several clinically significant antibiotics as well as widely used drugs against common diseases have been derived from this unique genus affiliated with the order actinomycetales .
diverse members of this order including streptomyces have been isolated from previously unexplored natural habitats to nourish the current microbial antibiotic searching programs [ 2 , 3 ] .
jaj06 is a gram - positive , moderately halophilic streptomyces strain , which has previously been isolated from hypersaline coastal solar saltern at tuticorin , india .
this strain produces an antimicrobial polyketide compound which has been reported to have potent antimicrobial activity against a set of bacteria and yeast with significant minimal inhibitory concentrations .
production of antibiotic compound in jaj06 has been reported to have seawater dependence . like many other complex medium components ,
seawater has nondefined composition which might affect the reproducibility of the production profile of microorganisms . to maintain a reproducible production profile ,
media components and their optimum levels are critical to the secondary metabolites produced by microorganisms . in the field of antibiotics , much effort was directed toward optimizing production rates and directing the product spectrum .
production of microbial secondary metabolites in a microbial system can be improved by optimization of physical parameters and nutritional constituents of production medium .
the optimization experiments are usually performed using nonstatistical one - factor - at - a - time [ 9 , 10 ] and statistical experimental design approaches [ 11 , 12 ] . however , the former one is highly laborious and time consuming than the statistical methods .
consequently , statistical experimental design techniques especially plackett - burman design ( pbd ) and response surface methodology ( rsm ) are widely used to select the significant variables and obtain the optimal levels , respectively [ 7 , 1118 ] .
the application of these statistical experimental design techniques in media optimization can result in improved product yields , reduced process variability , reduced time , and overall costs , compared with conventional practice of single factor optimization [ 7 , 13 ] .
plackett - burman design has been applied by several researchers to select influencing factors among the constituents of complex medium [ 1517 ] .
optimization of selected , highly influencing factors can be done using response surface methodology with either central composite design ( ccd )   or box - behnken design experiments . in present work , efforts taken to expel the seawater from the production medium of jaj06 , with incorporation of chemically defined salt formulations .
further , in order to enhance the antibiotic production by jaj06 , production medium was optimized using a successive optimization strategy , selection of media components that significantly influence the antibiotic production using plackett - burman design and optimization of these media components using rsm with box - behnken design .
jaj06 was previously isolated from a coastal solar saltern soil and extensively studied for their antibacterial secondary metabolite .
the strain was maintained over the surface of isp4 agar slants supplemented with 3% of sea salt ( w / v ) .
a nutrient medium contained 10  g of starch , 4  g of yeast extract , 2  g of peptone , 1  g of mgso4 , 2  g of caco3 , 0.04  g of feso47h2o , and 0.1  g of kbr in 1  l of sterile seawater was taken as basal production medium for further modification and statistical optimization . in order to replace the chemically nondefined seawater ,
two defined salt formulations were screened and compared with seawater to support antibiotic compound in jaj06 . according to previous reports
, two salt formulations were prepared with some modifications : salt formulation i   contained 12  g of nacl , 0.35  g of kcl , 0.22  g of cacl22h2o , 10.7  mg of h3bo3 , 7.3  mg of srcl2 , 1.3  mg of naf , and 26  g of cocl26h2o in 1 liter of deionized water ; salt formulation ii   contained 15  g of kcl , 0.22  g of cacl22h2o , 10.7  mg of h3bo3 , 7.5  mg of srcl2 , 1.3  mg of naf , and 26  g of cocl26h2o in 1 liter of deionized water .
spore suspension of jaj06 was prepared in distilled water from cultures grown on modified isp-4 medium supplemented with 3% of sea salt ( w / v ) at 30c for 7 days . the suspension
was then added to isp-2 broth in 250  ml erlenmeyer flask at a rate of 10 spores in 50  ml liquid medium .
cultures were incubated on a shaker at 120  rpm at 30c for 3 days and used as seed stocks . for antibiotic production
, strain jaj06 was inoculated into production medium and incubated on a shaker at 120  rpm for 8 days at 30c .
cell - free supernatant of fermentation broth was recovered by centrifuging it at 10000  rpm for 10  min .
ethyl acetate was added to the supernatant in 1  :  1 proportion and the mixture was agitated for 10  min .
