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

*New Advances on Fermentation Processes*

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

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

The percentage of initial sugar added to the medium (w/v) 0% 10% 20% Conversion efficiency 233% 46.60% 6.40%

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

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

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.

maximum activity levels of the microbes.

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**Table 2.**

**Figure 1.**

*medium.*

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 maximizes the production of acetic acid.

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

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

#### **Figure 2.**

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

#### **Figure 3.**

*Changes in the acetic acid contents and pH values with variations in the inoculum ratios between S. cerevisiae and A. aceti during the fermentation process.*

acetic acid levels to decrease. Moreover, the acetic acid that is already produced undergoes overoxidation by *A. aceti* via the tricarboxylic acid (TCA) cycle [27].

The highest level of acetic acid was achieved on day 8, using the 7:3 inoculum ratio of *S. cerevisiae* to *A. aceti*. At the beginning of the fermentation process, the acetic acid concentration for this ratio was lower than for the 3:7 inoculum ratio, due to the dominance of *S. cerevisiae*. However, under aerobic conditions, *S. cerevisiae* is still able to produce alcohol in small amounts, and the large population of *S. cerevisiae* cells can produce enough alcohol to meet the nutrient requirements of *A. aceti*. During the later stages, the low levels of oxygen consumption by *S. cerevisiae* during alcohol production cause the availability of oxygen in the medium to become sufficient for *A. aceti* growth, and the resulting acetic acid contents increase.

#### **3.3 Agitation speed for optimal mixing**

As explained above, under aerobic conditions, the fermentation process using mixed cultures can work well, as indicated by the greater than 100% conversion efficiency from sugar to acetic acid. These results were achieved using an agitation speed of 80 rpm. Agitation plays an important role in fermentation processes, causing surface renewal; aiding in the dissolution of oxygen found at the top of the fermentor; improving the transfer of oxygen, heat, and mass through the system; and maintaining homogeneous physical and chemical conditions within the medium [28, 29]. Thus, the effect of agitation speed on the production of acetic acid in this system was evaluated by examining agitation speeds of 80 and 160 rpm (**Figure 4**). The percentage of acetic acid produced from both treatments can be seen in **Figure 5**.

The results showed that faster agitation speeds consistently resulted in higher acetic acid contents. The highest acetic acid level, 6.47%, was achieved on day 10 using an agitation speed of 160 rpm. Agitation is an important parameter for all aerobic processes [29]. The purpose of agitation during a submerged fermentation process is to homogeneously increase the availability and solubility of oxygen in the medium. Increased dissolved oxygen concentrations, generated by increased

**197**

*Streamlining the Fermentation Process Using Mixed Cultures*

agitation speeds, resulted in a shortened lag time for cell growth and increased biomass formation [28]. Oxygen is needed not only by *A. aceti* but also by *S. cerevisiae* for growth [30, 31]. Massive oxygen consumption by both microbes simultaneously can create anaerobic conditions, causing *S. cerevisiae* to shift its metabolism from respiratory to fermentative and to produce alcohol. According to Navarro and Durand [32], during fermentation, yeast growth is rapidly stopped when the concentration of alcohol in the medium increases; however, fermentative activity is not entirely inhibited until high alcohol concentrations are reached. However, alcohol consumption by *A. aceti* prevents the concentration of alcohol in the medium from reaching the maximum value, preventing the inhibition of *S. cerevisiae* growth and activity, as indicated by the increase of glucose and alcohol contents in the medium. However, oxygen remains available in the medium, due to rapid agitation, allowing the growth and the activity of *A. aceti* to remain at high levels. *A. aceti* can directly use dissolved oxygen to grow and to produce acetic acid, and, simultaneously, the environment becomes anaerobic or microaerobic, allowing *S. cerevisiae* to produce alcohol, which is then used by *A. aceti* as a substrate for the production of acetic acid. According to Zhou et al. [29], agitation can cause shear forces that can influence changes in cell morphology, variations in the growth and formation of products, and damages to the cell structure. However, increasing the speed of agitation results in stronger mixing processes, more rapid contacts between nutrients and microbes,

