Enhanced production antibiotics using green gram husk medium by Streptomyces sp. SD1 using response surface methodology

2.1 Isolation of actinomycetes species

Sampling and isolation of actinomycetes

A total of 10 mangrove samples were collected from Manakudy mangrove at three different stations using pre-labelled plastic containers using a spatula and tightly closed. The collected samples were stored in an ice box and transferred to the laboratory. In this study, the physico-chemical properties of soil samples of three sampling sites were analyzed to study the nature of soil. The properties such as, texture, colour, salinity, nitrogen, phosphorus content, pH, magnesium, chloride and calcium content were analyzed. Actinomycetes were initially isolated from the sample using spread plate method using actinomycetes isolation agar (g/L) (sodium caseinate-2, L-Asparagine-0.1, sodium propionate-4, dipotassium phosphate-0.5, magnesium sulphate-0.001 and agar-15) and starch casein agar (g/L) soluble starch-10, casein-0.3, potassium nitrate-2, magnesium sulphate-0.05, di-potassium hydrogenphosphate-2, sodium chloride-2, ferrous sulphate-0.01 and agar-18), and incubated for 14 to 21 days at 28 °C. The culture media were supplemented with nystatin (50 µg/ml) and gentamycin sulphate (25 µg/ml) to reduce fungal and bacterial contamination, respectively. After 28 days of incubation, the culture was purified by standard method (Hayakawa and Nonomura, 1987; Crawford et al., 1993; Yu et al., 2013).

2.2 Primary screening of actinomycetes for antibiotics production

The antibacterial activity was tested against Staphylococcus aureus MTCC96, Bacillus subtilis MTCC 441 and Escherichia coli MTCC 64. The isolated Actinomycetes were screened for antagonistic activity by inoculating a single line streak of the Actinomycetes in the centre of the petri plate. Then the plates were incubated for 6 days at 28 °C and seeded with various bacterial strains by a single streak and the plates were further incubated at 28 °C for 1–2 days. The antagonistic activity was then determined as zone of inhibition (mm) (Wu, 1984). Streptomyces strain showing good activity against test bacteria was further tested by agar well method.

2.3 Secondary screening

The isolated actinomycetes which showed potent activity against B. subtilis MTCC 441 were inoculated into the Actinomycetes culture medium and incubated for 2 weeks at 175 rpm (Madigan et al., 1997). After 2 weeks, the secondary metabolites were extracted using solvent such as, methanol, ethanol, ethyl acetate, hexane and acetone. The solvent was evaporated and antibacterial assay was performed by Kirby-Bauer method. The ethyl acetate fraction was loaded on a sterile 6 mm disc, dried and placed on B. subtilis MTCC 441 culture plates. To the control disc, ethyl acetate was loaded and dried. The zone of diameter was obtained and the size of zone of inhibition was considered as antibiotic titre as described by Maxwell et al. (1994).

2.4 Morphological characterization and 16S rDNA sequencing

The maximum antibiotics producing Streptomyces strain was grown on ISP medium and the culture was identified based on morphological characters, including pigment production, morphology of spores and aerial hyphae. The isolated potent actinomycete was grown actinomycetes isolation agar medium. 16S rDNA gene sequencing was performed by standard method. Genomic DNA of the potent Streptomyces sp. was performed using DNA purification kit (Qiagen, USA). The 16S rRNA gene was amplified by using the forward (5′-GTTGGATCCAGAGTTTGATCMTGGCT-3′) and reverse (5′- GTTGGATCCACGGYTACCTTGTTA CG-3′), primer (Sambrook et al., 1989).

2.5 Agro-industrial wastes for the production of antibiotics

Pineapple peel, wheat bran, apple pomace, rice bran, tapioca powder, green gram husk and orange peel were dried at 60 °C for overnight. All agro residues were ground well and passed through using a standard mesh (30 mesh sieve). All substrates were directly used without any pretreatment.

2.6 Antibiotics production by solid-state fermentation (SSF)

About 5 g of substrate (pineapple peel, wheat bran, apple pomace, rice bran, tapioca powder, green gram husk and orange peel) was taken and the moisture content was adjusted to 65%. The flask was sterilized and 0.5 ml inoculum was introduced. The culture was mixed frequently and incubated for 8 days at 30 ± 2 °C. After 8 days, the crude secondary metabolites were extracted, filtered, centrifuged and used as the crude antibiotics.

