**3.1. Preliminary evaluation of complex nitrogen sources on AC production**

The study in **Figure 2** shows that CSL supports rapid utilization of the reducing sugar (from 150 to 84 g/L) within the first 60 h of fermentation [32], the resultant acetoin and biomass growth being maximum during this period. The maximum biomass growth is found to be fairly constant at ~ 8 g/L until the 144 h when it starts to decline. Likewise, the maximum acetoin concentration is found to be ~ 7 g/L after 60 h of fermentation and then declines. The decline in the acetoin concentration could be attributed to the complete metabolism of glucose in the fermentation medium. Although the biomass concentration remained constant after the 60 h of fermentation, it can be assumed that the energy derived from reducing sugar metabolism was channeled toward cell maintenance since biomass growth remained constant and acetoin was still produced till the fermentation lapse. It has been shown previously that CSL is a rich source of proteins, amino acids, minerals, vitamins, and trace elements and can be used as nutritional supplement [32]. The addition of CSL reduced the fermentation time and promoted the growth and fermentation of the strain.

media. A maximum acetoin of about 5.2 g/L was obtained during the first 12 h of the fermentation, after which there was no significant change in the biomass growth from that period until the lapse time of fermentation. About 50% (72.53 g/L) of sugar concentration was not consumed until the fermentation time lapse of 168 h, and the sluggish consumption of glucose could be traceable to the lack of rich nitrogen sources, which was needed as a nutritional supplement [28].

Statistical Optimization of Acetoin Production Using Corn Steep Liquor as a Low-Cost Nitrogen…

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It was observed in the preliminary evaluation of nitrogen sources discussed earlier (**Figure 2a–d**) that corn steep liquor produces acetoin at a short time interval when compared to other nitrogen sources. Therefore, the fermentation time interval of acetoin was reduced from the initial 168 h (**Figure 2a–d**) to 48 h (**Figure 3**) and sample taken at every 2-h interval, to investigate the biomass growth, utilization of glucose, and the acetoin accumulation when corn steep liquor was used as nitrogen source. The results show that the accumulated acetoin and the biomass growth were already at the peak of 7.03 and 7.83 g/L, respectively, in the first 36 h of fermentation (**Figure 3**). The rapid utilization of reducing sugar (from 150 to 66 g/L) within 36 h of fermentation was due to high amino acids and polypeptides, which are excellent sources of nitrogen in corn steep liquor and has been reported to support the growth of most microorganism [35]. CSL comprises a mixture of reducing sugars that contribute to the nutritional growth of the bacteria with a steady increase in biomass growth (7.7–7.8 g/L) from 18 to 46 h when it starts to decline [36]. It can be affirmed from the findings that corn steep liquor hastens fermentation of acetoin

at short time interval and this makes large-scale production of acetoin cost-effective.

After the preliminary studies, RSM coupled with Box-Behnken design (BBD) was used for the optimization of the fermentation process with respect to glucose concentration, CSL

**Figure 3.** Acetoin growth profile using corn steep liquor at short fermentation time interval. RS–reducing sugar; AC–acetoin;

**3.3. Optimization of AC production using response surface method**

BM–biomass.

**3.2. Metabolism of corn steep liquor on AC production**

On the other hand, when yeast extract was used as the nitrogen source in the fermentation media, as depicted in **Figure 2b**, after reducing 150 g/L of sugar, the results showed a rapid consumption of the reducing sugar when compared to **Figure 2a**. More than 97% of the reducing sugar was converted (during the first 84 h of fermentation), the acetoin concentration approached a maximum, and the biomass growth was constant at 8.6 and 7.7 g/L, respectively. The wide range of amino acids, peptides, vitamins, inorganic salts, and carbon in growth media of yeast extract supports the biomass growth and rapid sugar utilization [33].

In **Figure 2c**, the extract was used in place of CSL and yeast extract as earlier discussed (**Figure 2a** and **b**). The beef extract is a mixture of peptides and amino acids, nucleotide fractions, organic acids, minerals, and some vitamins. Its function can, therefore, be described as complementing the nutritive properties of peptone by contributing minerals, phosphates, energy sources, and those essential factors missing from peptone [34]. The glucose was almost depleted after 96 h till the end of the fermentation lapse. It can be deduced from the study that the beef extract supports the utilization of glucose consumption as more than 98% of the glucose has been consumed within 96 h of fermentation.

To see the clear difference of the complex media earlier considered, the acetoin fermentation was done without the nitrogen sources (**Figure 2d**) but with the simple salts in the fermentation

**Figure 2.** (a) Plots of AC fermentation using corn steep liquor as nitrogen source. (b) Plots of AC fermentation using yeast extract as nitrogen source. (c) Plots of AC fermentation using beef extract as nitrogen source. (d) Plots of AC fermentation using none of the complex media as the nitrogen source. (RS–reducing sugar; AC–acetoin; BM–biomass).

media. A maximum acetoin of about 5.2 g/L was obtained during the first 12 h of the fermentation, after which there was no significant change in the biomass growth from that period until the lapse time of fermentation. About 50% (72.53 g/L) of sugar concentration was not consumed until the fermentation time lapse of 168 h, and the sluggish consumption of glucose could be traceable to the lack of rich nitrogen sources, which was needed as a nutritional supplement [28].
