**2.6. Analytical methods**

**2.3. Batch fermentation study**

88 Renewable Resources and Biorefineries

0, and +1, respectively.

the predicted response:

coefficients of X<sup>i</sup>

*Y* = *b*<sup>0</sup> + ∑

is the first-order model coefficient, bij

. X<sup>i</sup>

represents the random error.

**2.5. Statistical analysis**

and Xj

model. The coefficient of determination (*R*<sup>2</sup>

**Table 1.** Factors and their levels for Box-Behnken design.

The preliminary experimental study of acetoin production was performed with 50 mL of glucose solution (100 g/L) measured into 250-mL Duran flask, and the growth effect of three complex nitrogen sources (yeast extract, beef extract, and corn steep liquor) were tested for acetoin fermentation nutrients. The pH of the medium was adjusted with 120 g/L of NaOH and 36.5 g/L of HCl buffer solutions. Subsequently, 5% volume fraction of inoculum size was added aseptically to the flask. The flasks were transferred into the environment-controlled

A three-level factor was employed to generate 17 experimental runs by considering the effect of glucose concentration (g/L), inoculum size (% v/v), and corn steep liquor (% w/v). The range and the levels of the independent variables investigated using the Box-Behnken experimental design (**Table 1**) were chosen based on variables previously reported to influence acetoin [11, 26]. The minimum, center point, and maximum levels of each variable were coded as −1,

A second-order mathematical equation, including all interaction terms, was used to calculate

The observed data were subjected to multiple regression analysis using Design-Expert versions 10.0 (Stat Ease Inc., Minneapolis, USA) to obtain the coefficients of the quadratic equation. The *F*-value and the probability *p*-value were used to appraise the significance of the

performance of the regression equation. The behavior of the model in the experimental area

**Factor Unit Symbols Coded factors**

Glucose (g/L) X<sup>1</sup> 50 100 150 CSL (% w/v) X<sup>2</sup> 5 10 15 Inoculum size (% v/v) X3 2 3.5 5

) and adjusted *R*<sup>2</sup>

*i*=1 *k*

where Y is response variable (acetoin concentration), b0

*bi Xi* + ∑ *i*=1 *k bii Xi* <sup>2</sup> + ∑ *i*<*j k*

C and 150 rpm. Fermentation

*bij Xi Xj* + *e* (1)

was calculated to evaluate the

**0 −1 1**

(i = 1, 2…k)

is the intercept value, b<sup>i</sup>

is the interaction effect, and bii represents the quadratic

are the input variables that influence the response variable and *e*

incubator shaker (platform shaker, model: FSIM SP016) at 30°

was performed for 168 h with 12-h sampling interval.

**2.4. Experimental design by Box-Behnken Design**

## *2.6.1. Reducing sugar analysis*

Reducing sugar concentration was analyzed using the dinitrosalicylic acid (DNS) method [27] and the results were expressed as glucose equivalent. To 1 mL of the supernatant, 3 mL of the DNS solution was added in the test tube and boiled for 15 min, cooled, and diluted appropriately after which the absorbance was measured at a wavelength of 540 nm using a UV–Visible Spectrometer (GBC Cintra 2020).
