*3.1.1 Analysis of ED factors*

The data obtained from the ED-*L*<sup>0</sup> was further used for regression analysis, and the polynomial model-derived parameters (*β*0–*β*22) are shown in **Table 2**. The β parameters of this polynomial model used to estimate the responses have the following meanings: *β*<sup>0</sup> represents the center of the experimental domain; *β*<sup>1</sup> and *β*<sup>2</sup> indicate the importance of the respective factors (factor 1: % glucose in a mixture glu + fru or glucose ratio, and factor 2: sulfate concentration, respectively) on the responses. The interaction parameter, *β*12, indicates how the effect of one factor is dependent on the level of the other factor. *β*<sup>11</sup> and *β*<sup>12</sup> values determine how the response surface folds downward (negative values) or upward (positive values) quadratically, more or less rapidly in accordance with the magnitude of the absolute value [23].


#### **Table 2.**

*Parameters of the polynomial model representing the responses studied (biomass production; total carotenoid production), with and without light (L400* v*s L0), at 72 h and 216 h.* β*0, response at the center of the experimental domain;* β*<sup>1</sup> and* β*2, parameters of the factors 1 (% glucose in a mix glu + fru) and 2 (sulfate concentration, mg/ L), respectively;* β*12, parameter of the interaction of the factors 1 and 2;* β*<sup>11</sup> and* β*22, self-interaction parameters of the factors 1 and 2, respectively.*

#### *New Insights on Carotenoid Production by* Gordonia alkanivorans *Strain 1B DOI: http://dx.doi.org/10.5772/intechopen.103919*

At 72 h, *β*<sup>1</sup> and *β*<sup>2</sup> have opposite influences. *β*<sup>1</sup> presents the greatest value, indicating that glucose % has the highest influence on biomass production. However, being negative, *β*<sup>1</sup> also indicates that increasing this factor leads to a decrease in response, meaning that an increase of glucose % leads to a decrease in biomass. On the other hand, *β*<sup>2</sup> has a smaller, positive value indicating that an increase in sulfate concentration leads to an increase in biomass production. Analyzing pigment production at 72 h, *β*1, *β*2, and *β*<sup>12</sup> presented positive values, indicating that the increase of each factor, individually or simultaneously, results in an increase of the response. Sulfate concentration was the factor with the greatest influence on pigment production.

At 216 h, there was a change in the response, as illustrated by the *β* parameters. Increasing each factor led to an increase in both biomass and pigment production. As shown in **Table 2**, for these conditions, sulfate concentration had a greater influence on biomass production (*β*<sup>2</sup> was 1.5-fold higher than *β*1), however, pigment production was mostly influenced by glucose ratio (*β*<sup>1</sup> almost two-fold higher than *β*2).

#### *3.1.2 Comparing light and dark influence on carotenoid production*

In a previous work [32], a similar experimental design was performed to evaluate the influence of light (400 lux). The results from that work, referred to as ED-L400, are presented in **Table 1** and **Figure 2A**–**D**. From the comparison of ED-L0 and ED-L400 results (**Figure 1** *versus* **Figure 2**), several differences become evident. In terms of biomass production, at 72 h optimum conditions do not differ substantially; however, the average results obtained under dark conditions were higher for every condition tested. This seems to indicate an inhibitory effect of the light source on growth rates, possibly resulting from the allocation of nutrients toward carotenoid production.

At 216 h, when sulfate concentration was 9.01 mg/L (**Table 1**: tests 9–12), there was no difference between light and dark cultures, and biomass did not surpass 3 g/L, reinforcing the observation that sulfate, at this concentration, was limiting. For the remaining conditions, there seemed to be a response in which cultures were grown with lower glucose %, and consequently with higher fructose concentrations, have lower biomass under dark conditions (**Table 1**: tests 5-6 and 13-14). As suggested above, this can be explained by the faster growth of these cultures under dark conditions, that by 216 h were already undergoing the cellular death phase. The inhibitory effect of the light source lowered the overall growth rates, to the point that, under light conditions, at 216 h these cultures were at an earlier stage of the growth, explaining the increased absorbance. For cultures with higher levels of glucose, since the growth rate is already slower, the inhibitory effects of light were not so evident with this two-point sampling (72 h and 216 h).

