## 4 Discussion  
In this study, out of 53 isolates, 12 were identified as lacase producers. Out of them, CDBT-F-G1 isolate was reported as potential lacase producer. This is the first report on isolation and characterization of lacase-producing strain from high altitudes of Nepal. Lacase secretion was monitored by visual color change in the plates. Reddish brown color with guaiacol and tannic acid, and a deep purple color with 1-naphthol, confirms lacase secretion [17; 18]. Morphological and microscopic characteristics of CDBT-F-G1 were similar to the characteristics of _Pestalotiopsis_ species as was described by [PERSON] and associates [19]. Isolate CDBT-F-G1 was an endophytic fungus with multi-septal spore morphology, with elongated flagella in spore cells (Figure 2b) [20]. According to Mycobank accession date 31 October, 2018, there are 334 associated records of _Pestalotiopsis_ species. More recently, some new species have been added to the list based on host occurrence, and morphological and molecular data. They are mostly endophytic and to some extent, host-specific [13; 15]. The optimum media pH for lacase secretion was different in various organisms and it was highly dependent on the substrate used. The observed optimum pH range of the culture media under which fungal species secrete lacases range from 3-7. This was consistent with the fact that _Pestalotiopsis_ species are mesophilic fungi and mesophilic fungi, e.g., _Ganoderma lucidum_, have been reported to secrete highest levels of lacase at temperatures between 25-30 \({}^{\circ}\)C [18; 21]. Similarly, the isolate CDBT-F-G1 has highest lacase production at pH 5 (Figure 5a) and a temperature optimum of 30 \({}^{\circ}\)C (Figure 5b). Copper ions, as a micronutrient, play an important role as a metal activator. It induces both lacase transcription as well as plays an important role in lacase secretion [22]. Magnesium sulfate (1 mM) was found to enhance lacase production in CDBT-F-G1 isolate (Figure 5c). These observations were consistent with literature reports wherein metal ions have been shown to enhance the growth of fungi, cause morphological and physiological changes, and affect the secretion of lacase [18; 23]. Further, it has been observed that various strains and species of fungi differ in their sensitivity towards metals during their growth on lignocellulosic substrates [24]. Increased agitation was also found to increase lacase production in CDBT-F-G1 isolate (Figure 5d). The latter was probably due to proper mixing and easy access to nutrients. However, agitation as a factor that affects lacase production has been controversial. [PERSON] and associates have reported that agitation did not play any role in the production of lacase in _Tranetes versicolour_ cultures. The decrease in lacase production was believed to be due to mycelial damage caused by agitation, which, in turn, probably led to a considerable decrease in lacase production [25]. As compared to other _Pestalotiopsis_ species, theCDBT-F-G1 strain reported herein was a better lacase producer, as well as a halotolerant fungus. Accordingly, we consider this as a novel fungus that is different from the other _Pestalotiopsis_ species reported by [PERSON] and associates [26]. In yet another study, cultivation of white-rot fungus, _viz._, _Bjerkandera adusta_[27], in a stirred tank reactor, resulted in decreased levels of lacase activity. Again, this is also believed to be mycelial damage caused during agitation. Surfactants, especially Tween-80, increase the bioavailability of less soluble substrates for fungi and stimulate the growth of fungal spores as well as increase the production of metabolic enzymes, such as lacases, as shown inCDBT-F-G1 isolate (Figure 5e) [28]. The specific mechanism by which surfactants enhance extracellular enzyme production in fungi has not been elucidated in detail [29]. Increased secretion of lacase by fungi when cultured in media supplemented with aromatic compounds, such as vanillin, guaiacol, catechol, and gallic acid, has been well documented.