Optimization of the Fermentation Process of Actinomycete Strain Hhs.015T

Strain Hhs.015T (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.015T. 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 MgSO4·7H2O 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 25°C, 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.015T on a large scale at low costs.


Introduction
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, rare actinomycetes appear as a promising source of new antibacterial and antifungal compounds. Strain Hhs.015 T , 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 [3].
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 [9]. 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 [10].
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 [11][12][13]. Crop pomaces are annually renewable sources of energy. Approximately 3.5 billion tons of agricultural pomaces are produced per annum in the world. But utilization of these 2 Journal of Biomedicine and Biotechnology pomaces was limited due to low protein content and poor digestibility [14]. 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 T in order to greatly reduce the production cost and improve the formation of components with antimicrobial activity.

Materials and Methods
2.1. Culture Media. 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 CaCO 3 , 0.5 g MgSO 4 ·7H 2 O, 0.5 g KH 2 PO 4 , 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 121 • C for 30 minutes.

Microorganisms and Culture Conditions. Strain
Hhs.015 T was kept on SSY solid medium at 4 • C. For spore production, the strain was transferred to SSY solid medium plates and incubated for 7 days in darkness at 28 • C. 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 T were prepared to inoculate a 250-mL flask containing 50 mL SSY medium. The flasks were incubated at 28 • C 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 28 • C 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 4 • C and was cultivated in PD liquid culture medium in darkness at 28 • C on a rotary shaker at 150 rpm for 2 days. Then, C. vulgaris culture medium (10 8 -10 9 spores/mL) was kept at 4 • C for the assay of the antimicrobial activity in the fermentation supernatant produced by Hhs.015 T .

Testing the Effect of Carbon, Nitrogen, and Microelement
Sources in the Medium on the Production of Antimicrobial Active Components. In order to investigate the effect of the carbon and nitrogen sources on the antimicrobial activity produced by strain Hhs.015 T , 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 30 • C (see Section 2.5). The prices of the different carbon and nitrogen sources are listed in Tables 1 and 2. 2.4. Experimental Design and Optimization. The alternative culture media were optimized according to orthogonal tests. In the orthogonal tests, an L 16 4 5 experimental design was employed for 4 independent variables each at 4 levels. A total of 16 experiments were carried out simultaneously, each experiment with three replicates. The variables involved in the study and their concentrations were given in Table 3.
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. Relative effects of two variables on response were identified from contour plots. An optimum value of the variables for maximum antimicrobial activity was determined by response optimizer tool of the software.

2.5.
Assay for Antimicrobial Activity. The antibacterial assay was carried out using the Oxford Cup method in 20cm-diameter Petri dishes. After autoclaving, 100 mL PDA medium were cooled to 40-50 • C, and 1% (v/v) of the C. vulgaris suspension (10 8 -10 9 spores/mL) was added to the PDA medium. After the medium in the Petri dishes solidified, the autoclaved Oxford cups were put onto the medium surface and 200 μL of the testing samples (supernatants) were added into the cups. One Petri dish was one replicate. The antimicrobial activity was evaluated by measuring the diameter of translucent inhibition zones after incubating the dishes 24 hours at 30 • C.

Data Calculation and Analysis.
The data from the orthogonal tests were analyzed by SAS software (Stat Soft, Inc., Tulsa, USA). 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:  Note: Inhibition zones with the same letter showed no significant difference analyzed by SAS statistics systems. 1.5% (w/v) of apple pomace was used as the carbon source replacing starch and sucrose in SSY medium except the control. The portion of nitrogen sources was about 0.7% (w/v). The control medium was the SSY medium. The Black: without soybean powder, yeast extract, and protease peptone in SSY medium where X i j 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. R i 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: where x i was the independent variable coded value, X i was the independent variable real value, X i was the independent variable real value on the centre point, and X i 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: where Y was the measured response, b 0 was the intercept term, b i , b i j , and b ii were the measures of the effects of variables x i , x i x j , and x 2 i , respectively. The variable x i x j represents the first-order interaction between x i and x j (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 2 that measured the goodness of  Note: fit of the regression model. The analysis also included the Student's t-value for the estimated coefficients and associated probabilities, P(t). For each variable, the quadratic models were represented as contour plots. The results of the nitrogen sources showed that strain Hhs.015 T 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 NaNO 3 , 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). No antimicrobial activity was found in the medium with NH 4 NO 3 . 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.

Screening the Components in the Alternative
The results showed (Figure 1) that the antimicrobial activity produced by strain Hhs.015 T enhanced in the presence of MgSO 4 ·7H 2 O concentrations increasing from 0.05% to 0.08% and KH 2 PO 4 levels increasing from 0 to 0.01%, but the activity decreased while the concentrations of the other microelements increased, for example, NaCl and CaCO 3 (Figure 1). Therefore, MgSO 4 ·7H 2 O and KH 2 PO 4 were chosen as the microelements for further experiments.

Orthogonal Design for the Optimization of the Alternative
Cultural Medium for Strain Hhs.015 T . 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 T . 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 < F 0.05 ), probably because in the experiments the concentration range of every variable was defined. Finally, the optimum values of apple pomace 15 g/L, rapeseed meal 4 g/L, KH 2 PO 4 0.1 g/L, and MgSO 4 ·7H 2 O 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%.

Optimization of Fermentation Conditions for Strain
Hhs.015 T . Applying multiple regression analysis on the experimental data, the results (Table 5) of the CCD design fitted with a second-order of polynomial equation: where Y was the response (the antimicrobial activity units) and X 1 , X 2 , X 3 , X 4 , and X 5 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 results of the regression analysis were shown in Table 8. 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 (P X3 = .001). Initial pH and media volume were also significant at 5% level (P X1 = .039, P X2 = .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.
This model resulted in ten 2D contour plots. 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 2 = 0.82) was also very high to advocate for a high significance of the model [17]. 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 = 25.0 • C, 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. This shows 98% validity of the predicted model. 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).

Discussion
Carbon sources could play an important role in the production of compounds with antimicrobial activity of strain Hhs.015 T . 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 [20], 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 [21].
For microbe growth and synthesis of the secondary metabolites, microelements (mineral and metal ions) Note: Candida vulgaris was used as the target microbe inoculated 24 hours at 30 • C on PDA medium in 20-cm-diameter Petri dish. One dish: one replication, the data in the table were the average of three replications  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., zinc, iron, and manganese were the most important  trace elements [22]. Yang et al. [23] [24]. 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. Finally, the price of fermentation material for strain Hhs.015 T was 0.1836 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 T .
In the optimization process of fermentation conditions, RSM proved to be a powerful tool. The approach allowed the determination of the culture conditions that yielded the highest antimicrobial activity by strain Hhs.015 T . In this study, there was a mutual interaction between the medium volume and rotary speed (Figure 3(d)) to influence the  Note: * Significant at 5%-level, * * Significant at 1%-level. 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 [1]. A proper DO level was beneficial to antibiotic production, which is in agreement with those obtained in a 5-L fermentor. Lai et al. [15] 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 T . 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)). Therefore, the optimal primary pH was around 7.0. Similarly, the optimal temperature was around 25 • C, 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.

Conclusions
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 T . 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 T . 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.