<table><caption>Table 2. *Cont.*</caption><thead><tr><th></th><th>Control</th><th>eCO<sub>2</sub></th><th>AMF</th><th>AMF + eCO<sub>2</sub></th></tr></thead><tbody><tr><td colspan="5"><strong>Essential amino acids (EAAs)</strong></td></tr><tr><td>Histidine</td><td>2.49 ± 0.19</td><td>2.75 ± 0.3</td><td>3.19 ± 0.23</td><td>2.95 ± 0.38</td></tr><tr><td>Isoleucine</td><td>0.15 ± 0.02</td><td>0.26 ± 0.03 *</td><td>0.28 ± 0.03 *</td><td>0.34 ± 0.01</td></tr><tr><td>Leucine</td><td>0.02 ± 0</td><td>0.03 ± 0</td><td>0.03 ± 0 *</td><td>0.04 ± 0</td></tr><tr><td>Lysine</td><td>4.2 ± 0.21</td><td>4.55 ± 0.29</td><td>4.97 ± 0.25</td><td>6.67 ± 0.5 <sup>a</sup></td></tr><tr><td>Methionine</td><td>0.02 ± 0</td><td>0.02 ± 0 *</td><td>0.03 ± 0 *</td><td>0.03 ± 0</td></tr><tr><td>Phenylalanine</td><td>0.33 ± 0.04</td><td>0.68 ± 0.03 **</td><td>0.76 ± 0.1 *</td><td>1.31 ± 0.08 <sup>a</sup></td></tr><tr><td>Valine</td><td>0.48 ± 0.05</td><td>0.58 ± 0.06</td><td>0.78 ± 0.08 *</td><td>1.72 ± 0.07 <sup>b</sup></td></tr><tr><td>Threonine</td><td>0.11 ± 0.01</td><td>0.16 ± 0.02</td><td>0.19 ± 0.02 *</td><td>0.21 ± 0.01</td></tr><tr><td>Arginine</td><td>1.86 ± 0.13</td><td>2.35 ± 0.25</td><td>3.14 ± 0.34 *</td><td>3.76 ± 0.17</td></tr><tr><td>Total EAAs</td><td>9.66</td><td>11.38</td><td>13.37</td><td>17.03</td></tr><tr><td colspan="5"><strong>Non-essential amino acids (NEAAs)</strong></td></tr><tr><td>Aspartate</td><td>0.03 ± 0</td><td>0.05 ± 0</td><td>0.06 ± 0.01 *</td><td>1.51 ± 0.1 <sup>b</sup></td></tr><tr><td>Cystine</td><td>0.03 ± 0</td><td>0.2 ± 0.02 **</td><td>0.12 ± 0.01 **</td><td>0.62 ± 0.06 <sup>b</sup></td></tr><tr><td>Glutamic acid</td><td>77.02 ± 5.21</td><td>97.05 ± 3.71 *</td><td>82.21 ± 5.27</td><td>132.44 ± 6.78 <sup>b</sup></td></tr><tr><td>Glutamine</td><td>96.66 ± 6.05</td><td>106.65 ± 0.98</td><td>159.99 ± 6.47 **</td><td>180.39 ± 9.12</td></tr><tr><td>Asparagine</td><td>1.23 ± 0.12</td><td>1.8 ± 0.19</td><td>1.84 ± 0.13 *</td><td>2.05 ± 0.09</td></tr><tr><td>Glycine</td><td>1.18 ± 0.13</td><td>2.03 ± 0.22 *</td><td>2.2 ± 0.24 *</td><td>2.67 ± 0.29</td></tr><tr><td>Ornithine</td><td>0.18 ± 0.03</td><td>0.21 ± 0.02</td><td>0.27 ± 0.04</td><td>0.32 ± 0.02</td></tr><tr><td>Proline</td><td>1.25 ± 0.06</td><td>2.24 ± 0.08 **</td><td>2.79 ± 0.15 **</td><td>3.52 ± 0.19 <sup>a</sup></td></tr><tr><td>Serine</td><td>0.35 ± 0.04</td><td>0.59 ± 0.06 *</td><td>0.64 ± 0.07 *</td><td>0.78 ± 0.08</td></tr><tr><td>Tyrosine</td><td>0.99 ± 0.11</td><td>1.3 ± 0.02 *</td><td>1.64 ± 0.06 **</td><td>1.41 ± 0.09</td></tr><tr><td>Alanine</td><td>18.65 ± 0.97</td><td>22.22 ± 0.76 *</td><td>25.09 ± 0.67 **</td><td>28.06 ± 0.53 <sup>a</sup></td></tr><tr><td>Total NEAAs</td><td>197.57</td><td>234.34</td><td>276.85</td><td>353.77</td></tr><tr><td colspan="5"><strong>Saturated fatty acids (SFA)</strong></td></tr><tr><td>Lauric (C12:0)</td><td>1.42 ± 0.17</td><td>1.6 ± 0.15</td><td>1.31 ± 0.26</td><td>2.24 ± 0.19 <sup>a</sup></td></tr><tr><td>Tetradecanoic (C14:0)</td><td>1.7 ± 0.15</td><td>2.6 ± 0.12 **</td><td>2.44 ± 0.14 *</td><td>3.05 ± 0.18</td></tr><tr><td>Hexadecanoic (C16:0)</td><td>10.18 ± 0.87</td><td>13.08 ± 0.73</td><td>13.38 ± 0.55 *</td><td>19.8 ± 1.14 <sup>b</sup></td></tr><tr><td>Heptadecanoic (C17:0)</td><td>0.62 ± 0.06</td><td>0.81 ± 0.09</td><td>0.95 ± 0.09 *</td><td>1.62 ± 0.09 <sup>b</sup></td></tr><tr><td>Octadecanoic (C18:0)</td><td>2.26 ± 0.21</td><td>2.96 ± 0.33</td><td>3.46 ± 0.34 *</td><td>5.9 ± 0.33 <sup>b</sup></td></tr><tr><td>Eicosanoic (C20:0)</td><td>2 ± 0.19</td><td>2.16 ± 0.19</td><td>2.9 ± 0.14 *</td><td>4.06 ± 0.4</td></tr><tr><td>Total SFA</td><td>18.18 ± 1.15</td><td>23.2 ± 0.68 *</td><td>24.43 ± 0.94 *</td><td>36.68 ± 0.38 <sup>b</sup></td></tr><tr><td colspan="5"><strong>Unsaturated fatty acids</strong></td></tr><tr><td>Myristoleic (C14:1)</td><td>0.61 ± 0.06</td><td>0.8 ± 0.09</td><td>0.94 ± 0.09 *</td><td>1.6 ± 0.09 <sup>b</sup></td></tr><tr><td>Palmitoleic (C16:1n-7)</td><td>1.61 ± 0.08</td><td>2.02 ± 0.1 *</td><td>1.48 ± 0.16</td><td>3.47 ± 0.19 <sup>b</sup></td></tr><tr><td>Octadecenoic (C18:1)</td><td>7.45 ± 0.7</td><td>11.29 ± 0.35 **</td><td>10.51 ± 0.24 *</td><td>15.82 ± 0.23 <sup>b</sup></td></tr><tr><td>Erucic acid (C22: 1)</td><td>12.19 ± 0.73</td><td>9.22 ± 0.28 *</td><td>12.93 ± 2.31</td><td>12.2 ± 0.54</td></tr><tr><td>Octadecadienoic (C18:2)</td><td>16.68 ± 1.57</td><td>24.65 ± 2.14 *</td><td>25.5 ± 1.48*</td><td>40.43 ± 0.78 <sup>b</sup></td></tr><tr><td>Octadecatrienoic (C18:3)</td><td>5.36 ± 0.5</td><td>7.01 ± 0.28 *</td><td>6.1 ± 0.28</td><td>8.97 ± 0.28 <sup>b</sup></td></tr><tr><td>USFA</td><td>43.9 ± 2.2</td><td>55 ± 2.55 *</td><td>57.46 ± 0.63 **</td><td>82.48 ± 0.68 <sup>b</sup></td></tr><tr><td>SFA/USFA</td><td>0.35</td><td>0.34</td><td>0.35</td><td>0.35</td></tr></tbody></table>

Besides, the current results revealed that the combined AMF and eCO₂ treatment induced a significant increase in the majority of the detected organic acids, amino acids (including both essential and non-essential amino acids) and fatty acids in *T. vulgare*, relative to AMF alone (Table 2, Figure 2). Regarding the individual treatments, AMF was more efficient in inducing the accumulation of these primary metabolites than eCO₂. All AMF and/or eCO₂ treatments did not affect the SFA/USFA ratio. Similarly, it was reported that AMF-inoculated maize plants, under low temperature, had higher amino acid concentrations than non-mycorrhizal ones, especially for Thr, Lys, Gly, Ala and His contents [55]. In contrast, proline content was reduced in mycorrhizal *Capsicum annuum* grown under saline conditions [56]. Moreover, different effects of eCO₂ on amino acids were reported, which were reduced in barley [57], increased in spring wheat [58], or were not affected in maize [59]. It was also shown that organic acid levels were increased in mycorrhizal *Pinus sylvestris* grown under
heavy metal concentrations [60], while they were not increased in *Portulacaria afra* under eCO₂ [61]. The concentration of most fatty acids of soybean was unchanged under higher levels of CO₂ [62]. On the other hand, a significant increase in the levels of individual fatty acids was reported in parsley and dill grown under eCO₂, which is more evident on UFA than SFA [9]. Therefore, the synchronous application of AMF and eCO₂ could be beneficial to avoid the negative impact of the individual treatment and/or to support their positive effects.

<figure><img src="image_55.png" /><figcaption>**Figure 2.** Heatmap of fold change in the contents of primary and secondary metabolites of *Thymus vulgare* grown under the effect of eCO₂ (620 ppm), AMF or their combination (AMF-eCO₂). Asterisks indicate significant (* p* < 0.05; ** * p* < 0.01) increased fold changes compared to control (untreated plants), as revealed by Student's t-test. Lowercase letters indicate significant differences (a * p* < 0.05; b * p* < 0.01) between AMF alone and the combined AMF+eCO₂ treatment.</figcaption></figure>

### 3.4. AMF and eCO₂ Promote the Accumulation of Phenolic Compounds and Volatile Oils in *T. vulgare*

Mycorrhizal symbiosis with medicinal plants has been recognized to induce the accumulation of secondary metabolites, especially phenolic compounds which play an important role in curing several ailments [63]. The present results showed that protocatechuic, p-coumaric and rosmarinic acids are the most abundant phenolic acids; while apigenin, kaempferol, quercetin and luteolin are the predominant flavonoid in *T. vulgare* (Table 3). Similarly, previous studies revealed the presence of some phenolic acids such as cinnamic, carnosic and rosmarinic acids, and also flavonoids such as luteolin and apigenin derivatives in *T. vulgare* [64]. There was a significant increment in the levels of the majority of the detected phenolic acids and flavonoids in *T. vulgare* under AMF and/or eCO₂ treatments, however the combined treatment was much more efficient than the individual ones (Figure 2). On the other hand,
in consistence with the previous studies [65], the present results revealed the presence of 16 volatile oils in *T. vulgare*, whereas 1,8-cineol, carvacrol and p-cymene are the most dominant followed by less amounts of linalol, α- and β-pineno, α-Phellandrene, myrecene and thymol (Table 3). There is also a significant increase in the volatile oils of *T. vulgare*, under the individual and combined treatments.

<table><caption>**Table 3.** Levels of phenolic compounds and volatile oils (mg g⁻¹ dry weight) and biological activities in *Thymus vulgare* grown under normal conditions (control) or the effect of eCO₂ (620 ppm), AMF or their combination (AMF-eCO₂). Values are mean ± standard error of five independent replicates. Asterisks indicate significant changes (* *p* < 0.05; ** *p* < 0.01) compared to control, as revealed by Student's t-test. Lowercase letters indicate significant differences (a *p* < 0.05; b *p* < 0.01) between AMF alone and the combined AMF+eCO₂ treatment.</caption><thead><tr><th>Metabolite</th><th>Control</th><th>eCO<sub>2</sub></th><th>AMF</th><th>AMF + eCO<sub>2</sub></th></tr></thead><tbody><tr><td><strong>Phenolic acids</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Caffeic acid</td><td>0.46 ± 0.04</td><td>0.71 ± 0.04*</td><td>0.66 ± 0.03*</td><td>0.73 ± 0.03</td></tr><tr><td>Chlorogenic acid</td><td>0.01 ± 0</td><td>0.01 ± 0</td><td>0.01 ± 0</td><td>0.01 ± 0<sup>a</sup></td></tr><tr><td>Protocatechuic acid</td><td>4.11 ± 0.32</td><td>4.47 ± 0.35</td><td>4.38 ± 0.45</td><td>6.42 ± 0.6</td></tr><tr><td>Gallic acid</td><td>0.34 ± 0.02</td><td>0.48 ± 0.06</td><td>0.39 ± 0.03</td><td>0.5 ± 0.02<sup>a</sup></td></tr><tr><td>p-Coumaric acid</td><td>2.66 ± 0.22</td><td>2.82 ± 0.35</td><td>2.32 ± 0.26</td><td>3.76 ± 0.36<sup>a</sup></td></tr><tr><td>Chicoric acid</td><td>1.28 ± 0.13</td><td>1.44 ± 0.15</td><td>1.23 ± 0.15</td><td>1.87 ± 0.16<sup>a</sup></td></tr><tr><td>Rosmarinic acid</td><td>1.72 ± 0.2</td><td>1.89 ± 0.1</td><td>1.59 ± 0.1</td><td>2.5 ± 0.11<sup>b</sup></td></tr><tr><td><strong>Flavonoids</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Quercetin</td><td>0.07 ± 0</td><td>0.07 ± 0</td><td>0.08 ± 0.01</td><td>0.08 ± 0.01</td></tr><tr><td>Naringenin</td><td>0.05 ± 0</td><td>0.06 ± 0</td><td>0.05 ± 0.01</td><td>0.06 ± 0.01</td></tr><tr><td>Kaempferol</td><td>0.08 ± 0</td><td>0.1 ± 0.01*</td><td>0.08 ± 0</td><td>0.13 ± 0.01<sup>b</sup></td></tr><tr><td>Luteolin</td><td>0.07 ± 0</td><td>0.1 ± 0.01*</td><td>0.06 ± 0</td><td>0.13 ± 0.01<sup>b</sup></td></tr><tr><td>Apigenin</td><td>0.11 ± 0.02</td><td>0.13 ± 0</td><td>0.14 ± 0.01</td><td>0.17 ± 0.01<sup>a</sup></td></tr><tr><td>Rutin</td><td>0.01 ± 0</td><td>0.01 ± 0</td><td>0.01 ± 0</td><td>0.01 ± 0</td></tr><tr><td><strong>Volatile oils</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>alpha-pineno</td><td>1.62 ± 0.21</td><td>2.1 ± 0.08</td><td>2 ± 0.03</td><td>2.97 ± 0.1<sup>a,b</sup></td></tr><tr><td>Beta-pineno</td><td>2.79 ± 0.36</td><td>3.57 ± 0.27</td><td>3.29 ± 0.43</td><td>5.13 ± 0.23<sup>a</sup></td></tr><tr><td>p-cymene</td><td>4.45 ± 0.32</td><td>5.94 ± 0.48</td><td>5.85 ± 0.51</td><td>8.31 ± 0.37<sup>a</sup></td></tr><tr><td>limonene</td><td>0.1 ± 0.03</td><td>0.08 ± 0.01</td><td>0.1 ± 0</td><td>0.12 ± 0.02</td></tr><tr><td>1,8-cineol</td><td>8.23 ± 1.04</td><td>8.54 ± 1.19</td><td>9.61 ± 1.34</td><td>8.51 ± 0.73</td></tr><tr><td>linalol</td><td>3.42 ± 0.44</td><td>3.54 ± 0.49</td><td>3.19 ± 0.7</td><td>3.96 ± 0.23</td></tr><tr><td>Camphor</td><td>0.64 ± 0.08</td><td>0.82 ± 0.09</td><td>0.76 ± 0.1</td><td>1.18 ± 0.09<sup>a</sup></td></tr><tr><td>Borneol</td><td>0.39 ± 0.05</td><td>0.4 ± 0.06</td><td>0.36 ± 0.08</td><td>0.45 ± 0.09</td></tr><tr><td>Thymol</td><td>1.5 ± 0.19</td><td>5.66 ± 0.48<sup>**</sup></td><td>2.47 ± 0.28<sup>*</sup></td><td>6.55 ± 0.48<sup>b</sup></td></tr><tr><td>Carvacrol</td><td>4.97 ± 0.63</td><td>6.35 ± 0.68</td><td>5.85 ± 0.76</td><td>9.13 ± 0.15<sup>a</sup></td></tr><tr><td>alpha-Phellandrene</td><td>2.16 ± 0.21</td><td>3.15 ± 0.34</td><td>2.9 ± 0.38</td><td>2.86 ± 0.17</td></tr><tr><td>beta-Phellandrene</td><td>0.25 ± 0.03</td><td>0.26 ± 0.03</td><td>0.26 ± 0.04</td><td>0.32 ± 0.04</td></tr><tr><td>Humuline</td><td>0.14 ± 0.01</td><td>0.17 ± 0.01</td><td>1.06 ± 0.05<sup>**</sup></td><td>0.7 ± 0.04<sup>b</sup></td></tr><tr><td>Myrcene</td><td>1.84 ± 0.12</td><td>2 ± 0.4</td><td>4.07 ± 0.41<sup>**</sup></td><td>2.64 ± 0.12<sup>a</sup></td></tr><tr><td>alpha-thyjone</td><td>1.1 ± 0.05</td><td>1.1 ± 0.04</td><td>1.09 ± 0.08</td><td>1.04 ± 0.03</td></tr><tr><td>Tricyclene</td><td>0.3 ± 0.02</td><td>0.66 ± 0.04<sup>**</sup></td><td>0.65 ± 0.07<sup>*</sup></td><td>1.21 ± 0.14<sup>a</sup></td></tr><tr><td><strong>Antioxidant capacity (FRAP)</strong></td><td>17.08 ± 1.8</td><td>27.57 ± 1<sup>**</sup></td><td>22.85 ± 1.13</td><td>35.91 ± 0.91<sup>b</sup></td></tr><tr><td><strong>Oxygen radical absorbance capacity (ORAC)</strong></td><td>743.41 ± 33.19</td><td>1034.6 ± 108.25</td><td>983.99 ± 35.17<sup>**</sup></td><td>1680.57 ± 81.71<sup>b</sup></td></tr><tr><td><strong>% inhibition of LDL oxidation</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>TBARS)</td><td>14.08 ± 0.99</td><td>26.56 ± 0.76<sup>**</sup></td><td>24.55 ± 0.98<sup>**</sup></td><td>35.32 ± 2.25<sup>a</sup></td></tr><tr><td>conjugated dienes</td><td>17.7 ± 2.1</td><td>32.56 ± 0.51<sup>**</sup></td><td>28.51 ± 1.16<sup>*</sup></td><td>43.53 ± 1.29<sup>b</sup></td></tr><tr><td><strong>% inhibition of hemolysis</strong></td><td>13.4 ± 0.83</td><td>22.02 ± 1.97<sup>*</sup></td><td>16.45 ± 1.05</td><td>28.28 ± 1.08<sup>b</sup></td></tr></tbody></table>

Supporting our results, several studies have investigated the potential effects of eCO₂ and AMF, separately and in combination, on the levels of phenolic compounds and antioxidant activity in a variety of plant species. For instance, AMF treatments caused an increase in phenolic compounds content of lettuce [53,66], and in the antioxidant capacity of sweet basil [67]. Moreover, the flavonoids of some wild plants, such as *Libidibia ferrea*, were found to be accumulated by mycorrhizal association [68]. Similarly, eCO₂ induced the accumulation of some phenolic compounds in birch [69], and *Zingiber officinale* [52]. However, a low phenolic content was reported for some plants such as rice [70] under eCO₂. Regarding the interaction between eCO₂ and AMF, it was reported that the induction of secondary metabolites
in lettuce and alfalfa by AMF was negatively affected under eCO₂, probably due to utilization of photoassimilates for increasing plant biomass and for AMF growth as well, at the expense of secondary metabolism [43,44]. Therefore, it could be suggested that climatic changes might have an impact on AMF, which in turn, affect the metabolic functions of their host plants.

