in recent years , the increasing prevalence of infectious diseases resistant to chemotherapy has caused an urgent need to discover and develop new antibiotics . in this context ,
strain hhs.015 , isolated from roots of cucumber plants , belongs to saccharothrix actinomycete [ 1 , 2 ] .
the isolate showed a broad antimicrobial spectrum in vitro against plant pathogenic ( e.g. , valsa ceratosperma and phytophthora capsici ) and nonplant pathogenic fungi , for example , candida vulgaris . in field experiments ,
fermentation supernatant of hhs.015 showed effective biocontrol to tomato leaf mould caused by fulvia fulva   and apple tree valsa canker caused by valsa ceratosperma .
up to date , many bioactive secondary metabolites have been reported from cultures of saccharothrix .
these metabolites are characterised by diverse chemical structures and may be suitable because of their wide range of activity in agriculture to control fungal diseases in cultivated plants and domestic animals .  
production of secondary metabolites by microorganisms differs qualitatively and quantitatively depending on the strains and species of microorganisms used as well as on their nutritional and cultural conditions . and
as fermentation moves into lower - value , higher - volume substrates , it becomes necessary to maximize the efficiency and minimize costs by using waste by - products to complete effectively with traditional high - value , low - volume compounds .   in recent years , more and more attention was paid to agricultural pomaces such as apple pomace , wheat / rice bran , corn steep liquor , and peanut meal , for their use as raw materials in the production of high value products [ 1113 ] .
but utilization of these pomaces was limited due to low protein content and poor digestibility .
studies on optimization of the production of antimicrobial active compounds using agricultural pomaces have been reported comparatively seldom .  
the objective of the present study was to produce an alternate culture medium using agricultural waste as medium component and optimize fermentation conditions of the strain hhs.015 in order to greatly reduce the production cost and improve the formation of components with antimicrobial activity .
ssy liquid medium consisted of : 20  g soybean powder , 10  g sucrose , 5  g soluble starch , 2  g yeast extract , 2  g protease peptone , 2  g nacl , 1  g caco3 , 0.5  g mgso47h2o , 0.5  g kh2po4 , and 1  l double distilled ( dd ) water .
ssy solid medium was prepared by adding 10  g agar to the liquid medium .
in addition , the following media were used : pda ( 200  g potato , 20  g dextrose , and 10  g agar in 1  l dd water ) , and pd ( 200  g potato and 20  g dextrose in 1  l dd water ) .
all cultural media , plates , and flasks used in the experiments were autoclaved at 121c for 30 minutes .
the strain was transferred to ssy solid medium plates and incubated for 7 days in darkness at 28c .
the spores produced on the plates were used for inoculation of the liquid fermentation media .
two spore cakes ( diameter 4  mm ) of strain hhs.015 were prepared to inoculate a 250-ml flask containing 50  ml ssy medium .
the flasks were incubated at 28c on a rotary shaker at 150  rpm for 48 hours in darkness .
for optimization of an alternative culture medium , submerged fermentation was carried out in 250-ml flasks containing 50  ml different test fermentation media .
these flasks were inoculated with 10% ( v / v ) of the spore culture and incubated in darkness at 28c on a rotary shaker at 150  rpm for 5  d. for optimization of fermentation conditions , the experiment was carried out according to the experimental design .
after fermentation , all of the test fermentation broths were centrifuged at 11,086    g for 10 minutes ; the supernatants ( test samples ) were used to evaluate the antimicrobial activity .  

candida vulgaris as the target microbe was kept on pda medium at 4c and was cultivated in pd liquid culture medium in darkness at 28c on a rotary shaker at 150  rpm for 2 days .
then , c. vulgaris culture medium ( 1010  spores / ml ) was kept at 4c for the assay of the antimicrobial activity in the fermentation supernatant produced by hhs.015 . in order to investigate the effect of the carbon and nitrogen sources on the antimicrobial activity produced by strain hhs.015 , ssy medium was used as a basal medium for the following optimization studies .
various single and complex carbon or nitrogen sources ( tables 1 and 2 ) were used to replace the carbon and nitrogen sources in the ssy medium , while all other components were kept constant .   in the selection of proper microelements ,
all of the microelements in ssy medium were replaced by one microelement at different concentrations .
the antimicrobial activity was evaluated by measurement of inhibition zones of the target organism after 24 hours of incubation at 30c ( see section 2.5 ) .
the prices of the different carbon and nitrogen sources are listed in tables 1 and 2 .
the alternative culture media were optimized according to orthogonal tests . in the orthogonal tests ,
a total of 16 experiments were carried out simultaneously , each experiment with three replicates .
