Collection of samples
Soil samples were collected from three different sources of grassland soils at a depth of 10 cm at Obanla, Federal University of Technology, Akure, (FUTA) in Ondo State, Nigeria using soil auger. The above soil samples were used for the isolation of Streptomyces species after been transported in sterile polyethylene bags to the Department of Microbiology laboratory, Obakekere, FUTA.
Determination of physicochemical properties of the soil samples
The physicochemical properties of the soil used for the isolation of Streptomyces were determined. The parameters measured included pH, Calcium, Magnesium, Sodium, Organic Carbon, Organic matter, Potassium, Sodium, Phosphorus and Nitrogen at O.B.C. laboratory, Akure, Ondo State.
Determination of the pH of soil samples
Twenty (20) grams of each soil sample was weighed and put in a 100 ml beaker. A 20 ml of distilled water was added to the sample. The suspension was stirred for fifteen minutes thrice using a stirrer, the electrode was rinsed with the distilled water and wiped with tissue paper, and the pH meter was calibrated using buffer 4 and 7. The pH of the suspension was read twice from the pH meter and the average was calculated (AOAC 2007).
Determination of metal concentrations
Air-dried, ground and sieved soil sample (0.5 g) was accurately weighed into a digestion tube and 6 ml aqua regia and 1.5 ml hydrogen peroxide were measured and added into the digestive tube. The digestion tubes were placed on digestive furnace (Model:KDN-20C, China) and heated at a temperature for 3 h at 180 °C. All the digests were cooled and filtered through Whatman filter paper in to 50 ml volumetric flask. Lanthanum chloride solution (1%) was added to the filtrate and the flask containing the filtrate was made up to the mark with double distilled water. Each sample was digested in replicates of five and transferred to acid washed stoppered glass bottle, labelled and kept for metal analysis. The concentrations of Calcium and Magnesium in the filtrate were determined by using flame atomic absorption Spectrophotometer, sodium and potassium was determined by flame photometer. Final concentration of the element present in soil samples was calculated as:
$$\begin{aligned} & {\text{Concentration}}\,{\text{of}}\,{\text{the}}\,{\text{element}}\,{\text{in}}\,{\text{soil}}\,({\text{mg}}/{\text{kg}}) \\ & = \frac{{Conc.\,({\text{mg}}/{\text{l}}) \times ({\text{ml}})}}{{({\text{g}})}} \\ \end{aligned}$$
where Conc.: concentration of the element obtained (mg/l), V is the final volume of the digested solution (50 ml) and W is the weight of the soil sample (0.5 g).
Determination of organic carbon and organic matter of soil samples
One gram finely ground soil sample was passed through 0.5 mm sieve without loss and was put into 500 ml conical flask, to which 10 ml of 1 N potassium dichromate and 20 ml conc. H2SO4 were added with measuring cylinder. The contents were shaken for a minute and allowed to stand for 30 min. Then 200 ml distilled water, 10 ml orthophosphoric acid and 1 ml diphenylamine indicator were added. The solution was titrated against 0.5 N ferrous ammonium sulfate till the colour changes from blue-violet to green after which the blank titration was carried at the beginning without soil. The results were calculated by making use of the following formulas:
$${\text{Organic}}\,{\text{carbon}}\,\% = {\text{N}} \times ({\text{V1}} - {\text{V2}}) \times 0.{39} \times {\text{mcf}}\quad \quad \quad {\text{S}}$$
where N is the Normality of ferrous ammonium sulfate.
V1 is the Volume of 0.5 N ferrous ammonium sulfate required to neutralize 10 ml of 1 N K2Cr2O7 i.e., blank reading (ml).
V2 is the Volume of 0.5 N ferrous ammonium sulfate needed for titration of soil sample (ml).
S = Weight of air-dry sample (g).
0.39 = 0.003 × 100% × 1.31 (0.003 is the milliequivalent weight of carbon in g). only 77% of the organic matter content is oxidized and a fraction of 100/77 = 1.31
Organic matter (%) is equal to Organic carbon(%) × 1.724.
1.724 is the average content of carbon in soil organic matter is equal to 58%)
Determination of moisture content
Using oven drying method as described by (AOAC 2007), ten (10) g of composite soil sample was taken. The samples were oven dried at 105 °C for 24 h. Dry weight of the sample was taken till it showed its constant weight. The amount of water present in the soil sample is equivalent to the amount of weight lost during the process. The formula below was used to calculate the percentage of moisture content in each of the soil samples (Joel and Amajuoyi 2009).