the solvent layer containing antibiotic substance was separated from broth and it was further centrifuged at 5000  rpm for 15  min to remove traces of fermentation broth . the antimicrobial crude extract was concentrated tenfold using a rotational vacuum concentrator and used for antibacterial assay .
the extracted crude substance was assayed in triplicate for their antimicrobial activity against bacillus subtilis mtcc 441 by agar diffusion plate assay [ 7 , 20 ] .
the crude extract was loaded on 6  mm sterile discs , dried , and placed on nutrient agar plate inoculated with b. subtilis suspension adjusted to a mcfarland standard of 0.5 , which is equivalent to 1.5  10  cfu / ml .
the plates were incubated at 37c for 24  h and the inhibition zone formed around the disc was measured with transparent ruler in millimeter .
.   confirmed that the size of the zones of inhibition can be considered as measure of antibiotic titre .
therefore , the antibiotic activity was expressed as units of activity per millilitre the crude substance of culture , where 1  u was defined as a 1.0  mm annular clearing around the antibiotic disk .
plackett - burman design ( pbd ) was employed for screening the most significant medium components for growth and antimicrobial compound production by streptomyces sp .
, pa , usa ) was used for the experimental designs and subsequent analysis of the experimental data . in the experimental design , 7 medium components ( independent variables )
were screened by representing them at two levels , low (  ) and high ( + ) in 12 trials .  
table 1 shows media components , symbol code , and actual low and high level of the variables .  
table 2 shows the detail of the design , each row represents a trial , and each column represents an independent variable .
the experiment was carried out in triplicate and the average antimicrobial activity against b. subtitles was taken as the response . the variables with confidence levels above 90% were considered to have significant effect on antimicrobial compound production and thus used for further optimization .
response surface methodology ( rsm ) was used with box - behnken design to optimize the selected media constituents : starch , kbr , and caco3 for enhanced antibiotic production in jaj06 .
the three medium components ( independent variables ) were studied at three different levels : (  ) , ( 0 ) , and ( + ) for low , intermediate , and high concentrations , respectively ( table 3 ) .
the experiment was carried out in 17 trials ( table 4 ) with five replicates at the centre point and the values of responses were the mean of two replications .
for statistical calculations , coding of the factors was done according to the following equation : 
 ( 1)xi = xix0xi ,  i=0,1,2,3,  ,n , 
 where xi was the coded value of an independent factor , xi was the actual value of an independent factor , x0 was the actual value of an independent factor at the center point , and xi was the step change value . for predicting the optimal point ,
a second - order model was fitted to correlate the relationship between independent variables and response .
the behaviour of the system was explained by the following quadratic equation : 
 ( 2)y=0+ixi+ijxixj+iixi2 , 
 where y is the predicted response , 0 is the intercept term , i is the linear coefficient , ij is the quadratic coefficient , ii is the interaction coefficient , and xixj represent the independent variables . the design expert trial package ( version 7.0 )
the statistical significance of the model was verified by applying the analysis of variance ( anova ) .
overall model significance was determined using fisher 's f - test and its associated probability p(f ) .
lack of fit values lower than 0.05 indicates that there might be a contribution to the variables - response relationship that the model does not take into account .
the quality of the polynomial model equation was judged statistically by coefficient of determination ( r ) and adjusted r. the fitted polynomial equation was then expressed in the form of three - dimensional response surface plots , to illustrate the relationship between the responses and the experimental levels of each independent variable .
the design expert 's numerical optimization method was employed to optimize the level of each variable for maximum response .
the combination of different optimized variables , which yielded the maximum response , was experimentally validated by culturing jaj06 in optimized and unoptimized production medium .
the cell - free culture broths were collected and extracted with equal volume of ethyl acetate and the top organic layer was dried for further analysis .
the dried ethyl acetate extracts were resuspended in methanol and assayed as above for antibiotic activity .
the maximal antibiotic activity of jaj06 in three different media prepared separately with nondefined sterile seawater and two chemically defined salt formulations was examined in shake flask cultures . growth and antibiotic activity exerted by streptomyces sp .
however growth rate in both seawater and sodium chloride based salt formulation seemed to be the same , while the growth rate and antibiotic activity are slightly lower in salt formulation ii than those of the other two .