In terms of total carotenoid production, in both cases, the best results were obtained after 216 h with 100% glucose and 22 mg/L sulfate. However, the best average results obtained under light (794 μg) were more than two-fold higher than those obtained in the dark conditions (311 μg). Regardless of the presence of a light source, there was an increase of total carotenoid production from 72 h to 216 h for every condition tested. In both studies, at 72 h, this response was negatively influenced by higher glucose percentages in the mix. This effect, which is especially evident under light, seems to be mitigated, in both cases, by a higher concentration of sulfates. At 216 h this effect is reversed, and response is stimulated by glucose, since, as explained above, glucose induces slower growth rates, and longer division times would benefit such cultures.

#### **Figure 2.**

*Response surfaces for the biomass production (g/L) at 72 h (A) and 216 h (B); and for the total carotenoid production (μg) at 72 h (C) and 216 h (D), obtained in ED-L400 for the factors % glucose in a mixture of fructose + glucose (0–100%) and sulfate concentration (7–37 mg/L). Reprinted from Fernandes et al. [32].*

Analyzing the beta parameters for both EDs (**Table 2**), the differences are again evident. At 72 h, in terms of biomass, each factor individually influences the response in a similar manner (*β*<sup>1</sup> and *β*2). However, when both factors were increased simultaneously (*β*12), the responses observed were opposite. Under dark conditions, this led to a decrease in biomass, while with light, it increased biomass production. This could indicate that, in the presence of light, the inhibitory effect that glucose has on biomass production can be partially reversed by increasing sulfate concentration, while under dark conditions, it can only be mitigated, maintaining the negative effect. The existence of this inhibitory effect was not previously observed, since, up to now, the works performed with sugar mixtures, did not take into account the conjugation of a lack of sulfur and light sources [17]. Nevertheless, Silva et al. [7] have already referred that strain 1B showed lower growth rates under the light. At 216 h the concentration of glucose has opposite effects depending on the presence of the light source. In the dark,

#### *New Insights on Carotenoid Production by* Gordonia alkanivorans *Strain 1B DOI: http://dx.doi.org/10.5772/intechopen.103919*

it has a positive effect, while with light, it has a slightly negative influence. Furthermore, the relative influence of sulfate (*β*2) was higher with light, while glucose (*β*1) was higher without.

In terms of total carotenoid production, at 72 h, comparing *β*1, *β*2, and *β*<sup>12</sup> to their respective *β*0, it becomes clear that the studied factors presented a greater relative influence under light. Moreover, the increase of the glucose % (*β*1) led to opposite responses. In the absence of light, an increase of glucose % resulted in an increase of total carotenoids, while in its presence, it greatly reduced total carotenoid production. At 216 h, total carotenoid production was positively influenced by all factors in both cases. However, all factors, especially glucose concentration, have greater relative importance under dark conditions. This indicates that in the absence of the stimulus of light, sulfate concentration, % of glucose in the sugar mix, and even time have greater importance for this response.

#### **3.2 Specific carotenoid production**

Total carotenoid production is an important parameter for the industrial process. Since it indicates the amount of carotenoids obtained in a certain volume of culture, it is deeply influenced by biomass production. The best conditions for total carotenoid production are obtained when there is a compromise between the highest carotenoid and highest biomass production. However, to better understand the mechanisms that influence carotenoid synthesis, it is fundamental to analyze specific carotenoid production (μg of carotenoids per g of dry cell weight - μg(Carotenoids)/g(DCW) or μg/g(DCW)). By evaluating the concentration of carotenoids per g of cells, it is possible to determine, which conditions induce greater cellular accumulation.

Using the results obtained in terms of biomass and total carotenoids, for each condition tested, the specific carotenoid concentrations were calculated, for both EDs, at dark and light conditions, at 72 h and 216 h and are presented in **Table 3,** with the corresponding response surfaces represented in **Figure 3A**–**D**.

Observing these results, it is possible to see that there are two very different behaviors in relation to time. Under the effects of light, the specific production of carotenoids is almost unchanged throughout the time period, from 72 h to 216 h (**Table 3**; **Figure 3A** and **B**). However, when the culture is grown in the darkness, time clearly has a significant influence, leading to a considerable increase of the carotenoids concentration present in the bacterial biomass, as well as to different influences of the studied factors, easily seen in the response surfaces in **Figure 3C** and **D**. In previous works, this feature had gone unnoticed since it is not observable when analyzing only the total carotenoid production.

From **Table 3**, it is also possible to determine that, for the same tests, specific carotenoid production was always greater in the presence of light, although this difference was more evident at 72 h. Tests 3 and 4, with glucose at 100% of the sugar mix and 22 mg/L of sulfates, always presented the highest response in each set of assays (L400/L0).

Further analyzing the results obtained in the presence of light, it is possible to see that, at both times, the two factors had a positive influence on specific carotenoid production. Maximum values were observed with 100% glucose and 22 mg/L of sulfate, while the lowest was registered with 9.01 mg/L of sulfates regardless of glucose percentage. As shown in **Figure 3A** and **B**, glucose % is the most influential factor, evidenced by the almost vertical lines, with sulfate having a smaller influence,


#### **Table 3.**

*Doehlert distribution for two factors: % of glucose in mixture glucose + fructose (0–100%) and sulfate concentration (7–37 mg/L), and the response in terms of specific carotenoid production (μg/g(DCW)), with and without light (L400* v*s L0), at 72 h and 216 h. seven conditions were tested in duplicates (14 tests), for statistical analysis.*

mostly for values below 22 mg/L. Optimum conditions for maximum specific carotenoid production are glucose at 100% of the sugar mix and sulfate concentration of at least 22 mg/L. These results also seem to reinforce the idea that the presence of fructose interferes with the cellular accumulation of carotenoids. Even at lower fructose concentrations (2.5 g/L in 10 g/L mix), specific carotenoid concentration only reaches 770 μg/g(DCW); however, when glucose represents 100% of 10 g/L mix, cellular accumulation of carotenoids has an increase of 140% and 94% at 72 h and 216 h, respectively, showing that a small concentration of fructose can have significant negative impacts on carotenoid production.

Under dark conditions, the responses obtained at both times were similar, but differences between tests were more pronounced at 72 h. As in previous results, the highest responses were obtained with glucose at 100% and 22 mg/L of sulfate (377 μg/ g(DCW)). While the lowest results were observed with glucose at 25% at either sulfate concentrations (21.3 and 36.9 μg/g(DCW)), followed by glucose at 0% (51.7 μg/g(DCW)). This indicates a lower influence of the factors, when fructose is the dominant sugar, as demonstrated by the left quadrants of **Figure 3C**, which show a reduced variation of the response, regardless of glucose % and sulfate concentration. When glucose % is above 50%, both factors influence the response, with glucose showing the highest influence, as shown by the almost vertical lines on the right quadrants. Sulfate concentration is mostly important up to 22 mg/L, with higher values showing reduced or

*New Insights on Carotenoid Production by* Gordonia alkanivorans *Strain 1B DOI: http://dx.doi.org/10.5772/intechopen.103919*

**Figure 3.**

*Response surfaces for the specific carotenoid production (μg/g(DCW)) with a light source (400 lux), at 72 h (A) and 216 h (B); and in the absence of light, at 72 h (C) and 216 h (D), obtained in ED-L0 and ED-L400 for the factors % glucose in a mixture of fructose + glucose (0–100%) and sulfate concentration (7–37 mg/L).*

even negative impact on specific carotenoid production. So, **Figure 3C** shows that maximum cellular carotenoids would be obtained with 100% glucose and 22 mg/L, as it was tested (**Table 3**, tests 3 and 4).