the optimization of the fermentation conditions was carried out according to the central composite tool ( ccd ) of rsm ( response surface methodology ) using minitab 14 software ( minitab inc . , 2003 ) for selected five environmental factor parameters [ 15 , 16 ] .
the crucial factors involved in the study and their concentrations are given in table 4 .
a total of 32 experiments were carried out simultaneously ( table 5 ) , with experiments 2 , 4 , 6 , 8 , and 17 as five replicates of the central point .
an optimum value of the variables for maximum antimicrobial activity was determined by response optimizer tool of the software .
the antibacterial assay was carried out using the oxford cup method in 20-cm - diameter petri dishes .
after autoclaving , 100  ml pda medium were cooled to 4050c , and 1% ( v / v ) of the c. vulgaris suspension ( 1010 
after the medium in the petri dishes solidified , the autoclaved oxford cups were put onto the medium surface and 200  l of the testing samples ( supernatants ) were added into the cups .
the antimicrobial activity was evaluated by measuring the diameter of translucent inhibition zones after incubating the dishes 24 hours at 30c .  
the data from the orthogonal tests were analyzed by sas software ( stat soft , inc .
the optimum value of the variables for the maximum antimicrobial activity was obtained by range analysis ( table 6 ) .
range of every variable can roughly explain the effect of every variable on the antimicrobial activity .
the range of every variable was calculated according to the following equation :  
 ( 1)xij = xijn ,  n=4 ,  j=1,2,3,4,ri = xijmax  xijmin  ,  i=1,2,3,4 , 
							 where xij is the average value of the j level of one variable in line , i , n is the number of replications of every level of one variable in one line .
ri is the difference of the maximum and the minor average value of all of the levels of one variable in line i.   the experimental results of rsm were fitted with the response surface regression procedure . in developing the regression equation , the test variables were coded according to the equation : 
 ( 2)xi=(xixi)xi ,  i=1,2,3,  ,k , 
							 where xi was the independent variable coded value , xi was the independent variable real value , xi was the independent variable real value on the centre point , and xi was the step change value .
the response variable ( antimicrobial activity unit ) was fitted by a second - order model in order to correlate the response variable to the independent variables .
the general form of the second degree of polynomial equation is : y = b0 + ibixi + ijbijxixj + biixi , where y was the measured response , b0 was the intercept term , bi , bij , and bii were the measures of the effects of variables xi , xixj , and xi , respectively .
the variable xixj represents the first - order interaction between xi and xj ( i < j ) .  
the statistical analysis of the model was performed in the form of analysis of variance ( anova ) .
this analysis included the fisher 's f - test ( overall model significance ) , its associated probability p(f ) , correlation coefficient r , and determination coefficient r that measured the goodness of fit of the regression model .
the analysis also included the student 's t - value for the estimated coefficients and associated probabilities , p(t ) .
the results indicated that the carbon sources obviously affected the synthesis of substances with antimicrobial activity by strain hhs.015 ( table 1 ) . among the various carbon sources tested , strain hhs.015 produced in the presence of glucose ( 18.65  mm diameter of growth inhibition zones ) , white sugar ( 18.33  mm ) , brown sugar ( 17.86  mm ) , apple pomace ( 17.45  mm ) , and starch ( 16.21  mm ) greater growth inhibition zones than in the control medium ( 15.28  mm ) .
statistical analysis indicated that the growth inhibition zones were significantly different from the control for glucose and white sugar , while the growth inhibition zones were not significantly different from glucose and white sugar for apple pomace .
significant lower antimicrobial activities were found in the vinegar residue ( 7.75  mm ) as well as in black ( 7.75  mm ) and bran ( 13.15  mm ) . taking the costs of the raw materials into consideration ,
the price of apple pomace ( 0.6  rmb / kg ) was much lower compared with other carbon sources showing similar antimicrobial activity .  
the results of the nitrogen sources showed that strain hhs.015 could utilize different forms both organic and inorganic nitrogen sources ( table 2 ) .
rapeseed meal favored production of compounds with antimicrobial activity ( 19.59  mm diameter of inhibition zones ) but not significantly compared to the control .
also in media containing urea and nano3 , components with low antimicrobial activity were produced ; these sources were not significantly different from the control and other sources ( e.g. , ammonium phosphate , cotton dregs , ammonium sulfate , and fish meal ) .
thus , considering the price of raw materials and the antimicrobial activity produced , apple pomace and rapeseed meal were used as carbon and nitrogen sources for further experiments .  
the results showed ( figure 1 ) that the antimicrobial activity produced by strain hhs.015 enhanced in the presence of mgso47h2o concentrations increasing from 0.05% to 0.08% and kh2po4 levels increasing from 0 to 0.01% , but the activity decreased while the concentrations of the other microelements increased , for example , nacl and caco3 ( figure 1 ) .