Several scenarios have been proposed to explain the induction of secondary metabolites in response to AMF associations and eCO₂. It was found that AMF could affect secondary metabolites through improved photosynthesis and mineral content of the host plants, activation of pathways involved in synthesis of secondary metabolites, or higher expression of some genes related to secondary metabolism [68]. On the other hand, the eCO₂-induced changes in plant secondary metabolism have been attributed to either excess amount of non-structural carbohydrates, resulting in an increment in carbon-based secondary metabolites [9,37,51].

### 3.5. AMF and eCO₂-Induced Changes in Secondary Metabolites Support the Biological Activities of T. vulgare

Reactive oxygen species and free radicals have been recognized to induce harmful effects on living organisms. In this aspect, antioxidants, such as phenolic compounds and volatile oils, could act as free radical scavengers [71]. The present results showed an increase in the total antioxidant capacities of *T. vulgare*, tested by different methods (FRAP, ORAC, inhibition of LDL oxidation (TBARS and conjugated dienes) and inhibition of hemolysis), under the effects of AMF and/or eCO₂ (Table 3). It was previously reported that the antioxidant properties exhibited by *T. vulgare* extracts have been attributed to their content of volatile oils, especially carvacrol and thymol [72], flavonoids such as apigenin and luteolin derivatives and phenolic acids such as cinnamic and rosmarinic acids [64]. Moreover, some phenolic compounds were previously isolated from *T. vulgare* and proved to inhibit oxidative hemolysis [73]. The decreased levels of lipid peroxidation products such as TBARS and conjugated dienes might be ascribed to some protective effects of thymol [74].

## 4. Conclusions

Based on the above results, it is clear that the tested plant, *T. vulgare*, has benefited from the independent and combined effects of both AMF and eCO₂, however their synchronous application is much more beneficial. Such positive impacts are being reflected on improved biomass production and higher accumulation of primary (sugars, amino acids, fatty acids and organic acids), and secondary (phenolic acids, flavonoids and volatile oils) metabolites. *T. vulgare* plants grown under synchronous application of AMF and eCO₂ have taken much advantage over those grown under the individual effects of both factors in terms of improved growth and bioactive components. Thus, the current study clearly shows that co-application of AMF and eCO₂ is a promising approach to improve the growth and the nutritional and health promoting values of *T. vulgare*. Further, the robust monitoring of primary and secondary metabolites presented herein could support our understanding about the mechanisms behind the positive impacts of AMF and eCO₂ on plants.

**Author Contributions:** Conceived and designed the experiments: A.M.S., H.A., A.M.A.K. Conducted the main experiment: T.H.H., R.S.Y, A.M.A.K. Measured the fungal related parameters: R.S.Y., A.M.A.K. Performed the metabolite profiling: H.A., A.M.S., M.A.-M. Measured the biological activities: M.A.-M., Analyzed the data: A.M.S., M.A.-M., A.M.A.K., T.H.H. Wrote the original draft: M.A.-M., T.H.H., Reviewed, edited and prepared the MS for submission: A.M.S., H.A., A.M.A.K. All authors have read and agreed to the published version of the manuscript.

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

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

# Alginate-Derived Elicitors Enhance β-Glucan Content and Antioxidant Activities in Culinary and Medicinal Mushroom, *Sparassis latifolia*

Yong-Woon Kim <fnref n="1" />,†, Yuanzheng Wu <fnref n="2" />,†, Moon-Hee Choi <fnref n="1" />, Hyun-Jae Shin <fnref n="1" />,* and Jishun Li <fnref n="2" />

Received: 9 June 2020; Accepted: 24 June 2020; Published: 25 June 2020

<figure><img src="image_58.png" /></figure>

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**Abstract:** This study aimed to investigate the elicitation effects of alginate oligosaccharides extracted from brown algae (*Sargassum* species) on β-glucan production in cauliflower mushroom (*Sparassis latifolia*). Sodium alginate was refined from *Sargassum fulvellum*, *S. fusiforme*, and *S. horneri*, and characterized by proton nuclear magnetic resonance spectroscopy (¹H NMR), resulting mannuronic acid to guluronic acid (M/G) rationes from 0.64 to 1.38. Three oligosaccharide fractions, ethanol fraction (EF), solid fraction (SF), and liquid fraction (LF), were prepared by acid hydrolysis and analyzed by Fourier transform infrared (FT-IR) spectra and high-performance anion-exchange chromatography with a pulsed amperometric detector (HPAEC-PAD). The samples of *S. fusiforme* resulted in the highest hydrolysate in SF and the lowest in LF, which was consistent with its highest M/G ratio. The SF of *S. fusiforme* and LF of *S. horneri* were chosen for elicitation on *S. latifolia*, yielding the highest β-glucan contents of 56.01 ± 3.45% and 59.74 ± 4.49% in the stalk, respectively. Total polyphenol content (TPC) and antioxidant activities (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging and Superoxide dismutase (SOD)-like activity) of aqueous extracts of *S. latifolia* were greatly stimulated by alginate elicitation. These results demonstrate that alginate oligosaccharides extracted from brown algae may be useful as an elicitor to enhance the nutritional value of mushrooms.

**Keywords:** Alginate; β-glucan; oligosaccharides; elicitation; *Sargassum* species; *Sparassis latifolia*; polyphenol; antioxidant

## 1. Introduction

Mushrooms have been recognized as medicine sources and functional foods since ancient times owing to their bioactive compounds and diverse health benefits [1]. Cauliflower mushrooms, species of *Sparassis* Fr., are culinary and medicinal mushrooms that primarily, but not exclusively, grow on the stumps of coniferous trees and are widely distributed throughout northern temperate forests [2]. The Asian *Sparassis* isolate was originally known as *S. crispa*, until morphological and molecular studies redefined it as *S. latifolia* [3,4]. Recently this mushroom has become cultivable in Japan and Korea using conifers [5]. The fruiting bodies of *S. crispa* and *S. latifolia* exhibit excellent effects for enhancing cytokine synthesis and preventing human diseases, such as gastric ulceration, oesophageal cancer, hypertension, and diabetes. Such effects are attributable to different compounds including polysaccharides, terpenoids, phenolic compounds, and glycoproteins [6–8].

<footnote n="1">¹ Department of Biochemical and Polymer Engineering, Chosun University, Gwangju 61452, Korea; ywkim1205@naver.com (Y.-W.K.); aamoony1222@naver.com (M.-H.C.)</footnote>

<footnote n="2">² Shandong Provincial Key Laboratory of Applied Microbiology, Ecology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250103, China; wuyzh@sdas.org (Y.W.); yewu2@sdas.org (J.L.)</footnote>

<footnote>* Correspondence: shinhj@chosun.ac.kr; Tel.: +82-62-230-7518</footnote>

<footnote>† These authors contributed equally to this work.</footnote>
As major constituents of fungal cell walls, β-glucans are present in all mushroom species and play important roles in their beneficial properties for human health [9]. Previous studies described a wide range of mushroom β-glucans with different structures, such as linear 1-3-β-glucans isolated from *Poria cocos*, linear 1-6-β-glucans from *Agaricus* spp., 6-branched 1,3-β-glucans from *Lentinus edodes* (designated lentinan) and *Grifola frondosa* (designated grifolan), and 3-branched 1,6-β-glucans from *Sarcodon aspratus* [10–12]. Among most studied fungal glucans, 6-branched 1,3-β-glucans were reported to be efficient as biological response modifiers (BRM) for the treatment of cancer and infectious diseases [13]. *Sparassis* species contain considerably higher contents of β-glucan than other mushrooms, up to 43.6% of dry weight in the fruiting bodies of *S. crispa* [14,15]. The primary structure of β-glucan isolated from *S. crispa* was 6-branched 1,3-β-glucan with one branch in every third main-chain unit, showing high water solubility and an estimated molecular weight of ca. 510 kDa [16,17]. Extraction and purification of β-glucans from mushroom mycelia and fruiting bodies have been established [18,19]. However, there are few studies about the enhancement of β-glucan contents in mushrooms.

To enhance the production of desirable compounds in plants or microorganisms, elicitation has been employed using biotic or abiotic elicitors to stimulate the accumulation of secondary metabolites [20,21]. For example, physical elicitation and enzyme treatments have been exploited for the growth enhancement and promotion of β-glucan contents in *S. latifolia* [22,23]. Alginate is a biotic elicitor that has been demonstrated to provide induced plant defense against pathogens, tolerance improvement to environmental stresses such as drought and salinity, and growth enhancement in *Eucomis autumnalis*, *Vitis vinifera*, *Phoenix dactylifera*, and *Olea europaea* [24–27]. Chemically, alginate is a linear copolymer consisting of homopolymeric blocks of residues of (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G), covalently linked together in different sequences [28]. As a biocompatible and immunogenic polysaccharide, alginate can be extracted from brown algae such as *Ecklonia* spp., *Laminaria* spp., or *Sargassum* spp. [29].

In this study, the physicochemical characteristics of alginate oligosaccharides extracted from *Sargassum* species from Korea and the enhancement of β-glucan content in *S. latifolia* by alginate elicitation were investigated. To the best of our knowledge, there has been no prior attempt to investigate alginate-derived elicitors for enhancement of β-glucan synthesis in mushrooms.

## 2. Materials and Methods

### 2.1. Collection of Marine Brown Algae

Three brown algae of *Sargassum* species were collected from the Korean coast. *S. fulvellum* (Turner) C. Agardh was collected in March 2017 at Jindo (34°22′18.0″ N, 126°08′09.4″ E), *S. fusiforme* (Harvey) Setchell was harvested in July 2017 at Wando (34°20′11.8″ N, 126°48′54.6″ E), and *S. horneri* (Turner) C. Agardh was collected in February 2018 at Jeju (33°31′08.3″ N, 126°31′16.7″ E). All *Sargassum* samples were cleaned by washed in distilled water to remove sand and excess salt. Samples were then oven dried at 60 °C. The samples were stored in plastic bags at room temperature (25 °C) until alginate extraction.

### 2.2. Extraction of Sodium Alginate

Sodium alginate was extracted as described by Davis et al. [30] In brief, 25 g of *Sargassum* samples were soaked in 800 mL of 2% formaldehyde for 24 h at room temperature, washed with distilled water, added to 800 mL of 0.2 M HCl, and left for 24 h. The samples were then washed with distilled water before extraction with 2% sodium carbonate ($Na_2CO_3$) for 3 h at 100 °C. The filtered extract was centrifuged (10,000 rpm, 30 min, 4 °C) and the supernatant was precipitated with 3 volumes of 95% ethanol. The precipitate was washed with acetone and dried at 60 °C.
## 2.3. Preparation of Oligosaccharide Fractions

Oligosaccharide separation from sodium alginate was performed as described by Asilonu et al. [31] This method gave three oligosaccharide fractions instead of two fractions from sodium alginate. In brief, 10 g of sodium alginate samples were acid hydrolyzed with 1 L of 0.3 M HCl in an autoclave at 121 °C for 1 h. The suspension was quickly cooled using ice water and neutralized with NaOH. Sodium chloride (NaCl) was added to a final concentration of 0.5% (w/v), and the solution mixture precipitated with 2 volumes of 95% ethanol and centrifuged (4000 rpm, 30 min, 4 °C). The supernatant was marked as ethanol fraction (EF). The precipitate was then washed with distilled water, centrifuged again, dissolved in distilled water, and adjusted to pH 2.85 with 1 M HCl. The solid fraction (SF) and liquid fraction (LF) were separated and lyophilized. Thus, three different oligosaccharide fractions (EF, SF, and LF) were obtained for further analysis.

## 2.4. Physicochemical Analysis of Oligosaccharide Fractions

### 2.4.1. Molecular Weight Analysis

The average molecular weight ($M_W$) of the EF, LF, and SF oligosaccharide fractions was determined using an SB-803 HQ column (6 µm, 8.0 mm × 300 mm, Shodex, Japan) and an SB-805 HQ column (13 µm, 8.0 mm × 300 mm, Shodex, Japan). A mobile phase of 0.1 mol/L NaCl solution was used after 0.22 µm filtration. The flow rate was 0.5 mL/min and analyses were performed at 50 °C. The sodium alginate sample (100 mg) was then resuspended in 10 mL distilled water. Molecular weights were determined by reference to a calibration curve using pullulan standards [32].

### 2.4.2. H NMR Spectroscopy Analysis

Proton nuclear magnetic resonance (¹H NMR) spectroscopy was acquired on alginate solution (1% w/v) in D₂O, with recordings at 80 °C using an AVANCE III HD 400 (Bruker Scientific Instruments, USA) spectrometer operating at a frequency of 400 MHz. The individual blocks of guluronic and mannuronic acids ($F_G$ and $F_M$, respectively), the homopolymeric ($F_{GG}$ and $F_{MM}$, respectively) blocks, and the heterogeneous ($F_{GM}$ or $F_{MG}$) blocks of alginate were calculated using the areas of I, II, and III ($A_I$, $A_{II}$, and $A_{III}$, respectively) signals according to the Grasdalen [33] method and Equations (1)–(3):

$$F_G = \frac{A_I}{A_{II} + A_{III}} \qquad (1)$$

$$F_M = 1 - F_G \qquad (2)$$

$$M/G = \frac{1 - F_G}{F_G} = \frac{F_M}{F_G} \qquad (3)$$

The double fractions ($F_{GM}$ and $F_{MM}$) were deduced by referring to Equations (4)–(6):

$$F_{GG} = \frac{A_{II}}{A_{II} + A_{III}} \qquad (4)$$

$$F_{GM} = F_{MG} = F_G - F_{GG} \qquad (5)$$

$$F_{MM} = F_M - F_{MG} \qquad (6)$$

### 2.4.3. FT-IR Spectroscopy Analysis

Fourier transform infrared (FT-IR) spectra were measured on a Nicolet 6700 FT-IR (Thermo Electron Co., Waltham, MA, USA). Spectra of the alginate samples in KBr pellets were recorded in the 4000–400 cm⁻¹ range.
### 2.4.4. Monosaccharide Analysis

Monosaccharide analysis for the three oligosaccharide fractions was performed as follows [34]. The first acid hydrolysis was performed in 3 mL of 72% H₂SO₄ with 0.3 g of sample for 2 h at 30 °C. The second hydrolysis was carried out by adding 84 mL of distilled water to the first hydrolysate followed by acid hydrolysis at 121 °C for 1 h in an autoclave. Analysis was performed using high performance anion exchange chromatography with a pulsed amperometric detector (HPAEC-PAD, ICS-5000, Dionex Co., Sunnyvale, CA, USA) equipped with a current detector, with CarboPac PA-4 (250 mm × 4 mm, Dionex Co., USA) used as the column at room temperature. An 18 mM NaOH solution was chosen as the mobile phase with a flowrate of 1.0 mL/min.

## 2.5. *Cultivation of *Sparassis Latifolia*

*Sparassis latifolia* JF02-06 was provided by Jeonnam Forest Research Institute (Naju, Korea). A sawdust-based medium was used for the cultivation of cauliflower mushrooms as adopted by Park et al. [23] Fermented sawdust of *Larix kaempferi*, corn, and wheat flour were mixed at a ratio of 8:1:1 (w/w/w), followed by the addition of 10% aqueous solution of starch syrup and an adjustment of the moisture content to 55–60%. The medium was packed in a 500 mL incubation bottle and sterilized at 121 °C for 90 min. The inoculum of *S. latifolia* was prepared in potato dextrose broth (PDB) for 3 weeks and 10 mL of liquid inoculum was inoculated into each bottle. The inoculated medium was then incubated at room temperature. Cultivation was completed after 50–70 days from inoculation. When mycelium was formed in the medium, the culture was transferred to the cultivation room at a temperature of 20 ± 2 °C and 95% humidity. Fruit-shaped fruiting bodies were formed after 1 week and then harvested after 40–45 days.

## 2.6. Elicitation by Alginate Oligosaccharide Fractions

Two of the oligosaccharide fractions, LF and SF (each 200 mg/L in distilled water), were applied to the surface of the sawdust medium for elicitation: 12 mL/bottle after sterilization of the medium (first application) and 20 mL/bottle after transfer to the cultivation room (second application). The same volume of distilled water was used as a control group. All treatments were arranged in a completely randomized block design with four replicates. Fruiting body and stalk of *S. latifolia* were harvested, frozen in liquid nitrogen, and stored in a freezer at -80 °C until extraction and analysis.

## 2.7. *Assay of Glucan Content*

Content of total and β-glucans was determined by a β-glucan assay kit (Cat. No. K-YBGL, Megazyme International, Wicklow, Ireland), following the H₂SO₄ acid hydrolysis procedure by McCleary and Draga [35]. Briefly, after being milled to pass through a 1.0 mm screen, 100 mg of the dried mushroom samples were added with 2 mL of ice-cold 12 M H₂SO₄, and then the mixture was stirred vigorously and incubated in an ice-cold water bath for 2 h. Then, 12 mL of distilled water was added to each sample and the suspension was kept in a boiling-water bath (~100 °C) for 2 h. After cooling to room temperature, 6 mL of 10 M KOH was added and the volume was adjusted to 100 mL with 200 mM sodium acetate buffer (pH 5.0). After centrifugation at 12,000 rpm for 10 min, an aliquot of the supernatant (0.1 mL) was mixed with 0.05 mL of exo-1,3-β-glucanase (20 U/mL) plus β-glucosidase (4 U/mL) and incubated at 40 °C for 60 min. Then, the mixture was incubated at 40 °C for 20 min with 3 mL of glucose-oxidase/peroxidase-reagent (GOPOD). Total glucan content was evaluated by a UV-Vis spectrophotometer (S-3100, SCINCO, Seoul, Korea) at λ = 510 nm with the reagent blank.

The α-glucan content was determined after incubation at 40 °C for 30 min of the suspension of 100 mg mushroom samples in 2 mL of 2 M KOH with an addition of 8 mL of 1.2 M sodium acetate buffer (pH 3.8) with 0.2 mL of amyloglucosidase (1630 U/mL) plus invertase (500 U/mL). Each sample was centrifuged at 12,000 rpm for 10 min, and 0.1 mL of the supernatant was analyzed for glucose
by mixing with 0.1 mL of 200 mM of sodium acetate buffer (pH 5.0) and 3 mL of GOPOD. α-Glucan content was evaluated by a UV-Vis spectrophotometer. β-Glucan content was determined as the difference between total and α-glucan contents.

