**3. Enhanced acetic acid production from raw materials using mixed cultures during batch-type fermentation**

Acetic acid fermentation has been studied using apples as a substrate. The fermentation was performed using submerged batch cultivation with mixed cultures of *S. cerevisiae* and *A. aceti*, which were inoculated simultaneously at the beginning of the process. These two microorganisms have different physiological properties: *S. cerevisiae* is a facultative anaerobe and requires anaerobic conditions to produce alcohol, and *A. aceti* is strictly aerobic. Because these microorganisms have opposing characteristics, using both cultures simultaneously is challenging, especially because acetic acid is a strong inhibitor of yeast, whereas yeast makes the medium anaerobic and unsuitable for AAB growth [5].

During this fermentation process, the first thing to be considered is the availability of sugar in the substrate, which represents a carbon source for the growth of the two microorganisms and the production of acetic acid. We must determine whether the sugar requirements are met by the substrate or whether sugar must be added, as described in previous studies [7, 8, 16]. Another factor that must be considered during this process is the inoculum ratio between the two microbes, as the strong competition between the two microbial groups must be anticipated and balanced to allow the production of acetic acid. Furthermore, because the different stages of acetic acid fermentation demand different oxygen requirements, the appropriate agitation speed is also important to consider.

#### **3.1 Availability of sugar for microbial growth and acetic acid production**

The microbial requirements for sugar during acetic acid fermentation and the availability of sugar in the substrate can be observed using different experiments, such as those shown in **Figure 1**. The system uses one-third of the working volume. Because this type of fermentation uses a batch culture with only one stage, all materials are added simultaneously at the beginning of the process, including sugar, at concentration of 0, 10, and 20% (w/v). Changes in the sugar, alcohol, and acetic acid contents and changes in the pH values during this process were evaluated, as previously described [9]. The results demonstrated that the conversions of sugar into alcohol and of alcohol into acetic acid were accompanied by decreases in the pH of the medium. This result indicates that the fermentation process has been successfully performed.

#### **Figure 1.**

*Schematic diagram of the submerged batch fermentation process to evaluate the availability of sugar in the medium.*


#### **Table 2.**

*The conversion efficiency from sugar to acetic acid during 10 days of the fermentation process.*

The sugar availability requirements during fermentation were determined by calculating the efficiency of the conversion of sugars into acetic acid (**Table 2**). According to **Table 2**, the conversion of sugar to acetic acid occurred with the highest efficiency when no sugar was added to the medium. Therefore, there is no need to add sugar to the medium because the substrate itself is sufficient to meet the needs of the microbes involved in the fermentation process and still produce a high acetic acid yield. The conversion efficiency of sugar into acetic acid during the fermentation process without the addition of sugar was greater than 100%. This result can be the result of the hydrolysis of starches contained in the medium, either chemically due to the decrease in pH [19, 20] or by *S. cerevisiae* to support growth and metabolic activity [21]. In addition to using glucose as its primary substrate, *S. cerevisiae* is able to grow on a wide range of carbon compounds, is able to metabolize some carbohydrates after they have undergone extracellular hydrolysis, and is able to ensure the efficient metabolism of those hydrolyzed carbohydrates [22]. Therefore, the addition of sugar to the fermentation medium is not required because *S. cerevisiae* is able to decompose and utilize the sugars that already exist in apples, which is evident from the relatively stable fermentative sugar content found in the fermentation medium during the fermentation process [9]. These results suggest that the nutrients contained in the apples were sufficient to support the maximum activity levels of the microbes.

In general, a higher sugar concentration in the medium results in the formation of a greater acetic acid content. However, excess sugar in the fermentation medium will not increase the microbial activity above its maximum threshold, and high sugar concentrations can limit the production of yeast biomass [23]. In addition, high levels of sugar can create anaerobic or microaerobic environmental conditions, which can inhibit the growth and activity of aerobic obligate bacteria, such as *A. aceti*, which is not optimal for acetic acid production. Thus, the availability of complex forms of sugar within the natural medium presents the advantage of providing a gradual carbon source to meet the needs of microbes.

**195**

**Figure 2.**

*the cultures used.*

*Streamlining the Fermentation Process Using Mixed Cultures*

In addition to the availability of sugar in the medium, the other factor that must be considered when performing acetic acid fermentations using mixed cultures is the optimal inoculum ratio of all cultures involved; in this case, *S. cerevisiae* and *A. aceti* were used. Because the two groups of microbes have different physiological properties, especially in terms of oxygen requirements, they also have different needs for carbon, different metabolic properties, and different growth rates. As mentioned above, *S. cerevisiae* is a facultative anaerobe that is able to grow on a wide range of carbon compounds and is able to produce alcohol under anaerobic conditions, whereas *A. aceti* is an obligate aerobe that is able to use ethanol, glycerol, and glucose as carbon sources for growth but is unable to hydrolyze lactose and starch and can oxidize ethanol to acetic acid and acetate to CO2 and H2O [4, 24]. Moreover, *S. cerevisiae* has a longer growth rate than *A. aceti* [9]*.* The metabolism and physiology of these two microbes have been described previously, in detail [4, 22, 24–26]. With these differences, the regulation of species dominance in mixed cultures by adjusting the inoculum ratios is expected to result in a syntrophic state that maxi-

An example of an experimental design to determine the best ratio of the cultures used during acetic acid fermentation is shown in **Figure 2**. The ratios of *S. cerevisiae* and *A. aceti* cultures used were 3:7, 1:1, and 7:3. The performances of these microbes when used at different ratios during acetic acid production can be observed by measuring the changes in acetic acid contents and pH values during the process (**Figure 3**). The results showed that the highest acetic acid concentration with the lowest pH value was achieved on day 8 using mixed cultures of *S. cerevisiae* and

According to **Figure 3**, the acetic acid levels produced by the ratio of the 3:7 of *S. cerevisiae* to *A. aceti* are higher at the beginning of the process than those produced by the other ratios. In this period, the dominance of *A. aceti* over *S. cerevisiae* results in *A. aceti* rapidly utilizing glucose to convert the ethanol produced by *S. cerevisiae* into acetic acid. According to Maier [26], the initial inoculum size controls the length of the lag phase. However, during the next stage, the resulting acetic acid contents decreased. The larger ratio of *A. aceti* causes this microbe to require more nutrients, which the smaller ratio of *S. cerevisiae* cannot provide. The limited nutrients available to *A. aceti* result in suboptimal cell growth and enzymatic activity, causing the metabolic processes of *A. aceti* to not work properly and the resulting

*Schematic diagram of the submerged batch fermentation process to determine the optimum inoculum ratio for* 

**3.2 Inoculum ratio of** *S. cerevisiae* **and** *A. aceti*

*DOI: http://dx.doi.org/10.5772/intechopen.87205*

mizes the production of acetic acid.

*A. aceti* at a 7:3 ratio.