the main effect of each variable on antibiotic activity was calculated to determine the medium components which influence the antimicrobial compound production by streptomyces sp .
table 6 represents the effect , standard error , t - value , p value , and confidence level of each component from the result of antibiotic assay given in table 2 .
the medium components were screened at the confidence level of 92.5% on the basis of their effects .
it was indicated that starch ( x1 ) , kbr ( x5 ) , and caco3 ( x7 ) had apparent influences in antibiotic production by jaj06 .
confidence levels of these three variables were higher than 90% which indicates their significant contributions than those of other media components .
the same was confirmed from the pareto graph ( figure 1 ) in which higher effects were presented in the upper portion and then progress down to the lower effects .
it directly shows that the most important factors influencing antimicrobial compound production were starch , kbr , and caco3 .
based on the results of the plackett - burman design , starch , kbr , and caco3 were chosen as most influencing media components and further optimised using response surface methodology . in this approach ,
the batch runs were conducted in box - behnken design experiments and results to determine the effect of independent factors on the response along with the predicted values are shown in table 4 .
the regression equation coefficients were calculated and the data was fitted to a second - order polynomial equation .
the response of antibiotic activity ( y ) can be expressed in terms of the following regression equation : 
 ( 3)y  ( antibiotic  activity )  = 165.34 + 13.54a+7.29b  5.83c0.43ab+2.50ac+0.85bc  14.13a221.63b218.71c2 , 
 where y represented the response of antibiotic activity , and a , b , and c were the coded values of starch , kbr , and caco3 , respectively .
the anova was performed to inspect the second - order response surface model and results are given in table 7 .
the anova showed that the model was highly significant , as it was evident from the low p value ( < 0.0001 ) of the fisher 's f - test .
significance of model was also supported by statistically insignificant lack of fit , as was evident from the lower calculated f value ( 4.01 ) .
the model r of 0.9831 implied that model equation could explain 98.31% of the total variation which suggested a good agreement between predicated values and experimental data .
diagnostic plots were drawn to judge the model adequacy and clarify the signs of any problems in the experimental data .
plot of observed response ( antibiotic activity ) versus predicted reponse is shown in figure 2(a ) . in this case , predicted values were in agreement with observed ones in the range of the operating variables .
the normal probability plot of the studentized residuals was used to check for normality of residuals ( figure 2(b ) ) .
a linear pattern observed in this plot suggests that there was no sign of any problem in the experimental data .  
figure 2(c ) represents a plot of studentized residuals versus predicted values to check for constant error .
residuals displayed randomness in scattering and suggested that the variance of the original observation was constant .
the three - dimensional ( 3d ) response surface plots were drawn to illustrate the individual and interactive effects of starch , kbr , and caco3 on antibiotic production by jaj06 ( figure 3 ) .
each 3d plot presented the effects of two variables while the rest one was held at middle level .
there was insignificant mutual interaction between starch and kbr as well as starch and caco3 ( figures 3(a ) and 3(b ) ) , respectively . with the increase of the concentration of starch from 2 to 17.5  g /
l ( coded value , 1.0 to 0.75 ) , the antibiotic activity significantly increased at moderate concentration of kbr ( coded value , 0.5 ) ; however , decreased antibiotic activity was observed with any further increase of kbr even at high concentration of starch ( figure 3(a ) ) .
when the concentration of caco3 was just beyond the middle level ( coded value , 0.25 ) , the antibiotic activity increased with increasing concentration of starch from 2 to 15  g / l ( coded value , 0.1 to 0.5 ) and further increase of starch to its high level resulted mild decrease in antibiotic production ( figure 3(b ) ) . with the increase of the concentration of kbr from 0.02 to 1.1  g / l ( coded value , 1 to 0.15 ) , the antibiotic activity significantly increased to certain level at a low concentration of caco3 ( coded value , 1 ) and further slightly increased at a moderate level of caco3 ( coded value , 0.25 ) .
lower concentration of caco3 was found to be supportive for antibiotic activity ; however the activity was suppressed when the concentration of caco3 was higher in production medium .
on the basis of numerical optimization , the quadratic model predicted that the maximum antibiotic activity was 169.07  u / ml , when the optimal values of test factors in the coded units were starch = 0.37 , kbr = 0.24 , and caco3 = 0.20 ( figure 4 ) , which were 7.4  g / l starch , 0.048  g / l kbr , and 0.8  g / l caco3 , respectively .