## 2.8. Determination of Total Polyphenol Content

Total polyphenol content (TPC) of aqueous extracts of S. latifolia after alginate elicitation was determined by the modified Folin-Ciocalteu method [36]. An ramount of 0.5 mL of the extract was mixed with 0.5 mL of 0.2 N Folin-Ciocalteu reagent and 0.5 mL of 2% (w/v) sodium carbonate. The mixture was vortexed and incubated at room temperature for 30 min. The absorbance of the mixture was measured at λ = 750 nm with a UV-Vis spectrophotometer. Total polyphenol content was expressed as of gallic acid equivalent (GAE) mg/100 g.

## 2.9. Measurement of Antioxidant Activities

The antioxidant activities of aqueous extracts of S. latifolia were evaluated by scavenging activity of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation and superoxide dismutase (SOD)-like activity.

### 2.9.1. Assay of ABTS Radical Scavenging Activity

2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was measured using a modified method of Re et al. [37] The ABTS stock solution was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate and then kept in the dark for 12 h. The stock solution was then diluted with phosphate buffered saline (PBS, pH 7.4) until absorbance reached 0.80 ± 0.02 at λ = 730 nm using a UV-Vis spectrophotometer. Of the extracts of S. latifolia, 0.2 mL was mixed with 1 mL of diluted ABTS solution and left for 15 min in the dark. ABTS radical scavenging activity (%) was calculated as in Equation (7) as follows:

$$ \text{ABTS radical scavenging activity (\%)} = \frac{A_0 - A_1}{A_0} \times 100, \qquad (7) $$

where A₀ is the absorbance of the blank sample using water, and A₁ is the absorbance of the aqueous extracts of S. latifolia.

### 2.9.2. Assay of SOD-Like Activity

Superoxide dismutase (SOD)-like activity was measured by reference using an SOD kit (Cat. No. 19160, Sigma-Aldrich, St. Louis, MO, USA) [38]. Of the aqueous extracts of S. latifolia, 20 µL was mixed with 200 µL of water-soluble tetrazolium salt (WST) working solution and 20 µL of enzyme working solution to each well in a 96-well microplate. Then the microplate was incubated at 37 °C for 20 min. The absorbance of each sample was measured at λ = 450 nm using a microplate reader. The SOD-like activity (%) was calculated as in Equation (8) as follows:

$$ \text{SOD-like activity (\%)} = \frac{(\text{Blank 1} - \text{Blank 3}) - (\text{Sample} - \text{Blank 2})}{(\text{Blank 1} - \text{Blank 3})} \times 100 \qquad (8) $$

where Blank 1 is the absorbance of the water with WST working solution and enzyme working solution, Blank 2 is the absorbance of sample with WST working solution and dilution buffer, Blank 3 is the absorbance of water with WST working solution and dilution buffer, and Sample is the absorbance of the aqueous extracts of S. latifolia.
## 2.10. Statistical Analysis

Data were analyzed using analysis of variance (ANOVA) followed by Duncan's multiple-range test ($p < 0.05$) using SPSS software (version 23, SPSS, Chicago, IL, USA). Error bars indicate the mean ± SD and different letters describe significant differences within the same application data group.

# 3. Results

## 3.1. Physicochemical Properties of Alginate

Sodium alginate was extracted and refined from three *Sargassum* species, namely *S. fulvellum*, *S. fusiforme*, and *S. horneri*. The composition of extracted alginate is summarized in Table 1. The highest alginate yield was obtained from *S. fusiforme* (37.84 ± 0.48%), followed by 34.11 ± 1.65% from *S. horneri* and 30.88 ± 1.51% from *S. fulvellum*. The size of the extracted alginates was relatively high in *S. fusiforme* and *S. fulvellum* with an average $M_W$ of 504.65 and 461.07 kDa, respectively. However, the alginate from *S. horneri* had a much lower $M_W$ (138.10 kDa) than that of the other two algae. This indicates that different ranges of alginates are obtained from different *Sargassum* species.

<table><caption>**Table 1.** Composition of sodium alginate extracted from *Sargassum* species.</caption><thead><tr><th>Species</th><th>Yield (%)</th><th>Mw (kDa)</th><th>M/G</th><th>F<sub>M</sub></th><th>F<sub>G</sub></th><th>F<sub>MM</sub></th><th>F<sub>MG</sub></th><th>F<sub>GG</sub></th></tr></thead><tbody><tr><td><i>Sargassum fusiforme</i></td><td>37.84 ± 0.48</td><td>504.65</td><td>1.38</td><td>0.58</td><td>0.42</td><td>0.55</td><td>0.03</td><td>0.39</td></tr><tr><td><i>S. fulvellum</i></td><td>30.88 ± 1.51</td><td>461.07</td><td>0.88</td><td>0.47</td><td>0.53</td><td>0.43</td><td>0.04</td><td>0.49</td></tr><tr><td><i>S. horneri</i></td><td>34.11 ± 1.65</td><td>138.10</td><td>0.64</td><td>0.39</td><td>0.61</td><td>0.30</td><td>0.09</td><td>0.52</td></tr></tbody></table>

*Mw*: average molecular weight; *F*<sub>M</sub>: Mannuronic acid block fractions; *F*<sub>G</sub>: Guluronic acid block fractions; *F*<sub>MM</sub>: Homopolymeric mannuronic acid block fractions; *F*<sub>MG</sub>: Heterogeneous block fractions (mannuronic acid and guluronic acid); *F*<sub>GG</sub>: Homopolymeric guluronic acid block fractions.

The ratio of mannuronic acid to guluronic acid (M/G) of the structural blocks were determined by ¹H NMR analysis. As shown in Table 1, the M/G ratio of *Sargassum* species ranged from 0.64 to 1.38, with the highest ratio of 1.38 from *S. fusiforme* and similar M/G rationes between *S. fusiforme* and *S. horneri*. The guluronic acid anomeric proton (G-1) occurred at 5.56–5.58 ppm (peak I), guluronic acid H-5 (G-5) occurred at 4.96–4.98 ppm (peak III), and mannuronic acid anomeric proton (M-1) and the C-5 of alternating blocks (GM-5) overlapped at 5.21–5.23 ppm (peak II), as shown in Figure 1.

The FT-IR spectrum of sodium alginate of *Sargassum* species is presented in Figure 2. A broad band at 3466.25 cm⁻¹ was assigned to the hydrogen-bonded (O-H) stretching vibrations, and a weak signal at 2928.43 cm⁻¹ was attributed to C-H stretching vibrations. The peaks at 1619.79 cm⁻¹ and 1427.52 cm⁻¹ were attributed to asymmetric stretching of carboxylate O-C-O vibrations. The characteristic peaks of alginate include 1096.76 cm⁻¹ assigned to β-mannuronic acid and 1029.66 cm⁻¹ assigned to α-L-guluronic acid. Moreover, signals of glucuronic acid (1737.01, 1629.72, and 1144.66-937.31 cm⁻¹) could be detected in Figure 2 (LF and EF of a,b,c).
<figure><img src="image_59.png" /><figcaption>**Figure 1.** ¹H NMR spectra of sodium alginates extracted from (a) *Sargassum fulvellum*, (b) *S. fusiforme*, and (c) *S. horneri* (*A*₁: Area of guluronic acid anomeric proton peak I; *A*₂: Area of mannuronic acid anomeric proton and the C-5 of alternating blocks peak II; *A*₃: Area of guluronic acid peak III).</figcaption></figure>

<figure><img src="image_60.png" /><figcaption>**Figure 2.** Fourier transform infrared (FT-IR) spectra of sodium alginate, solid fraction (SF), liquid fraction (LF), and ethanol fraction (EF) from (a) *Sargassum fulvellum*, (b) *S. fusiforme*, and (c) *S. horneri*.</figcaption></figure>

## 3.2. Oligosaccharide Analysis

Sodium alginates extracted from *Sargassum* species were submitted to a two-step H₂SO₄ acid hydrolysis process to obtain partial hydrolysates for monosaccharide analysis. The block distribution of partial acid hydrolysates in EF, SF, and LF were 13.9–20.4%, 15.8–22.8%, and 61.1–65.0%, respectively.
The block distribution of this study was compared with that of previous reports by Haug et al. and Leal et al. [39,40], as summarized in Table 2. The comparison data showed a great difference in partial hydrolysis yield according to species used for the preparation of alginate oligosaccharides. The samples of *S. fusiforme* showed the highest hydrolysate yields in SF and the lowest in LF among *Sargassum* species, which was consistent with the highest M/G ratio of 1.38 and might be explained by the high content of polymannuronic acid and low content of polyguluronic acid. *S. fulvellum* and *S. horneri* both produced lower hydrolysate yields in SF and higher yields in LF, as indicated by their low M/G rationes. All three *Sargassum* species yielded similar percentages of hydrolysates in EF.

<table><caption>**Table 2.** Block distribution of sodium alginate from different species of brown algae.</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">M/G</th><th colspan="3">Partial Hydrolysis (Yields)</th><th rowspan="2">Reference</th></tr><tr><th>EF (%)</th><th>SF (%)</th><th>LF (%)</th></tr></thead><tbody><tr><td><i>Sargassum fusiforme</i></td><td>1.38</td><td>63.8</td><td>20.4</td><td>15.8</td><td>This study</td></tr><tr><td><i>S. fulvellum</i></td><td>0.88</td><td>65.0</td><td>13.9</td><td>21.1</td><td>This study</td></tr><tr><td><i>S. horneri</i></td><td>0.64</td><td>61.1</td><td>16.1</td><td>22.8</td><td>This study</td></tr><tr><td><i>Asclephyllum nodosum</i></td><td>1.85</td><td>52</td><td>35</td><td>13</td><td>[39]</td></tr><tr><td><i>Chordaria flagelliformis</i></td><td>0.90</td><td>21</td><td>28</td><td>51</td><td>[39]</td></tr><tr><td><i>Desmarestia aculeata</i></td><td>0.85</td><td>27</td><td>23</td><td>50</td><td>[39]</td></tr><tr><td><i>Dictyosiphon foeniculaceus</i></td><td>0.85</td><td>25</td><td>25</td><td>50</td><td>[39]</td></tr><tr><td><i>Fucus serratus</i></td><td>1.30</td><td>35</td><td>34</td><td>31</td><td>[39]</td></tr><tr><td><i>Laminaria digitata</i></td><td>1.45</td><td>34</td><td>43</td><td>23</td><td>[39]</td></tr><tr><td><i>L. hyperborea, fronds</i></td><td>1.35</td><td>26</td><td>43</td><td>31</td><td>[39]</td></tr><tr><td><i>L. hyperborea, stripes</i></td><td>0.65</td><td>25</td><td>15</td><td>60</td><td>[39]</td></tr><tr><td><i>Pelvetia canaliculata</i></td><td>1.50</td><td>38</td><td>37</td><td>25</td><td>[38]</td></tr><tr><td><i>Pylaiella</i></td><td>0.75</td><td>40</td><td>18</td><td>42</td><td>[39]</td></tr><tr><td><i>Scytosiphon lomentaria</i></td><td>1.15</td><td>25</td><td>35</td><td>40</td><td>[39]</td></tr><tr><td><i>Spermatochnus paradoxus</i></td><td>1.30</td><td>35</td><td>32</td><td>33</td><td>[39]</td></tr><tr><td><i>Desmarestia ligulata</i></td><td>0.58</td><td>3.7</td><td>25.1</td><td>56.4</td><td>[40]</td></tr><tr><td><i>D. ligulata</i></td><td>0.77</td><td>3.5</td><td>37.0</td><td>47.1</td><td>[40]</td></tr><tr><td><i>Lessonia flavicans</i></td><td>1.03</td><td>8.5</td><td>41.3</td><td>22.2</td><td>[40]</td></tr></tbody></table>

EF: Ethanol fraction of acid-hydrolyzed sodium alginate; SF: Solid fraction at pH 2.85 (fraction enriched in polymannuronic acid); LF: Liquid fraction at pH 2.85 (fraction enriched in polyguluronic acid).

HPAEC-PAD analysis of the H₂SO₄ hydrolysate of sodium alginate showed that monosaccharides were dominated by the weight of fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, mannuronic acid, guluronic acid, and glucuronic acid (Table 3). The major alginate contents comprised mannuronic acid and guluronic acid, as expected. The content of uronic acids was almost the same as that of *S. turbinarioides* as reported by Fenoradosoa et al. [41] The main monosaccharides of alginate from EF, SF, and LF were mannuronic acid, guluronic acid, and glucuronic acid. The size of the extracted ethanol fraction ranged from 650 to 5500 Da.
<table><caption>**Table 3.** Monomeric carbohydrate contents from *Sargassum* species determined by high-performance anion-exchange chromatography with a pulsed amperometric detector (HPAEC-PAD) analysis after two-step sulfuric acid hydrolysis (mg·g⁻¹).</caption><thead><tr><th>Species</th><th>Fraction</th><th>Fuc</th><th>Rham</th><th>Arab</th><th>Gal</th><th>Glu</th><th>Man</th><th>Xyl</th><th>Mannu</th><th>Gulur</th><th>Glucu</th></tr></thead><tbody><tr><td rowspan="4">Sargassum fusiforme</td><td>SA</td><td>26.38 ± 0.88</td><td>ND</td><td>ND</td><td>6.21 ± 0.16</td><td>0.29 ± 0.02</td><td>7.17 ± 0.25</td><td>4.50 ± 0.18</td><td>179.64 ± 2.37</td><td>167.57 ± 1.88</td><td>10.67 ± 0.29</td></tr><tr><td>SF</td><td>0.32 ± 0.01</td><td>ND</td><td>ND</td><td>0.10 ± 0.01</td><td>ND</td><td>ND</td><td>ND</td><td>54.29 ± 0.14</td><td>13.18 ± 0.51</td><td>ND</td></tr><tr><td>LF</td><td>1.84 ± 0.03</td><td>ND</td><td>ND</td><td>0.69 ± 0.01</td><td>ND</td><td>0.082 ± 0.04</td><td>0.35</td><td>8.47 ± 0.09</td><td>107.57 ± 1.88</td><td>ND</td></tr><tr><td>EF</td><td>7.68 ± 0.10</td><td>ND</td><td>ND</td><td>2.8 ± 0.04</td><td>ND</td><td>3.59 ± 0.04</td><td>0.87 ± 0.02</td><td>5.46 ± 0.02</td><td>11.38 ± 0.11</td><td>6.39 ± 0.06</td></tr><tr><td rowspan="4">S. fulvellum</td><td>SA</td><td>24.21 ± 0.60</td><td>0.12 ± 0.01</td><td>0.03 ± 0.00</td><td>15.07 ± 0.32</td><td>0.35 ± 0.00</td><td>8.59 ± 0.36</td><td>4.60 ± 0.05</td><td>136.79 ± 1.82</td><td>167.19 ± 1.10</td><td>20.27 ± 0.22</td></tr><tr><td>SF</td><td>0.17 ± 0.00</td><td>ND</td><td>ND</td><td>ND</td><td>0.11 ± 0.00</td><td>0.48 ± 0.00</td><td>0.05 ± 0.00</td><td>42.07 ± 2.82</td><td>7.53 ± 6.79</td><td>ND</td></tr><tr><td>LF</td><td>3.88 ± 0.05</td><td>0.03 ± 0.00</td><td>0.05 ± 0.00</td><td>2.40 ± 0.03</td><td>1.77 ± 0.02</td><td>2.09 ± 0.04</td><td>1.08 ± 0.03</td><td>26.18 ± 0.77</td><td>100.16 ± 0.54</td><td>4.11 ± 0.31</td></tr><tr><td>EF</td><td>8.86 ± 0.06</td><td>0.05 ± 0.00</td><td>0.01 ± 0.00</td><td>4.96 ± 0.04</td><td>0.10 ± 0.03</td><td>4.42 ± 0.04</td><td>1.03 ± 0.04</td><td>9.11 ± 0.18</td><td>3.56 ± 0.20</td><td>4.24 ± 0.05</td></tr><tr><td rowspan="4">S. horneri</td><td>SA</td><td>20.14 ± 0.20</td><td>ND</td><td>ND</td><td>13.45 ± 0.09</td><td>ND</td><td>5.70 ± 0.13</td><td>4.40 ± 0.24</td><td>127.37 ± 0.39</td><td>189.99 ± 0.36</td><td>16.93 ± 0.32</td></tr><tr><td>SF</td><td>0.24 ± 0.00</td><td>ND</td><td>ND</td><td>ND</td><td>0.19 ± 0.00</td><td>0.14 ± 0.00</td><td>0.09 ± 0.00</td><td>43.98 ± 5.70</td><td>7.89 ± 1.05</td><td>ND</td></tr><tr><td>LF</td><td>3.26 ± 0.02</td><td>0.03 ± 0.00</td><td>0.01 ± 0.00</td><td>2.51 ± 0.02</td><td>0.16 ± 0.02</td><td>2.11 ± 0.05</td><td>0.34 ± 0.02</td><td>22.21 ± 1.08</td><td>135.61 ± 3.18</td><td>3.44 ± 0.31</td></tr><tr><td>EF</td><td>9.26 ± 0.12</td><td>0.08 ± 0.00</td><td>ND</td><td>6.57 ± 0.04</td><td>0.16 ± 0.00</td><td>5.52 ± 0.08</td><td>1.03 ± 0.02</td><td>11.71 ± 0.39</td><td>4.77 ± 0.76</td><td>5.65 ± 0.32</td></tr></tbody></table>

Fuc: Fucose; Rham: Rhamnose; Arab: Arabinose; Gal: Galactose; Glu: Glucose; Man: Mannose; Xyl: Xylose; Mannu: Mannuronic acid; Gulur: Guluronic acid; Glucu: Glucuronic acid; SA: Sodium alginate; EF: Ethanol fraction; SF: Solid fraction; LF: Liquid fraction; ND: Not detected.

### 3.3. Elicitation by Alginate on β-Glucan Contents

From the results of oligosaccharide analysis of different fractions of *Sargassum* species, the solid fraction (SF) of *S. fusiforme* and liquid fraction (LF) of *S. horneri* were chosen as elicitors because of their high content of mannuronic acid and guluronic acid, respectively. The elicitation on *Sparassis latifolia* showed that SF treatment required a cultivation period of 47 days for mycelial growth, whereas LF treatment and the control group required a cultivation period of 43 days (Figure 3a). SF treatment provided higher mushroom production (197.30 ± 2.64 g) than the control group (192.04 ± 1.58 g), whereas LF treatment (191.05 ± 2.43 g) showed slightly lower results than the control group (Figure 3b). However, an additional spray of SF and LF elicitors resulted in lower yields of mushroom production, and this may be caused by excessive humidity as the control group also showed a decline with the second application.

These alginate-derived elicitors presented different effects on β-glucan content in the fruiting body and stalk of *S. latifolia*. SF elicitor was more effective for β-glucan enhancement in the fruiting body, whereas LF elicitor was more effective in the stalk (Figure 3c). For the second application, both SF (56.01 ± 3.45%) and LF (59.74 ± 4.49%) treatments showed obvious high increments of β-glucan contents in the stalk compared with the control (21.65%), which indicates that the additional spray of alginate oligosaccharide fractions apparently stimulated the β-glucan synthesis process in the stalk compared with that in the fruiting body (Figure 3d).
<figure><img src="image_61.png" /><figcaption>**Figure 3.** Elicitation of *Sparassis latifolia* by alginate oligosaccharide fractions on (a) cultivation period, (b) mushroom production, (c) β-glucan content of the fruiting body, and (d) β-glucan content of the stalk. SF: Solid fraction of *Sargassum fusiforme*; LF: Liquid fraction of *S. horneri*; Control: distilled water. (All data are presented as the mean ± SD. A t-test was used to compare the control and treated samples. *p* < 0.05, vs. the control in first application; #p < 0.05, vs. the control in second application).</figcaption></figure>

3.4. Alginate Effects on Total Polyphenol Content

After the second application of alginate oligosaccharide fractions, total polyphenol content (TPC)
in the fruit body and in the stalk of S. latifolia was determined, and gallic acid was used as standard.
The TPC of the samples ranged from 140.11 ± 6.72 to 420.87 ± 16.99 GAE mg/100g (Figure 4). There were
significant differences in TPC (p < 0.05) depending on the mushroom part and treated elicitor. The TPC
of stalk samples was higher than for the corresponding fruit body samples. LF treatment exhibited a
higher TPC value than SF treatment, both in the fruit body and stalk samples.

<figure><img src="image_62.png" /><figcaption>**Figure 4.** Total polyphenol contents of aqueous extracts of *Sparassis latifolia* after alginate elicitation. SF: Solid fraction of *Sargassum fusiforme*; LF: Liquid fraction of *S. horneri*; Control: distilled water. The data were analyzed using analysis of variance (ANOVA) followed by Duncan's multiple-range test (p < 0.05) using SPSS software (SPSS, Chicago, IL). Error bars indicate the mean ± SD and different letters describe greater differences within the same part of *S. latifolia* (fruit body and stalk).</figcaption></figure>
### 3.5. Alginate Effects on Antioxidant Activities

The antioxidant activities of aqueous extracts of *S. latifolia* after alginate elicitation were measured by ABTS radical scavenging and SOD-like activity. ABTS radical scavenging ability was calculated by IC₅₀ value. The results ranged from 926.67 ± 56.26 to 4002.19 ± 149.53 µg·mL⁻¹ (Figure 5a). SOD-like activity was measured by a colorimetric method and calculated by IC₅₀ value. IC₅₀ values of SOD-like activity ranged from 6.74 ± 0.29 to 18.70 ± 0.57 mg·mL⁻¹ (Figure 5b). The highest ABTS radical scavenging and SOD-like activity were recorded with LF elicitor treated stalk and the lowest were from the fruit body of the control group. Similar to the TPC results, both fruit body and stalk samples of LF treatment exhibited higher antioxidant activities than the corresponding samples treated by SF elicitor. The results of antioxidant activities were significantly (*p* < 0.05) stimulated with the alginate elicitation.

<figure><img src="image_63.png" /><figcaption>**Figure 5.** IC₅₀ values of ABTS radical scavenging (a) and SOD-like activity (b) of aqueous extracts of *Sparassis latifolia* after alginate elicitation. SF: Solid fraction of *Sargassum fusiforme*; LF: Liquid fraction of *S. horneri*; Control: distilled water. The data were analyzed using analysis of variance (ANOVA) followed by Duncan's multiple-range test (*p* < 0.05) using SPSS software (SPSS, Chicago, IL, USA). Error bars indicate the mean ± SD and different letters describe great differences within the same part of *S. latifolia* (fruit body and stalk).</figcaption></figure>

## 4. Discussion

*Sparassis* species are widely used medicinal mushrooms in traditional Chinese medicine. However, despite its commercial potential, cultivation of *S. latifolia* has been limited to a few high-tech commercial farms because of its slow mycelial propagation into solid medium [42]. In addition, its slow rate of growth is a major obstacle to the employment of *S. latifolia* as a producer of β-glucan. Biotic or abiotic elicitors have been employed to enhance the production of β-glucan in *S. latifolia*. Ryoo et al. exploited the effects of physical stimulation of UV irradiation and temperature shock on β-glucan contents in *S. latifolia* and found that β-glucan yields reached 41.36 ± 2.96% of flabella and 42.16 ± 2.90% of stipe after UV irradiation for 10 min [22]. Park et al. utilized chitinase, β-glucuronidase, and lysing enzyme complex as elicitors to enhance the β-glucan content of *S. latifolia* and produced an increase in β-glucan concentration of 31%, although the treatment caused a decrease in mushroom yield [23]. These results indicate that the elicitation technique needs to be evaluated for application in high-value mushroom cultivation.

This study aimed to increase the contents of β-glucan in *S. latifolia* through alginate elicitation, which was extracted from *Sargassum* species. Marine algae are an excellent biomass source used as a fertilizer for soil reformation because they are rich in carbohydrates and mineral content [43]. However, although *Sargassum* species contain a large amount of alginate and fucoidan, the high content of arsenic makes them unsuitable for consumption [44,45]. Polysaccharide-derived hydrocolloids in marine algae such as alginate, carrageenan, fucoidan, laminarin, ulvan, and glucuronan have been suggested as biotic elicitors to induce several different mechanisms including host defense mechanisms and growth enhancement in plant and fungi [46–49].

Alginate is the major structural polysaccharide of *Sargassum* species, consisting of MM, GG, and MG blocks arranged in various proportions in block units. The alginate M/G ratio and yield may vary in accordance with the algae harvest season and geographical location [41]. Leal et al. studied
block fractions of alginate in the brown algae *Lessonia flavicans* and *Desmarestia ligulata* [40]. The ethanol fraction obtained by partial acid hydrolysis of alginate was mainly composed of a heteropolymer block (MG). In addition, the solid fraction was rich in polymannuronic acid (MM) and the liquid fraction was rich in polyguluronic acid (GG) [28]. These were consistent with the results found in this study (see Table 2).

Alginate elicitors have been employed to modify cell metabolism in order to enhance the productivity of useful metabolites in plants and microorganisms [50]. The addition of chitosan, chitosan oligosaccharide, and alginate oligosaccharide to a culture of *Panax ginseng* C. A. Mey hairy roots caused growth inhibition and rises in total ginseng saponin accumulation with elicitor concentration [51]. Mannuronic acid of alginate may have a potent stimulatory effect on cytokine production, and it appeared to affect β-glucan content because of increased specific immunity owing to immunological effects [52]. In contrast, guluronic acid suppresses tissue damage caused by cytokines produced in response to inflammatory stimuli [53]. Our group investigated the supplement of sodium alginate to *S. latifolia* for the induced expression of γ-aminobutyric acid (GABA) both in the mycelia and fruiting bodies, which inhibited the dendrite outgrowth of excitatory neurons, but not that of inhibitory neurons [54]. Genome sequencing and genomics studies are underway to determine the underlying effect of alginate elicitors on β-glucan content of *S. latifolia* [55].

The effects of alginate elicitors on antioxidant activity and polyphenol synthesis in plants and microorganisms were also verified. Chitosan, pectin, and alginate promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in *Vitis vinifera*, which was positively correlated with increased accumulation of anthocyanin [25]. Ulvan, carrageenan, alginate, and laminarin were examined for any elicitation effect in twigs of olive trees to elicit phenolic metabolism and control against *Verticillium* wilt of olive caused by *V. dahlia*, and the results showed increased phenylalanine ammonia-lyase (PAL) activity and total polyphenol content combined with the decline of wilt symptoms [27]. Sodium alginate extracted from Moroccan brown algae *Fucus spiralis* and *Bifurcaria bifurcata* was evaluated for elicitation on phenolic metabolism including PAL activity and total polyphenol content in seedling roots of date palm, and the results showed that PAL activity and phenolic compound content were stimulated with 1 mg·mL⁻¹ sodium alginate [26].

The present study suggested that treatment with *Sargassum* alginate exhibited elicitation effects on the growth and β-glucan contents in *S. latifolia*. Elicitors derived from algae alginate can be widely used in the cultivation process of other culinary and medicinal mushrooms.

**5. Conclusions**

Based on our results, the products of partial acid hydrolysis of alginate oligosaccharides could act
as elicitors for stimulated growth of *S. latifolia* and production of useful metabolites such as β-glucan
and polyphenols.

**Author Contributions:** Conceptualization and methodology, Y.-W.K. and H.-J.S.; formal analysis and investigation, Y.-W.K. and M.-H.C.; software and validation, Y.W. and J.L.; resources, M.-H.C.; data curation, Y.-W.K.; writing—original draft preparation, Y.-W.K.; writing—review and editing, Y.W.; supervision, H.-J.S.; project administration, H.-J.S.; funding acquisition, H.-J.S. and J.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a National Research Foundation of Korea (NRF) grant funded by the Korean government, Grant No. NRF-2017R1A2B4006204; Shandong Provincial Key Research and Development Program (International Science and Technology Cooperation), Grant No. 2019GHZ033; and International Science and Technology Cooperation Program of Shandong Academy of Sciences, Grant No. 2019GHPY05.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
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Article

# Secondary Metabolites and Antioxidant Activity of the Solid-State Fermentation in Apple (Pirus malus L.) and Agave Mezcalero (Agave angustifolia H.) Bagasse

Diego Ibarra-Cantún <fnref n="1" /> ID, María Elena Ramos-Cassellis <fnref n="2" />,*, ID, Marco Antonio Marín-Castro <fnref n="3" /> ID and Rosalía del Carmen Castelán-Vega <fnref n="3" /> ID

Received: 30 June 2020; Accepted: 3 August 2020; Published: 18 August 2020

<figure><img src="image_67.png" /></figure>

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**Abstract:** Solid-state fermentation (SSF) is used in enzyme and antibiotic production, bioethanol and biodiesel as an alternative energy source, biosurfactants with environmental goals, and the production of organic acids and bioactive compounds. The present project determined the quantity of secondary metabolites and the antioxidant activity of the extracts obtained by the solid-state fermentation of apple and agave mezcalero bagasse over 28 days, inoculated with the *Pleurotus ostreatus* strain. The extraction was carried out with three solvents: acetone and water (80:20 v/v), 100% methanol and 100% water. The results showed a higher presence of phenolic compounds, flavonoids, total triterpenes and antioxidant activity in the apple bagasse from the SSF on day 21 in the extract of acetone and water (80:20 v/v), 100% methanol and aqueous; while the agave bagasse showed a significant presence of phenolic compounds and flavonoids only in the aqueous extract. In conclusion, the presence of secondary metabolites exhibiting antioxidant activities from the solid-state fermentation in the residues of the cider and mezcal industry is an alternative use for wasted raw material, plus, it reduces the pollution generated from the agroindustrial residues.

**Keywords:** agave mezcalero bagasse; apple bagasse; solid-state fermentation; secondary metabolites; *Pleurotus ostreatus*

## 1. Introduction

The residues generated by the agroindustry are, in most cases, not processed, causing environmental pollution and adverse effects on health [1]. In Mexico, this situation is mainly reflected in the production of the cider industry in Puebla and the production of mezcal in the state of Oaxaca. The former reported an annual production of 150 thousand bottles [2], where the processed fruit residues fluctuate between 15% and 30% of said production, depending on the state of maturation and the used technology [3]. The latter generated 122,696 tons of bagasse in 2012, which is usually dumped in rivers or streams, endangering the region's environment [4].

Most of these wastes degrade slowly due to their solid and organic nature, which corresponds to their lignocellulosic biomass, rich in cellulose, hemicellulose and lignin. In addition, they are not subjected to an adequate process of exploitation, giving as a result an utterly deficient disposal of the waste [5,6]. Therefore, the aim of the present investigation was to determine the content of the total phenolic compounds,

<footnote n="1">¹ Posgrado en Ciencias Ambientales, Benemérita Universidad Autónoma de Puebla, Col. Jardines de San Manuel, Edificio IC6, 72570 Puebla, Mexico; diego.ibarrac@alumno.buap.mx</footnote>

<footnote n="2">² Facultad de Ingeniería Química, Benemérita Universidad Autónoma de Puebla, Av. San Claudio y 18 Sur, 72570 Puebla, Mexico</footnote>

<footnote n="3">³ Departamento de Investigación en Ciencias Agrícolas, Benemérita Universidad Autónoma de Puebla, 14 Sur 6301 Edificio IC1, 72570 Puebla, Mexico; marco.marin@ correo.buap.mx (M.A.M.-C.); rosalia. castelan@ correo.buap.mx (R.d.C.C.-V.)</footnote>

<footnote>* Correspondence: elena.ramos@ correo.buap.mx; Tel.: +52-222-229-55-00 (ext. 7356)</footnote>
total flavonoids and total triterpenes, and the antioxidant activity, by 2,2-diphenyl-1-picrylhydrazyl (DPPH●) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS●+) test methods resulting from the solid-state fermentation of apple bagasse and agave mezcalero bagasse substrates inoculated with the *Pleurotus ostreatus* strain, in order to optimize the utilization of residues of the cider and mezcalero industry in the attaining of secondary metabolites.

*Pleurotus ostreatus* synthesizes bioactive compounds, such as: polysaccharides (alpha and beta glucan) and polyphenols [7,8]. The macromycetes, such as *Pleurotus* and *Agaricus*, contain tetracyclic triterpenoids, with the sterols being the most abundant ones. At the same time, in *Pleurotus mutilis* and *Clitopilus passeckerianus* (called *Pleurotus passeckerianus* in the past), pleuromutilin and diterpene were identified as having antibiotic action against microplasmatic infections in animals [9,10].

Singhania et al. [11] and Thomas et al. [12] describe solid-state fermentation (SSF) as a process that takes place in a solid matrix that is within an inert support or substrate, or near free water, with just enough to promote the growth and metabolic activity of microorganisms. The SSF reduces the production costs due to the lower energy consumption and production area (smaller reactors), proving that it is an economic, interesting and environmentally friendly process for the production of secondary metabolites from agroindustrial and forest residues [13]. The SSF has generated great interest in recent years due to its variety of uses, such as bioremediation, biofuels (biodiesel, bioethanol, biobutanol, and biohydrogen among others) and the production or extraction of bioactive compounds [14].

One of the main applications of the solid fermentation of agroindustrial wastes is the extraction of secondary metabolites, also called phytochemicals, which include carotenes, terpenes, sterols, tocopherols and polyphenols. These can be used in the treatment and prevention of various diseases [15], since they have antitumor, anti-inflammatory, antithrombotic, antimicrobial, immune system modulating, and hypoglycemic properties; also, they reduce the concentration of lipids in the blood and display antioxidant activities [16,17]. Waste from agribusiness, such as the peel of fruits and vegetables, has been employed recently as a potential source of production of bioactive compounds that are commonly known as phenolic compounds [18]. Therefore, the secondary metabolites obtained from the solid fermentation of residues can be used as natural bioactive triggers for the formulation of functional foods or serve as additives in food products to extend their shelf life [19].

## 2. Materials and Methods

### 2.1. Obtaining Plant Material

The apple bagasse was collected from Huejotzingo's cider industry, a town located in the state of Puebla, Mexico, and the agave mezcalero bagasse was collected from San Pablo Villa de Mitla's mezcal industry, a town located in the state of Oaxaca, Mexico.

These bagasses were transported in airtight containers at 20 °C in less than 4 h. They were subsequently bleached at 70 °C for 5 min and dried in a forced air dryer (HTP 72 Lumistell Celaya, Gto. México) for 8 h at 60 °C until the moisture content was less than 10%. Once dried, the bagasse was crushed in a mill to obtain a particle of 0.841 mm to be used during solid fermentation.

Physicochemical tests were performed on the crushed bagasse in order to discover its attributes and adjust its physicochemical properties to make them suitable for the growth of the *Pleurotus* fungus. The bagasse reconditioning was carried out according to Staments and Chilton [20] where it is mentioned that *Pleurotus ostreatus* culture must have the following conditions: a humidity percentage of 80-90%, temperature from 23 to 26 °C, and pH from 6.5 to 7.5. The temperature and relative humidity were controlled during the process. The physicochemical tests performed were: total soluble solids (°Brix) [21], pH [21], moisture percentage [21], water activity (Aw) [21], and titratable acidity percentage [21]; the meq value of malic acid were 0.067, the meq value of acetic acid were 0.060 and direct reducing sugars were also used [21].
## 2.2. Solid-State Fermentation

This process was accomplished with the contribution of the Mycology Laboratory of the Departamento de Investigación de Ciencias Agrícolas (Agricultural Sciences Research Department) of the Benemérita Universidad Autónoma de Puebla (Benemeritus Autonomous University of Puebla), which provided the *Pleurotus ostreatus* strains.

The solid-state fermentation was carried out in 9.7 × 2.6 cm glass tubes with a Bakelite cork, and each contained 10 g of each residue adjusted to a humidity percentage of 80% and a pH of 6.5, which was then sterilized in an autoclave ((CV300 AESA Tecámac, Edo. México, México)) at 120 °C and 15 psi for 45 min.

Then, a 1 cm diameter mycelium-agar circle of the *Pleurotus ostreatus* strain, corresponding to 3 mg of dry weight biomass, was incorporated into each tube and incubated in an oven (E51 Riossa Monterrey, N.L., México) at 25 °C. Samples were taken at 0, 7, 14, 21 and 28 days.

## 2.3. Solid Waste Extraction

After the fermentation days, 12 mL of each solvent was added to each glass tube for extraction. The used solvents were acetone and water (80:20 *v/v*), 100% methanol and distilled water. The samples were placed in a shaker (KJ-201BD Orbital Shaker Westtune Hangzhou, Zhejiang, China) at 80 rpm for 1 h, filtered and placed in a centrifuge (Z200A Hermle Wehingen, Germany) at 47 g, and the ensuing supernatant was stored in Eppendorf tubes at -20 °C for further analysis.

## 2.4. Quantification of Total Phenolic Compounds (TPC)

The analysis was performed using the method described by Singleton and Rossi [22], with some modifications. A total of 250 µL of 50% Folin-Ciocalteu reagent was added to 50 µL of the extract; then it was stirred and allowed to stand in the dark for 8 min. Subsequently, 1.25 mL of 5% $CaCO_3$ (*w/v*) was added and allowed to stand again for 30 min in the dark at room temperature. An absorbance at 725 nm was read afterwards on a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). A calibration curve for gallic acid (Sigma Aldrich, CAS 149-91-7, Steinheim, Germany) was used in the range of 0 to 0.3 g/L. The results were expressed in mg gallic acid equivalent per g substrate on dry weight (mg GAE/g substrate dw). These analyses were performed in triplicate.

## 2.5. Total Flavonoid Quantification (TF)

The methodology proposed by Chang et al. [23] was used, with some modifications. A total of 500 µL of the extract, 1.5 mL of 80% ethanol (*v/v*), 100 µL of 10% hexahydrate aluminum chloride solution, 100 µL of 1 M potassium acetate, and 2.8 mL of distilled water were placed, stirred and incubated for 30 min at room temperature.

Subsequently, an absorbance at 415 nm was read on a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). A quercetin calibration curve (Sigma Aldrich, CAS 117-39-5, Germany) was used at a concentration of 0 to 0.1 g/L. The results were expressed in mg quercetin equivalents per g substrate on dry weight (mg QE/g substrate dw). These analyses were performed in triplicate.

## 2.6. Total Triterpenes Quantification (TT)

The vanillin-acetic acid method was used, with some modifications [24]. A total of 120 µL of each extract, 100 µL of vanillin (Sigma Aldrich, CAS 121-33-5, Germany) 5% (*w/v*), and 400 µL of perchloric acid in 0.1 N glacial acetic acid were placed together in a test tube at 60 °C for 15 min.

Then, the mixture was cooled down to room temperature and 2.5 mL of glacial acetic acid were added. The absorbance was measured at 550 nm in a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). The results were expressed as mg ursolic acid per g substrate on dry weight (mg UA/g substrate dw). A calibration curve for uric acid (Sigma Aldrich, CAS 77-52-1, Germany) was used with a concentration from 0 to 120 g/L. These analyses were performed in triplicate.
## 2.7. Antioxidant Activity by 1,1-Diphenyl-2-Picril Hydracil (DPPH●)

The DPPH● (2,2-Diphenyl-1-picrilhydrazyl) radical in methanol solution was developed by Brand-Williams et al. [25], and was used with some alterations. A total of 990 µL of DPPH (Sigma Aldrich, CAS 1898-66-4, Germany) 0.188 mM was added to 10 µL of the extract. The mixture was then homogenized and kept in the dark for 30 min at room temperature.

The absorbance at 517 nm was measured in both the control (methanol (A0)) and the test samples (A1) on a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). In order to calculate the percentage of inhibition, Equation (1) was used. These analyses were performed in triplicate.

Subsequently, the results were expressed as the inhibitory concentration (IC₅₀) and the percentage of inhibition of four different concentrations was calculated to obtain a linear regression curve. These analyses were performed in triplicate.

$$ \text{Percent inhibition} = \left[ \frac{A0 - A1}{A0} \right] * 100 \qquad (1) $$

A0 = Control absorbance

A1 = Sample absorbance.

## 2.8. Antioxidant Activity by 2,2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid Radical Cation (ABTS●+)

The method proposed by Re et al. [26] was used. An ABTS●+ radical cation was formed from 0.0033 g of potassium persulfate and 0.0194 g of ABTS reagent (2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid (Sigma Aldrich, CAS 30931-67-0, St. Louise, MO, USA) mixed in 5 mL of distilled water. The mixture was stirred and kept in the dark for 16 h at room temperature. After this, a mixture was made with absolute ethanol and the ABTS●+ radical cation until an absorbance of 0.70 ± 0.02 at 754 nm was obtained.

A total of 3920 µL of the ABTS radical solution were then added and the initial absorbance (Ai) was recorded; 80 µL of extract was afterwards added and mixed. After 7 min, the final absorbance (Af) was recorded with the use of a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). The inhibition percentage was calculated according to Equation (2) to obtain the medium inhibitory concentration (IC₅₀) by a curve of lineal regression using the inhibitory percentage of the four different concentrations. These analyses were performed in triplicate.

$$ \text{Percent inhibition (\%)} = \left[ \frac{\text{Ai} - \text{Af}}{\text{Ai}} \right] * 100 \qquad (2) $$

## 2.9. Statistic Analysis

The data analysis was performed through a completely randomized design. The experiment consisted of the inoculation of *Pleurotus ostreatus* in two bagasses (two treatments with five replicas) at 25 °C for 28 days. Samples of the substrates were taken at 0, 7, 14, 21 and 28 days of fermentation (5 periods) to obtain the extracts with three organic solvents: acetone and water (80:20 v/v), 100% methanol, and water at 100% (3 extracts); the secondary metabolite content and antioxidant activity were determined as well. Samples were analyzed in quadruplicate (n = 4).

The results were expressed as a mean and standard deviation, and were examined through the Statistical Analysis System (SAS) version 9.0 statistical package [27]. Additionally, an analysis of variance (ANOVA) means test (Tukey α = 0.05) and Pearson correlation were performed.
# 3. Results

## 3.1. Physicochemical Characterization

Table 1 shows the physicochemical characterization of the residues before fermentation. The values in percent humidity and Aw are similar since both bagasses were subjected to the same drying process at 60 °C for 12 h and then they were crushed.

<table><caption>**Table 1.** Physicochemical characteristics of apple bagasse and agave mezcalero bagasse.</caption><thead><tr><th>Agroindustrial Residue</th><th>°Brix</th><th>pH</th><th>Moisture Percentage (%)</th><th>Aw</th><th>Titratable Acid (%)</th><th>Direct Reducing Sugars <sup>1</sup> (%)</th><th>Direct Reducing Sugars <sup>2</sup> (%)</th></tr></thead><tbody><tr><td>Apple bagasse</td><td>8.90 ± 0.28 <sup>a</sup></td><td>4.04 ± 0.15 <sup>b</sup></td><td>8.67 ± 0.20 <sup>a</sup></td><td>0.98 ± 0.05 <sup>a</sup></td><td>0.97 ± 0.23 <sup>a</sup></td><td>86.20 ± 7.11 <sup>a</sup></td><td>9.98 ± 0.01 <sup>a</sup></td></tr><tr><td>Agave mezcalero bagasse</td><td>0.36 ± 0.20 <sup>b</sup></td><td>5.00 ± 0.10 <sup>a</sup></td><td>8.68 ± 0.20 <sup>a</sup></td><td>0.97 ± 0.01 <sup>a</sup></td><td>0.87 ± 0.21 <sup>a</sup></td><td>6.79 ± 0.37 <sup>b</sup></td><td>6.79 ± 0.37 <sup>b</sup></td></tr></tbody></table>

Values are the mean ± standard deviation. Means with the same letters within each column do not differ statistically by Tukey's test (p ≤ 0.05). ¹ Direct reducing sugars before washing apple bagasse; ² direct reducing sugars after washing apple bagasse.

## 3.2. Secondary Metabolites

### 3.2.1. Quantification of Total Phenolic Compounds (TPC)

In Figure 1, it is shown the total phenolic compounds resulting from the solid-state fermentation of agroindustrial wastes. The TPC had its highest concentration at the beginning of the experiment (day 0) in both residues, and after fermentation it decreased, increasing again after day 21.

<figure><img src="image_68.png" /><figcaption>**Figure 1.** Total phenolic compounds (mg gallic acid equivalent per g substrate on dry weight (mg GAE/g substrate dw)) of the solid-state fermentation of agroindustrial residues: (a) Apple bagasse; (b) Agave mezcalero bagasse. Acetone and water (80:20 v/v (●-●)); methanol extract (○-○); aqueous extract (▼). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.</figcaption></figure>

Apple bagasse extract (Figure 1a) increased its concentration 21 days after fermentation, with intervals of 0.385 and 0.587 mg gallic acid equivalent/g substrate dw. The highest concentration was present in the aqueous extract with a value of 0.587 ± 0.013 mg gallic acid equivalent/g substrate dw. The TPC content of agave mezcalero bagasse from the acetone and water (80:20 v/v) and methanol extracts tended to decrease during fermentation, while the aqueous extract increased its concentration after day 21, until it reached 0.976 ± 0.126 mg gallic acid equivalent/g substrate dw at 28 days.

### 3.2.2. Total Flavonoid Quantification (TF)

In Figure 2, the total flavonoids in apple bagasse and mezcalero agave are shown. First, the apple increased its TF content from day 21, giving final values (on day 28) of 0.015 ± 0.018, 0.013 ± 0.02, and 0.023 ± 0.012 mg QE/g substrate dw in extracts of acetone and water (80:20 v/v), methanol and the
aqueous extract, respectively; the latter was the extract with the highest amount of flavonoids. Then, the agave mezcalero bagasse registered a similar tendency to the content of TPC. The total flavonoid concentrations in extracts of acetone and water (80:20 *v*/*v*) and methanol decreased during fermentation, while in the aqueous extract there was an increase in the TF content at the end of the fermentation time, with a final concentration of 0.012 mg QE/g substrate dw.

<figure><img src="image_69.png" /><figcaption>**Figure 2.** Total flavonoids (mg QE/g substrate dw) of the solid-state fermentation of agroindustrial residues: (a) apple bagasse; (b) agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* (●-·)); methanol extract (○-·); aqueous extract (▼-·). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.</figcaption></figure>

### 3.2.3. Total Triterpenes Quantification (TT)

In Figure 3, the total triterpenes content is shown. Apple bagasse presented TT contents which increased from day 21 in extracts of acetone and water (80:20 *v*/*v*) and methanol, with final values (on day 28) of 8.043 ± 0.696, and 9.411 ± 2.512 mg ursolic acid/g substrate dw, respectively. The aqueous extract showed steady growth from the beginning of the solid-state fermentation of triterpenes, and reached a concentration of 26.440 ± 0.949 mg ursolic acid/g substrate dw at 28 days. Despite the fact that the TT in agave mezcalero bagasse had the highest value at the beginning of the fermentation, its concentration decreased in all extracts during this process.

<figure><img src="image_70.png" /><figcaption>**Figure 3.** Total triterpenes (mg UA/g substrate dw) of the solid-state fermentation of agroindustrial residues: (a) apple bagasse; (b) agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* (●-·)); methanol extract (○-·); aqueous extract (▼-·). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.</figcaption></figure>
### 3.3. Antioxidant Activities

The antioxidant activity of the extracts obtained by the solid-state fermentation of *Pleurotus* from apple and agave mezcalero bagasses was expressed as the 50% inhibitory capacity of the radical in question (IC₅₀), which refers to the concentration of antioxidant required to reduce the initial amount of the DPPH and/or ABTS radical by 50%. This is the parameter used to measure the antioxidant properties of a substance [28], that is, the lower the IC₅₀ value, the greater its antioxidant activity.

#### 3.3.1. Antioxidant Activity by 1,1-Diphenyl-2-Picril Hydracil (DPPH●)

In apple bagasse, the IC₅₀ of the DPPH assay ranged from 14.67 to 385.01 g/L residue. On the other hand, the agave mezcalero bagasse presented IC₅₀ values from 61.80 to 377.75 g/L residue (Figure 4a,b). A tendency for IC₅₀ to increase was observed from day 0 to day 21, however, a decrease was perceived from day 28 in different extracts.

<figure><img src="image_71.png" /><figcaption>**Figure 4.** 50% inhibitory concentration in 2,2-diphenyl-1-picrylhydrazyl (DPPH (IC₅₀/DPPH)) of the solid-state fermentation of agroindustrial residues: (a) apple bagasse; (b) agave mezcalero bagasse. Acetone and water (80:20 v/v (●-)); methanol extract (○-); aqueous extract (▼). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.</figcaption></figure>

#### 3.3.2. Antioxidant Activity by 2,2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid Radical Cation (ABTS●+)

In the ABTS test, the apple bagasse presented a range of residue values from 60.20 to 423.09 g/L; meanwhile, the agave mezcalero bagasse ranged from 74.65 to 390.86 g/L (Figure 5a,b). The obtained results in the apple bagasse showed that the initial values (on day 0) were higher than the agave mezcalero bagasse with intervals of 61.80 to 91.42 g/L of residue.
<figure><img src="image_72.png" /><figcaption>**Figure 5.** A 50% inhibitory concentration in 2, 2'-azino-bis (3-ethylbenzothiazoline)-6 ammonium sulfonate (ABTS (IC<sub>50</sub>/ABTS)) of the solid-state fermentation of agroindustrial residues: (a) apple bagasse; (b) agave mezcalero bagasse. Acetone and water (80:20 *v*/*v* (−●−)); methanol extract (−○−); aqueous extract (−▼−). Time SSF = time solid-state fermentation. Values are the mean ± standard deviation.</figcaption></figure>

The acetone and water (80:20 *v*/*v*) and aqueous extracts increased in IC<sub>50</sub> during the days of fermentation. It should be noted that the methanol extract displayed a decrease in IC<sub>50</sub> at day 28, which means that the secondary metabolites produced at the same time of solid fermentation denoted the antioxidant property of this residue.

**4. Discussion**

In both residues, the pH showed values lower than 5.0, lower than the value recommended for the adequate growth of *Pleurotus* (5.5–6.5) [29]. The pH was adjusted for proper fungus growth and fermentation development. The highest amounts of total soluble solids (°Brix), acidity percentage and reducing sugars were found in the apple bagasse because the residue had a significant amount of polysaccharides and organic acids [30]. This limited the growth of *Pleurotus*, thus, bagasse was subjected to a wash with tridestilated water. After washing the apple bagasse released sugars, it was observed that its content of reducing sugars decreased (Table 1), which was resulted in an adequate growth of the *Pleurotus* strain in the residue.

Apple bagasse presented a significant difference in the aqueous extract of the total triterpenes test, having the highest value; the other extracts showed no significant difference in the other tests performed. In the agave mezcalero bagasse, the significant difference between extracts was manifested in the tests of total flavonoids and total triterpenes; the aqueous extract had the highest value, while the others did not show significant differences in the rest of the performed tests.

The highest phenolic content was at the beginning, since the phenolic compounds are shown in the subtracts (bagasses). In addition, it contains lignin, which is a phenolic polymer made up of three principal monomerics: paracoumaryl alcohol, synapilic alcohol and conyferyl alcohol [31,32]. This acts as a carbon source for the *Pleurotus* fungi for its growth and development (trophophase) after the mycelial growth. However, the nutrient exhaustion produces the biosynthesis of secondary metabolites in the idiophase. In this period (T21) there is a metabolite increase according to Ferrer-Romero et al. [33] and Vamanu [34] which supports the presence of *Pleurotus* and its survival in the residue [35]. Therefore, to observe a higher antioxidant activity, idiophase time must be increased in order.

The TPC values generated from *Pleurotous* in other residues, such as the mixture of pineapple and rice straw residues [36], reported values of 0.179–0.650 mg GAE/g substrate dw. Other authors reported greater values than the ones obtained in this research: Hamdipour et al. [37] showed values of 5.53 to 11.6 mg GAE/g dry weight in the fermentation of apple residues with *Rhizopus oligosporus*, and Ajila et al. [38] obtained values of 4.6 to 16.12 mg GAE/g dry weight during the solid-state
fermentation of apple residues with *Phanerocheate chrysosporium*. Meanwhile, Ferrer-Romero et al. [33] obtained 0.1 g of total phenols/g of glucose by submerged fermentation from the mycelial biomass of *Pleurotus ostreatus*.

The *Pleurotus ostreatus* strain of this study led to the degradation of lignin to obtain the necessary nutrients for its development, given that growth was observed in both lignocellulosic substrates: apple bagasse and mezcalero agave bagasse [39]. In addition, the genus *Pleurotus* has the capacity to produce laccase enzymes, which have a fundamental role both in the degradation of lignin and in the biosynthesis of phenolic compounds in white-rot fungi [40].

Boonsong et al. [41] conducted an investigation with the purpose of evidencing the high nutritional value of edible fungi. This investigation reported intervals from 2.29 to 2.61 mg QE/g in aqueous extracts of *Pleurotus* strains, which are higher values compared to the ones obtained in this project.

Furthermore, Braga et al. [42] reported significant concentrations of total flavonoids (1.76 mg QE/g) in grape marc residue, and 1.70 mg quercetin equivalent/g in mango residue; Dulf et al. [43] found slightly lower values of 0.29 and 0.36 mg QE/g, resulting from the solid-state fermentation of apricot residue with *Aspergillus niger* and *Rhizopus oligosporus* for 14 days; while Lessa et al. [44] found concentrations of TF with values of 31.5 mg QE/100 g in fermented cocoa flour by means of *Penicillium roqueforti*. Therefore, the low concentration of this type of metabolites maybe be determined, once again, by the used strain, that degrades lignin during solid-state fermentation.

Yang et al. [45] found a higher concentration of triterpenes, 47.10 mg/g, 30 days after the solid-state fermentation of citrus residues with the fungus *Antrodia cinnamomea*. In this investigation, the total triterpenes values have great importance, especially those found during solid fermentation in the apple bagasse because this type of secondary metabolite has antibacterial properties against plant pathogens; in addition, it has demonstrated effectiveness against oxidative damage through the elimination of free radicals and the modulation of enzymatic activity [46]. The authors mention solvents with low polarity for the extraction of triterpenes, such as ethyl acetate [47,48]. Nevertheless, aqueous extracts have been used to identify secondary metabolites, like triterpenes, in other macromycete fungi, *Phellinus* and *Ganoderma* [49]. As a consequence, it was decided to use this type of solvent, due to polyphenols, sterols, triterpenes and anthracenes being moderately abundant [50].

The production of secondary metabolites can be considered similar to the tendency of antioxidant activity, due to the fact that since day 21 there was an increase in antioxidant activity in the apple bagasse, which can be attributed to a higher concentration of polyphenols present in acetone and water (80:20 *v/v*) and aqueous extracts. As mentioned by Lu and Foo [51], the antioxidant activity is caused by polyphenols in apple residues. Meanwhile, Ajila et al. [38] report $IC_{50}$ values of 12.24 ± 5.20 to 14.27 ± 2.50 µg in acetone and water (80:20 *v/v*) extracts and 40.16 ± 2.40 to 50.18 ± 2.00 µg in aqueous extracts in fermented apple residue.

While there is little information on antioxidant activity in agave mezcalero through solid-state fermentation, some authors have found this property in certain agave species. For example, Carmona et al. [52] mention the antioxidant activities of *Agave lechuguilla*, and Araldi et al. [53] mention it regarding *Agave sisalana*. Both species are used as a source of raw material in the production of biofuels from solid fermentation.

Although the values obtained can be considered low, the obtained methanol extract from the solid-state fermentation of the agave mezcalero can be used as an alternative for the production of compounds with antioxidant properties.

Overall, the $IC_{50}$ values of apple bagasse are lower; they displayed greater inhibitory activity against the DPPH radicals and ABTS radicals in comparison to those of the agave mezcalero bagasse. On the other hand, Ignat et al. [54] express that the different results in the metabolite content may be due to the extraction conditions, type of solvent, and its susceptibility to degradation. This way, it is possible to indicate the variability of secondary metabolites and antioxidant activity in agroindustrial waste.
**5. Conclusions**

The growth of *Pleurotus ostreatus* was observed in both the apple bagasse and agave mezcalero bagasse residues due to their ability to secrete extracellular, non-specific ligninolytic enzymes during secondary metabolism. Spectrophotometric studies have demonstrated that, because of solid-state fermentation, there was presence of secondary metabolites—phenolic compounds, flavonoids and triterpenes.

The highest concentration of compounds of biological interest occurred in apple bagasse after 21 days, with the aqueous extract presenting the highest extraction of the different phytochemical compounds. This behavior was reflected in the antioxidant activity of the different extracts during the same fermentation time. Minor amounts of phenolic and flavonoid compounds were found in the aqueous extract of the agave bagasse from day 21 of fermentation.

The biosynthesis of secondary metabolites displaying antioxidant activities in the solid-state fermentation of *Pleurotus ostreatus* in the apple residues of the cider industry and in the agave residues of the mezcalera industry can contribute to the use of these wastes, as they can be used in functional food formulation or, due to their medical properties, in the pharmaceutical industry.

**Author Contributions:** Conceptualization, D.I.-C., M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; methodology, D.I.-C., M.A.M.-C., and M.E.R.-C.; validation, D.I.-C., M.A.M.-C., and M.E.R.-C.; formal analysis, D.I.-C., M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; investigation, D.I.-C.; resources, M.A.M.-C. and M.E.R.-C.; data curation, D.I.-C., M.A.M.-C. and M.E.R.-C.; writing—original draft preparation, D.I.-C.; writing—review and editing, D.I.-C., M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; visualization, D.I.-C., M.A.M.-C. and M.E.R.-C.; supervision, M.A.M.-C. and M.E.R.-C.; project administration, M.A.M.-C., R.d.C.C.-V. and M.E.R.-C.; funding acquisition, M.A.M.-C., R.d.C.C.-V. and M.E.R.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Programa de Desarrollo Profesional Docente para el Tipo Superior (PRODEP) Folio PTC-BUAP.PTC-560.

**Acknowledgments:** The authors thank the Posgrado de Ciencias Ambientales—BUAP and CONACYT for the post-doctoral of Diego Ibarra-Cantún.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Article

# Metal and Phosphate Ions Show Remarkable Influence on the Biomass Production and Lipid Accumulation in Oleaginous *Mucor circinelloides*

Simona Dzurendova <fnref n="1" />*, Boris Zimmermann <fnref n="1" />, Valeria Tafintseva <fnref n="1" />, Achim Kohler <fnref n="1" />, Svein Jarle Horn <fnref n="2" />* and Volha Shapaval <fnref n="1" />

Received: 6 October 2020; Accepted: 27 October 2020; Published: 30 October 2020

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**Abstract:** The biomass of *Mucor circinelloides*, a dimorphic oleaginous filamentous fungus, has a significant nutritional value and can be used for single cell oil production. Metal ions are micronutrients supporting fungal growth and metabolic activity of cellular processes. We investigated the effect of 140 different substrates, with varying amounts of metal and phosphate ions concentration, on the growth, cell chemistry, lipid accumulation, and lipid profile of *M. circinelloides*. A high-throughput set-up consisting of a Duetz microcultivation system coupled to Fourier transform infrared spectroscopy was utilized. Lipids were extracted by a modified Lewis method and analyzed using gas chromatography. It was observed that Mg and Zn ions were essential for the growth and metabolic activity of *M. circinelloides*. An increase in Fe ion concentration inhibited fungal growth, while higher concentrations of Cu, Co, and Zn ions enhanced the growth and lipid accumulation. Lack of Ca and Cu ions, as well as higher amounts of Zn and Mn ions, enhanced lipid accumulation in *M. circinelloides*. Generally, the fatty acid profile of *M. circinelloides* lipids was quite consistent, irrespective of media composition. Increasing the amount of Ca ions enhanced polyphosphates accumulation, while lack of it showed fall in polyphosphate.

**Keywords:** *Mucor circinelloides*; high-throughput screening; metal ions; phosphorus; lipids; biofuel; FTIR spectroscopy; bioremediation; co-production

## 1. Introduction

*Mucor circinelloides* is a dimorphic oleaginous filamentous fungus with a fully sequenced genome [1]. It has a versatile metabolism, allowing utilization of a variety of feedstocks, making this fungus widely applicable in a range of biotechnological processes [2]. *M. circinelloides* is well known as a robust cell factory, where extracellular products include enzymes (cellulases, lipases, proteases, phytases, and amylases) [2,3] and ethanol [4]. Further, *M. circinelloides* can synthesize and accumulate a number of valuable intracellular components, such as lipids, polyphosphates, carotenoids, and chitin/chitosan [5-9]. The biomass of *M. circinelloides* has a significant nutritional value and can be used as a feed ingredient [10]. Chitosan exhibits great chelating properties, mainly due to the low level of acetylation and the abundance of hydroxyl groups [9,11,12]. Due to the presence of chitin and chitosan in the cell wall of *M. circinelloides*, the biomass of this fungus can be used as bioabsorbent for heavy metals and applied as a bioremediation agent, for example in the wastewater treatment [13].

<footnote n="1">¹ Faculty of Science and Technology, Norwegian University of Life Sciences, Drøbakveien 31, 1430 As, Norway; boris.zimmermann@nmbu.no (B.Z.); valeria.tafintseva@nmbu.no (V.T.); achim.kohler@nmbu.no (A.K.); volha.shapaval@nmbu.no (V.S.)</footnote>

<footnote n="2">² Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Christian Magnus Falsens vei 1, 1433 As, Norway; svein.horn@nmbu.no</footnote>

<footnote>* Correspondence: simona.dzurendova@gmail.com or simona.dzurendova@nmbu.no</footnote>
*M. circinelloides* has been extensively studied for the production of lipids for different applications [14–16]. The lipids are mainly in the form of triacylglycerides (TAGs) and contain palmitic, stearic, oleic, linoleic acid, and y-linoleic acids that make it particularly suitable for biodiesel production [17]. Therefore, the biomass of *M. circinelloides* could be considered as an important alternative feedstock for the biodiesel industry [18]. However, the cost of the *M. circinelloides* biomass production for biodiesel as a sole product is still too high compared to competitive bioprocesses. Thus, there is a need to further optimize lipid and biomass yield for *M. circinelloides*, and develop a coproduction concept, where other valuable components could be produced along with lipids in a single fermentation process [19,20].

Lipid accumulation, as well as biomass formation, can be affected by many different cultivation parameters, such as the nutrient composition of the growth medium, temperature, pH, aeration, or parameter shift during the fermentation (temperature/pH) [2,21,22]. Optimization of the nutrient composition of the growth medium is one of the most important aspects in improving fungal lipid production. In order to increase lipid and biomass yields, it is crucial to understand the role and effect of all media components. Many studies have assessed the effect of different carbon (C) and nitrogen (N) sources on the fungal lipid production in *Mucoromycota* fungi [23–29], where nitrogen limitation in carbon-rich media is the most frequently used strategy for inducing lipid accumulation and achieving high lipid yields. Macro- and micronutrients, such as phosphorus (P), potassium (K), sulfur (S), calcium (Ca), sodium (Na), iron (Fe), and magnesium (Mg), have previously been reported as essential for optimal fungal growth and metabolic activity [30].

Metal ions play an important role in fungal metabolism as they provide necessary redox and catalytic activities for the cellular processes [31]. The role of metal ions in yeast metabolism has been widely studied [32–35]. Bivalent metal ions are often reported as cofactors for different enzymes [36,37]. Metal ions are usually examined in the context of bioremediation capabilities of *M. cicrcinelloides* [9,38], while the role of metal ions in lipid accumulation of *Mucoromycota* fungi have been examined only to a limited extent. Different metal ions have shown strain-specific influence on lipid accumulation in *Mucoromycota* fungi, where either fatty acid profiles or total lipid content is affected. For example, manganese (Mn) has shown positive effects on the lipogenesis in *Mucor plumbeus* and *Mortierella* sp. [39,40]. Iron had an inhibiting effect on the arachidonic acid production in *Mortierella* sp. [39], while together with magnesium and zinc (Zn), it was enhancing lipid accumulation in *Cunninghamella* sp. Furthermore, zinc increased the gamma-linoleic acid production in *Cunninghamella* sp. [41], while iron, zinc, and copper (Cu) were reported as enhancers of arachidonic acid production in *Mortierella alpina* [42]. To the authors' knowledge, the effect of calcium and cobalt (Co) on the lipid accumulation in the oleaginous *Mucoromycota* fungi has not been investigated before. Moreover, there have been no studies reporting the effect of metal ions on the lipogenesis in *M. circinelloides*.

In our previous studies, we have assessed the chemical composition of nine different oleaginous filamentous fungi (including *M. circinelloides*) and revealed nutrient-induced coproduction of lipids, chitin/chitosan, and polyphosphates [43,44]. We reported that *M. circinelloides* has an ability to coproduce lipids, polyphosphate, and chitin/chitosan at different phosphorus concentrations and showed a versatile metabolism with a high adaptability level to different stress conditions. Thus, this fungus can be utilized in phosphorus recovery processes, while the co-production concept greatly contributes to the economic feasibility of such processes.

The aim of this study was to assess the effect of 140 different substrates, with varying amounts of metal and phosphate ions, on the growth, cell chemistry, and lipid production in *M. circinelloides*. Different concentrations of phosphorus source were used in order to study the effect of metal ions on the co-production of lipids, polyphosphates and cell wall polysaccharides, such as chitin/chitosan, triggered by phosphorus availability. Analogous to our previous studies, the study was performed in a high-throughput set-up using a Duetz microtiter plate system (Duetz-MTPs) combined with Fourier transform infrared (FTIR) spectroscopy [43–46]. FTIR spectroscopy was applied to obtain a biochemical
fingerprint of the microbial cells [43,47–52], while gas chromatography was used to analyze the lipid yield and fatty acid profiles of the extracted lipids.

## 2. Materials and Methods

### 2.1. Growth Media and Cultivation Conditions

Fungal strain *M. circinelloides* VI04473, provided by the Veterinary Institute, Oslo, Norway was selected based on the previous study of 100 oleaginous fungal strains, as it showed the highest lipid and biomass yield of all tested *Mucor* strains [50]. Moreover, this strain has also shown coproduction potential for lipids, chitin/chitosan, and polyphosphates [43]. *M. circinelloides* was cultivated on malt extract agar (MEA) for 7 days at 25 °C in order to obtain fresh spores for the inoculation into nitrogen-limited broth media with various metal and phosphorus ion concentrations. Spores were collected from agar plates using 10 ml of saline solution and a bacteriological loop. The composition of the reference medium, used and modified in our previous studies [27,43–45], was the following: 80 g/L of glucose, 1.5 g/L of (NH₄)₂SO₄, 7 g/L of KH₂PO₄, 2 g/L of Na₂HPO₄, 1.5 g/L of MgSO₄·7H₂O, 0.1 g/L of CaCl₂·2H₂O, 0.008 g/L of FeCl₃·6H₂O, 0.001 g/L of ZnSO₄·7H₂O, 0.0001 g/L of CoSO₄·7H₂O, 0.0001 g/L of CuSO₄·5H₂O, 0.0001 g/L of MnSO₄·5H₂O, where the listed concentrations of the metal ions Ca, Cu, Co, Fe, Mg, Mn, and Zn were assigned as reference concentration and marked as “R” (Table 1). The higher—1000; 100; 10 × R and lower—0.1; 0.01 and 0 × R concentrations of the metal ions were assessed in the study as described in Table 1. The reference medium was modified by using four relative levels of metal and phosphate ions (Table 1). KH₂PO₄ and Na₂HPO₄ were used as phosphates substrate, and their total concentration is hereafter referred as “phosphates concentration” (Pi). Phosphate concentrations KH₂PO₄ 7 g/L, Na₂HPO₄ 2 g/L have been assigned as Pi1. In addition to Pi1 concentration, the higher—4 and 2 × Pi1 and lower—0.5 and 0.25 × Pi1 concentrations of phosphates were assessed in the study as described in Table 1. Broth media, with the lower than Pi1 amount of Pi—Pi0.5 and Pi0.25, contained KCl and NaCl in a corresponding concentration in order to have equal amounts of K⁺ and Na⁺ ions as in the Pi1 condition. Different media were prepared by modifying one metal ion concentration at a time for every level of Pi ions. The only exception was for the condition 0Mg10Ca, which was tested in order to examine a possibility of substitution of Mg by Ca. Thus, in total, 140 different media were prepared. The concrete concentrations of all media components can be found in the Supplementary Materials (Table S1).

<table><caption>**Table 1.** Overview over the relative levels of concentration of metal ions and inorganic phosphate in the media. The exact concentrations can be found in the Supplementary Materials (Table S1).</caption><thead><tr><th>Ca</th><th>Mg</th><th>Cu</th><th>Co</th><th>Fe</th><th>Mn</th><th>Zn</th><th>Pi</th></tr></thead><tbody><tr><td>0</td><td>0Mg 10Ca</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0.25</td></tr><tr><td>0.01</td><td>0.01</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>0.5</td></tr><tr><td>0.1</td><td>0.1</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>Pi1</td></tr><tr><td>R</td><td>R</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>2</td></tr><tr><td>10</td><td></td><td>1000</td><td>1000</td><td>1000</td><td>1000</td><td>1000</td><td>4</td></tr></tbody></table>

Considering the high-throughput set-up of the study and consequently high number of samples, the reproducibility of the *M. circinelloides* growth was controlled by four biological replicates for the reference medium R with Pi1 concentration, two biological replicates for the reference metal ion concentrations, and the following phosphate ion concentrations: Pi4, Pi2, Pi0.5, and Pi0.25, and two biological replicates for the medium with 0Ca under all tested phosphate concentrations. The reproducibility of lipid accumulation was controlled under the reference metal concentrations by four biological replicates for the reference medium R-Pi1, two biological replicates for Pi4, Pi2, Pi0.5, and Pi0.25 media, and two biological replicates for the media with 0Ca-Pi2 and Pi1 levels. The biological variability is represented by error bars in Figure 1 and standard deviation in Table 3.
Cultivation was performed in Duetz-MTPS (Enzyscreen, Heemstede, The Netherlands) [43,45,47,50, 53,54], consisting of 24-square well polypropylene deep well microtiter plates (MTPs), low evaporation sandwich covers with a clamp system. A total of 7 ml of sterile media broth was transferred into the autoclaved microtiter plates and each well was inoculated with 50 µL of the spore suspension. MTPs were placed on the shaking platform of the incubator MAXQ 4000 (Thermo Scientific, Oslo, Norway). Cultivations were performed for 7 days at 25 °C and 400 rpm agitation speed (1.9 cm circular orbit).

## 2.2. Lipid Extraction and GC-FID Analysis of Lipid Concentration and Fatty Acid Profile

Direct transesterification was performed according to Lewis et al. [55], with some modifications [44]: 2 mL screw-cap polypropylene (PP) tubes were filled with 30 ± 3 mg of freeze-dried biomass, 250 ± 30 mg of acid-washed glass beads, and 500 µL of methanol. Further, the fungal biomass was disrupted in a tissue homogenizer (Bertin Technologies Percellys Evolution, Montigny-le-Bretonneux, France). The disrupted fungal biomass was transferred into glass reaction tubes by washing the PP tube with 2400 µL of a methanol-chloroform-hydrochloric acid solvent mixture (7.6:1:1 *v/v*). Then, 1 mg of C13:0 TAG internal standard in 100 µL of hexane was added to the glass reaction tubes (100 µL from a 10.2 mg/mL⁻¹ glyceryl tritridecanoate (C₄₂H₈₀O₆, C13:0 TAG (13:0/13:0/13:0), Sigma-Aldrich, St. Louis, Missouri, USA). Reaction tubes were incubated at 90 °C for 1 h, followed by cooling to room temperature and the addition of 1 mL distilled water. The fatty acid methyl esters (FAMEs) were extracted by the addition of 2 mL of a hexane-chloroform mixture (4:1 *v/v*) and applying 10 s of vortex mixing. The reaction tubes were centrifuged at 3000 g for 5 min at 4 °C and the upper hexane phase was collected in glass tubes. The extraction step was repeated three times for each sample. Subsequently, the solvent was evaporated under nitrogen at 30 °C and FAMEs were dissolved in 1.5 mL of hexane containing 0.01% of butylated hydroxytoluene (BHT, Sigma-Aldrich, St. Louis, Missouri, USA) and a small amount of anhydrous sodium sulfate (to remove traces of water in the sample). Samples were mixed by vortexing and, finally, dissolved FAMEs were transferred to the GC vials.

Fatty acid profile analysis was performed using a gas chromatography system with flame ionization detector (GC-FID) 7820A GC System, Agilent Technologies, controlled by Agilent OpenLAB software (Agilent Technologies, Santa Clara, CA, USA). Agilent J and W GC column 121–2323, DB-23, 20 m length; 0.180 mm diameter; 0.20 µm film was used for the separation of FAMEs. Then, 1 µL of the sample was injected in the 30:1 split mode with the split flow 30 mL/min. The inlet heater temperature was set on 250 °C and helium was used as the carrier gas. The flow of helium through the column was 1 mL/min. The total runtime for one sample was 36 min with the following oven temperature increase: initial temperature 70 °C for 2 min, after 8 min to 150 °C with no hold time, 230 °C in 16 min with 5 min hold time, and 245 °C in 1 min with 4 min hold time. For identification and quantification of fatty acids, the C4–C24 FAME mixture (Supelco, St. Louis, MO, USA) was used as an external standard, in addition to C13:0 TAG internal standard. The weight of individual FAs was calculated based on the peak areas, relative response factors (RRF), and C13 internal standard. The total lipids in the fungal biomass were the sum of FA (the weight of C13 IS was subtracted) divided by the weight of dry biomass.

## 2.3. Fourier Transform Infrared Spectroscopy of Fungal Biomass

Fourier transform infrared (FTIR) spectroscopy analysis of fungal biomass was performed according to Kosa et al. [45], with some modifications [43]. The biomass was separated from the growth media by centrifugation and washed with distilled water. Approximately 5 mg of washed biomass was transferred into a 2 ml polypropylene tube containing 250 ± 30 mg of acid washed glass beads and 0.5 ml of distilled water for further homogenization. The remaining washed biomass was freeze-dried for 24 h for determining biomass yield. The homogenization of fungal biomass was performed by using Percellys Evolution tissue homogenizer (Bertin Technologies, Aix-en-Provence, France) with the following set-up: 5500 rpm, 6 × 20 s cycle. Then, 10 µL of homogenized fungal biomass was pipetted
onto an IR transparent 384-well silica microplate. Samples were dried at room temperature for 2 h. In total, 140 biomass samples were analyzed in three technical replicates by FTIR spectroscopy.

FTIR spectra were recorded in a transmission mode using the high throughput screening extension (HTS-XT) unit coupled to the Vertex 70 FTIR spectrometer (both Bruker Optik, Leipzig, Germany). Spectra were recorded as the ratio of the sample spectrum to the spectrum of the empty IR transparent microplate in the region between 4000 cm⁻¹ and 500 cm⁻¹, with a spectral resolution of 6 cm⁻¹, a digital spacing of 1.928 cm⁻¹, and an aperture of 5 mm. For each spectrum, 64 scans were averaged. In total, 420 biomass spectra were obtained.

The OPUS software (Bruker Optik GmbH, Leipzig, Germany) was used for data acquisition and instrument control.

## 2.4. Data Analysis

The following software packages were used for the data analysis: Unscrambler X version 10.5.1 (CAMO Analytics, Oslo, Norway) and Matlab R2019a (The Mathworks Inc., Natick, MA, USA).

### 2.4.1. Analysis of FTIR Spectral Data

To evaluate the correlation of lipid content results obtained from FTIR and GC data and, further, investigate media associated changes of the lipid content in biomass, FTIR spectra were preprocessed by 2nd derivative using Savitzky-Golay algorithm with 2nd order polynomial and windows size 13, followed by spectral region of interest (SROI) selection 3050-2800 and 1800-1700 cm⁻¹ and normalization by extended multiplicative signal correction (EMSC) [56] with linear and quadratic terms. Preprocessed FTIR data were then analyzed by principal component analysis (PCA) and score plots were used to compare the lipid related information in FTIR and GC data.

Different preprocessing was applied in order to evaluate the cell chemistry changes that occurred under different media conditions. To do so, SROI 3300-2800 and 1800-800 cm⁻¹ was selected and normalized by EMSC using linear and quadratic terms and up-weighting the region 2800-1800 cm⁻¹. The up-weighting of the inactive region 2800-1800 cm⁻¹ helped in reducing baselines by EMSC in the SROI. Afterwards, the dataset was split according to the concentrations of inorganic phosphorus (Pi) and separately analyzed by PCA. Correlation loading plots were obtained for Pi1, Pi2, and Pi4 in order to analyze the most pronounced correlation patterns in the data. To obtain such a plot, we used scores of each separate PCA model corresponding to one Pi concentration and projected on them variables of interest such as certain relevant peaks of FTIR data and other reference variables, such as pH, biomass yield, and lipid content from GC data, in addition to experimental design factors. The maxima of the corresponding chemical bonds selected for correlation loading plots based on the FTIR spectra of reference materials were pure sodium polyphosphate, chitin, and glyceryl trioleate (Table 2, Figure S3 in the Supplementary Materials). These compounds were the main cell components of interest. For plotting the peaks on the correlation loading plots, the preselected peaks of the preprocessed spectra were used (Table 2).

### 2.4.2. Analysis of GC Data

The detailed fatty acid profiles from the GC analysis were analyzed and compared to FTIR data. Fatty acid profiles were represented by both single fatty acids and sum of fatty acids—saturated fatty acids (SAT), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). Each data column was standardized (x/std (x)) and then analyzed by PCA. The scatter plot was used to compare the information related to total lipid content and profile obtained by GC and FTIR.

To find differences in fatty acid composition at different Pi concentrations, after standardization, the GC dataset was split into three datasets: Pi1, Pi2, Pi4, and analyzed separately by PCA. To learn about the correlation patterns in the data, the correlation loading plots for GC data were obtained using scores of GC based PCA models. The fatty acid profile of *M. circinelloides* is dominated by
myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1n9), linoleic (C18:2n6), and γ-linolenic (C18:3n6) acid, therefore these were presented in the correlation loading plots.

<table><caption>**Table 2.** The maxima of the corresponding chemical bonds selected for correlation loading plots based on FTIR data.</caption><thead><tr><th>Cell Component</th><th>Peak Maxima</th><th>Molecular Vibration</th></tr></thead><tbody><tr><td rowspan="10">Chitin/chitosan</td><td>3261</td><td>N-H stretching</td></tr><tr><td>3105</td><td>N-H stretching</td></tr><tr><td>2879</td><td>-C-H stretching</td></tr><tr><td>1656</td><td>-C=O stretching (Amide I)</td></tr><tr><td>1620</td><td>-C=O stretching (Amide I)</td></tr><tr><td>1554</td><td>C-N-H deformation (Amide II)</td></tr><tr><td>1375</td><td>-CH<sub>3</sub> deformation</td></tr><tr><td>1305</td><td>C-N-H deformation (Amide III)</td></tr><tr><td>1027</td><td>C-O-C str., C-O-H def.</td></tr><tr><td>950</td><td>C-O-C def.</td></tr><tr><td rowspan="7">Lipids</td><td>3004</td><td>-CH<sub>3</sub> def.</td></tr><tr><td>2921</td><td>=C-H stretching</td></tr><tr><td>2852</td><td>-C-H stretching</td></tr><tr><td>1743</td><td>-C=O stretching</td></tr><tr><td>1463</td><td>-CH<sub>2</sub> bending</td></tr><tr><td>723</td><td>&gt;CH<sub>2</sub> rocking</td></tr><tr><td>1263</td><td>P=O stretching</td></tr><tr><td>Polyphosphates</td><td>885</td><td>P-O-P stretching</td></tr></tbody></table>

## 3. Results

### 3.1. Growth of Oleaginous *M. Circinelloides* in Metal Ion-Regulated Media with Different Pi Levels

In order to assess the variation between bioreplicates, cultivation of *M. circinelloides* in the reference medium R with Pi1 concentration was performed 11 times (part of the data were published previously [43]). Cultivations were performed at different timepoints and by using different MTPs. The results of statistical analysis of 11 bioreplicates show that the mean for the biomass production in the reference medium (R-Pi1) was 10.08 g/L (range: 9.35–11.52 g/L, median: 9.90 g/L), with 0.62 g/L standard deviation, and 0.19 g/L standard error. Therefore, it can be considered that all deviations higher than two standard deviations (13% of the average biomass concentration) are statistically significant and can be assigned as the effect of various metal and phosphates concentrations.

Growth of *M. circinelloides* in different media was strongly affected by the availability of phosphates. Low availability of phosphates (Pi0.25 and Pi0.5) led to a substantial decrease in pH (Table S2, Supplementary Materials) causing a significantly reduced growth of *M. circinelloides* (Figure 1, Table S3). Biomass yields in all Pi0.25 and Pi0.5 media were in the range from 0.51 g/L to 2.79 g/L and from 0.73 g/L to 4.70 g/L, respectively (Figure 1). The biomass production under moderate and high levels of phosphates (Pi1–Pi4) was substantially higher and in the range from 7.84 g/L to 12.90 g/L for Pi1, from 7.54 g/L to 12.47 g/L for Pi2, and from 7.89 g/L to 13.23 g/L for Pi4 (Figure 1). The biomass yield of *M. circinelloides* was, in several cases, higher than in the reference medium with Pi1 concentration (Figure 1, Table S3).
<figure><img src="image_77.png" /><figcaption>**Figure 1.** Final biomass concentrations after 7 days of incubation of *M. circinelloides* in the media with different concentrations of metal and phosphorus ions. * Empty slots indicate no growth.</figcaption></figure>
Metal ions affected the growth of *M. circinelloides* differently and the strongest effect was observed at moderate and high levels of phosphates (Pi1, Pi2, and Pi4). Metal ions' starvation for most of the tested metal ions led to a reduced fungal growth. Thus, Zn and Mg starvation resulted in a low or no growth of *M. ciricinelloides* in all tested Pi conditions (Figure 1, Table S3). Further, removal of Cu ions led to a slight biomass decrease. Removal and low concentrations of Ca ions resulted in a slight biomass decrease at Pi2 and Pi4 and increase at Pi0.25, Pi0.5, and Pi1 levels (Figure 1, Table S3). Similar increase in the biomass yield was observed for the media without Fe ions at Pi0.25, and it was significantly decreased at higher Pi levels -44% for Pi1, -30% Pi2, and -16% for Pi4 compared to the reference amount of Fe (Figure 1, Table S3). Deficiency of Co ions did not result in any significant change in biomass formation of *M. circinelloides* in comparison with high concentrations of Co ions (100Co).

Generally, increased availability of metal ions showed either no effect or both growth-stimulating and -inhibiting effects, depending on the metal type and phosphates concentration. Thus, high metal ions concentration in the media with low Pi levels did not lead to any significant changes in the biomass formation of *M. ciricinelloides* (Figure 1), with the exception of Fe and Zn ions. High amount of Fe ions in a combination with the low concentration of phosphates showed a negative effect on the biomass formation, and no or very limited growth was observed for the following Fe conditions: 1000Fe-Pi 0.25, 1000Fe-Pi1, and 100Fe-Pi0.25. Low growth under these conditions can be connected to the acidic pH ranging from 2.79 for Pi4 to 1.62 for Pi0.25 (Table S2 in the supplementary materials). The opposite effect was observed for the media with elevated concentration of Zn ions, which enhanced growth of *M. circinelloides*, resulting in a higher biomass yield under Pi limitation, with the highest biomass yield of 4.44 g/L observed for 1000Zn condition (1 g/L ZnSO₄·7H₂O) (Figure 1, Table S3).

A considerable effect of the increased metal ions availability was recorded for the media with moderate and high Pi levels (Figure 1). For example, high concentrations of Mn provided higher biomass at Pi4, with the highest yield of 12.93 g/L for 1000Mn condition, while the effect of other Mn conditions on the growth of *M. circinelloides* was generally negligible. Increasing amount of Fe ions up to the 100Fe condition positively affected the biomass production of *M. circinelloides* in the media with Pi4 and Pi2, and the highest yield of 11.84 g/L was observed for the condition 100Fe with Pi4 (Figure 1). However, very high iron concentration (1000Fe) showed an inhibiting effect at Pi1 and significantly decreased biomass at Pi2 and Pi4 (Figure 1). While increased concentration of Zn ions positively affected the growth of *M. circinelloides* in the media with low phosphates concentration (Pi0.5 and Pi0.25), it slightly decreased the growth in media with high phosphates concentration (Pi2 and Pi4). Generally, it can be concluded that increased concentrations of Zn ions (10Zn and 100Zn) in the media have beneficial effects, since under all Pi levels the biomass yield was increased compared to the standard conditions (R) (Figure 1). A similar effect can be seen for Cu and Co ions, where media with 10Cu condition provided the highest biomass yield for moderate and high Pi levels (Figure 1). Moreover, the highest biomass yield of 13.23 g/L of all tested conditions was observed for 10Co (0.001 g/L CoSO₄·7H₂O) with Pi4 level of phosphorus substrate (Figure 1, Table S3).

## 3.2. Effect of Metal Ions on Lipid Accumulation and Fatty Acid Profile of M. Circinelloides TAGs

Lipid content in oleaginous *M. circinelloides* biomass grown in the different media is reported in Table 3. Due to the low growth and not sufficient amount of biomass for lipid extraction, samples Pi0.25 and Pi0.5 for all metal ions conditions, and samples 1000Fe, 0Mg, 0Zn, 10Ca0Mg were excluded from the lipid extraction and further data analysis.
<table><caption>**Table 3.** Lipid accumulation (% of lipids per dry cell weight) for *M. circinelloides* grown in nitrogen-limited metal ion-regulated media with different amounts of inorganic phosphorus substrates (Pi1, Pi2, and Pi4).</caption><thead><tr><th>Metal Ion Condition</th><th>Pi1</th><th>Pi2</th><th>Pi4</th></tr></thead><tbody><tr><td>Reference medium</td><td>41.13 ± 1.19</td><td>33.44 ± 1.28</td><td>33.15 ± 0.01</td></tr><tr><td>0Ca</td><td>61.16 ± 0.16</td><td>40.15 ± 2.31</td><td>31.51</td></tr><tr><td>0.01Ca</td><td>34.00</td><td>39.61</td><td>34.93</td></tr><tr><td>0.1Ca</td><td>60.55</td><td>37.22</td><td>43.70</td></tr><tr><td>10Ca</td><td>44.37</td><td>33.50</td><td>27.95</td></tr><tr><td>0.01Mg</td><td>11.43</td><td>20.57</td><td>22.80</td></tr><tr><td>0.1Mg</td><td>30.38</td><td>32.90</td><td>39.40</td></tr><tr><td>0Co</td><td>38.78</td><td>37.24</td><td>31.46</td></tr><tr><td>10Co</td><td>30.25</td><td>34.40</td><td>29.87</td></tr><tr><td>100Co</td><td>30.31</td><td>38.52</td><td>29.60</td></tr><tr><td>1000Co</td><td>31.49</td><td>35.08</td><td>29.38</td></tr><tr><td>0Cu</td><td>61.27</td><td>53.80</td><td>52.24</td></tr><tr><td>10Cu</td><td>47.11</td><td>37.75</td><td>35.63</td></tr><tr><td>100Cu</td><td>46.70</td><td>41.27</td><td>38.27</td></tr><tr><td>1000Cu</td><td>43.11</td><td>42.46</td><td>38.36</td></tr><tr><td>0Fe</td><td>37.27</td><td>37.00</td><td>30.62</td></tr><tr><td>10Fe</td><td>36.73</td><td>34.36</td><td>29.19</td></tr><tr><td>100Fe</td><td>30.77</td><td>33.00</td><td>27.11</td></tr><tr><td>0Mn</td><td>46.78</td><td>37.94</td><td>38.74</td></tr><tr><td>10Mn</td><td>34.52</td><td>31.67</td><td>33.21</td></tr><tr><td>100Mn</td><td>35.16</td><td>39.13</td><td>34.22</td></tr><tr><td>1000Mn</td><td>33.23</td><td>30.58</td><td>33.61</td></tr><tr><td>10Zn</td><td>49.78</td><td>38.31</td><td>37.65</td></tr><tr><td>100Zn</td><td>43.84</td><td>41.94</td><td>34.72</td></tr><tr><td>1000Zn</td><td>42.36</td><td>41.85</td><td>38.04</td></tr></tbody></table>

Lipid accumulation in *M. circinelloides* grown under the reference metal ion conditions reached approximately 41% for Pi1 and 33% for Pi2 and Pi4 (Table 3). Lack of several metals resulted in an increase of lipid content for several Pi conditions. For example, removal of Ca, Co, and Fe ions in the media with Pi2 and Pi1 and Cu and Mn ions in the media with Pi1, Pi2, and Pi4 resulted in an increase in lipid accumulation in *M. circinelloides*, compared to the reference conditions. The most significant increase in lipid accumulation was recorded for 0Ca-Pi1 and 0Cu-Pi1 conditions. Interestingly, the removal of some metal ions, such as Ca, Co, and Fe, enhanced lipid accumulation only at moderate phosphate concentrations in the media (Pi1 and Pi2), and decreased lipid accumulation at high phosphate concentrations (Pi4). Removal of Mn, and especially Cu, ions resulted in increased lipid accumulation at moderate and high phosphate concentrations (Pi1, Pi2, and Pi4) (Table 3).

Variation in the availability of metal ions showed diverse and metal-specific effects on the lipid accumulation in *M. circinelloides* (Table 3). Lack of Mn ions has resulted in relatively high lipid accumulation in *M. circinelloides* for all tested Pi levels. The inhibiting effect of higher Mn ion concentrations was more visible in the media with reference amounts of phosphates (Pi1). Similar results were recorded for the media with increased levels of Fe ions, where lipid yield was lower than under the reference Fe condition (R). Two tested concentrations of Mg ions provided low lipid yield in *M. circinelloides* with the lowest values of 11.43% at 0.01Mg-Pi1 condition, while lipid yield of 39.4% was observed in the biomass grown in the 0.1Mg-Pi4 condition. When increasing the amount of Co ions in the media with Pi4 and Pi2, a decrease in lipid accumulation was recorded, while the opposite effect was seen for the medium with Pi4. An increase in the concentration of Zn ions showed a triggering effect on lipid accumulation in *M. circinelloides* grown at different levels of phosphorus substrate, with the highest lipid yield of 49.78% at 10Zn-Pi1 (0.01 g/L ZnSO₄·7H₂O), which was 9% higher than for the reference condition. A similar lipogenesis triggering effect was observed for increasing concentration of Cu ions at all tested Pi levels, while the highest lipid yield was recorded
when Cu ions were removed. The most diverse effect on lipid accumulation in *M. circinelloides* was observed for different concentrations of Ca ions. Increase in Ca ion availability from 0Ca to 0.01Ca resulted in the decrease of lipid yield for condition 0.01Ca-Pi1, while a further increase in Ca ions to 0.1Ca resulted in the increase of lipid yield for biomass grown in the media with Pi1, Pi2, and Pi4. A high concentration of Ca ions (10Ca) resulted in the decrease in lipid accumulation in the medium with Pi4 and slight increase in the media with Pi2 and Pi1 (Table 3).

The fatty acid profile of *M. circinelloides* grown under the reference condition was dominated by oleic acid (C18:1n9; 38%), followed by palmitic (C16:0; 22%), linoleic (C18:2n6; 14%), and γ-linolenic (C18:3n6; 12%) acids. Further, stearic (C18:0; 5%), palmitoleic (C16:1; 1.75%), and myristic (C14:0; 1.5%) acids were recorded in smaller amounts (Figure 2, Table S4). An example chromatogram can be found in the Supplementary materials (Figure S1). The fatty acid profile of *M. circinelloides*, grown under reference metal ion conditions, slightly changed depending on the phosphorus availability in the media. Thus, we observed an increase in the unsaturation and amount of palmitoleic acid with the increasing amount of phosphorus (Figure 2, Table S4). An opposite effect of phosphorus availability (and the associated changes in pH of media) was recorded for the unsaturation of stearic acid into oleic and γ-linolenic acid, where decreasing unsaturation was evident with increasing Pi concentrations and higher pH. This pattern can be visible through all the samples, with some exceptions for 10Fe-Pi1/Pi2/Pi4, 100Fe-Pi1/Pi2, 1000Zn, 1000Co, 10Cu-Pi2, and 0.01Mg-Pi1 conditions (Figure 2, Table S4). Minimal content of myristic acid (C14:0) was observed in 10Fe and 100Fe conditions, except for the 100Fe-Pi4 sample (Figure 2). Further, media with high amounts of Zn (1000Zn) and Co (1000Co) ions led to the synthesis of TAGs with the increased relative amount of stearic acid (Figure 2, Table S4).

To reveal underlying correlations among certain fatty acids, design variables, and reference variables, as well as sum of saturated (SAT), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids, PCA analysis of fatty acid (FA) profiles was done for each Pi substrate level separately. The separation of data into different Pi concentrations was done in order to focus on the effect of metal ions only on FA profile, excluding the effect of phosphorus substrate availability. The results, in the form of correlation loading plots, are presented in Figure 3. Generally, the fatty acid profile of *M. circinelloides* was quite consistent, irrespective of media composition.

In the biomass obtained from the media with Pi1 and Pi2 amounts of phosphorus, high concentrations of Co (1000 Co) and Zn (1000 Zn) ions were positively correlated with the saturated fatty acids (SAT) (Figure 3A). This was also evident from the detailed FA profiles, where the relative amount of palmitic and stearic acid was increased under these conditions (Figure 2). Further, some tendency of positive correlation between increasing concentration of Fe ions and content of polyunsaturated fatty acids (PUFA) was observed in the media with Pi1 level (Figure 3A). In the media with high amounts of phosphorus substrate (Pi4 and Pi2), 1000 Mn and 0.01 Mg conditions were positively correlated with the polyunsaturated fatty acids (PUFAs) (Figure 3B,C).
<figure><img src="image_78.png" /><figcaption>**Figure 2.** Fatty acid profile of lipids accumulated in *M. circinelloides* grown in media with Pi1, Pi2, and Pi4 levels of phosphorus. Only fatty acids present in amounts of more than 1% are displayed. The rest is summed up as ‘others’. An example chromatogram can be found in the Supplementary Materials (Figure S1).</figcaption></figure>
<figure><img src="image_79.png" /><figcaption>**Figure 3.** Correlation loading plots based on the PCA analysis of fatty acid (FA) profiles of lipids accumulated in *M. circinelloides* grown in metal ion-regulated media under Pi1 (A), Pi2 (B), and Pi4 (C) levels of phosphorus substrate.</figcaption></figure>

### 3.3. Chemical Composition of *M. Circinelloides* Biomass

In order to study differences in the compositional profile of the *M. circinelloides* biomass, high-throughput Fourier transform infrared (FTIR-HTS) spectroscopy was used. Spectral regions and peaks related to three types of metabolites—lipids, chitin/chitosan, and polyphosphates were used in the analysis. The FTIR-HTS spectra (Figures S2 and S3 in the Supplementary Materials) showed that fungal biomass was dominated by signals of these intracellular metabolites. The spectra of reference materials can be found in the supplementary materials (Figure S2). The maxima of the peaks selected for the correlation loading plots are listed in Table 2. Due to the insufficient growth (Figure 1), the following samples have been disregarded from the FTIR-HTS spectral data analysis: (i) all samples grown under Pi0.25 and Pi0.5 levels; (ii) samples grown in the media with 1000Fe, 0Mg, 0.01Mg, 0Zn, and 10Ca0Mg.

First, we examined the lipid region of FTIR-HTS spectra (3050–2800 and 1800–1700 cm⁻¹) and analyzed the correspondence of it with GC data by PCA analysis (Figure 4). On the PCA score plots
we can observe similar pattern for FTIR and GC data, indicating similarity in the obtained information about lipid yield and profile from the data of these analytical techniques.

<figure><img src="image_80.png" /><figcaption>**Figure 4.** PCA score plots of FTIR-HTS (A) and GC (B) data. PCA analysis was performed on the preprocessed FTIR-HTS data (2nd derivative, polynomial order 2, window size 13; SROI: 3050–2800 and 1800–1700 cm⁻¹, EMSC) and normalized GC data.</figcaption></figure>

The correlation loading plots from the PCA analysis visualize the relation between the presence of lipids, chitin/chitosan, and polyphosphate and different media (Figure 5). PCA analysis of EMSC preprocessed spectra was performed separately for different Pi concentrations in order to emphasize the effect of different metal ions and disconnect it from the effect of inorganic phosphorus substrate. The loading vectors and FTIR spectral scores are displayed in Figures S4–S6 in the Supplementary materials. The first principle component (PC1), which explained the highest variance in the FTIR data, was represented by lipids to proteins and to chitin and chitosan ratio. The second principle component (PC2) was represented by the polyphosphate peaks, which were strongly visible in the cases of polyphosphate accumulation triggering conditions when Pi4 and Pi2 phosphate concentrations were used in the media (Figures S5A and S6A), while no strong characteristic signals representing any of the studied metabolite were visible in the PC2 for Pi1 condition (Figure S4A).

Lipids and chitin/chitosan are both carbon-rich metabolites, therefore their synthesis processes are competing for the C source. In all correlation loading plots, we can observe that lipids and chitin/chitosan were anticorrelated, indicating that these metabolites cannot be produced simultaneously at high yields, while they still can be coproduced with one of them dominating (Figure 5). Further, we see that peaks 2879 cm⁻¹ (-C-H stretching) and 950 cm⁻¹ (C-O str, C-C str., C-O-H def. C-O-C def), responsible for chitin/chitosan, have been shown to be correlated with the lipid-related peaks (Figure 4). The reason is that the chemical bonds, represented by these peaks, are also present in lipids and the contribution of the lipid associated peaks was stronger than the contribution of chitin/chitosan peaks. The chitin/chitosan formation could also be negatively affected by the N-limitation. The biomass concentration was correlated with the lipid peaks, revealing that a good lipid accumulation can be achieved only with the optimal growth conditions providing good growth and biomass formation and that high biomass concentration was the result of the increased lipid accumulation (Figure 5).
<figure><img src="image_81.png" /><figcaption>**Figure 5.** Correlation loading plots based on the PCA analysis of FTIR-HTS spectra of *M. circinelloides* biomass grown in metal ion-regulated media with Pi1 (A), Pi2 (B), and Pi4 (C) levels. Main peaks corresponding to the lipids (purple), chitin/chitosan (green), and polyphosphates (yellow) are presented.</figcaption></figure>

The effect of metal ions on the *M. circinelloides* biomass composition in the media with reference level of phosphorus (Pi1) is displayed in a PCA score plot with loading vectors (Figure S4 in the Supplementary materials). Due to the fact that PC2 was not representing any clear relation between FTIR-HTS peaks and studied metabolites, we analyzed only PC1 representing the ratio of lipids to protein and to chitin and chitosan peaks. PCA score plots show that lipid content correlated with the absence of Ca ions (0Ca) (Figure S4B), which is in agreement with the lipid yield data presented in Table 3. Further, the PCA score plot shows that increasing Co amount displayed an inhibiting effect on the lipid accumulation (Figure S4B). The same results were observed for the lipid yield, where the highest lipid production was recorded for 0Co-Pi1 condition (Table 3). FTIR-HTS spectra of biomass grown in the media with reference level of inorganic phosphorus substrate (Pi1) did not exhibit significant absorbance for polyphosphate peaks. The correlation loading plot (Figure 5A) shows some effect of two metal ions—Fe and Co. Namely, a decrease in the concentration of Fe ions was correlated with the chitin/chitosan peaks, indicating that higher amounts of this metabolites is expected in the fungal biomass when Fe ions are in a low availability, while the absence of Fe ions (0Fe) was anticorrelated with the lipid peaks and biomass concentration (Figure 5A). This corresponds well to the biomass production results, where it is obvious that lack of Fe ions caused a significant decrease of biomass formation (Figure 1). A high concentration of Co ions (1000 Co) showed correlation with
chitin/chitosan peaks of FTIR-HTS spectra, indicating that the relative content of this metabolite in the fungal biomass increased with the increased concentration of Co ions. Absence of Ca ions (0Ca) was correlated with the high lipid and biomass concentration. This is in agreement with the reference biomass and lipid concentration results, where 0Ca-Pi1 condition provided the highest biomass and lipid production from all the tested Ca ions conditions (Figures 1 and 2).

The effect of metal ions on the synthesis of studied *M. circinelloides* metabolites in the media with Pi2 level of phosphorus is displayed on the Figure 5B, where we can see that increased amounts of Zn and Cu ions were correlated with the lipid yield. Similar correlation results were observed for Co ions, except for the condition of 1000Co-Pi2, which was slightly anticorrelated with lipid peaks. Correlation between polyphosphate spectral peaks and the highest tested Ca ions amount (10Ca) was observed (Figure 4B). Further, we can see correlation between amount of Zn ions and polyphosphate peaks. Finally, metal ions conditions 0.1Ca, 1000Zn, 100Zn, 10Zn, 10Co, 100Co, and 10Cu correlated with the lipid yield.

When examining the effect of metal ions under the high amounts of inorganic phosphorus substrate level (Pi4), on the PCA score plot it is seen that increasing amount of Ca ions corelated with the decrease of the relative lipid content and increase of the polyphosphate content (Figure S6 in the Supplementary materials). Further, in Figure 5C we observed that: (i) decreasing Cu ion availability and high concentration of Zn ions correlated with the increase of relative lipid content; (ii) low amount of Ca ions (0,1Ca) correlated with the lipid peaks and lipid yield and anticorrelated with polyphosphates peaks, while high amount of Ca ions (10Ca- 1 g/L CaCl₂·2H₂O) correlated with polyphosphate and chitin/chitosan peaks; (iii) low concentration of Mg ions (0,1Mg) correlated with lipid peaks and anticorrelates with polyphosphate peaks; (iv) there was no correlation observed for Co and Mn ions (Figure 5C).

## 4. Discussion

For half of a century, *M. circinelloides* has been studied as a microbial cell factory for production of a series of metabolites and valorization of different substrates. Today, this dimorphic oleaginous fungus is positioned as one of the most robust fungal cell factories for the biotech, biorefinery, and bioremediation industries [2].

Despite the deep understanding of *M. circinelloides* physiology and metabolic processes, the role and the effect of metal ions on the lipid accumulation and the cellular composition of this fungus have not been systematically investigated. The effect of metal ions on the growth and metabolic activity of *M. circinelloides* has, to the authors knowledge, only been assessed in the connection to bioremediation abilities of this fungus [11,12,57]. Therefore, in this study we performed an extensive screening of the growth, lipid accumulation, and compositional profile of *M. circinelloides* on 140 different media with variations in the concentrations of metals ions and phosphorus. Lipid accumulation and fatty acid profiles were determined by the GC-FID. The composition of the fungal biomass was investigated by the quantification of lipids, polyphosphates, and chitin/chitosan, as these components previously have been suggested for a coproduction concept involving *M. circinelloides* [43]. For the evaluation of biomass composition, the modern high-throughput analytical technique FTIR spectroscopy was applied. The main advantage of FTIR spectroscopy is that all biochemical components of the sample can be profiled in a single measurement run, without tedious extraction procedures [58–62]. FTIR spectroscopy provides detailed relative quantitative information about different chemical components of the samples and it has been previously utilized for the characterization of lipids [50,51,63], polyphosphates [64], and chitin/chitosan [65,66]. In this study, we have demonstrated that the FTIR analysis as a sole method coupled to multivariate data analysis can be applied for a fast and simple analysis of microbial biomass.

Efficiency of microbial biomass production, the yield of the targeted metabolite(s), and a coproduction potential are important assessment parameters in bioprocess development [19,20]. Biomass production is affected by factors such as pH, temperature, aeration, media composition, and cultivation mode [2]. For example, culture volume and mode of cultivation were reported by
Carvalho et al. as factors strongly affecting the final biomass yield of *M. circinelloides* [67]. The reported biomass production of *M. circinelloides*, depending on the culture conditions, varied greatly, from 5 g/L to 20 g/L [10,17,68]. In our previous studies, the biomass concentration for different *M. circinelloides* strains was between 10 g/L and 15 g/L for the cultivations performed in microtiter plates, and 15.8 g/L in bioreactors [54]. The biomass production of the *M. circinelloides* VI04473 strain in this study varied from 0.5 to 13.2 g/L (Figure 1). The standard growth medium containing reference amount of inorganic phosphorus substrate (Pi1) and the reference amounts of metal ions resulted in 9.8 g/L of biomass, significantly lower than in our previous screening study [50]. The reason for the lower biomass production in the reference medium in the present study was probably due to utilization of ammonium sulphate as a nitrogen source, instead of yeast extract as in the previous study [50]. Ammonium sulphate is a pure inorganic source of nitrogen, lacking any additional macro- and micronutrients, vitamins, and growth factors that are present in yeast extract. Buffering capacity of ammonium sulfate is lower than for yeast extract and, as it has been previously reported, the uptake of ammonium ions causes the release of H⁺ by the fungal cells into the media, which accelerates pH lowering [69,70]. Further, possible formation of sulfuric acid during the uptake of ammonium could occur [71]. In addition, formation of organic acids by fungal cells either during exponential or during the stationary growth phase [72] significantly contributes to the acidity of the growth media. Thus, in the media with the low Pi levels we detected considerably low pH and suppressed growth and lipid accumulation in *M. circinelloides* (Figure 1). Acidic pH is a stress factor for many cellular organelles, especially for endoplasmic reticulum (ER), which is connected to protein folding and lipogenesis in fungal cells. It has been previously reported that acidic pH causes ER stress and induces unfolded protein response (UPR). This results in the accumulation of misfolded proteins in the ER and activation of the ER-stress sensor (Ire1p) and ER stress-responsive transcription factor (Hac1p), leading to the inhibition of growth and metabolic activity [73]. In our previous studies, we have showed that acidic pH affects cell wall and increasing chitin/chitosan production in *M. circinelloides* [43]. Cultivation in Duetz-MTPS does not allow continuous adjustment of pH and only start- and end-point measurements are possible, therefore the effect of low phosphorus concentrations was directly linked to drop in pH. Due to the fact, that acidic pH is quite an aggressive stress factor inhibiting fungal growth, the effect of metal ions on the growth and lipid accumulation under low phosphorus substrate availability was difficult to assess. Only two observations could be considered as significant—increase of biomass under higher Zn ion availability and Ca deficiency. Moreover, under Pi conditions lower than the reference (Pi0.5 and Pi0.25), K and Na ions were compensated with KCl and NaCl salts in order to provide the same Na and K amounts as in the reference Pi1 condition [43,44]. It has been reported that chlorides could have negative effect on the mycelium formation of some fungi [74,75]. Moreover, much higher concentrations of Cl⁻ (10–15% NaCl) than used in our study (KCl and NaCl in total below 5%) have shown some negative impact on fungal growth [76]. No negative impact of increased Cl⁻ on the biomass and lipid production has been observed when yeast extract was used as N-source [43,44]. Thus, we can hypothesize that in addition to pH-stress, increased Cl⁻ ions possibly negatively impacted the growth under low pH conditions. Therefore, these samples were excluded from further data analysis.

Variation in metal ion availability showed diverse and often metal- and pH-specific effects on biomass production and biomass composition of *M. circinelloides*. Growth of *M. circinelloides* was severely inhibited in media lacking Zn and Mg, indicating that these metal ions are essential for the growth and metabolic activity of the fungus. Inhibition of fungal growth in the media lacking Zn ions can be related to the fact that Zn plays an important role in the regulation of all genes in the eukaryotic cells [31]. Deficiency of Zn is detrimental for the fungal spore germination and further cell proliferation. Our study shows that elevated concentration of Zn ions has a beneficial effect on the biomass formation under phosphorus limitation. Low concentrations of Mg (0.01Mg condition) led to a decrease in biomass production and lipid yield, especially for Pi1 condition, where a lipid content of only 11.43% was reached (Figure 1, Table 3, Table S4). This can be explained by the fact that magnesium deficiency in eukaryotic cells can result in the decrease of glucose-6-phosphate, total content of phospholipids, and a
remarkable decrease in oxygen and substrate delivery to the cells with further concomitant changes in membrane phospholipids, leading to the reduced cell growth, delay in the cell cycle, and metabolic activity [77]. It has been shown that long-term Mg deficiency for yeast may result in distortion of cell division, production of aberrant cell forms, and a decrease in viability that can lead to a delay or change of cell cycle [78]. Therefore, the difference in the FA profile of *M. circinelloides* grown under the Mg deficiency (0.01Mg condition) could be explained by disruption of the cell cycle [79].

In addition to Zn and Mg, Ca and Fe are known to be essential for fungal growth [31,80]. In our study, an absence of Fe ions in the medium suppressed the growth of *M. circinelloides* under conditions of moderate and high phosphorus concentrations. While these metals did not affect lipid accumulation. This is an interesting observation, due to the fact that Fe is an important cofactor of many enzymes, it is essential during DNA synthesis and cleavage, and, thus, Fe deficiency should strongly affect growth and metabolic activity of fungal cells.

An absence of Ca ions affected growth of *M. circinelloides* depending on the phosphorus concentration and associated pH of the growth media. A considerable increase in the biomass production of *M. circinelloides* was observed in the media lacking Ca ions and containing moderate (Pi1) and low concentrations of phosphates (Pi0.5 and Pi0.25). Elevated biomass production under the condition Ca0-Pi1 could be partially explained by the fact that the absence of Ca ions in the medium enhanced lipid yield up to 61% (w/w). Increase in lipid accumulation with the decrease of concentration of Ca ions was observed also for media containing Pi2 and Pi4 levels of phosphates. Calcium starvation enhancing lipid accumulation in oleaginous microorganisms has been reported for algae [81], where the lipid production was increased by 30% in Ca deprived media. To the authors knowledge, a similar effect of Ca ions deficiency on lipid accumulation has never been reported for oleaginous fungi. Currently, there is no clear understanding of the mechanisms behind Ca deficiency-induced lipid accumulation in oleaginous microorganisms, and the direct link between calcium and lipid accumulation and TAGs synthesis has not been clearly demonstrated yet. Similar observations have been reported for adipocyte cells, where low cellular availability of Ca ions mediated antilipolytic pathways through a calcium-sensing receptor (CaSR), resulting in enhancing of lipid content in adipose tissue [82]. Due to the fact that lipolytic pathways are functionally conserved from mammalian cells to fungi [83], we suggest that Ca deficiency is mediating similar antilipolytic pathways in oleaginous microorganisms. Further, Wang. W.A. et al. [83] showed that Ca ions are important for the basal sensitivity of the sterol sensing mechanism of the sterol response element binding proteins (SREBPs) pathway. Wang W.A. et al. discovered that reduction of Ca concentration in endoplasmic reticulum changes the distribution of intracellular sterol/cholesterol, resulting in the enhancement of SREBPs activation and triggering synthesis of neutral lipids. Sterol response element binding proteins (SREBP) are transcription factors that are synthesized on endoplasmic reticulum (ER) and considered as ER-associated integral membrane proteins [83]. SREBP were reported for eukaryotic cells, including mammalian and fungal cells [84]. The studies show that SREBP are involved in lipid homeostasis, while SREBP isoforms control the expression of genes responsible for the biosynthesis of sterol/cholesterol, fatty acids, triacylglycerols, and phospholipid in the cell [85]. Further, detailed studies would be needed to confirm if these two events are valid also for oleaginous fungi grown under calcium deficiency.

Increase in *M. circinelloides* biomass yield was observed also at high concentrations of Ca ions in the media with high phosphate concentrations (Pi2). Infrared spectra of *M. circinelloides* biomass grown in this medium showed strong absorbance values for polyphosphate peaks (Figure S5 in the Supplementary Materials). Thus, we can assume that increase in biomass production is associated with the intracellular accumulation of available inorganic phosphorus substrate in the form of polyphosphate. It has been previously reported that, in media with excess phosphorus source, *M. circinelloides* is able to perform so called luxury uptake of phosphorus and accumulate it in the form of polyphosphates either in the cell wall or in the form of intracellular polyphosphate granules [6]. Polyphosphate (polyP) is a polyanionic compound, and it has been reported by Kikuchi Y. et al. that in the fully dissociated
form, polyP has one negative charge per Pi residue and two extra charges of terminal residues [85]. Therefore, accumulation of polyP in the cell results in the accumulation of a large amount of negative charge, which is probably compensated by an existence of a regulatory mechanism for maintaining charge neutrality of the cell. The studies involving temporal and quantitative analyses of cationic components of the fungal cells revealed that Na, K, Ca, and Mg ions were taken up by polyP, providing strong evidence that these ions play a major role in the neutralization of the negative charge of polyP in the fungal cell [85,86]. Thus, it is likely that with the higher availability of calcium ions in the medium, the neutralization of the polyP negative charge is more efficient and a higher amount of phosphorus can be stored intracellularly in the form of polyP. Due to fact that polyphosphate accumulation takes place in the exponential growth phase, while lipid accumulation in the stationary growth phase [87,88], it could be possible to perform a coproduction of these two components by manipulation of the availability of calcium and phosphorus substrate in the medium. Therefore, *M. circinelloides* can be utilized in the phosphorus recycling processes.

In addition to Ca-deprived media, lack of Cu and higher amounts of Zn and Mn considerably enhanced lipid accumulation in *M. circinelloides*. While elevated lipid production observed due to Ca deficiency could be explained by the above-mentioned hypothesis, there is no clear explanation of the high lipid accumulation under the copper deficiency condition that was significantly higher at all Pi levels. It has to be noted that the highest lipid yield was obtained under deficiency of Ca and Cu ions. In the literature, there has only been only one study, conducted on the liver cells, reporting Cu deficiency enhancing lipid storage [89], while metabolic pathways linking copper to lipid homeostasis have not been reported for fungal and any other microbial cells.

The FA profile of the accumulated in *M. circinelloides* TAGs was not significantly affected by the availability of metal ions and phosphorus. Only some tendency in increase of saturation with high Co and Zn amount was observed, but further enzymatic study would be needed to assess the activity of desaturases at these conditions.

By applying FTIR spectroscopy, we revealed that Ca, Co, and Zn ions at different concentrations correlated with lipid peaks; Ca and Zn correlated with polyphosphate, while Fe and Co with chitin/chitosan peaks of *M. circinelloides* biomass spectra. Thus, these ions could be considered as important components in optimizing and developing coproduction of lipids, polyphosphate, and chitin/chitosan by *M. circinelloides*. However, further studies are needed to fully understand the role of these metal ions in the metabolic pathways of *M. circinelloides* metabolites.

**5. Conclusions**

The aim of the study was to evaluate the effect of different metal ions and their concentration on biomass production, composition, and the lipid production in the oleaginous fungus *M. circinelloides*. Moreover, the growth experiments were conducted at different concentrations of phosphates. It can be concluded that, among tested metals, Mg and Zn are essential metals required for the optimal growth of *M. circinelloides*. Calcium availability is important for optimizing polyphosphate accumulation, while calcium and copper deficiency is important for lipid accumulation in *M. circinelloides*. Tested metal ions did not affect fatty acid profile of the accumulated TAGs. However, Ca, Co, Mg, and Zn ions have affected the cellular biochemical profile of *M. circinelloides*. Thus, metal ions are an important tool for optimizing lipid accumulation and coproduction of lipids, polyphosphate, and chitin/chitosan in *M. circinelloides*.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2309-608X/6/4/260/s1.
Table S1: Concentrations of salts used for regulating metal ions and inorganic phosphorus levels in the growth media. Table S2: pH of culture supernatant. Table S3: Biomass concentration (g/L). Table S4: Fatty acid profiles (%). Figure S1: Example chromatogram, *Mucor circinelloides* grown in Pi1-R condition. Figure S2: EMSC corrected FTIR-HTS spectra of *Mucor circinelloides* biomass. Figure S3: FTIR spectra of reference materials. Adapted from Dzurendova et al. Figure S4: PCA analysis of FTIR-HTS spectra of *Mucor circinelloides* biomass grown under Pi1 level. The loadings of spectral PCA (A), the score plot (B). Figure S5: PCA analysis of FTIR-HTS spectra of *Mucor circinelloides* biomass grown under Pi2 level. The loadings of spectral PCA (A), the score plot (B). Figure S6: PCA