет у Новом Саду, Технолошки факултет,

Булевар цара Лазара 1, Нови Сад 21000, Србија

2 Универзитет у Новом Саду, Пољопривредни факултет, Трг Доситеја Обрадовића 8, Нови Сад 21000, Србија

Плодови јабуке су током читаве године присутни у исхрани деце и одраслих те је квалитет и здравствена безбедност ових намирница од изузетног значаја. Током вегетације и читавог периода складиштења, транспорта и продаје плодови јабуке су подложни инфекцији различитим фитопатогеним гљивама које плод користе као супстрат за раст, развој и размножавање те узрокују губитке који могу да износе од 50 до 80%. Међу значајним проузроковачима болести ускладиштених плодова јабу-ке налазе се и гљиве из родова Alternaria и Fusarium. Alternaria врсте проузрокују промене, како на листовима, тако и на плодовима јабука, док поједине врсте (A. arborescens, A. tenuissima и A. alternata) могу паразитирати и листове и плодове. Најчешћи проузроковачи трулежи плодова јабуке из рода Fusarium су: F. avena-ceum, F. culmorum, F. lateritium и F. solani. Зараза плодова врстом F. avenaceum може изазвати контаминацију сокова јабуке и осталих производа микотоксинима, секундарним метаболитима ових гљива, штетних за људско здравље. За заштиту плодова јабуке од фитопатогених гљива данас се углавном користе синтетички фунгициди. Међутим, због штетних екотоксиколошких особина и појаве резистент-ности, употреба хемијских фунгицида након бербе плодова забрањена је у великом броју земаља. Загађење животне средине, појава резистентних врста, неселек-тивност и остаци пестицида у храни довели су до потребе проналажења и кориш-ћења природних антагониста за биолошко сузбијање биљних патогена. Актиноми-цете рода Streptomyces представљају потенцијално значајну групу микроорганизама за производњу биоактивних компоненти значајних за пољопривреду. Услови кул-тивације, пре свега интензитет мешања, имају веома важну улогу у производњи антифунгалних агенаса применом Streptomyces врста, с обзиром да значајно утичу на пренос кисеоника у субмерзним култивацијама и на морфологију ћелија произ-водног микроорганизма. У овом раду испитан је утицај различитих интензитета мешања на производњу антифунгалних метаболита актиномицете Streptomyces hyg-roscopicus на пораст два изолата врсте Alternariа alternata и два изолата врсте Fusarium avenaceum. Култивација је изведена у лабораторијском биореактору (Bio-stat® Aplus, Sartorius AG, Germany) запремине 3 l на 27 °C са брзинама мешања од 100 о/мин и 200 о/мин током 7 дана. Циљ је био да се анализирају параметри био-процеса производње антифунгалних агенаса у подлози са глицеролом и испита у којим условима је производња антифунгалних метаболита већа. Ефикасност проду-



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кованих антифунгалних метаболита на тест гљиве A. alternata (КА10 и Т1Јg3) и F. avenaceum (КА12 и КА13) испитивана је након свака 24 h култивације in vitro, дифузионом методом бунарчића. У експериментима који су спроведени у биореак-тору при различитим брзинама мешања (100 и 200 о/мин) утврђено је да се макси-мална производња антифунгалнихг метаболита уочава у 96 сату култивације. Већи пречници зона инхибиције код тестираних фитопатогених гљива и већа потрошња субстрата који се јављају у биореактору са већом брзином мешања, показују да ме-шање са 200 о/мин омогућава боље преношење кисеоника до ћелија S. Hygrosco-picus и има позитивне ефекте на морфологију ћелија производног микроорганизма, чинећи га продуктивнијим. Kључне речи: Streptomyces hygroscopicus, биолошка контрола, интензитет меша-

ња, Alternariа alternata, Fusarium avenaceum.

Received: 07 September 2017. Accepted:04 October 2017.

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