2.7 Screening of variables by traditional method

Antibiotics production was performed using green gram husk as the solid substrate until otherwise stated. The process parameters such as, carbon source, nitrogen sources, divalent ions and minerals were evaluated. The selected medium components were supplemented into the Erlenmeyer flask containing green gram husk as the major nutrient medium. After sterilization, cooled the culture medium, 0.5 ml inoculum (10%, v/w) was inoculated in the Erlenmeyer flasks. The antibiotics production of Streptomyces sp. was measured after 8 days of incubation at 28 ± 2 °C.

2.8 Optimization of medium components

CCD and Response Surface Methodology (RSM) were used to screen maltose, CaCl₂ and NaH₂PO₄ for enhanced production of antibiotics based on the activities obtained from traditional method of optimization. The selected medium components were studied to determine the optimum value. The experiment was performed in 20 different experiment trials with triplicate experiment analysis. For the calculation of statistical model, the matrix was designed based on the following equation −1.$$x_i=\frac{X_i-X_0}{\delta X_i},i=0,1,2,3,\dots ,n,$$where xi – denotes selected independent variables for coded value, Xi – original value of the selected variable, X0 – centre point value of the actual value of the independent variable, and –Xi step change value.

Central composite design and response surface methodology was employed to optimize the process variables. The quadratic model equation is useful to analyze the behaviour of quadratic equation.$$Y={\beta }_0+\mathrm{\Sigma }{\beta }_i{X}_i+\mathrm{\Sigma }{\beta }_{\mathit{ij}}{X}_i{X}_j+\mathrm{\Sigma }{\beta }_{\mathit{ii}}{X}_i^2$$where Y = predicted response, β0 = intercept term, βi = linear coefficient, βij = quadratic coefficient, β = interaction coefficient, XiXi = independent variables.

Analysis of variance (ANOVA) was used to verify the designed model. The significance of the designed model was determined using F-test and the p-value < 0.05 was considered as the designed CCD model was significant. In this experiment, the response which was predicted was validated by triplicate experiment.

3.1 Antibiotics producing actinomycete

Totally, 36 mangrove actinomycetes were screened from three selected sites in Manakudy mangrove forest, Southern India (Fig. 1). The physico-chemical parameters of these three sites were varied widely and the results were tabulated in Table 1. The number of isolated actinomycetes varied among stations. Among all the selected sites, site 3 showed 71% of actinomycetes. Ethyl acetate extract of these isolates effectively inhibited the growth of bacterial pathogens. Actinomycetes isolated from the mangrove soil sediments showed diverse colony shape, colour and texture. Initially, the antimicrobial activity screening was evaluated from the selected Actinomycetes against Staphylococcus aureus MTCC96, Bacillus subtilis MTCC 441 and Escherichia coli MTCC 64. About 59% of the isolates showed antimicrobial properties and most of the samples were found to be effective against Bacillus subtilis MTCC 441. Among the actinomycetes, Streptomyces sp. SD1 showed hyper production; hence this was retained for enhanced production of antimicrobial agents. The morphological characters revealed that it comes under Streptomyces sp. The selected isolate was identified by molecular approach. The growth characteristics of the selected Streptomyces sp. was carried out at various pH and temperature levels. The present finding revealed that this isolate was able to grow well between 22 and 37 °C and at pH ranges from 6.5 to 8.0. The maximum growth was obtained at 28 °C and pH 7.2. This isolate was positive for chitinase, catalase and oxidase test, starch hydrolysis, and showed negative result for indole test and positive for the production of melanin. Actinomyces have been used to produce various antimicrobial lead molecules. The threat of various multiple drug resistant organisms is a serious concern in various countries (Hamers et al., 2012). The present study highlighted on the isolation and screening of Streptomyces from mangrove originating from three locations was screened for potent antimicrobials. The isolated actinomycetes mainly from the genus Streptomyces sp. showed various biological properties. Previously, many Streptomyces sp. were screened for antimicrobials isolated from aquatic environment (Bruntner et al., 2005; Rao et al., 2012). Based on the present finding, Streptomyces sp. SD1 was selected for optimized production of antibiotics in SSF. SSF is effective to synthesize broad spectrum of antimicrobial agents with novel properties, because the process conditions are very closely resemble with natural habitats of Streptomyces sp. than submerged fermentation.

