**3.1 Experimental design (ED-L0)**

**Table 1** shows the set of tests performed within the experimental design (ED), to study carotenoid and biomass production by strain 1B in the absence of light (L0), and the responses obtained (biomass and total carotenoid production, both after 72 h and 216 h). **Figure 1** shows the response surfaces for the variation of biomass (**Figure 1A** and **B**) and total carotenoid production (**Figure 1C** and **D**), within the experimental domain, based on the responses observed.

In terms of biomass production, it is possible to see that at 72 h both factors influenced the response. The highest results were obtained with glucose ≤50% and sulfate at 22 mg/L. Further increases in glucose % resulted in a significant reduction of biomass regardless of sulfate concentration, with the lowest value being registered with 100% glucose, having a difference of more than six-fold when compared to the best results (0.72 vs. 4.66 g/L). This indicates that, at this time, when glucose % is above 50% it becomes the most influential factor, as corroborated by the more vertical lines of the left quadrants of the response surfaces (**Figure 1A**)**.** Sulfate concentrations below and above 22 mg/L also resulted in lower biomass production,


#### **Table 1.**

*Doehlert distribution for two factors: % of glucose in mixture glucose + fructose (0–100%) and sulfate concentration (7–37 mg/L), and the responses evaluated (biomass and total carotenoids) in absence of light (ED-L0)* versus *with light (400 lux, ED-L400). Seven conditions were tested in duplicates (14 tests), for statistical analysis.*

**Figure 1.**

*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-L0 for the factors % glucose in a mixture of fructose + glucose (0–100%) and sulfate concentration (7–37 mg/L).*

even with glucose at 25%. This was especially evident with 9.01 mg/L of sulfate, as biomass never reached 3 g/L regardless of glucose concentration. In fact, sulfate was the most influential factor up to 20 mg/L, when glucose was below 50%, clearly demonstrated by the horizontal lines in the lower left quadrants of the response surfaces.

After 216 h, both factors continue to influence biomass production. However, contrary to what was observed at 72 h, an increase of glucose % was shown to have a positive effect on biomass, especially for values above 25%, regardless of sulfate concentration. The highest biomass concentration was observed with 100% glucose and 22 mg/L of sulfate (**Table 1**; tests 3–4: 3.98 g/L DCW), while the lowest results were registered with 9.01 mg/L of sulfate (**Table 1**; tests 9–10: 2.16 g/L DCW and tests 11–12: 2.38 g/L DCW). Increasing sulfate concentration resulted in a significant

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

increase of biomass up to 22 mg/L, after which its influence is greatly reduced, regardless of glucose %, as seen in the lower half of **Figure 1B**.

The results obtained at both times (72 h and 216 h) are in accordance with the fructophilic nature of this strain, as described by Alves and Paixão [17]. Up to 72 h, when glucose and fructose are at a 50:50 ratio in a 10 g/L mix, there is no significant difference from growth with 0% glucose (100% fructose); therefore, by this time and within this range of glucose %, the growth is mostly finished, achieving the highest results. Further increases of glucose % result in lower biomass production, confirming that fructose has a stimulant effect on growth rates. When glucose represents 100% of the 10 g/L mix, and there is no fructose to induce biomass formation, the lag phase becomes longer, and the culture presents its lowest results. At 216 h, glucose seems to have a stimulant effect toward biomass production, however, this is also the result of its fructophilic nature. Between 72 h and 216 h, cultures with higher glucose % were at an earlier stage of their growth, and as such continued to increase biomass production. On the other hand, cultures with lower glucose %, which had already finished their development at 72 h, entered the stationary, or even cell death phase of the growth, resulting in stagnation, and/or reduction of biomass production, thus explaining this apparent contradiction.

Finally, it becomes evident that, under these conditions, a minimum sulfate concentration between 20 and 22 mg/L is needed to achieve significant biomass production. When sulfate was at 9.01 mg/L, biomass never reached a concentration of 3 g/L regardless of time or glucose %. On the other hand, an increase to 34.99 mg/L was mostly shown to have a small effect on biomass, thus reinforcing the notion that 22 mg/L of sulfate is sufficient for the consumption of 10 g/L of glucose/fructose.

In terms of total carotenoid production (μg of carotenoids per shake-flask), at 72 h, it is clear that both factors equally influence the response. In fact, at this time the optimum conditions were found at the center of the experimental domain. As seen in both **Table 1** and **Figure 1C**, this response is at its highest (65.4 μg) when glucose % is at 50%, and sulfate is of about 22 mg/L. Any significant deviation from these values results in a reduction of total carotenoid production.

At 216 h, total carotenoid production was positively influenced by both factors. Glucose % is shown to have the highest influence, increasing its impact for higher values of this parameter. This is especially true above 75%, since an increase to 100% glucose in the sugar mix resulted in an increase of 75% of total carotenoid production, from 178.2 to 311.1 μg, the highest observed in this ED. The lowest values were observed for conditions with sulfate at 9.01 mg/L (**Table 1**; tests 9-10: 32.8 μg and tests 11-12: 52.2 μg). As seen with biomass, increasing sulfate up to 22 mg/L resulted in an increase in response to more than double. Further increases in sulfate concentration have a smaller but positive impact. **Figure 1D** indicates that this production could be further increased by combining the maximum of both factors with glucose % at 100% and sulfate at 37.0 mg/L reaching a value close to 350 μg per culture flask.

At 72 h, it is possible to see equilibrium between biomass production and carotenoid induction, guaranteed by the presence of both sugars and enough sulfate to ensure complete carbon consumption. While glucose stimulates pigment production, it induces slower biomass formation, so if there is no fructose in the sugar mix, there will be fewer cells, resulting in less carotenoids.

Changing any of these conditions would result in a reduction of response, as demonstrated by the concentric lines of the response surface (**Figure 1C**). After 216 h of growth, the response changes and both factors presented a positive influence on the total carotenoid concentration, reaching a theoretical maximum at 100% glucose and

37 mg/L of sulfate. Overall, for lower glucose % in mix and/or lower sulfate concentration, sulfate concentration appeared to have greater influence, as seen in horizontal and diagonal lines in the lower quadrants. As for higher sulfate and glucose concentrations, the percentage of glucose/total sugars presented the highest influence, evidenced by the much more vertical lines in the upper quadrants.