\({\text{Moisture content }}\left( {{\text{MC}}} \right){ }\left( {\text{\% }} \right){ } = \frac{{{\text{Loss in weight on drying}}\left( {\text{g}} \right)}}{{{\text{Initial sample weight }}\left( {\text{g}} \right)}}\) × 100.
The corresponding moisture correction factor (mcf) for analytical results is calculated as following: \(X\)\({\text{Moisture}}\,{\text{correction}}\,{\text{factor}}\,({\text{mcf}}) = {1}00 + \% {\text{moisture}}\quad \quad \quad {1}00\)\({\text{Moisture}}\,{\text{correction}}\,{\text{factor}}\,({\text{mcf}}) = {1}00 + \% \,{\text{moisture}}\quad \quad \quad {1}00\).
Determination of available phosphorus of soil samples
5 g of air-dried soil samples was weighed into a baker and 35 ml of Phosphorus extracting solution, NH4Cl was measured and added to the content of the baker. The mixture was well stirred for 5 min before filtered using 11 μm Whatsman filter paper of which 4 ml of the filtrate was pipetted into a test tube and 4 ml of ascorbic acid was also added. The resulting mixture was allowed to stand for 30 min on a test tube rack for colour development. The colour developed was blue and the procedure was repeated for the other samples. The standard was also prepared by measuring 0.5 ml of 100 ppm Phosphorus standards and adding 4 ml of indicator M and R solution. Twenty-five (25) milliliters of distilled water were added. The prepared solution was transferred into another test tube, after which a blank was prepared by measuring 4 ml of the ascorbic acid reagent and 25 ml of distilled water into another test tube, after which the available soil Phosphorus absorbance at 660 mm wavelength was read by making use of coming colorimeter model 253 (AOAC 2007).
Determination of total nitrogen content of soil samples
Soil sample (1 g), which previously has been grinded and sieved in a 2 mm wire mesh was weighed and transferred into a 500 ml micro-kjeldahl flask and 20 ml distilled water was added, the mixture was shaken gently for a few minutes and was allowed to stand for about 30 min. A twenty (20) ml concentrated copper oxide catalyst was added to the mixture. The flask was then transferred to a mantle for mechanical heating, the heater was placed in the fume cupboard connected to the electrical outlet socket and was switched. The flask was then left to boil for about 5 h in the fume cupboard. After the digest has been observed to be clear of H2SO4 fumes in the flask, the heater was then switched off. The micro-kejdahl flask was then removed from the heater and allowed to cool, follow by decanting the digest into another flask after which 100 ml of distilled water was added to the content of the flask. The micro-kejdahl flask was then attached to the distillation apparatus. After which a fifty (50) ml boric acid consisting of indicator solution was transferred into 25 ml conical flask. The flask was then placed under the condenser of the distillation apparatus. The tip of the condenser was positioned such that it was about 4 mm above the surface of the boric (H3BO3) solution in the conical flask. The digest was then distilled by allowing hot steam to pass from the steam pot into the digest in the micro-Kjedahl flask, thereby causing the digest to boil and distill over into boric acid. After about 150 ml of the distillate had been collected in boric acid, the distillation was stopped. After which distilled water was titrated with 0.5 m standard hydrogen chloride, colour change at the end of the reaction was from green to pink (AOAC 2007).
Sterilization of glass wares and culture media
Test tubes, beakers, conical flasks and other glass wares used were thoroughly washed, rinsed and drained. Prepared media were corked with cotton wool and foil paper. The glass wares and the media were then sterilized using an autoclave at 121 °C for 15 min in order to avoid contamination. The water used for serial dilution was also sterilized using the autoclave. Inoculating loops were thoroughly and appropriately flamed using a Bunsen burner. Work benches were also swabbed using cotton wool and ethanol to make it free of contamination (Olusola-makinde et al. 2021).
Isolation of Streptomyces species from grass land soils
Ten-fold serial dilution was carried out on each of the soil samples, starch casein agar used for the isolation of Streptomyces species from grass land soils was prepared according to manufacturer’s specification and was sterilized in an autoclave at 121 °C for fifteen minutes, after sterilization, it was allowed to cool. The diluent from dilution factor of five was inoculated into the plates; 20 ml of the cooled agar was then poured into the inoculated plates and was allowed solidified after which the plates were incubated at 28 °C for 5 days.