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Fig. 1

Soil sampling station of Manakudy mangrove forest. Samples were collected from these three stations at 10 places. The colour of the soil was black and was collected using a sterile spatula.

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3.2 Agro-industrial residues and antibiotics

The important factors for bacterial growth and production of various biomolecules are particle size, substrate type, and moisture holding capacity of the medium and nutrient value of the raw material in SSF. The small substrate material provides very large surface area for anchoring of microbial cells. At the same time, very small powdery substrates may heavily interfere with respiration of microbial cells, and thus leads to poor growth (Vijayaraghavan and Vincent, 2012; Pandey, 1991). The antibacterial activity of crude extract from Streptomyces sp. SD1 cultured in various substrates was tested against B. subtilis MTCC 441 and the zone of inhibition was determined (Fig. 2a). In the present study, green gram husk stimulated the production of antibiotics than other selected substrates (212 ± 8 U/g substrate) (Fig. 2b). In Streptomyces sp. strain MAR01, wheat bran supported Meroparamycin production than the substrate such as, quaker, bread and ground corn (El-Naggar et al., 2009). Earlier, many Streptomyces sp. have been used for the production of antimicrobial agents using peanut shell, corncob, cassava peels and corn pomace. Among the selected substrates, peanut enhanced tetracycline production (Asagbra et al., 2005).

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Fig. 2a

Antibacterial activity of Streptomyces sp. SD1 against B. subtilis MTCC 441. The strain was cultured in various agro-industrial wastes in solid state fermentation and antibacterial activity was assayed (WB – wheat bran; AP – Apple peel; RB – Rice bran; GGH – Green gram husk; OP – Orange peel; TP – Topioca powder and PAP – Pine apple peel). About 20 µl sample was loaded and incubated for 24 h.

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Fig. 2b

Effect of agro-industrial residues for the production of antibiotics. Streptomyces sp. SD1 was grown on these wastes and antibiotic activity was assayed.

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3.3 Screening of nutrient factors by one-variable at a time approach

Carbon sources influenced on antibiotics production and the results were analyzed. Starch, and maltose maximized antibiotics production, however, sucrose and glucose negatively influenced on the production of antibiotics. The zone of inhibition against the tested bacterial strain was described in Fig. 3a and the total enzyme yield was described in Fig. 3b. The stimulatory effect of various nitrogen sources at 1% (w/w) level on antibiotics synthesis was analyzed. Among the nitrogen sources, beef extract, peptone and yeast extract positively influenced on antibiotics production. The zone of inhibition of secondary metabolites against indicator bacterium was described in Fig. 4a. The antibiotics yield vary widely based on the nitrogen sources used in SSF (Fig. 4b). The green gram husk medium was supplemented with divalent ions and minerals, individually to evaluate the stimulatory effect on antibiotics production. The effect of supplementation of various divalent ions at 0.1% (w/w) concentration was tested and the zone of inhibition was described in Fig. 5a. Ions, such as, calcium chloride, magnesium sulphate and manganese chloride enhanced the production of antibiotics, however supplementation of cobalt chloride and mercuric chloride affected antibiotics production (Fig. 5b). Antibiotics production was critically enhanced by the supplementation of various carbon sources as nutrient supplements in fermentation medium (Mahalaxmi et al., 2010). All selected mineral sources, enhanced the production of antibiotics and the yield was higher than control and the zone of inhibition was calculated (Fig. 5c). The production of secondary metabolite was maximum in the culture medium containing sodium dihydrogen phosphate, followed by potassium dihydrogen phosphate (Fig. 5d). In Streptomyces fradiae NCIM 2418, neomycin production was enhanced by copper sulphate and zinc sulphate with the coconut oil cake (Vastrad and Neelagund, 2014). Also, sodium nitrate and ammonium chloride positively influenced on neomycin production, however, the carbon sources, glucose, sucrose, maltose, starch, ferrous sulphate showed less impact on neomycin production. In S. aureofaciens, production of tetracycline was critically influenced when 10% soluble starch was enriched with sweet potato residues (Yang and Ling, 1989). In Streptomyces rimosus, supplementation of 1% (w/w) CaCO₃, enhance the production of tracycline in SSF when corncob was used as the solid medium (Yang and Swei, 1996).

