Experimental design data for the biosynthesis of citric acid using Central Composite Design method

In the present investigation, we report that statistical design and optimization of significant variables for the microbial production of citric acid from sucrose in presence of filamentous fungi A. niger NCIM 705. Various combinations of experiments were designed with Central Composite Design (CCD) of Response Surface Methodology (RSM) for the production of citric acid as a function of six variables. The variables are; initial sucrose concentration, initial pH of medium, fermentation temperature, incubation time, stirrer rotational speed, and oxygen flow rate. From experimental data, a statistical model for this process has been developed. The optimum conditions reported in the present article are initial concentration of sucrose of 163.6 g/L, initial pH of medium 5.26, stirrer rotational speed of 247.78 rpm, incubation time of 8.18 days, fermentation temperature of 30.06 °C and flow rate of oxygen of 1.35 lpm. Under optimum conditions the predicted maximum citric acid is 86.42 g/L. The experimental validation carried out under the optimal values and reported citric acid to be 82.0 g/L. The model is able to represent the experimental data and the agreement between the model and experimental data is good.


Subject area
Biology More specific subject area

Microbial biosynthesis
Type of data Table, text file, graph, figure How data was acquired

Analytical method
Data format Analyze Experimental factors Determination of the yield of Citric acid

Experimental features
The sterilization for fermentation experiment was carried out for 25% sucrose solution by supplying the water and oxygen to the feremetror. After adding the nutrients, clear supernatant liquid was diluted to 15% sucrose level. The solution and growth medium were sterilized. The prepared culture was poured to fermentor and thereafter fermentation experiments were carried out for different operating conditions.

Data source location
Data of experimental and model provided in Table 2.
Data accessibility Data provided in the article

Value of the data
The data presented for the production of citric acid using the A. niger using the batch solid state fermentor.
The significant factors which influence on the growth of the citric acid are given in Table 3 both the CCD regression model and experimental data.
The data presented in the article is useful to the industry as well as researchers. The detailed data of CCD and experimental data are useful to researchers for the production of citric acid with other strains.

Data
Citric acid is one of the most important biochemical products that are extensively used in many industrial processes like in the food technology to various fields of chemical industry [1]. Citric acid is in highly demandable product. It is produced by the extraction of citrus fruits, chemical synthesis and fermentation. Due to the limited supply of natural citric acid, it is produced commercially by using A. niger from the fermentation process of bulk hydrated materials and the byproducts of sugar production [2][3][4][5].
Citric acid is produced mainly by A. niger from the fermentation process [6,7]. The growth and production rate of A. niger are very much affected by the medium composition, fermentation variables and stimulators.
The worldwide demand for citric acid is increasing faster than its production and it requires more economical process models [3,8]. The raw materials for citric acid production include; brewery wastes, corn starch, beet molasses, coconut oil, carob pod extract, glycerol date syrup, and pure sugars such as glucose and sucrose [9]. In addition to molds, several yeast strains are now known to produce large amounts of citric acid [10,11].
The experimental plans were obtained from the Design expert soft ware. From that a set of combinations the experiments were performed. The experimental results were used to find a statistical mathematical model as a function of all the influenced factors or variables.

Materials and methods
A. niger NCIM 705 was purchased from National Chemical Laboratory, Pune, India. The substrate, sucrose, potato dextrose agar medium comprising dextrose, yeast extract and agar-agar for subculture preparation, growth medium consisting of glucose, NH 4 NO 3 , MgSO 4 Á 7H 2 O, KH 2 PO 4 , (NH 4 )SO 4 , Fe (SO 4 ) 2 Á 24H 2 O and ZnSO 4 Á 7H 2 O, phenolphthalein indicator and 0.1 N NaOH for citric acid estimation [12] and di-nitro salicylic acid for sucrose estimation were procured M/s Hichem Chemicals, Warangal, India. Autoclave for sterilization of medium and fermentor, laminar flow chamber for inoculation and the glass fermentor of Scigenics India Ltd. make were used for experimentation.

Plackett-Burman design
The optimizations for the fermentation process are very important because of their influence on the economy and practicability of the process [13]. Plackett-Burman design is two level fractional factorial designs for N À 1 variables in N experimental runs. The design considers only the main effects of the variables but not their interactive effects [14].
Citric acid yield is the response variable and the actual number of independent variables for the present system are eleven; initial sucrose concentration, methanol concentration, inoculums density, initial medium pH, spore age, stirrer rotational speed, incubation time, fermentation temperature, particle size distribution, oxygen flow rate and moisture content. Twelve experiments were recommended for eleven variables by Plackett-Burman design [15]. The twelve experiments were conducted accordingly to find the influence of each variable on the citric acid yield. If the variables have confidence levels greater than 95%, then their influence is more on the citric acid yield. From this design, it has been found that six most significant variables are namely initial sucrose concentration, initial medium pH, stirrer rotational speed, incubation time, fermentation temperature, and oxygen flow rate which strongly influence the citric acid yield.