the validation of the statistical results using the optimized medium was accomplished by carrying out shake - flask experiments in triplicate .
the maximum antibiotic activity unit obtained experimentally was found to be 173.3  u / ml , ( table 8) which is in close agreement with the prediction value ( 169.07  u / ml ) .
therefore , the developed model was considered to be accurate and reliable for predicting the production of antibiotic by streptomyces sp .
the final optimized medium contained 7.4  g of starch , 4  g of yeast extract , 2  g of peptone , 1  g of mgso4 , 0.8  g of caco3 , 0.04  g of feso47h2o , and 0.048  g of kbr in 1  l of sodium chloride based salt formulation i.
their antibiotic producing capability is not a static property and it can be significantly affected by constituents of production medium [ 24 , 25 ] .
. undefined nature of seawater can affect the reproducibility of the production profile of actinomycetes . in this study
, the seawater has been expelled from the production medium with incorporation of chemically defined salt formulations . among the two defined salt formulation ,
this type of chemically defined salt formulation has already been reported for consistent production of bioactive compounds from seawater dependent salinispora tropica strain nps21184 .
small manipulations in the culture medium composition can exert significant effect on secondary metabolites biosynthesis in microorganisms [ 7 , 26 ] .
several researchers working on antibiotics discovery programs have applied pbd and rsm as statistical tools to recognize , manipulate and optimize influencing medium constituents and recorded the increased antibiotic production .
for instance , wang et al . ,   applied rsm approach for medium optimization for antibiotic production by xenorhabdus bovienii and recorded 37.8% increase in antibiotic activity .
recently , chen et al .   reported 2.7-fold increase in antibiotic production by bacillus sp .
zjuibe-076 using rsm approach . in the present study , pbd and rsm were applied for medium optimization for antibiotic production by streptomyces sp . jaj06 and recorded 26.8% increase in antibiotic activity .
the results of pbd revealed that the crucial media components related to the antibiotic production by jaj06 were starch , kbr , and caco3 .
raytapadar and paul   reported starch as a significant media component for production of antibiotic from streptomyces aburaviensis 1da-28 .
similarly , caco3 has been identified as a crucial ingredients related to the production of cyclic hexapeptide antibiotic by streptomyces alboflavus .
rsm was found to be very effective in optimizing the selected medium components evident from positive diagnostic plots ( figure 2 ) and r value 0.9831 which was comparable with the earlier reports [ 7 , 27 ] .
jaj06 as a function of various salt compositions and levels of ingredients in production medium .
pbd and rsm were found to be very effective in selecting and optimizing the medium components in manageable number of experimental trials with overall 26.8% increase in antibiotic activity .
moreover , the optimum culture medium obtained in this experiment will be useful for further study with large scale fermentation in a fermenter for the efficient production of antibiotic from this streptomyces sp .
 
 streptomyces sp . jaj06 is a seawater - dependent antibiotic producer , previously isolated and characterised from an indian coastal solar saltern . this paper reports replacement of seawater with a defined salt formulation in production medium and subsequent statistical media optimization to ensure consistent as well as improved antibiotic production by streptomyces sp . 
 jaj06 . 
 this strain was observed to be proficient to produce antibiotic compound with incorporation of chemically defined sodium - chloride - based salt formulation instead of seawater into the production medium . 
 plackett - burman design experiment was applied , and three media constituents , starch , kbr , and caco3 , were recognised to have significant effect on the antibiotic production of streptomyces jaj06 at their individual levels . 
 subsequently , response surface methodology with box - behnken design was employed to optimize these influencing medium constituents for the improved antibiotic production of streptomyces sp . 
 jaj06 
 . a total of 17 experiments were conducted towards the construction of a quadratic model and a second - order polynomial equation . 
 optimum levels of medium constituents were obtained by analysis of the model and numerical optimization method . when the strain jaj06 was cultivated in the optimized medium , the antibiotic activity 
 was increased to 173.3  u / ml , 26.8% increase as compared to the original ( 136.7  u / ml ) . 
 this study found a useful way to cultivate streptomyces sp . 
 jaj06 for enhanced production of antibiotic compound . 