the effects of concentrations of the components in the medium on the antimicrobial activity were further investigated by tests of orthogonal design . from the range analysis , the apple pomace as the main variable affected the antimicrobial activity , indicating that the carbon source was essential for the production of compounds with antimicrobial activity by strain hhs.015 . from the results of the analysis of variance of orthogonal design , ( table 7 ) showed that each single variable had no significant effect on the antimicrobial activity ( f < f0.05 ) , probably because in the experiments the concentration range of every variable
g / l , rapeseed meal 4  g / l , kh2po4 0.1  g / l , and mgso47h2o 0.6  g / l were determined according to the range analysis ( table 6 ) .
the antimicrobial activity produced in the alternative culture medium was not significantly different from the ssy medium ( figure 2a , ck ) , while the price of the materials of 0.1836  rmb / l was drastically lower than the price of the ssy medium , 2.1624  rmb / l ; thus , the price was reduced by 91.5% . applying multiple regression analysis on the experimental data , the results ( table 5 ) of the ccd design fitted with a second - order of polynomial equation : 
 ( 3)y=15.7443  +  1.4375x11.6625x2 + 3.8792x3    2.0443x122.9743x222.2193x323.9943x42  + 1.5313x1x5 + 1.5313x2x4 , 
							 where y was the response ( the antimicrobial activity units ) and x1 , x2 , x3 , x4 , and x5 were the coded values of the independent variables , initial ph , medium volume in the flask , rotary speed , temperature , and inoculation volume , respectively .  
the student 's t - test and p - values were used to check the significance of each coefficient ; the results also indicated interaction strength between each independent variable .
the degree of significance ( table 8) shows that the linear term regression coefficients of rotary speed were highly significant as demonstrated by the p - values ( px3 = .001 ) .
initial ph and media volume were also significant at 5% level ( px1 = .039 , px2 = .020 ) .
quadratic coefficients of primary ph , media volume , rotary speed , and temperature were highly significant at 1% level , and showed negative effect on the antimicrobial activity .  
the few response contour plots of the calculated model for antimicrobial activity were shown in figures 3(a)3(f ) by keeping the other three variables constant at their middle level .
the analysis of variance of regression for antimicrobial activity was summarized in table 8 . in the case of antimicrobial activity , this calculated model was able to explain 93.6% of the results ( table 9 ) .
the value of the adjusted determination coefficient ( adj r = 0.82 ) was also very high to advocate for a high significance of the model .
the coefficient for quadratic effect of ph , medium volume , temperature , and rotary speed may be significant to some extent .  
the response surfaces 2d contour could be analyzed for determining the optimized value of the variables , but it was difficult to analyze all these simultaneously . finally , the optimum culture condition was obtained as follows : initial ph = 7.0 , medium volume = 90.0  ml/250  ml plate , rotary speed = 100  rpm , temperature = 25.0c , and inoculation volume = 15.8% .  
these values predict a 17.5  mm growth inhibition zone caused by the antimicrobial activity .
these optimized values of nutrient parameters were validated in a duplicate flask study and an average of 17.3  0.25  mm growth inhibition zone was obtained .
the result proved that the production of ingredients with antimicrobial activity had been improved by 20% when compared with the basal fermentation conditions ( 14.4  0.15  mm ) ( figures 2a and b ) .
carbon sources could play an important role in the production of compounds with antimicrobial activity of strain hhs.015 .
nutrition plays an important role in the onset and intensity of secondary metabolism , not only because of limiting the supply of an essential nutrient is an effective means of restricting growth but also because the choice of limiting nutrients can have specific metabolic and regulatory effects [ 18 , 19 ] .  
the results of this study showed that production of compounds with antimicrobial activity was drastically reduced when a carbon source such as vinegar residue or no carbon source ( black ) was present in the culture medium .
a similar catabolic repression mechanism for antibiotic production had been described for bacteria , suggesting that the synthesis of the antibiotic was related to phosphoenolpyruvate sugar phosphotransferase system , and controlling the concentration of carbon sources could regulate the synthesis of antibiotics . in the optimization of the culture medium , apple pomace was selected as the carbon source .
apple pomace , a by - product from a juice production factory in qianxian , shaanxi province , contained numerous kinds of nutrients , such as proteins , fats , soluble sugars , ca , p , and many other microelements , vitamins , and so forth .   for microbe growth and synthesis of the secondary metabolites , microelements ( mineral and metal ions )
were necessary , which are an important part of enzyme reactive centers or could maintain the structure stability of biomacromolecules and the balance of cell osmotic pressure . for the secondary metabolites in phoma sp .
yang et al .   found that mgso4 , mgcl2 , nacl , kh2po4 , kno3 , and ( nh4)2so4 favored antibiotic production by xenorhabdus spp .