After incubation, the growth of the organisms was observed, sub culturing was done until pure cultures were obtained, the pure isolates were kept on starch casein agar slants for the screening of chitinase production ability.
Biochemical tests
Biochemical tests were carried out to identify, differentiate and characterize organisms. Gram staining technique was carried out to ascertain the morphology and gram reaction behavior of the isolates, other biochemical tests carried out include: sugar fermentation test, catalase and citrate (Olusola-makinde et al. 2021).
Gram staining
A drop of water was put on a grease free slide using a sterile syringe; an inoculum from a cultured plate was picked using a flamed inoculating loop and mixed with the water on the slide to make a smear. The smear was allowed to dry and passed through flame thrice in order to heat fix the slide. Crystal violet was added for one minute and then rinsed off with water. lugol´s Iodine was added which is the mordant and left for 60 s, then washed with water and allowed to dry. 70% ethanol was then added to decolorize and was washed with water immediately. Counter staining was done with safranin for one minute. It was rinsed with water and then air dried. Immersion oil was added which was then viewed with × 100 lens of the light microscope (Ibisanmi and Aribisala 2022).
Catalase test
A drop of hydrogen peroxide was added on a clean glass slide using a sterile syringe; an inoculum of the test organism was picked from the cultured plate using a flamed inoculating loop and dropped on the hydrogen peroxide. Production of bubbles indicates that the test organism is catalase positive while absence of bubbles indicates that the organism is catalase negative (Olusola-makinde et al. 2021).
Citrate utilization test
Simmons citrate agar was prepared following manufacturer´s specification, i.e., 24 g in 1 L of distilled water; it was sterilized in an autoclave at 121 °C for 15 min. Citrate slope was prepared in sterilized bijou bottle, few colonies of the test organism was picked using a sterile wire loop and inoculated into the citrate slope, It was incubated at 37 °C for 24 h and observed for change in colouration, If the organism has the ability to use citrate, the medium changes its colour from green to blue (Ibisanmi and Aribisala 2022).
Sugar fermentation test
Sugar fermentation test was carried out using maltose, sucrose, fructose and glucose sugars. Peptone broth was prepared following the manufacturer´s specification (i.e., 15 g in 1L of distilled water) and poured into different conical flasks. 1 g of each was measured from carbohydrate into the prepared peptone water then phenol red was added as indicator for acid production, the solution was prepared into universal bottles, Durham tubes were inserted in an inverted position and the media were sterilized. After sterilization, each bottle was aseptically inoculated with the test microorganism using a flamed inoculating loop and incubated at 37 °C for 72 h. Colour change from orange to yellow indicates that there is a drop in the pH because of the production of acid by the fermentation of the sugar present in the media making it positive. gas production is observed as bubble in the inverted Durham tube and noted as a positive result, absence of bubble in the inverted Durham tube and no colour change indicating the bacteria cannot ferment that particular carbohydrate source present in the media which shows it is negative (Olusola-makinde et al. 2021).
Production of colloidal chitin
30 g of chitin flakes were suspended in 200 ml concentrated hydrochloric acid and then incubated in rotary shaker at room temperature for 2 h until the chitin flakes are completely dissolved. With rigid stirring, the resulting solution was poured into 1 l deionized water to form precipitates of colloidal chitin, which were subsequently collected by centrifugation at 7000 rpm for ten minutes at 4 °C. The precipitates were washed with sterile distilled water (30 ml each) several times to bring the pH value of the colloidal chitin suspension to 2.0–3.0. the suspension was then neutralized with 1 m sodium hydroxide and centrifuged again at 7000 rpm for ten minutes and washed with sterile water for two to three times to obtain the low-salt colloidal chitin, the colloidal chitin was then dried in an oven at 60 °C for 24 h in order to obtain chitin pellets. The chitin pellet was kept at 4 °C for further application (Wu et al. 2009).
Screening of Streptomyces isolates for chitinase
Different isolates were screened for the production of chitinase by plate assay method. Nutrient agar medium enriched with 0.1% colloidal chitin as sterilized after which the organism was introduced and incubated at 30 °C for 4 days. Following incubation period, 0.1% Congo red solution was fed over the plates after which it was distained with 1% NaCl, the plates were observed for zone of clearance. Formation of clear utilization zone around the organism was considered as positive (chitin utilizers) (Wu et al. 2009).
