**4. Effect of process parameters in VFAs production**

The production of VFAs through FW fermentation using mixed microbial cultures, as well as the VFA composition and yield, are influenced by numerous factors, such as carbon source, pH, temperature, HRT, inoculum, and OLR [2, 3, 6, 7].

#### **4.1 Carbon source**

The type of carbon source, namely the complexity and composition, affects the acidification degree, since this depends on the readily fermentable organic fraction, and consequently, influences the production of VFAs [3, 25]. The content of carbohydrates, lipids and proteins influences the production of VFAs, since each fraction present different biodegradability and hydrolysis efficiency [6]. Moreover, the type of acids produced depends on the waste composition. The fermentation of carbohydrates, proteins and lipids form directly acetic, propionic, and butyric acids, while valeric and iso-valeric acids are related to the fermentation of proteins [15]. Lipids are the fraction more resistant to the biodegradation, being less suitable for AF than the other FW fractions, despite their high contribution for COD [6, 20]. For that reason, usually, lipids are separated and used to produce biodiesel instead of using to VFAs production [20].

Commonly, carbohydrates are easily converted into glucose and then fermented to VFAs [20]. However, the carbohydrate fraction, that is not readily degraded (cellulose, lignin or hemicellulose), can affect the VFA production rate, as well as the VFA concentration and yield (**Table 1**).

Zhang et al. [25] observed that high content of readily degradable carbohydrates promotes a faster production of VFAs, while the presence of not readily degraded fraction (e.g., cellulose) can delay the production of VFAs or even lead to a lower production. Another study, showed that different FW (cheese whey, sugarcane molasses and olive mill effluent) resulted in different VFAs production (**Table 1**) and also different acidification degree (cheese whey and sugarcane molasses: up to 40%; olive mill effluent: up to 12%) [39].

The type of carbon source and its composition (e.g. carbohydrates, lipids, proteins) lead to different acidogenic metabolic pathways and, consequently, different VFAs composition. Indeed, in Zhang's study [25], according to the VFA profile, different fermentation type were obtained (**Table 1**). Silva et al., [39] observed that for the readily fermentable wastes (cheese whey and sugarcane molasses), acetic


#### **Table 1.**

*VFA production from FW using mixed microbial cultures.*

acid was the main acid produced, followed by butyric acid (26–28%) and caproic and iso-valeric acids (10–14%), while for the waste with low acidogenic potential (olive mill effluent), acetic acid was followed by propionic acid (21–24%) and the production of heavier VFA (e.g., butyric, valeric acids) was not detected.

Proteins have a complex structure, making them less suitable to protease action and, consequently, a lower hydrolysis efficiency [6, 20]. Indeed, the hydrolysis of carbohydrates can be up to 80%, while protein hydrolysis is between 40% to 70%, which leads the latter to be considered the rate-limiting step in AF [6]. As carbohydrates, the origin of proteins affects the production and composition of VFAs. Shen et al., [20] obtained a high production of VFA using animal protein when compared with vegetal protein (**Table 1**). Independently of the protein, animal and vegetal, acetic, propionic, butyric and valeric acids were produced. However, the acids profile is different in the fermented vegetal and animal protein. This difference in composition can be related to the type of amino acids present in tofu and egg white. Even though it has been demonstrated that the carbon source affects the VFA production, FW feedstocks with similar compositions must still be individually investigated, as other factors, like operational conditions, should be considered.

#### **4.2 pH**

pH is one of the most important and critical parameters in VFA production, since it affects VFA concentration and composition due to its influence in hydrolysis and acidogenic process [3, 5]. pH affects the microorganisms activity since most of the enzymes cannot tolerate low or high pH environments (pH < 3 or pH > 12) [2, 5]. Moreover, pH can also promote the inhibition of methanogenic activity, since operating the reactor out of their optimal range (7.0–8.2), the methane production can be inhibited [33]. The optimal pH for VFA production depends on the type of waste and should fit both hydrolysis and acidogenic steps [2, 5]. Indeed, different wastes present different optimal pH for VFA production (**Table 2**).


*From Food Waste to Volatile Fatty Acids towards a Circular Economy DOI: http://dx.doi.org/10.5772/intechopen.96542*

*RT – retention time; HAc – acetic acid; HPr – propionic acid; HBut – butyric acid; others can include valeric acid, iso-butyric and iso-valeric acid, caproic acid or not VFA; Ref. - reference.*

#### **Table 2.**

*Optimal pH for VFA production from different carbon source.*

Bermúdez-Penabad et al. [15] studied the effect of low (5 and 6), neutral (7) and alkaline (8, 9 and 10) pH in the acidogenic fermentation of tuna waste and obtained the highest VFA production under alkaline conditions, achieving the highest production at pH 8. Although, the highest hydrolysis was also obtained under alkaline conditions, indicating that more substrate is available for acidification, strong alkaline conditions (pH 10) seem to affect the acidogenic bacteria activity since the ratio VFA/sCOD was the lowest one. Acetic and butyric acids were the main VFAs produced (except for pH 10). The increase of pH from 7 to 10 led to a decrease of the butyric acid content, while the change of pH from 5 to 8 led to an increase of acetic acid. Another study, using FW from canteen as feedstock, also observed the highest VFA production at alkaline pH (9), being acetic acid the dominant acid (60.5%) [33]. Hussain et al., [14], using solid FW from cafeteria as feedstock, operated four thermophilic leach bed reactor in batch mode under pH 4–7 and observed an increase of VFA production with pH increase (from 6 gCOD/L to 36.5 gCOD/L, corresponding a maximum yield of 247 g COD/kg TVSadded). At all pH, acetic and butyric acids accounted to 80–85% of the total VFA, being acetic acid the main produced acid at pH 4 and 5 (55–61%) and butyric acid the dominant compound at pH 6 and 7 (48–54%). On the other hand, Ma et al., [38], using FW from cafeteria, obtained the highest production of VFA at pH 6 (53.87 g/L), with a significant production of propionic acid. The fermentation of potato peel waste showed a different trend of VFA composition, since butyric acid was the main compound at acidic (pH 5) and uncontrolled pH, while acetic acid was the main compound produced at alkaline and neutral pH [19]. Moreover, the highest production of VFA was achieved at pH 7 (41.9 gCOD/L and 0.63 gCOD/gVSfed). Stein et al., [34] studied the effect of pH, temperature and HRT on the maximization of the butyric acid production. pH 9 led to the highest production of VFA, as well as the highest VFA yield (0.726 gVFA/gVSfed), which was about 80% higher than the yield obtained at pH 7. Besides butyric acid, that was the dominant compound at pH 9, acetic acid was also produced as the dominant compound (**Table 2**) and was the dominant

compound at pH 5.5 and 7 (34.8% and 47.9%, respectively). Propionic and isobutyric acids were also produced. However, their content decreased at the highest pH tested. Wang et al., [23] studied the effect of pH (4, 5, 6 and uncontrolled) on the VFA production with two different inoculums (aerobic and anaerobic activated sludge). Regardless of the inoculum, VFA production was highest at pH 6, achieving a maximum of 30.8 gCOD/L (0.482 g/gVSSremoval, after 4 days of fermentation) and 51.3 gCOD/L (0.918 g/gVSSremoval, after 20 days of fermentation) for aerobic and anaerobic inoculum, respectively. Butyric acid was the dominant acid at pH 6, followed by acetic and propionic acids. Moreover, acetic and butyric acids were the predominant acids in all the pH tested, except for pH 4 and uncontrolled pH for aerobic inoculum, where acetic and propionic acids were the prevalent acids. Gouveia et al. [8] studied the impact of a dynamic variation of pH from 4 to 7, returning to pH 6 after each pH variation, in cheese whey derived-VFA production process. The production of organic acids was quite constant for all pH tested (about 13 gCOD/L), apart from pH 4 (about 4 gCOD/L). At pH 6, acetic acid was the main VFA, comprising 22–44%, and regardless of the dynamic variation of pH, the composition was always similar at pH 6. At high pH, the production of acetic acid was favored. Feng et al., [26] tested different pH (4–11) at room temperature and observed the highest concentration of VFAs at pH 8 (8.24 gCOD/L at 4 days of fermentation) during the co-digestion of FW with WAS. Acetic, propionic and butyric acids were the most prevalent VFA produced, achieving a total content of 82.5%, 91.9% and 92.9% at pH 5, 7 and 8, respectively. From pH 6 to 10, propionic acid was the main compound produced, while for pH 4 and 5, acetic acid was the main compound.

The pH effect on VFA production and profile using mixed cultures and FW does not present a direct relationship, being also dependent of the type of substrate used.

#### **4.3 Temperature**

Temperature is a key parameter that impacts the growth of microorganisms and their metabolism. Each microbial taxon has an optimal temperature range for its growth, and, therefore, a change on the operating temperature can affect the microbial population involved in the acidogenic fermentation [5, 6]. Mesophilic condition (25–45 °C) showed a similar or even higher VFA yield than thermophilic (50–60 °C) or hyperthermophilic (>65 °C) conditions [5]. On the other hand, thermophilic and hyperthermophilic conditions results in a higher hydrolysis and solubilization in comparison with mesophilic condition, which can lead to an increase of VFA production if an adequate microbial community is present [2, 13]. The composition of VFAs is also affected by the temperature, but in a less extent than that was observed for pH [2, 5]. The increase of temperature from mesophilic to thermophilic conditions leads to the metabolic shift from acetic acid to butyric acid [13]. Jiang et al. [36] reported that sCOD increased with temperature increase. However, the VFA concentration and yield at 55 °C was much lower than that obtained at 35 °C and 45 °C (14.90 g/L and 0.137 g/gVSfed, 41.34 g/L and 0.379 g/gVSfed, and 47.89 g/L and 0.440 g/gVSfed, for 55, 35 and 45 °C, respectively), indicating a higher solubilization but a lower acidogenesis of FW at thermophilic condition (55 °C). The temperature increase resulted in a decrease of acetic and valeric acids content and an increase of butyric acid content. Acetic and propionic acid were the major compounds at 35 and 45 °C, representing ca. 70% of the total of VFAs. At 55 °C, butyric acid was the main compound, comprising more than 81% of the total VFAs and valeric acid was not detected. Although a higher ratio of VFA/COD and VFA concentration was observed at 45 °C, a high amount of energy is necessary to operate at this temperature. As such, 35 °C is considered as being the most cost-effective temperature

for FW derived-VFA production process [36]. Similarly, He et al., [37] observed a decrease of VFA concentration, from 17 to 11 g/L, when the temperature increase from 35 to 55 °C. At 70 °C, the VFA production (about 13 g/L) was higher than 45 °C but lower than 35 °C. The hydrolysis rate was directly affected by temperature, having increased with temperature increase, indicating that higher temperatures promote the hydrolysis of FW. Comparing the three temperatures, acetic acid was the main compound at 70 °C, iso-butyric and butyric acids were the main compounds at 55 °C, while ethanol was the main compound at 35 °C, showing that the increase of temperature can inhibit ethanol production, favoring the production of VFAs, namely acetic and butyric acids. Zhang et al. [35], who have studied the effect of two temperatures (35 and 55 °C) at different pH (5, 6 and 7), reached the maximum VFA yield of 11.8 gCOD/L at pH 7 and 35 °C, being acetic, propionic and butyric acids the main produced VFAs (about 80% of total VFAs). For each initial pH applied, mesophilic conditions led to higher VFA concentration. When comparing all the conditions tested, except for pH 7 and 55 °C, thermophilic temperatures led to lower VFA production than mesophilic conditions. Applying the optimal conditions to a continuous reactor, an average VFA concentration and yield of 6.3 gCOD/L and 0.29 gVFA/gVSadded was obtained.

Temperature (37, 55 and 70 °C) also affected the maximization of the butyric acid production [34]. Higher temperatures led to a decrease of VFA concentration, namely in butyric acid concentration, except at pH 7 and 55 °C, where an 280% increase of butyric acid concentration was achieved when compared to mesophilic conditions. The maximum concentration of butyric acid was achieved at pH 7 and 55 °C (10.55 g/L ± 0.17) and pH 9 and 37 °C (8.52 g/L ± 0.10).

Considering operating costs, mesophilic conditions (25–45 °C) are the most economical and efficient temperatures to produce VFAs.