Response Surface Methodology (RSM)
Response surface methodology is useful method for the modeling and analysis of all the industrial processes by using mathematical and statistical techniques. The output is influenced by various input variables. The main objective is to optimize the output by selecting the most significant variables which influence on the output or the response [15].
CCD method with most significant independent process variables (six variables) was used to find the effect of these variables on the yield of citric acid. From the CCD; sequential experiments are obtained for approximate information for testing the lack of fit [16][17][18][19]. All the coded values are shown in Table 1. Hence the six factors investigated are initial sucrose concentration (X 1 ), initial medium pH(X 2 ), stirrer rotational speed(X 3 ), incubation period(X 4 ), fermentation temperature(X 5 ), and oxygen flow rate(X 6 ). Table 1 Range of parameters selected for optimization.

Parameter
Code Units Low High Initial sucrose concentration

. Experimental setup and procedure
The pure culture of A. niger NCIM 705 was procured and preserved in a refrigerator by periodic subculture on potato dextrose agar medium. A fermentor capacity of 1.2 L contains a standard control and instrumentation panel. It was cleaned with pure water after that sterilized in an autoclave for 20 min. The sterilized fermentor was placed in the main assembly. The water as well as O 2 is supplied by the tube connections. A 25% of sucrose solution was taken. After adding 35 mL of 1 N H 2 SO 4 , it was boiled for half an hour, cooled, neutralized with lime water and was left overnight for clarification. The clear supernatant liquid was diluted to 15% sucrose level. The solution and growth medium were sterilized, inoculated and the mixture was kept in an incubator for 24 h. The prepared culture was poured into the fermentor in the first run; thereafter the fermentor was put into operation for 7 days for batch operation [12].

Analysis
All the samples were collected from the fermentor periodically for every 24 h and analyzed for sucrose, biomass and citric acid using standard analytical methods [20,21].

Statistical analysis
The Pareto analysis was used for the selection of a minimum number of tasks that gives a significant overall effect. The Pareto chart used for the contributory effect of each variable on citric acid fermentation.
After selecting the significant parameters; the CCD method used to find the effect of each significant parameter on the citric acid yield. A total of 86 sets of experiments were suggested by CCD    Table 2. Table 3 shows the ANOVA results and regression analysis. The probability test values (P-values) less than 0.0500 indicates that the model terms are more significant on the citric acid yield. The higher the F value; better is the certainty, that the factors explain adequately the variation in the data about its mean and that the estimated factor effects are real. The ANOVA of the regression model demonstrates that the model is highly significant as is evident from a very high value of F (28.54) and a very low value of P ( o0.0001). Based on p-value, it can be stated from the above Table 3 that initial sucrose concentration (X 1 ), incubation time (X 4 ), combined effect of initial sucrose concentration and stirrer rotational speed (X 1 X 3 ), combined effect of initial sucrose concentration and incubation time (X 1 X 4 ), square of the initial sucrose concentration ðX 2 1 Þ and square of the stirrer rotational speed ðX 2 3 Þ are significant model terms. Values greater than 0.1000 indicate that the model terms are not significant. F-value of 1.68 implies that the Lack of Fit is not significant relative to the error. There is a 20.55% chance that a large lack of fit F-value could occur due to noise. Non-significant lack of fit is desirable if the model is to be fit and it is true for the present study from the above Table 3. The predicted R 2 is in reasonable agreement with the adjusted R 2 (0.8974). It indicates that, the mathematical model is very reliable for the prediction of citric acid yield.

The RSM curves
The citric acid yield response surface figure is shown in the Fig. 1. It gives the information that the yield of citric acid with initial concentration of the sugar and pH of the medium, the other variables are speed of 240 rpm, incubation time of 5.5 days, temperature of 30°C and oxygen flow rate of 1.5 lpm. The maximum citric acid concentration is 86 g/L found at a pH of 6.26 and initial sucrose concentration of 140 g/L. The experimental citric acid yield obtained the present study is 82 g/L.  Fig. 2 shows the citric acid yield with respect to medium pH and stirrer speed and the other parameters are sucrose concentration of 140 g/L, incubation time of 5.5 days, temperature of 30°C and oxygen flow rate of 1.5 lpm. The maximum predicted citric acid yield is 86 g/L.

Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2017.03.049.