*The percentage of acetic acid produced from fermentation at different agitation speeds.*

*Schematic diagram of the submerged batch fermentation process to determine the optimum agitation speed.*

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

**Figure 4.**

**Figure 5.**

*Streamlining the Fermentation Process Using Mixed Cultures DOI: http://dx.doi.org/10.5772/intechopen.87205*

**Figure 4.**

*New Advances on Fermentation Processes*

acetic acid levels to decrease. Moreover, the acetic acid that is already produced undergoes overoxidation by *A. aceti* via the tricarboxylic acid (TCA) cycle [27]. The highest level of acetic acid was achieved on day 8, using the 7:3 inoculum ratio of *S. cerevisiae* to *A. aceti*. At the beginning of the fermentation process, the acetic acid concentration for this ratio was lower than for the 3:7 inoculum ratio, due to the dominance of *S. cerevisiae*. However, under aerobic conditions, *S. cerevisiae* is

*Changes in the acetic acid contents and pH values with variations in the inoculum ratios between S. cerevisiae* 

still able to produce alcohol in small amounts, and the large population of

*A. aceti*. During the later stages, the low levels of oxygen consumption by *S. cerevisiae* during alcohol production cause the availability of oxygen in the medium to become sufficient for *A. aceti* growth, and the resulting acetic acid

*S. cerevisiae* cells can produce enough alcohol to meet the nutrient requirements of

As explained above, under aerobic conditions, the fermentation process using mixed cultures can work well, as indicated by the greater than 100% conversion efficiency from sugar to acetic acid. These results were achieved using an agitation speed of 80 rpm. Agitation plays an important role in fermentation processes, causing surface renewal; aiding in the dissolution of oxygen found at the top of the fermentor; improving the transfer of oxygen, heat, and mass through the system; and maintaining homogeneous physical and chemical conditions within the medium [28, 29]. Thus, the effect of agitation speed on the production of acetic acid in this system was evaluated by examining agitation speeds of 80 and 160 rpm (**Figure 4**). The percentage of acetic acid produced from both treatments can be

The results showed that faster agitation speeds consistently resulted in higher acetic acid contents. The highest acetic acid level, 6.47%, was achieved on day 10 using an agitation speed of 160 rpm. Agitation is an important parameter for all aerobic processes [29]. The purpose of agitation during a submerged fermentation process is to homogeneously increase the availability and solubility of oxygen in the medium. Increased dissolved oxygen concentrations, generated by increased

**196**

contents increase.

**Figure 3.**

seen in **Figure 5**.

**3.3 Agitation speed for optimal mixing**

*and A. aceti during the fermentation process.*

*Schematic diagram of the submerged batch fermentation process to determine the optimum agitation speed.*

#### **Figure 5.**

*The percentage of acetic acid produced from fermentation at different agitation speeds.*

agitation speeds, resulted in a shortened lag time for cell growth and increased biomass formation [28]. Oxygen is needed not only by *A. aceti* but also by *S. cerevisiae* for growth [30, 31]. Massive oxygen consumption by both microbes simultaneously can create anaerobic conditions, causing *S. cerevisiae* to shift its metabolism from respiratory to fermentative and to produce alcohol. According to Navarro and Durand [32], during fermentation, yeast growth is rapidly stopped when the concentration of alcohol in the medium increases; however, fermentative activity is not entirely inhibited until high alcohol concentrations are reached. However, alcohol consumption by *A. aceti* prevents the concentration of alcohol in the medium from reaching the maximum value, preventing the inhibition of *S. cerevisiae* growth and activity, as indicated by the increase of glucose and alcohol contents in the medium. However, oxygen remains available in the medium, due to rapid agitation, allowing the growth and the activity of *A. aceti* to remain at high levels. *A. aceti* can directly use dissolved oxygen to grow and to produce acetic acid, and, simultaneously, the environment becomes anaerobic or microaerobic, allowing *S. cerevisiae* to produce alcohol, which is then used by *A. aceti* as a substrate for the production of acetic acid.

