*3.8. Statistical Analysis*

The experiments were carried out in triplicate. The results shown in Figures 1 and 4–6 were analyzed by a one-way analysis of variance (one-way ANOVA) followed by Tukey's post-hoc test for multiple comparisons. Data from the comparison of the control strain and the 1–6 mutant were analyzed statistically using the unpaired Student's t-test (Figure 7). The statistical analysis was carried out using GraphPad Prism for Windows version 5.03 (GraphPad Software, San Diego, CA, USA). All results are presented as mean +/- SEM. *p* < 0.05 was considered statistically significant with a confidence level of 95%.

#### **4. Conclusions**

The present results demonstrate that the adapted mutants of *C. pannorum* A-1 are characterized by an enhanced resistance to stressful conditions. So far, however, they have shown moderate biocatalytic activities, giving relatively high *trans*-pinocarveol yields, which means they are still subject to optimization. The maximum *trans*-pinocarveol concentrations obtained in the experiment reported in the present paper were in the range of 147.2–314.7 mg/g d.w. × L. In our non-optimized flask system, we were able to biotransform of β-pinene worth US\$ 0.36 to *trans*-pinocarveol with a value of US\$ 67 (per 1 L of batch culture), using the most active mutant. This result indicates that *C. pannorum* A-1 and its mutants are efficient biocatalysts for the conversion of β-pinene to this valuable product. Further improvement in β-pinene biotransformation may be achieved by selecting blocked mutants of the isolated fungal strains, which would prevent further metabolization of the main product (*trans*-pinocarveol). Filtration-enrichment methods for selecting auxotrophs may be used for this purpose [75]. Moreover, optimization of the culture conditions in bioreactors is necessary for the strain to be utilized in larger scale bioconversion processes, as some parameters, such as oxygen supply or pH, cannot be controlled in a flask culture.

The results show that it is possible to increase the rate of positive mutations by using media with a gradient of the toxic substrate and performing rapid initial evaluation of the GMA of the examined mutants of *C. pannorum* A-1. The use of the gradient plate method and subsequent determination of the GMA of the isolated mutants greatly simplifies the selection of substrate-resistant strains. Direct screening has the advantage of significantly reducing the number of cultures isolated from plates, which would normally require productivity to be tested in shake flask cultures. This method may considerably improve the selection of β-pinene biotransformation-active microbial strains and also other cultures showing biotransformation activity toward toxic substrates.

**Supplementary Materials:** The following are available online, Figure S1: Agar plate with linear gradient of β-pinene concentration, Figure S2: Flowchart of the procedure for improving *C. pannorum* A-1 as a biocatalyst for β-pinene biotransformation, Figure S3: Mass spectra of unknown compounds; RI = 1275, RT = 20.1 min, Table S1: Variants of mutagenesis of the psychrotrophic fungus *C. pannorum* A-1 and their impact on survivability of conidia. Survivability was expressed as the number of colonies formed compared to control plates with non-treated spores after 2 days of incubation at 20 ◦C on agarized BM, Table S2: Oxygen uptake rate *<sup>k</sup>* [% <sup>×</sup> <sup>s</sup><sup>−</sup>1] for the 12 most active GMA mutants and parental strain. The standard deviation was approximately 3%

**Author Contributions:** Conceptualization, J.F. and M.T.; methodology, M.K., J.F., A.G. and M.T.; software, M.K., J.F., K.J. and M.T.; validation, M.K., A.G. and M.T.; formal analysis, M.K., K.J. and M.T.; investigation, M.K., K.J. and A.G.; resources, M.K., J.F., A.G., and M.T.; data curation, M.K. and M.T.; writing—original draft preparation, J.F. and M.T.; writing—review and editing, M.K., J.F. and M.T.; visualization, M.K. and K.J.; supervision, J.F. and M.T.; project administration, J.F.; funding acquisition, M.K, J.F., A.G., K.J. and M.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank Maria Curie-Skłodowska University in Lublin, Poland, for providing institutional funds to support this work.

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