At 216 h, carotenoid concentrations were higher for every condition tested. The highest value was recorded with 100% glucose and 22 mg/L of sulfate (520 μg/g DCW). The lowest values were observed when sulfate was at 9.01 mg/L with glucose at 25% (101 μg/g(DCW)) and 75% (146.10 μg/g(DCW)). Both factors have a positive influence on the cellular accumulation of carotenoids; however, when sulfate is 22 mg/L or higher, glucose has the highest influence, shown by the vertical lines in the upper right corner of **Figure 3D**. Results indicate that the highest specific carotenoid production could be achieved with glucose 100% and sulfate >35 mg/L.

These results also indicate that a concentration of 9.01 mg/L of sulfate hinders specific carotenoid production, not only biomass production, and 22 mg/L of sulfate are sufficient for high carotenoid production, at earlier stages of the growth (72 h), with further increases having minimal impact. At later times of growth, increasing sulfate concentration to 34.99 mg/L leads to increases in cellular accumulation of carotenoids, most likely because cellular needs for growth are met and it can be diverted toward secondary metabolite production.

Most importantly, fructose seems to have an inhibitory effect on carotenogenesis. Under these conditions, since the tests were performed with a mix of fructose and glucose, with different percentages while maintaining 10 g/L of total sugars, when glucose % was increased, there was a proportional decrease in fructose. So, when glucose was increased from 25 to 50% (and sulfate from 9.01 to 22 mg/L) there was a 25% reduction in fructose, which caused a very significant increase in the accumulation of carotenoids (five-fold at 72 h and two-fold at 216 h). A further increase of both glucose and sulfate values to 75% and 34.99 mg/L, respectively, resulted in a much lower or nonexistent increase in the response, especially at 72 h. However, by completely removing fructose from the mix, from 50–0% fructose (**Table 3**; tests 1-2 and 5-6), even without increasing sulfate concentration, there was an increase in response (μg(Carotenoids)/g(DCW)) of more than three-fold at 72 h and more than twofold at 216 h. This seems to indicate an inhibitory effect, which is slightly lower at 216 h, since most fructose has been completely consumed within 72 h to 76 h, possibly attenuating its inhibitory effects. Furthermore, cells grown with glucose at 25% were those that had the highest increase over time, raising cellular concentration of carotenoids 4.75- and five-fold; while for higher glucose %, the increase was under threefold, further reinforcing the theory that carotenoid production is increased after fructose disappears, especially when glucose is present.

Sugars having inhibitory effects on carotenoid synthesis is a known phenomenon, mostly observed for glucose. Some researchers proposed the use of alternative sugars, or alcohols, as C-sources, to achieve higher carotenoid concentrations. In fact, a similar phenomenon was described for *Xanthophyllomyces dendrorhous*, a yeast that produces astaxanthin. This microorganism starts to produce carotenoids at the stationary phase of the growth if grown with glucose but starts the production at the beginning of the growth if cultivated with succinate [34]. Since *G. alkanivorans* strain 1B is a fructophilic bacterium [17], it could be the case that similar mechanisms are being applied to its preferred C-source, explaining the reduced carotenoid production in the presence of this sugar.

**Table 4** presents the beta parameters for the polynomial model used for the specific carotenoid production, showing the influence of each factor studied (% glucose in a mix glu + fru; sulfate concentration). It is possible to note that both factors have a positive influence on the response. It is also clear that glucose percentage is the most influential factor on the conditions tested (**Figure 3**), being several times more influential than sulfate concentration in all cases.

Considering the responses at the center of the domain (*β*0), it becomes clear that both factors have a greater relative influence under dark conditions, despite having significantly lower responses. Glucose concentration is especially influential at 72 h, as *β*<sup>1</sup> is greater than *β*0. Moreover, under light, the relative importance of the individual factors reduces with time, while under dark conditions, the relative influence of sulfate increases, and that of the glucose percentage is reduced.

Comparing the overall results for specific carotenoid production (**Table 3**; **Figure 3**) with those obtained for total carotenoid production (**Table 1**, **Figure 2**), it


*New Insights on Carotenoid Production by* Gordonia alkanivorans *Strain 1B DOI: http://dx.doi.org/10.5772/intechopen.103919*

#### **Table 4.**

*Parameters of the polynomial model representing specific carotenoid production (μg/g(DCW)), with and without light, at 72 h and 216 h.* β*0, response at the center of the experimental domain;* β*<sup>1</sup> and* β*2, parameters of the factors 1 (% glucose in a mix glu + fru) and 2 (sulfate concentration, mg/L), respectively;* β*12, parameter of the interaction of the factors 1 and 2;* β*<sup>11</sup> and* β*22, self-interaction parameters of the factors 1 and 2, respectively.*

becomes visible that, under light, cellular carotenoid production is not influenced by time above 72 h, meaning that total carotenoid production increased due to an increase in biomass concentration and not in the cellular carotenoid concentration. In the same way, the sulfate concentration had a greater influence on total carotenoid production, due to its importance for biomass production. Values above 22 mg/L had little effect on cellular carotenoid production, despite increasing total carotenoid production. Under dark conditions, by comparing the response surfaces obtained for specific carotenoid production (**Figure 3C** and **D**) with those obtained for total carotenoids (**Figure 1C** and **D**), it is possible to observe that there are significant differences at 72 h since at this time the biomass produced is very different depending on the conditions tested. So, the conditions for the highest total carotenoid production will lead to more biomass, but not the cells at the highest pigmentation. At 216 h, the figures are much similar, since the differences between biomass were much smaller.

#### **3.3 Influence of the carbon source**

#### *3.3.1 Carbon consumption*

Sugar consumption was greatly affected by the influence of light, under some conditions, sugars were fully consumed, while in others sugar consumption was residual. Due to this range of results, carbon consumption could not be properly represented by the models previously applied. However, given their importance to understanding the metabolic response, total sugar consumption results were displayed in **Figure 4A**–**D**, to better illustrate how they were influenced by the factors studied in EDs (L0 and L400).

Under dark conditions, at 72 h (**Figure 4C**), it is possible to see that the tests with more glucose and less sulfate were those in which sugar consumption was the lowest. Except for the conditions when glucose was at 75%, fructose was always completely consumed, which seems to indicate a slower consumption of glucose, expected from this fructophilic strain. At 216 h (**Figure 4D**), sugars were completely consumed, apart from the tests with lower sulfate concentration, which maintained the same concentration values presented at 72 h, and one of the replicates with 100% glucose. This reinforces the concept that sulfate levels were limiting the growth of the culture, and that a value higher than 9.01 mg/L is needed to consume 10 g/L of sugars.

Under light, at 72 h, none of the tests resulted in the complete consumption of sugars. The highest result (8.81 g/L) was observed when glucose was at 0% and fructose was the sole C-source. As shown in **Figure 4A**, increasing glucose % resulted in a decrease in sugar consumption that was especially evident above 50% glucose with 22–34.99 mg/L of sulfate. The lowest sugar consumption was observed when glucose was 100% of the 10 g/L sugar mix, where the culture was still in its lag phase, and sugar consumption was residual (0.0042 g/L). At 216 h (**Figure 4B**), sugars were fully consumed when glucose percentage was up to 50% and sulfate concentration was ≥22 mg/L. At 75% glucose with 34.99 mg/L sulfate, less than 2.2 g/L were left as residual, and at 100% glucose, less than 5 of the 10 g/L of initial glucose were consumed.

Comparing both light and dark results, there was a clear inhibitory effect of the light source, in terms of consumption of both sugars, this had only been described for glucose, mostly because assays with fructose ended before 72 h. Moreover, these results also point out that, with sufficient sulfur source, carbon source consumption is

#### **Figure 4.**

*Doehlert distribution for two factors: % of glucose in a mix glu + fru (0–100%) and sulfate concentration (7– 37 mg/L), and the responses in terms of total sugar consumed (g/L), with light at 72 h (A) and 216 h (B) and in dark at 72 h (C) and 216 h (D), respectively. Seven conditions were tested in duplicates (14 tests), for statistical analysis.*

reduced, there is an accentuated fructophilic behavior and a significant effect of the glucose concentration under light conditions. The presence of fructose, greatly accelerated sugar consumption, as seen when comparing the results of 75% glucose and 100% glucose on every assay.

#### *3.3.2 Carbon conversion to biomass*

To better understand and prepare a future biorefinery it is important to evaluate how carbon is diverted toward biomass production. As such, based on the results for biomass production and carbon consumption, **Figure 5A**–**D** was created, illustrating biomass produced (g/L DCW) per total sugar consumed (g/L), that is, (g/g), for the different conditions tested.

At 72 h, with light (**Figure 5A**), the highest result was achieved with 75% glucose and 34.99 mg/L of sulfate (0.52 g/g), any reduction in glucose % or sulfate concentration resulted in a decrease in response. The remaining conditions had values between 0.33 and 0.25 g/g. Results with glucose at 100% of the mix were disregarded, since as previously stated, under these conditions the culture was still in its lag phase, there was residual carbon consumption and residual biomass production. At 216 h (**Figure 5B**), carbon conversion to biomass increased on every condition tested, nonetheless, the general trends remained similar. The highest result was registered with 100% glucose (0.76 g/g), followed by 75% glucose with 34.99 mg/L of sulfate (0.65 g/g), the lowest was also observed with 75% glucose but with 9.01 mg/L (0.3 g/g). The remaining results varied between 0.44 g/g and 0.51 g/g.

#### **Figure 5.**

*Doehlert distribution for two factors: % of glucose in a mix gluc + fru (0–100%) and sulfate concentration (7–37 mg/L), and the responses in terms of biomass produced/sugar consumed (g/g), with light at 72 h (A) and 216 h (B) and in dark at 72 h (C) and 216 h (D), respectively. Seven conditions were tested in duplicates (14 tests), for statistical analysis.*

This shows that higher concentrations of glucose and sulfate result in a higher carbon conversion to biomass. However, since fructose is needed to induce faster biomass production, 100% glucose leads to lower initial results, this can be surpassed by maintaining a small concentration of fructose, no more than 25% of total sugars. Furthermore, it also seems to indicate that carbon conversion to biomass increases with time. This could result from the accumulation of reserve substances at later times, in response to the stimulus of light, coupled with a slower metabolic activity which results in less carbon being converted to CO2.

Under dark conditions, at 72 h (**Figure 5C**), significant differences were mostly observed at both extremes of glucose %, with 22 mg/L of sulfate. The highest result occurred with 0% glucose (0.47 g/g) and the lowest with 100% glucose (0.22 g/g). The remaining conditions resulted in values between 0.32 g/g and 0.43 g/g without a defined tendency. At 72 h sulfate appears to be less influential to how cells allocate carbon, most influence is centered on the presence/absence of fructose and glucose. Indicating that the nature of carbon source leads to different metabolic responses, which could be related to the fructophilic behavior of *G. alkanivorans* strain 1B. After 216 h (**Figure 5D**), the lowest results were observed with glucose <50% or sulfate <22 mg/L (0.27–0.32 g/g). The highest result was achieved with 100% glucose and 22 mg/L of sulfates (0.48 g/g). Overall, carbon conversion to biomass appears to be stimulated by higher glucose %. Sulfate concentration also stimulates this response, up to 22 mg/L, greater increases seem to have no influence. This could indicate that fructose stimulates the production of extracellular compounds, such as biosurfactants [26], and glucose the accumulation of reserve substances, such as lipids [35], thus diverting carbon to different pathways.

Comparing the results obtained, at 72 h, carbon conversion to biomass was mostly higher under dark conditions, however, at 216 h this is reversed. Moreover, higher glucose and sulfate concentrations at later times, also present higher carbon conversion rates to biomass, with the highest results being obtained at 216 h, with light, 100% glucose, and 22 mg/L of sulfate.

Without light, when there was lower glucose %, time led to a reduction in carbon conversion to biomass, even if there was very few or no carbon left at 72 h. With greater glucose percentages, there was an increase of carbon conversion to biomass from 72 h to 216 h, even when sugar was already consumed at 72 h. Comparing each individual value obtained at 216 h and 72 h (**Figure 5C** and **D**), it becomes clear that, over time, carbon conversion to biomass increases with the increase in glucose % (0 < 25 < 50 < 75 < 100%). This increase with time could be the result of the accumulation of reserve substances, such as lipids, sugars, or PHA's, induced by light and/or glucose, resulting in slower growth rates, and higher carbon conversion yields. Conversely, without the stimulus of light, especially at greater fructose concentrations, growth rates are higher, but there is a loss of biomass over time, which could indicate that cells produce fewer reserve substances, or that these are converted, or released into the medium in the form of biosurfactants or other exopolysaccharides, resulting in loss of dry weight. Alternatively, this could be the result of a higher metabolic activity induced by fructose, as highlighted by Alves and Paixão [17], which could result in cells with lower abundance of reserve substances, leading to greater cellular lysis over time.

Other fructophilic bacteria and yeast have been shown to convert fructose to mannitol, which is accumulated and used as an osmolyte, a carbon reserve, or an antioxidant, substituting carotenoids in the latter function. However, the same phenomenon is not observed when glucose is the C-source [36, 37]. A similar mechanism could be occurring here, in which fructose is converted into mannitol or another intermediary and then consumed for the cell metabolism, while glucose is consumed at a slower speed, and further converted to carotenoids and other reserve substances, which would be consumed after the 216 h evaluated in this work (**Figure 4**).

#### *3.3.3 Specific carotenoid production per gram of sugar consumed*

A final response was still obtained by combining the responses in terms of specific carotenoid production (μg(Carotenoids)/g(DCW)) per total sugar consumed (g/L), that is, specific carotenoid production per sugar consumed (μg(Carotenoids)/g(DCW)/g(Sugars)/L). This combined response indicates how efficiently sugars are converted into carotenoids in each cell. The results obtained are illustrated in **Figure 6A**–**D**.

After 72 h under light, the lowest results were registered when glucose was at 0% or sulfate was 9.01 mg/L (72.31–84.17 μg(Carotenoids)/g(DCW)/g(Sugars)/L). Increases in glucose % or sulfate concentration always resulted in an increased response. The highest result calculated was obtained for 75% glucose and 34.99 mg/L of sulfate (165.71 μg(Carotenoids)/g(DCW)/g(Sugars)/L). It should be noted that cells grown on 100% glucose and 22 mg/L of sulfate revealed much greater specific carotenoid production, and since carbon consumption at this point was still residual, the calculated result was several thousand times greater than any other, losing most of its comparative meaning (**Figure 6A**). However, this is not devoid of biological meaning, since it indicates that under these conditions, before biomass production begins, or sugar consumption starts, carotenoid production is already occurring, and as seen above at the same specific concentration, as later times.

#### **Figure 6.**

*Doehlert distribution for two factors: % of glucose in a mix glu + fru (0–100%) and sulfate concentration (7–37 mg/L), and the response in terms of specific carotenoid production per sugar consumed (μg(Carotenoids)/ g(DCW)/g(Sugars)/L), with light at 72 h (A) and 216 h (B) and in dark at 72 h (C) and 216 h (D), respectively. Seven conditions were tested in duplicates (14 tests), for statistical analysis.*

After 216 h, as demonstrated in **Figure 6B**, the highest values were achieved when glucose was at 100% of the 10 g/L sugar mix and sulfate at 22 mg/L (293 μg(Carotenoids)/ g(DCW)/g(Sugars)/L). The lowest values were obtained when glucose % was between 25 and 50% with sulfate ≥22 mg/L (60.4–63 μg(Carotenoids)/g(DCW)/g(Sugars)/L).

These results show that, at both times, glucose is the factor with most influence. At 72 h, the factors studied had a greater influence at higher concentrations of sulfate and glucose %, and both glucose and sulfate have a positive effect. At 216 h, the response presented two behaviors. For lower concentrations of sulfate, lower glucose % benefits the response, while for higher concentrations of sulfates, greater glucose % benefits the response. This could be due to the fact that lower concentrations of sulfate result in less biomass, leading to an excess carbon source, which could help induce the synthesis of carotenoids, as a response to the light. But, with greater sulfate concentration glucose acts as the inducer. Moreover, both at 72 h and 216 h, 100% glucose proved to be the best condition, even at lower sulfate concentration.

In the absence of light, at 72 h (**Figure 6C**), the highest results were obtained with glucose at 100% and 22 mg/L of sulfate, while the lowest were observed with glucose at 25% and sulfate at 9.01 mg/L (130 and 2.82 μg(Carotenoids)/g(DCW)/g(Sugars)/L, respectively). At 216 h (**Figure 6D**), the maximum and minimum values were registered at the same conditions (62.7 and 12.6 μg(Carotenoids)/g(DCW)/g(Sugars)/L, respectively). This clearly demonstrates that both factors have a positive effect on the response, also showing that under dark conditions, especially at 72 h, glucose is the most influential factor. At 216 h response values were higher, apart from the tests with 100% glucose; however, the relative importance of glucose % was lower, as shown by the closer response values, and sulfate gained influence, when glucose % was <100%. Furthermore, as previously stated, fructose seems to have an inhibitory effect on the carotenoid production, and on the efficiency of converting carbon into carotenoids, as evidenced by the difference in response values between 75% and 100% glucose, especially significant at 72 h (**Figure 6C**).

Comparing the results obtained for specific carotenoid production per sugar consumed (μg(Carotenoids)/g(DCW)/g(Sugars)/L) at both times, with and without light (**Figure 6**), it becomes clear that time, for most conditions, had opposite influences on the response. With light, carbon conversion to carotenoids mostly decreased over time, while without light, it seems to increase, except when glucose is used at 100% of the sugar mix. This fact could be a conjugation of several different factors. At earlier stages of the growth, the cells were fewer in number and had greater exposure to light, due to a lower cell density. As the cultures progressed, the number of cells increased and there was an increase in shade effect. Since under dark conditions the stimulus of light is not present, the values are lower, but increase over time. Other factors, such as oxygen and nutrient concentration vary in a similar fashion in both assays, and as such, should play a smaller role in this difference. The result observed for 100% glucose at 72 h under light is an extreme example, where the culture was only starting to grow at that point, however, the cells were heavily stimulated to produce carotenoids. The cells present at this time were mostly from the initial inoculum, and they were induced to produce carotenoids, by the presence of light, and high concentrations of glucose (100% of the 10 g/L mix) and sulfate (22 mg/L), before starting to grow and consume sugar in a significant manner.

The presence of light enhances the conversion of carbon into carotenoids in each cell, as demonstrated by the smaller response under dark conditions. Moreover, carotenoid production seems to be induced by sugar but is not dependent on it. As stated above, with light, at 72 h and 100% glucose (10 g/L), the cells were extremely rich in

carotenoids, but sugar consumption was minimal. This indicates that strain 1B could be producing the carotenoids based on cellular reserve substances present in the cells of the inoculum. This is reinforced by the fact that, in the dark, with 0% glucose, and 25% glucose, there is a complete sugar consumption by 72 h (**Figure 4C** and **D**), and the specific carotenoid production increases at 216 h (**Figure 6C** and **D**). This evidence confirms what had already been reported in the work by Silva et al. [7], in which strain 1B cells grown in the dark with fructose and DBT were placed in the light after the end of the growth and have developed coloration, without additional extracellular sugar.