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Fig. 3a

Antibacterial activity of Streptomyces sp. SD1 against B. subtilis MTCC 441. The strain was cultured in green gram husk medium containing various carbon sources in SSF and antibacterial activity was assayed (ST – Starch; MA – Maltose; GL – Glucose; XY – Xylose and SU – Sucrose). About 20 µl sample was loaded and zone of inhibition was measured.

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Fig. 3b

Effect of carbon source (1%) for the production of antibiotics from green gram husk by using Streptomyces sp. SD1 in SSF.

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Fig. 4a

Antibacterial activity of Streptomyces sp. SD1 against B. subtilis MTCC 441. The strain was cultured in green gram husk medium containing various nitrogen sources in SSF and antibacterial activity was assayed (BE – Beef extract; YE – Yeast extract; PE – Peptone; CA – Casein and OM – Oat meal). About 20 µl sample was loaded and zone of inhibition was measured.

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Fig. 4b

Effect of nitrogen source (1%) for the production of antibiotics from green gram husk by using Streptomyces sp. SD1 in SSF.

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Fig. 5a

Antibacterial activity of Streptomyces sp. SD1 against B. subtilis MTCC 441. The strain was cultured in green gram husk medium containing various ionic sources in SSF and antibacterial activity was assayed. The divalent ions, Co²⁺, Ca²⁺, Mg²⁺, Hg²⁺ and Mn²⁺ were incorporated with the culture medium. About 20 µl sample was loaded and zone of inhibition was measured.

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Fig. 5b

Effect of ionic source (1%) for the production of antibiotics from green gram husk by using Streptomyces sp. SD1 in SSF.

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Fig. 5c

Antibacterial activity of Streptomyces sp. SD1 against B. subtilis MTCC 441. The strain was cultured in green gram husk medium containing various mineral sources (Na₂H – disodium hydrogen phosphate; K₂H – dipotassium hydrogen phosphate; KH₂P – potassium dihydrogen phosphate; NaH – sodium dihydrogen phosphate; C – control). About 20 µl sample was loaded and zone of inhibition was measured.

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Fig. 5d

Effect of minerals (1%) for the production of antibiotics using green gram husk by Streptomyces sp. SD1 in SSF.

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3.4 Central composite design and response surface methodology

The selected nutrient factors enhanced the production of antibiotics and the yield varies from 246 U/g to 1459 U/g. The designed matrix and the response of CCD were tabulated (Table 2a). The F-value of the fitted quadratic design was 167.79 implies the designed quadratic model is statistically significant. In this designed model all factors and interactive effects were statistically significant. The predicted R² value was 0.9663 and the adjusted R² value was 0.987. The lack of fit value was 0.3654 and was statistically insignificant (Table 2b). As described in the results, RSM are key tool for identifying and determining the concentration of nutrients for antibiotics production. The 3D graph described the interaction between Maltose and CaCl₂, Maltose and NaH₂PO₄, Maltose and NaH₂PO₄. All three 3D response surface graphs showed maximum yield of antibiotics at higher concentration of nutrient sources (Fig. 6a–c). The predicted response was 1468 U/g and this was almost similar with experimental value (1473 U/g) validate the designed model. In this study, antibiotics production enhanced 2.9 fold by Streptomyces sp. SD1 in optimized medium than unoptimized medium. In recent years, RSM has been extensively used for the production of various antibiotics. RSM was used for the synthesis of neomycin (Vastrad and Neelagund, 2014), fibrinolytic enzymes (Vijayaraghavan et al., 2016) and streptomycin (Saval et al., 1993).

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Fig. 6

a–c. Response surface plots explaining the individual and interactive effects of factors on the antimicrobial activity of Streptomyces sp. SD1.

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