According to Zhou et al. [29], agitation can cause shear forces that can influence changes in cell morphology, variations in the growth and formation of products, and damages to the cell structure. However, increasing the speed of agitation results in stronger mixing processes, more rapid contacts between nutrients and microbes,

and higher oxygen transfer rates (OTR) and oxygen uptake rates (OUR); therefore, aerobic and anaerobic environmental conditions are created simultaneously over a short period of time. Therefore, increasing agitation speed, up to a certain level, can lead to the production of larger amounts of acetic acid over shorter periods of time. In addition, the high dissolved oxygen content caused by the increased agitation speed in this system does not appear to cause oxidative stress or damage to proteins in cells, which could inhibit *A. aceti* growth [33]. As a whole, under conditions using an optimal inoculum ratio and an optimal agitation speed, the conversion efficiency from sugar to acetic acid increased to 362%.

### **3.4 The dynamics of changes in the sugar, alcohol, and acetic acid contents and in the pH value during fermentation under optimal conditions**

The dynamics of changes in the sugar, alcohol, and acetic acid contents and in the pH values during the fermentation of acetic acid from apple juice under optimal conditions can be observed in **Figure 6**. In the beginning, when the sugar level is high, *S. cerevisiae* works to produce alcohol, increasing the alcohol contents. In conjunction with the production of alcohol, *A. aceti* began to produce acetic acid, causing the acetic acid level to increase. As *S. cerevisiae* produces alcohol, *A. aceti* simultaneously grows until the alcohol contents produced by *S. cerevisiae* are sufficient for *A. aceti* to produce acetic acid. In the mixed culture fermentation, *A. aceti* which is an obligate aerobic microbe uses dissolved oxygen for growth and for the oxidation of alcohol into acetic acid. However, the medium also undergoes an anaerobic state due to a lack of oxygen, allowing *S. cerevisiae* to convert sugar into alcohol. Another advantage of the use of mixed cultures is that the continuous consumption of oxygen by *A. aceti* appears to cause *S. cerevisiae* to grow without the multiplication of cell mass. Thus, the sugar present in the substrate can maximally be converted into alcohol by *S. cerevisiae*, and the alcohol can subsequently maximally be converted into acetic acid by *A. aceti*. However, at the end of the process, a decrease in the resulting acetic acid levels was observed. This decrease may be due to the unfavorable pH of the medium, which could inhibit the microbes from metabolizing substrates and producing acetic acid, or may be due to acetic acid overoxidation due to the limited

#### **Figure 6.**

*The dynamics of the changes in glucose, alcohol, and acetic acid contents and pH values under optimal fermentation conditions.*

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*Streamlining the Fermentation Process Using Mixed Cultures*

tion process can be stopped when the highest yield is achieved.

availability of nutrients. For the purposes of harvesting the products, the fermenta-

Under the right conditions, the production of acetic acid can be maximized by using a simple system, such as submerged batch fermentation using a mixed culture that acts synchronously. Optimization can be performed by considering the character and needs of all microbes involved, which are the nutritional adequacy of the medium, the microbial proportions in the inoculum, and the agitation speed. The use of a mixed culture could shorten the fermentation time, reduce fermentation losses, and increase the acetic acid yields [16]. Some other advantages of this system compared with a gradual system are the relatively simple operation and easy handling of this system, which no particular control is required during the fermentation process, and the low risk of contamination. Thus, the application of this system for industrial purposes can be considered. However, the future scaling up of this process should consider other factors, including automation systems and the use of cutting-edge technologies in both the production and monitoring processes, to further improve the productivity and product quality without increasing produc-

The efficiency of the acetic acid fermentation process can be assessed using a simplified system with mixed cultures. Some of the aspects evaluated in this system were the availability of sugar in the medium, the inoculum ratio of the cultures used, and the speed of agitation. By optimizing this system, the resulting acetic acid

Department of Biology, Padjadjaran University, Sumedang, Indonesia

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: keukeu@unpad.ac.id

provided the original work is properly cited.

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

**3.5 Future outlook**

tion costs.

**4. Conclusion**

levels can be increased.

**Author details**

Keukeu Kaniawati Rosada

availability of nutrients. For the purposes of harvesting the products, the fermentation process can be stopped when the highest yield is achieved.