The basis of their chitinase activity determined quantitatively (chitinase assay) and qualitatively (Measuring clearance zone) was used in the selection of working strain. Good visible growth of the organism was observed after incubation for 7 days, they showed clear zone around the colonies which indicates extra cellular chitinase production, the specie with the widest zone of inhibition was selected for chitinase production and assayed for the effect of cultural conditions such as carbon source, incubation, pH, temperature, etc.
Production of chitinase
Minimal synthetic medium (MSM) was supplemented with chitin as the carbon source, after that the pH was adjusted to 6.3 with 50 mM phosphate buffer and autoclaved at 121 °C, 15 atm for 20 min, the medium was also subjected to the required environmental factors for maximum chitinase production. The medium was inoculated with a suspension of the best chitinolytic Streptomyces species to give a final suspension and placed in a rotary shaker at 4000 rpm for 20 min and then incubated at 30 °C for 48 h. The culture was harvested at the 36th hour of incubation (Duzhak et al. 2012).
Antifungal activity of the crude enzyme
Antifungal activity of the crude enzyme was determined by agar well diffusion method as described by (Olutiola et al. 2000) Potato dextrose agar (PDA) was prepared following the manufacturer’s specification, it was sterilized and allowed to cool. The mycelium of the test fungi was placed in the center of the petri plates containing PDA and wells were bored at equidistant from each other in the agar plates with the use of a cork borer (diameter 10 mm). The wells were then filled with 0.1 ml of the culture filtrate and 50% mancozeb. Distilled water was used as negative Control. The plates were allowed to stand for one hour to allow diffusion of the metabolite in the filtrate, then incubated at 25 °C for 72 h and observed for zones of inhibition. Three replicates of the experiment were performed and the diameters of the inhibition zones were measured and recorded (Narayana and Vijayalakshmi 2009).
Assay and optimization for chitinase production
Chitinase activity was assayed using a mixture containing 1 ml of 0.5% pure chitin (sigma suspended in 50 Mm acetate buffer pH of 5.2) and 1 ml of enzyme solution. The reaction mixture was then incubated at 370C for 1 h with shaking and was stopped by centrifugation (5000 g/min) for 10 min, 1 ml of dinitrosalicylate (DNS) reagent was added followed by heating at 100 ºC for five minutes. Absorbance of mixture was measured at 540 nm against the blank prepared with sterile production medium without the enzyme (Sadasivam and Manickam 2014).
Effect of temperature on chitinase production
A 50 ml of production medium was prepared, sterilized and 2 ml inoculum was added then incubated at different temperatures at 30 °C, 40 °C, 50 °C, 60 °C, 70 °C and 80 °C after which culture filtrate was harvested for every 24 h. The enzyme activity and protein content were studied for up to 6 days (Gao et al. 2012).
Effect of pH on chitinase production
A 50 ml of production medium was prepared and pH of the medium was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. The sterilized production media were inoculated with 2 ml suspension and incubated under the shaking condition. The enzyme activity and protein content study were done for every day up to 6 days (Gao et al. 2012).
Effect of incubation time
To determine the optimum incubation period for chitinase production, inoculated flasks were incubated in a rotary shaker in 100 rpm at room temperature for about 6 days. Every 24 h, the culture filtrate was harvested and checked for the enzyme activity as well as the total protein content (Gao et al. 2012).
Determination of the effect of carbon source on chitinase production
50 ml of production medium was prepared and then sterilized after which 2 ml inoculum was introduced and incubated for each carbon source. The culture filtrate was harvested for every 6 h each for 2% glucose, fructose, maltose, galactose and sucrose, then the enzyme activity was studied for 36 h (Gao et al. 2012).
Determination of effect of nitrogen source on chitinase production
50 ml of production medium was prepared and then sterilized after which 2 ml inoculum was introduced and incubated for each nitrogen source. The culture filtrate was harvested for every 6 h for NH4Cl, NH4CO3, NH4NO3, NH4 (SO4)2 NH4 (NO3)2 and (NH4) PO4, then the enzyme activity was studied for 36 h (Gao et al. 2012).
Effect of metal ion on chitinase production
50 ml of production medium was prepared and sterilized after which 2 ml inoculum was added and then incubated for the different for metal ions. The culture filtrate was harvested for every 6 h each for the following metal ions; KCl, CaCl2, FeCl3, NaCl, MgCl2, ZnSO4 and MnCl2, then the enzyme activity was studied for 36 h (Gao et al. 2012).