d43 , but antibiotic production by addition of zn(no3)2 and cuso4 decreased . in this experiment , the results showed that 0.06% mgso47h2o and 0.01% kh2po4 were proper for strain hhs.015 to produce antimicrobial activity . increasing or reducing the concentrations of mgso47h2o and kh2po4 , the production of compounds with antimicrobial activity by strain hhs.015 decreased .
microelements at different concentrations showed different effects on the microbe physiological activity , so , the suitable concentrations are changing depending on microbe properties and fermentation medium .  
the apple pomace and rapeseed meal were chosen as two main components in the optimum culture medium for their low market price , 598.4  rmb / t , while soybean in the ssy medium was 3774  rmb / t in the china market .
rmb / l in the optimum culture medium ; 91.5% of the production cost were saved compared with the ssy medium , 2.1624  rmb / l , with similar antimicrobial activity .
hence , there are a high possibility and feasibility to use the modified cultural media in the scale - up fermentation of strain hhs.015 .   in the optimization process of fermentation conditions ,
the approach allowed the determination of the culture conditions that yielded the highest antimicrobial activity by strain hhs.015 . in this study
, there was a mutual interaction between the medium volume and rotary speed ( figure 3(d ) ) to influence the production of antimicrobial active components .
the optimal medium volume in the flask and the rotary speed were around 90  ml and 100  rpm , respectively .
our study revealed that rotary speeds , medium volume in the flask are related to the dissolved oxygen ( do ) in shaken flasks .
a proper do level was beneficial to antibiotic production , which is in agreement with those obtained in a 5-l fermentor .
lai et al .   found that the shear effect on cell morphology such as mycelium growth or pellet formation was found closely influenced by the do level and antibiotic production was maximized under suboptimal conditions for cell growth .  
the analysis of significance of each coefficient of the quadratic regression model indicated that initial ph and temperature also have a significant effect on antimicrobial activity of strain hhs.015 .
it could be observed that the antimicrobial activity increased upon increasing the initial ph from 2.0 to 7.0 , but any further increase of ph resulted in decreased production of antimicrobial active components ( figures 3(a ) and 3(b ) ) .
similarly , the optimal temperature was around 25c , lower or higher temperatures reduced the production of compounds with antimicrobial activity ( figures 3(c ) , 3(e ) , and 3(f ) ) .
the results suggested that the broth ph could play a crucial role in the production of secondary metabolites ; this finding is in good agreement with previous studies using for example xenorhabdus species [ 23 , 25 , 26 ] .
the authors suggested as possible reason that the key enzymes essential for synthesis of antibiotics become inactive at suboptimal ph - values .
from the present study , the authors conclude that apple pomace and rapeseed meal were identified as the most suitable medium components for low - cost production of antimicrobial compounds by strain hhs.015 .
this is the first report about the fermentation of saccharothrix actinomycete using apple pomace as the main medium component .
statistical optimization methods for fermentation process could overcome the limitations of classical empirical methods and proved to be a powerful tool for the optimization of conditions of production of antibiotics by strain hhs.015 .
the optimization of the fermentation process resulted not only in a 91.5% reduction of the costs of raw material compared to the conventional medium but also in a 20% higher antimicrobial activity .
furthermore , the information obtained is considered fundamental and useful for developing a cultivation process for efficient production of antibiotics on a large scale .
 strain hhs.015 t ( saccharothrix yanglingensis sp . nov . ) , an antagonistic endophytic saccharothrix actinomycete isolated from roots of cucumber plants , exhibited a broad antimicrobial spectrum in vitro and was active as a biocontrol against plant diseases in field trials . 
 the ssy medium was used for production of antimicrobial   metabolites by strain hhs.015 t . however 
 , this 
 medium is too expensive for large - scale production . in this study , an alternative culture medium , based on agricultural waste 
 products ( e.g. , apple pomace ) , was optimized . 
 the results showed that the alternative medium contained 15  g apple pomace , 4  g rapeseed meal , 0.1  g kh2po4 , and 0.6  g mgso47h2o in 1  l distilled water . 
 this medium reduced the material costs by 91.5% compared to ssy medium . 
 response surface methodology ( rsm ) was used to investigate the influence of environmental variables on production of compounds of antimicrobial metabolites . 
 the optimal conditions achieved were initial ph 7.0 , medium volume of 90  ml in 250  ml flasks , rotary speed of 100 rpm , temperature 25c , and 
 inoculation volume of 15.8% . 
 the antimicrobial activity was increased by 20% by optimizing the environmental parameters . 
 the results obtained allow an efficient production of components with antimicrobial activity by strain hhs.015 t on a large scale at low costs . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions