4.1 Effect of raw glycerol concentration
Table 1 presents the results of the impact of glycerol and raw glycerol concentration on various parameters of R. toruloides growth. No growth inhibition was observed within the examined range. Notably, an increase in biomass concentration biomass/substrate yield (Yx/s), and maximum specific growth rate (µ) was observed when raw glycerol was utilized compared to pure glycerol under identical conditions.
Table 1
The effect of glycerol and raw glycerol concentration on dry cell concentration (DCC), biomass/substrate yield (Yx/s) and maximum specific growth rate (µ) of R. toruloides when grown at pH 5.0, 30°C and 150 rpm.
|
Glycerol
|
Raw glycerol
|
Conc
|
DCC
|
Yx/s
|
µ
|
DCC
|
Yx/s
|
µ
|
(g/L)
|
( g/L)
|
(g/g)
|
(h− 1)
|
(g/L)
|
(g/g)
|
(h− 1)
|
20
|
7,34 ± 0,3a
|
0,28 ± 0,04d
|
0,15 ± 0,0g
|
10,2 ± 1,77j
|
0,51 ± 0,09n
|
0,190 ± 0,01q
|
40
|
9,15 ± 0,2a
|
0,19 ± 0,03e
|
0,16 ± 0,0g
|
13,5 ± 0,38k
|
0,34 ± 0,01ñ
|
0,200 ± 0,01q
|
60
|
10,3 ± 1,6a
|
0,14 ± 0,02f
|
0,18 ± 0,0g
|
15,9 ± 1,20k
|
0,26 ± 0,02o
|
0,237 ± 0,01q
|
80
|
12,1 ± 1,9b
|
0,13 ± 0,02f
|
0,22 ± 0,0h
|
17,4 ± 1,80kl
|
0,21 ± 0,02o
|
0,222 ± 0,01q
|
100
|
15,7 ± 1,4c
|
0,14 ± 0,01f
|
0,20 ± 0,0h
|
16,2 ± 1,45k
|
0,16 ± 0,01p
|
0,253 ± 0,01r
|
120
|
17,7 ± 2,1c
|
0,14 ± 0,04f
|
0,26 ± 0,0i
|
21,4 ± 1,62m
|
0,18 ± 0,01p
|
0,270 ± 0,03r
|
Different letters in the same column in the table indicate that there are significant differences according to the Tukey test (p ≤ 0.05). Data are the mean values of 3 samples ± standart deviation (SD)
This behavior aligns with findings by Xu et al [65] using the R. toruloides AS 21389; although a latency period during the initial hours was observed in R toruloides Y-1091. Specific growth rates in glycerol were comparable to those reported for Rhodotorula mucilaginosa DISVA6094 (0.14 h− 1) and DISVAC7.1 (0.16 h− 1) [66].
High substrate concentration, even in the case of raw glycerol, can inhibit cell growth due to medium acidification. Although there is evidence of this effect during growth adaptation phase, specific growth rates remain similar than those observed with pure glycerol. The composition of the raw glycerol impurities, dependent of biodiesel preparation methods, plays a crucial role in this effects [67].
The observed increase of biomass concentration, biomass yield and specific growth rate with raw glycerol can be attributed to the due presence of macroelements as potassium, calcium, sulfur and magnesium in raw glycerol, favoring cellular growth and lipid accumulation. Certain impurities, including inorganic salts, glycerides and soaps, positively impact biomass and lipid production, while methyl esters exhibit no significant effect. Studies indicate that methanol, a common impurity, has the largest inhibitor effect [68–69].
4.2 Effect of Nitrogen Source
The utilization of either ammonium sulpate or urea in media with glycerol, as well as raw glycerol, revealed no significant differences in maximal cell growth. However, a noteworthy increase in lipid content was observed when ammonium sulfate was replaced by urea in both substrates, with higher values attained in raw glycerol media (Fig. 1).
Higher results were obtained using raw glycerol and urea, reaching 7.9 ± 0.4 g/L of biomass concentration, 51.8 ± 1.6% of lipids and productivity of 0.034 ± 0.0005 g/Lh. Similar behavior was obtained with R. toruloides CBS 14 in different nitrogen sources such as ammonium sulphate and L-glutamate chloride, reaching 18 and 51%, respectively [70].
Figure 1: The effect of the nitrogen source on biomass concentration, lipid concentration and productivity grown at initial pH 5.0, 30°C and 150 rpm. Data are shown as mean ± SD from three experiments (n = 3).
Several researchers obtained similar contents of lipids using R. mucilaginous (55.1%) and Lipomyces Lipofer NRRL Y-11555 (57.8%) cultured in raw glycerol [71–72]. On the other hand, results of Xu et al [67] were even higher when cultured R. toruloides AS2.1389 in 5L fermenter.
Research indicate that organic and inorganic nitrogen have varying effects on cell growth and lipid accumulation. Inorganic nitrogen enhances both, while organic nitrogen containing amino acid and vitamins, may specifically support the growth and production of lipids in yeasts [73–76].
4.3 Effect of C/N molar ratio
Under established conditions, biomass concentration and biomass/substrate yield remained constant at an average value of 13.47 ± 0.8 g/L and 25,88 ± 1.6 g/g, respectively, in C/N 50–400 interval. However, lipid content and productivity reached maximum values of 39.3 ± 2.5% and 0.033 ± 0.004 g/Lh in a same C/N interval 250–400. These results suggest that, under the conditions evaluated (50 ≤ C/N ≤ 400), the metabolism of R. toruloides, is directed towards lipid biosynthesis rather than cell proliferation. C/N = 250 was selected to continue the next studies to establish the limited culture condition. Figure 2 shows the behavior of the main growth and lipid production parameters obtained.
Cell biomass concentration, lipid content and productivity in 20–120 g/L glycerol interval were determined once the C/N value with the highest productivity (C/N 250) was established. The results showed that the biomass concentration increased until 40 g/L glycerol and then remained constant (11.1 ± 0.4 g/L). Lipid content increased to 80 g/L (48.1 ± 1,3%) and lipid productivity had a parabolic behavior reaching maximal values (0.033 ± 0.002 g/Lh) at 60–80 g/L glycerol range (Fig. 3).
Figure 2: The effect of C/N ratio on biomass concentration, lipid content, biomass performance/substrate and productivity of R. toruloides grown at initial pH 5.0, 30°C and 150 rpm. Each point represents the mean value ± SD of three samples of a representative experiment of three runs made (* p < 0.05, Fisher test).
Figure 3: The effect of glycerol concentration in biomass concentration, lipid content, biomass/substrate yield and productivity of R. toruloides using nitrogen limited media (C/N 250). Each point represents the mean value ± SD of three samples from a representative experiment of three runs performed (p < 0.05, Fisher's test).
The biomass/substrate and product/substrate yields decreased with increasing glycerol concentration. This behavior is similar to that observed during the evaluation of Torulaspora maleeae Y30 using 40–100 g/L glucose concentrations [77] and to those obtained by Kuntzler et al. with R. mucilaginosa and R. glutinis strains using raw glycerol as substrate [71].
4.4 Effect of Initial Temperature
The results of this trial showed that the maximum value of biomass concentration was reached at 25°C (26.6 ± 0.7 g/L) however, the lipid content did not vary significantly in the evaluated temperature range. Therefore, the highest lipid productivity was obtained at 25°C (0.027 ± 0.001 g/L). These results are similar to those reported by Want et al [78] with Rhodosporidium sp. TJUWZ4 cultured at 20–35°C interval. Figure 4 shows the behavior of biomass concentration, lipid content, and productivity in this interval using a medium composed by 50 g/L glycerol diluted in vinasse B (factor 2).
Figure 4: The effect of temperature on biomass concentration, lipid content and productivity of R. toruloides using vinasse B (factor 2) supplemented with glycerol 50 g/L at pH 6.0 and150 rpm. Each point represents the mean ± SD of three samples from a representative experiment of three runs (p < 0.05, Dunn's multiple test).
4.5 Effect of initial pH
The results revealed that the specific maximum growth rate and lipid content increased significantly from pH 5.0. A significant increase of these parameters (35.8 and 24.11%, respectively) was observed at pH 6.0 compare to pH 4.0. Specific growth rate and lipid productivity reached maximal value of 0.144 ± 0.003 h-1 and 0.033 ± 0.001 g/Lh, respectively. Table 2 provides a summary of the results.
Table 2
The effect of pH on biomass concentration, maximum specific growth rate, lipid content and productivity of R. toruloides grown in pH interval 4.0–6.0 using glycerol and urea in limited medium (C/N 250).
pH
|
Biomass
(g/L)
|
µ
(h-1)
|
Lipids
(%)
|
Lipids
(g/L)
|
Productivity
(g/Lh)
|
4,0
|
12,32 ± 0,16a
|
0,106 ± 0,004a
|
25,3 ± 1,3a
|
3,12 ± 0,001a
|
0,022 ± 0,001a
|
4,5
|
12,61 ± 0,16a
|
0,111 ± 0,001a
|
25,6 ± 1,5a
|
3,23 ± 0,001a
|
0,023 ± 0,001a
|
5,0
|
13,08 ± 0,15a
|
0,127 ± 0,002b
|
26,1 ± 2,2a
|
3,42 ± 0,002a
|
0,023 ± 0,001a
|
5,5
|
14,50 ± 0,11a
|
0,132 ± 0,002b
|
29,4 ± 1,8b
|
4,26 ± 0,001b
|
0,030 ± 0,001b
|
6,0
|
14,80 ± 0,20a
|
0,144 ± 0,003c
|
31,4 ± 1,7b
|
4,65 ± 0,002b
|
0,033 ± 0,001b
|
Different letters in the same column of the table indicate that there are significant differences according to the Tukey test (p ≤ 0.05). Data are shown as mean value of three replicates ± SD.
Similar studies in nitrogen-limited media have shown that the highest values of lipid content and glucose consumption were obtained in pH interval 5.0–6.0 [77, 79]. Other authors have reported different behaviors stating that optimal pH of R. toruloides NCYC 921 was 4.0.
In fermenters without pH control, a decrease in this variable occurs due to the production of some acid compounds. pH control by adding sodium hydroxide produces an increase in growth rate and biomass production, probably due to a decrease in the inhibitory effect of low pH values. With pH control, a higher rate of lipid accumulation is also achieved, but at the end of the culture, no significant differences are obtained in the production of lipids compared to the culture without pH control [80].
4.6 Effects of Vinasse Composition
The kinetic behavior of cell growth using vinasses A and B undiluted (factor 1) and diluted (factor 2) showed that no cell growth inhibition occurred on undiluted vinasse A. Nevertheless, culture on undiluted vinasse B showed a lag phase of approximately 24 hours, and after slow cells growth rate, a maximum specific growth rate of 0.034 h− 1 was reached (Fig. 5).The dilution of vinasse B (factor 2) allowed a significant reduction in adaptation phase time and a significant increase in growth-specific rate up to 0.143 h− 1. It is possible that vinasse B contains some compounds with toxic concentration that produce growth inhibition.
Studies have reported that initial COD of vinasses is the factor with the greatest effect on growth and lipid accumulation, compared to other parameters such as inoculum size, temperature, and culture time. The best results in terms of cell density, lipid content, and organic load removal of R. toruloides SA 2.1389 in vinasse from rice wine distilleries with COD 53 000 mg/L, were achieved using a dilution factor 3 [65]. Generally, researchers suggest that vinasses should be diluted until they reach an organic load below 25 000 mg/L due to the presence of some growth-inhibiting compounds such as furfural and HMF, in order to reduce their toxic effects [81]. Table 3 summarizes the results of biomass concentration, maximum specific growth rate and removal of organic load at the end of the culture.
The results obtained on undiluted vinasse A and diluted vinasse B with an organic load greater than 35 000 mg/L contradict the previous approach, since no inhibition was produced under these conditions. This result suggests that the determining variable or element for the inhibition of cell growth to occur in the vinasses is not COD value but the composition in terms of the presence and concentration of toxic compounds that affect the inhibition of the yeast´s growth, in particular.
Table 3
Biomass concentration, maximal specific rate and COD remotion of R. toruloides cultured in vinasses A y B without dilution (factor 1) and diluted (factor 2)
Factor
|
COD (g/L)
|
t (h)
|
X (g/L)
|
µmax (h− 1)
|
COD remotion (%)
|
Vinasse A
|
1
|
40,5 ± 4,90
|
74
|
14, 5 ± 0,04
|
0,116 ± 0,015
|
70,5 ± 1,33
|
2
|
21,2 ± 1,31
|
51
|
9,40 ± 0,16
|
0,116 ± 0,021
|
55,3 ± 0,45
|
Vinasse B
|
1
|
69,8 ± 2,30
|
96
|
11,5 ± 0,51
|
0,034 ± 0,006
|
68,6 ± 0,52
|
2
|
34,5 ± 2,45
|
24
|
13,9 ± 0,41
|
0,143 ± 0,022
|
71,3 ± 0,76
|
Several studies have reported the effect of organic acids on yeasts and have established some concentration values of compounds considered as alarms of the industrial process that indicate the presence of problems in the metabolic performance of yeasts, leading to a decrease in yields and besides may also modify the fatty acid profile of microbial oils. Most inhibitory events in microorganisms are due to the simultaneous presence of two or more toxic compounds, which produces a synergistic effect in the inhibition of growth and lipid synthesis, tolerance to inhibitors, and the synergistic effect depends of particular yeast strain [82–83]. Among the most studied inhibitory compounds are furfural, 5-hydroxymethyl furfural (HMF), vanillin and some acids as acetic, formic and levulinic [10, 84]. In studies of Hu et al [85] with R. toruloides Y4, acetic acid could be used as a single substrate up to 40 g/L, while it is capable of completely inhibiting growth when other inhibitory compounds are present. According to these researchers, the presence of 5-HMF (16 mmol) and acetic acid (100 mmol) in the medium have a very slight effect. However, their growth practically ceases in presence of furfural and vanillin in the range of 8–12 mmol. They also suggest that the synergistic effect of acetic acid and vanillin is greater on cell growth than other synergistic effects.
According to the composition of the vinasse B used in this study, it is likely that the cause of the growth inhibition found was the synergistic effect of 1.23 g/L acetic acid, 0.02 g/L vanillin and others toxic compounds that were not determined.
4.7 Effect of the Impurities of Raw Glycerol in Vinasse
The results shown in Table 4 demonstrate that biomass concentration and lipid content in the presence of raw glycerol after 148 hours was higher in 16.1 and 25.7%, respectively. Both parameters caused a significant increment of lipid productivity (59.6%).
Table 4
Fermentation parameters of R. toruloides cultured in vinasse B (factor 2) supplemented with glycerol and raw glycerol
Parameter
|
Vinasse/glycerol
|
Vinasse/raw glycerol
|
DCC (gL− 1)
|
36,60 ± 2,40
|
42,5 ± 0,40
|
Maximum specific growth rate (h− 1)
|
0,16 ± 0,01
|
0,18 ± 0,02
|
Lipids content (%)
|
20,60 ± 2,40
|
25,9 ± 3,10
|
Lipids productivity (gL− 1h− 1)
|
0,047 ± 0,008
|
0,075 ± 0,012
|
The profile of the methyl esters composition of yeast oils cultured in both media showed that the percentage of C16-C18 in both media was greater than 85%, with predominance of oleic (C18:1), palmitic (C16:0), stearic (C18:0) and linoleic (C18:2) acids (Fig. 6). Raw glycerol produced an increase in palmitic and linoleic acids by 22.3 and 48.8%, respectively. Nevertheless, a reduction in stearic and oleic acids of 21.5 and 12.5% was also determined.
Figure 6: Average percentage of select fatty acids of oils extracted from R. toruloides biomass grown on diluted vinasse B (factor 2) supplemented with pure and raw glycerol. Each bar represents the average of each sample.
Raw glycerol produced an increase in palmitic and linoleic acids by 22.3 and 48.8%, respectively. Nevertheless, a reduction in stearic and oleic acids of 21.5 and 12.5% was also determined. These results differ from those obtained by Signori et al [86], where no significant changes were detected in the composition of methyl esters when R. toruloides (DSM 4444), C. Curvatus (DSM 70022) and L. Starkeyi (DSM70295) were cultured in glycerol and raw glycerol as unique substrates.
In general, although lipid content and yields may vary between different proportions of pure or raw glycerol, it was demonstrated that fatty acids profiles (FAP) in the evaluated conditions are usually similar to those found by other authors in other species of oleaginous yeasts [51, 87–91]. Therefore, it can be concluded that substitution of raw glycerol for glycerol in medium compound by vinasse didn´t modify significantly the microbial oils quality and can be used as raw materials for biodiesel production.
4.8 Batch and Fed Batch Strategies
4.8.1 Batch system
The maximal biomass concentration of batch cultures in 4L fermenter reached 57.3 ± 4.1 g/L after 196 h with a maximal specific growth rate of 0,092 h− 1 and glycerol consumption rate of 0.72 g/Lh. At the end of batch culture, the lipid concentration reached 34.7 ± 1.8%, which is higher than the results obtained in 1000 mL frasks under same conditions. Specific lipid production rate was 0.10 g/L, and the lipid productivity was 0.12 ± 0.015 g/Lh.
Although the lipid content in this study was similar to other tests carried out using undiluted or diluted stillage from distilleries, the biomass concentration results obtained in this system are higher than those reported by other authors. Ling et al [55] obtained lipid contents of 25–35% using R. toruloides AS 2.1389 grown in vinasse from rice wine factories as the only substrate and C/N 25, however the biomass concentration was much lower than those obtained in this trial. Zhou et al [92] used stillage from ethanol distilleries from sweet potato hydrolyzate and also managed to obtain lipid contents around 35%, but the biomass concentration was less than 10 g/L. It is possible that the cause of the limited cell growth in these two vinasses is the presence of toxic compounds from lignin degradation during rice fermentation or from sweet potato hydrolysis that inhibit cell proliferation.
Reports using R. toruloides strains grown on raw glycerol as the sole substrate in nitrogen-limited media have achieved higher lipid contents than the present assay, but lower cell densities [68]; therefore, it is possible that the use of mixtures of vinasse and raw glycerol as initial medium in fed-batch systems will allow obtaining higher biomass concentrations in a shorter time and with it, the increase in productivity.
Fed batch strategies began after the first 48 h of exponential growth of the yeast culture where more than 85% of the initial glycerol was consumed.
4.8.2 Strategy I
This strategy consisted in add pulses of glycerol each time that glycerol concentration decrease lower than 5 g/L until reach 50–60 g/L. Once the glycerol increase was carried out, there was an exponential increase in cell growth reaching a maximum specific growth rate of 0.15 h− 1. This result was similar to that obtained with the R. toruloides Y4 strain in a medium using glucose 60 g/L [9]. The rate of glycerol consumption during the first 160 hours of culture was 0.75 ± 0.02 g/Lh, however, after the third glycerol increase, it drops to 0.55 g/Lh, while the rate of lipid accumulation increased in this stage from 0.14 to 0.39 g/Lh. In this strategy it was necessary to increase the agitation of the fermenter due to a decrease in pO2 below 30%. Biomass, lipid concentrations and lipid productivity at the end of culture (286 h) reached values of 129.0 ± 5.6 g/L, 66.2 ± 4.5 g/L (51.3%) and 0.23 ± 0.01 g/Lh respectively.
The kinetic behavior of these parameters (Fig. 7) can be explained if we consider that other substrates from vinasse, such as glucose, fructose, among others, were consumed in the first hours of the culture, so after the first glycerol increment, it was the unique substrate available. The pO2 gradually decreased, being less than 30% at 120 h, so the stirring speed was increased to 1000 rpm. This operation allowed the pO2 to remain above 30% until the end of the culture.
Figure 7: Cultivation of R. toruloides at 4 L fermenter according strategy I with addition of raw glycerol.
4.8.3 Estrategy II
This strategy was inspired by the results achieved by Wieber et al [93] in increased systems, where the highest accumulation of total lipids and the highest consumption of substrate occurred when the increased medium was composed of the carbon source and a small supply of nitrogen.
Therefore, once the batch stage was completed, 400 mL increments of diluted vinasse and raw glycerol were made, adjusting glycerol concentration to 60 g/L. The addition of diluted vinasse is a valid option if it is considered that it manages to provide the adequate nitrogen concentration to achieve an increase in lipid accumulation. On the other hand, the added volume of vinasse allowed a decrease of the high media viscosity reached by the increase of cell density, as well as a decrease of the energy consumption required because it was not required the use of higher agitation speeds or higher air flow. Similar increments were added each time the glycerol concentration decreased to 5 g/L.
The biomass concentration reached a maximum value of 145.8 ± 5.8 g/Lh at 170 h of culture. The maximum specific growth rate in the exponential stage was 0.15 ± 0.002 h− 1. At the end of this study (306 h), the lipid content was 41.3% for a productivity of 0.2 ± 0.02 g/Lh and the accumulated biomass reached up to 429.6 g in 3 ± 0.2 L. The rate of lipid accumulation increased from 0.12 to 0.29 g/Lh after 170 h. Figure 8 shows the k inetic behavior of the parameters in this strategy.
Figure 8: Cultivation of R. toruloides at 4 L fermenter according strategy II with addition of diluted vinasse B (factor 10) and raw glycerol.
4.8.4 Strategy III
This strategy consisted of continuously supplementing a medium composed of diluted Vinasse B and raw glycerol (VDG Medium), after the glycerol in the initial medium was exhausted. The composition of this medium was adjusted so that the C/N ratio was 220 (glycerol 500 g/L and vinasse diluted, factor 3) to favor the accumulation of lipids in the cells.
Unlike strategies I and II, Strategy III used the addition of continuous flow of media composed of diluted vinasse B (factor 3) and raw glycerol adjusted to 500 g/L (VDG Media, C/N = 220). Once the glycerol in the batch stage was exhausted and the addition of VDG medium started. Cell concentration continued increasing until the specific growth rate began to decrease because substrate supply was not sufficient for the high cell culture density reached on the fermenter.
Glycerol concentration increased until maximum value of 72.3 ± 3.8 g/L at 145 h, and accumulation lipid rate reached 0.98 g/Lh. Next, glycerol accumulation stopped, and glycerol concentration began to decrease as a result of the higher consumption rate of glycerol. As a result of this strategy, 136.0 ± 1.9 g/L of biomass concentration and 52.5 ± 5.5% of lipids was reached allowing lipid productivity 0.30 ± 0.04 g/Lh and 523.6 ± 2.4 of total dry biomass. Figure 9 shows the kinetic behavior of biomass, glycerol and lipid concentration in this strategy.
Figure 9: Cultivation of R. toruloides at 4 L fermenter according strategy III using VDG medium.
With a view to carrying out industrial-scale production of SCO through a fed-batch system, a fermentation time of 240 h was set to compare the productivity of the three evaluated strategies and select the most appropriate one for this purpose. Table 5 shows a summary of the results obtained during this time.
Table 5
Biomass concentration, lipid content, lipid productivity and total biomass after 240 h of fed bath strategies I – III
|
Suplement
|
|
MSG
|
Lipids
|
Prod
|
Biomass
|
Mode
|
(gL− 1)
|
(%)
|
(gL− 1h− 1)
|
(g)
|
I
|
50 g/L Glycerol
|
Pulses
|
132,3 ± 7,7
|
34,3 ± 2,5
|
0,19 ± 0,02
|
330,8 ± 7,7
|
II
|
50 g/L Glycerol + Vinasse B (1:2, v/v)
|
Pulses
|
143,6 ± 6,2
|
28,8 ± 3,5
|
0,17 ± 0,08
|
402,1 ± 2,9
|
III
|
500 g/L Glycerol + Vinasse B (1:3, v/v)
|
Constant flow
|
136,0 ± 1,9
|
52,5 ± 7,5
|
0,30 ± 0,04
|
523,6 ± 2,4
|
The results showed that the higher productivity was obtained using strategy III. Lipid productivity was 1,6 ± 0,1 times higher than others strategies and lipid content was 1,5 and 1,8 times than strategy I and II, respectively.
The lipid content was higher 1.5 and 1.8 times compared to strategies I and II, respectively. The strategies evaluated confirm that the fed-batch mode is suitable to shift cellular metabolism towards lipid accumulation and prevent substrate inhibition that can occur at discontinuous mode and it was possible to achieve significant increases in biomass production, lipid accumulation and productivity.
Others researchers had obtained biomass concentration higher than 100 g/L and similar lipid content with L. starkeyi, Candida curvatus and R. toruloides strains in fed batch system using glucose [93–96]. In the studies carried out by Zhou et al [93] with R. toruloides Y2 using stillages from distilleries, the lipid content was increased from 39.5 to 53.8% by adding glucose at a flow rate of 1.2 g/Ld. Productivity in this study was considerably lower than these cases, which is reasonable if it is consider that the specific growth rate in the presence of glucose is higher than that achieved in the presence of the vinasse and raw glycerol [97–98]. Comparing the results obtained in this study, it can be stated that the use of vinasse from ethanol distilleries and raw glycerol allows achieving similar or superior results to that obtained using glucose [9, 26, 80, 92, 99–104].