Int.J.Curr.Microbiol.App.Sci (2020) 9(4): 284-302 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 4 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.904.035 Optimization of Nutritional Variables Using Response Surface Methodology for Enhanced Antifungal Metabolite Production by Janibacter sp. RC18 from Turmeric Rhizosphere Ruth Chiamaka Osaro-Matthew*, Francis Sopuruchukwu Ire and Nnenna Frank-peterside Department of Microbiology University of Port Harcourt Choba, Nigeria *Corresponding author ABSTRACT Antifungal metabolites from rare actinomycetes have been found attractive for application due to its novelty, potency and environmental friendliness. K e yw or ds Optimization of process variables for enhanced production of antifungal metabolite in Janibacter sp. RC18 was carried out in this study. One factor at a Janibacter sp. time (OFAT) was used for preliminary optimization of fermentation variables RC18, Antifungal (time, temperature, initial pH, carbon and nitrogen sources). A three factor central metabolite , OFAT, composite design (CCD) and response surface methodology (RSM) were Central composite design; Response employed for optimization of the selected significant nutritional variable (starch, surface soybean meal) and CaCO3. The optimum antifungal metabolite production was methodology o obtained at day 7 incubation period inhibition, temperature 30 C, pH 8, starch as carbon source and soybean meal as nitrogen source. Response surface analysis Article Info revealed that the optimum value of the variables were 10.76 g/l of starch, 11.95 g/l Accepted: of soybean meal and 1.57 g/l of CaCO . Under this optimal condition antifungal 04 March 2020 3 Available Online: metabolite production was 23.7 ± 0.09 mm which increased by 22.33 % compared 10 April 2020 to OFAT optimized medium (18.4 ± 0.06 mm). The present study has proved CCD and RSM is a reliable statistical tool for optimization of antifungal metabolite production in actinomycetes. Introduction agrochemicals are recalcitrant xenobiotics persisting in the environment thereby causing The recent call for green or sustainable environmental degradation. It also agriculture is a path to profitable and accumulates in the agricultural produce and environmentally benign agriculture (FAO, can be passed to humans through food chain 2017). The persistent use of synthetic hence leading to health problems (EEA, agrochemicals leads to environmental and 2005). health problem. Most of these synthetic 284 Int.J.Curr.Microbiol.App.Sci (2020) 9(4): 284-302 Microorganisms have gained global attention a strain by OFAT is important. But for as agroactive agents which are alternative to sustainability, variables especially nutritional synthetic agrochemicals. Actinomycetes factors should be incorporated at the correct which are Gram positive bacteria have proved levels, also relationship between the to be a promising microorganisms for dependent and independent variables must be production of various bioactive agent of established hence the need for RSM agricultural importance (Ahmad et al., 2008). optimization. Response surface methodology The genus Janibacter which Janibacter (RSM) which is a collection of mathematical limosus was the first specie reported by and statistical tools is useful for analysing and Martin et al., (1997) belongs to the family optimizing response of multivariate system intrasporangiaceae in the actinomycetales (Kocheki, 2009). order. Some other species of this genus that have been reported are: J. terrae (Yoon et al., Hence the aim of this study is to use OFAT 2000), J. brevis (Imamura et al., 2000), J. for preliminary optimization investigations, melonis (Yoon et al., 2004), J. corallicola RSM and central composite design (CCD) to (Kageyama et al., 2007), J. hoylei (Shivaji et model and statistically optimize process al., 2009), J. alkaliphilus (Li et al., 2012), J. variables for maximum antifungal production cremeus (Hamada et al., 2013), and J. indicus in Janibacter sp strain RC18. Zhang et al., 2014). Some investigations have shown Janibacter as potential biocontrol Materials and Methods agents, Janibacter melonis was reported to be antagonistic against Ralstonia solanacearum Actinomycetes and inoculum preparation by Achari and Ramesh, (2014), and Nimaichand et al., (2015) reported Janibacter Janibacter sp strain RC18 (Genbank antagonistic against Fusarium oxysporum and Accession number MK473882) isolated from Rhizoctonia oryzae-sativea. turmeric rhizosphere was described in precious studies (Osaro-Matthew et al., In bioprospecting, one thing is to source for 2020). Strain RC18 was inoculated into 50ml novel and potent strain another thing is Starch Casein broth in a 100 ml Erlenmeyer