*3.2. Growth Experiments with Fertilizer Addition through DoE*

With the aims of finding an optimal LED lights combination, balancing the number of hyper red and deep blue LED lights, that is capable of enhancing all the growth parameters, and investigating the possible effects in combination with fertilizer, a new experimental set-up was managed through an ad hoc DoE experimental plan. Table 4 shows the complete experimental plan and the obtained results, in which the average values of each response among three repetitions of the same experiment are indicated.

As previously stated, with the aim to evaluate only fertilizer and LED lights effects, all the other parameters were kept as constant; thereafter, temperature and humidity were controlled during all the experiments to avoid environmental conditioning. Temperature and humidity were kept almost constant near 30 ◦C and 60% respectively, with restrained variation around 5% due to the employment of an indoor environment not perfectly conditioned. From a general observation of the results, germination occurred for almost all the basil seeds; in fact, only two of them did not germinate, and thereafter, the number is not statistically relevant. In addition, it must be noted that all the cultivation conditions applied according to this experiment plan were proved to be appropriate for basil, as it was confirmed by the absence of morphological and developmental abnormalities during plant growth. Observing only the data in Table 4, a general comparison can be stated with the results obtained without fertilizer, as shown in Table 5. From this comparison, it is clear that the fertilizer addition, by soil or by controlled-release fertilizer addition, is capable of improving the basil growth performances. In particular, it can be observed that plant height is always greater with fertilizer as well as the NoL and SLA parameters. Figures 5–7 show examples of the plants obtained at the end of the experimentation, respectively those grown under 3HR:1DB, 1HR:1DB, and 1HR:3DB, thereafter moving from a higher to lower proportion of hyper red LED light in relation to deep blue. As can be seen observing Figures 5–7 and the data in Table 4, the introduction of NPK controlled-release fertilizer has a positive effect on the plants' growth by increasing a considerable number of plants parameters (such as Height, TFM, TDM, LAI, and SLA), and in general, this effect is not influenced by the type of LED light. Nevertheless, from Figure 7, it appears clear that a more distinguishable difference among plants with and without NPK is appreciable, suggesting a possible synergic effect between NPK addition and 1HR:3DB LED lights combination that is capable of possibly promoting plant height and TFM. For these reasons, it is essential to study the ANOVA analysis to mathematically identify the possible correlation among NPK and LED lights combination as a function of the different measured parameters.

**Figure 5.** Basil plants obtained under 3HR:1DB LED lights.

**Figure 6.** Basil plants obtained under 1HR:1DB LED lights.

**Figure 7.** Basil plants obtained under 1HR:3DB LED lights.

The normal distribution of the residuals, as well as their homogeneity, has been analyzed (data not reported) for each response, confirming that the mathematical models derived can be used to explore the region of interest. The resulting models allowed describing the relationships between growth condition and the measured responses. The ANOVA results have been presented in Tables 6 and 7, where the quantification of the significance of factors and their interactions were reported as well as the fitting quality parameters as well as the effect sizes for each significant model. An inverse square root transformation was required to normalize the data and codify the hierarchy of the factors for some responses. The model correlating the factors (in single or interaction) to the panel data evaluation is significant, as confirmed by the *p*-value < 0.0001 for all the responses, which means that the probability of the data variation due to unknown factors is statistically irrelevant. Moreover, it is relevant that the curvature is not significant, and therefore, the

central points can be treated as additional data in the regression model, augmenting the design plan. R2 and Pred-R2 (Table 6) confirm the sufficient fit of the data and a quite fair predictive power for four responses, having R<sup>2</sup> > 0.45, with a particular high fitting for SLA parameters with R<sup>2</sup> = 0.75. Considering all the models' equations in Table 6 (where the coefficients of the variables in the model are reported), it is clear that NPK controlled-release fertilizer plays the main role in the performance of the responses, and this is consistent with the previous hypothesis, but a synergic effect has been shown and calculated for the SLA responses, which also corresponds to the model with higher fitting quality (higher R2).


**Table 6.** ANOVA results.

**Table 7.** Effect sizes for significant responses.


Figure 8 shows interaction graphs representing graphically each calculated model. A quite similar behavior can be detected for NoL, TFM, and LAI (Figure 8a–c), where an increasing of these responses is observed by introducing the NPK fertilizer independently of the LED lights combination. In contrast, the SLA parameter (Figure 8d) is clearly influenced by a synergic effect of NPK and LED lights: a restrained quantity of hyper red (1HR:3DB) is favorable to the SLA increase, independently of the presence of NPK, even if the highest value of SLA is obtained employing also controlled release NPK. The overall result is that a high quantity of hyper red LED light should be avoided to promote the basil growth. This result is in agreement with previous literature related to other plants' growth in an indoor and black environment such as lettuce, tomato, and flowers, where the employment of a high quantity of red LED light leads to reduced dry weight, plant height, and leaf area [22,23,41,42].

**Figure 8.** Interaction plots of different responses: in black color, the interaction lines related to 1HR:3DB; in red color, the interaction lines related to 3HR:1DB: (**a**) NoL; (**b**) TFM; (**c**) LAI; (**d**) SLA. Error bars indicate the standard deviation on the average values. LAI: Leaf Area Index, NoL: Number of Leaves, SLA: Specific Leaf Area, TFM: Total Fresh Mass.

#### **4. Conclusions**

In this study, it has been demonstrated that the implementation of various combination of LED lights is generally favorable to basil (*Ocimum Basilicum)* germination and growth with respect to daylight (e.g., Height: +30%, Total fresh mass: +50%), although no differences among hyper red and deep blue LED lights combination can be noticed when a soil without any fertilizer is employed as growing media. Applying a design of experiments approach, mathematically reliable information has been derived concerning a possible synergic effect with a soil enriched in macronutrients and with an NPK controlledrelease fertilizer derived from agro-residues in a circular economy perspective. It has been demonstrated that the use of NPK controlled-release fertilizer provides better performance, for the cumulative values for individual pots, than the use of fertilized soil alone. In addition, for the SLA parameter, the highest value is obtained by employing controlledrelease NPK fertilizer coupled with a specific LED light combination (one hyper red: three deep blue). It should be noted that the present study analyzed the influence of different factors on basil growth considering only one moment of collection without considering the variation of the light recipe in the different phases of life of the same. Thereafter, for a future perspective, the variation of the light recipe during the different phases of life of the basil plant should be investigated. Moreover, by taking some leaves or part of them and storing them in liquid nitrogen, it would be possible to perform biochemical measurements, such as determination of the content of phenolic compounds, flavonoids, antioxidant capacity, nitrate content, chlorophyll content, and carotenoids. Finally, it must be noted that basil is an edible aromatic herb that is extensively employed in the Mediterranean diet; therefore, panel test judging the possible different tastes should be proposed as a future perspective.

**Author Contributions:** Conceptualization, M.M. and A.B.; software, S.B.; investigation, F.B. and A.B.; resources, L.B. and M.M.; writing—original draft preparation, S.B.; writing—review and editing, F.B. and A.B.; supervision, M.M. and L.B.; funding acquisition, M.M. and L.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Università di Modena e Reggio Emilia under the FAR program supported by Fondazione Cassa di Risparmio di Modena, project title "GREW (Garden from Recycling & Wastes)—New integrated system for house and vertical gardens cultures by synergic application of innovative fertilizer and LED lighting: A circular economy strategy giving to waste materials a new second life".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to founding regulation.

**Acknowledgments:** The authors thank Giovanni Verzellesi (University of Modena and Reggio Emilia) for the support and the fruitful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