sustainable production of bioactive flask. The flask was then incubated in a rotary metabolites by the strain. Antifungal incubator for 150 rpm at temperature 30oC metabolite production is affected by some until absorbance of 0.2 was recorded at OD600 physical (temperature, pH and incubation and 20 µl of this culture was used as seed period) and nutritional factors (carbon, inoculum. nitrogen and mineral) (Bundale et al., 2015). These factors are responsible to make Test phytopathogens fermentation conditions suitable for bacterial growth and metabolites production. One- Alternaria pimpriana DSM 62023 and factor-at-a-time (OFAT) technique which is a Colletotrichum coccodes DSM 2492 were traditional method of optimization that fails to obtained from DSMZ (German collection of describe interactions between variables and microorganism and cell cultures). response is suitable for preliminary study in Collectotricum coccodes DMS 2924 was order to select significant factors before selected as the test organism for RSM proceeding to optimization. experiment since it is a commonly found phytopathogen in Nigeria. Although, identifying significant variables for optimal production of bioactive compound in 285 Int.J.Curr.Microbiol.App.Sci (2020) 9(4): 284-302 Experimental set up Initial pH The experiment was carried out in 100 ml Optimum pH was studied by adjusting initial Erlenmeryer flask containing 50 ml Glycerol fermentation media pH to different pH levels beef extract broth of (Glycerol 10 g/l, Beef (4, 5, 6, 7, 8, 9, 10) using 1 M HCl and 1 M extract 10 g/l, NaCl 5 g/l, CaCO3 2 g/l and NaOH. The fermentation broth was K2HPO4 2.5 g/l,). Fermentation was carried inoculated with strain RC18 and incubated at out in a rotary incubator at 150 rpm, all optimized temperature (30oC) and optimized experiment was done in triplicates. incubation period (7 days). Bioassay Carbon sources At the end of fermentation, antifungal activity To select the best carbon source for maximum was determined using Agar well diffusion antifungal metabolite production five technique (Barry and Thornsberry, 1985), on different carbon sources (glucose, lactose, a PDA (Potato Dextrose Agar). A 5 mm fructose, sucrose and starch) were used in diameter well was made by punching the agar substituting glycerol which was the carbon (inoculated with test organisms) with a sterile source in the primary basal medium one at a steel borer and 50 µl of the culture time. Afterward, fermentation was carried out supernatant obtained after centrifuging at at optimum temperature (30 oC), incubation 5000 rpm for 10 minutes was poured in the time (7 days) and pH 8 and bioassay was well. Incubation was carried out at 25 oC ± 2 carried as previously described. oC for 5 days and the diameter zone of inhibition was measured and recorded in Nitrogen sources millimetres (mm). In order to select the best nitrogen sources for Selection of fermentation conditions enhancing the antifungal activity of the strain Incubation period RC18. Various sources of nitrogen (malt extract, yeast extract, peptone, ammonium Fermentation media inoculated with strain sulphate, and soybean meal) were used to RC18 were incubated at 28 oC on a rotary substitute one by one for beef extract shaker for 10 days for maximum antifungal (nitrogen source) in the primary basal metabolite production and antifungal assay medium and fermentation was carried out was carried out every 24 hours till the day 10. using the best carbon source (starch), at the optimum temperature (30 oC), incubation time Incubation temperature (7 days) and pH 8, thereafter, bioassay was carried as previously described Effect of incubation temperatures on antifungal production of strain RC18 were Experimental design for optimization by studied at different temperature (25 oC, 30 oC, RSM 35 oC, 40 oC and 45 oC). Fermentation was carried out in a Glycerol Beef extract (GBE) Central composite design broth at the optimized fermentation period (day 7) and at the end bioassay was carried The optimization of antifungal production out as previously described. was carried out according RSM. The nutritional variables were optimized for 286 Int.J.Curr.Microbiol.App.Sci (2020) 9(4): 284-302 enhanced antifungal activity using the central βii is the coefficient squared effect, is the composite design, the three selected variables coefficient of interaction effect, and is were carbon, nitrogen and CaCO3. For carbon independent variables under study. This and nitrogen sources, starch and soybean meal design was used to study the effects of the were selected based on the one factor at a variables (main, interaction, and quadratic). It time result, for CaCO3 it was selected based was also used to optimize