A newly isolated Cerrena unicolor capable of laccase production and lignin degradation in agricultural wastes

Lignin is main residue of agro-industrial biomass which can be decomposed through enzymatic hydrolysis by fungi. In this study, a strain was isolated from birch forest and identied as Cerrena unicolor GC.u01 by 18S rDNA gene-sequencing technology. The activity of laccase (Lac) reached maximum 1605.28 ± 32.21 U·L-1 at 8th day via submerged fermentation, while the highest Lac activity by solid-state fermentation 1280.04 ± 48.11 U·g-1 with rice stalks and 566.83 ± 47.02 U·g-1 with wheat stalks were both obtained at 10th day, and 2677.50 ± 49.38 U·g-1 with corn stalks at 12th day. Then the lignin degradation ratios were up to 24.3%, 34.3% and 26.2% in wheat stalks, rice stalks and corn stalks, respectively, suggesting that the newly isolated Cerrena unicolor GC.u01 is potential for laccase production and lignin degradation by solid-state fermentation.


Introduction
Straw is mainly composed of cellulose, hemicellulose and lignin, which can be used as raw materials of high value-added products (Mathew et al. 2013; ). Cellulose and hemicellulose make up the entire biomass and are rmly linked to the lignin molecules via covalent and hydrogenic linkages, thus pretreatments must be performed to break down the compact structure into simple organic compounds primarily for the e cient utilization of straw, where the degradation of lignin is the key obstacle. Amount of studies have proved that pretreatment works for deconstructing the lignocellulose and expanding enzyme accessibility to cellulose in biomass are valid (Li et (Scott et al. 2002). In order to seek high e ciency industrial applications, large screening programs have been carried out to discover the most suitable strains, especially white rot fungi (Krogh et al. 2004;Hakala et al. 2005; Cianchetta et al. 2012). On the other hand, many researchers have studied on the catalytic mechanism of lignin degradation by the enzyme system secreted by white rot fungi. While growing on wood, white rot fungi produce extracellular laccase (Lac, EC1.10.3.2), lignin peroxidase (LiP, EC1. 11.1.14) and manganese peroxidase (MnP, EC1.11.1.13) due to the large structure of lignin polymers (Chandra et al. 2007;Chen et al. 2012). And the degradation enzymes liberated lignin oxidation reaction rather than hydrolysis. Some white rot fungi produce all three of these enzymes, but most produce two or even only one (Hatakka 1994;Eggert et al. 1996a;Eggert et al. 1996b), suggesting that the presence of all three enzymes is not necessary. Of the three mentioned enzymes, laccase belongs to the copper-containing oxidase family, which can be produced by several plants, insects, fungi and bacteria. Fungal laccase with higher redox (0.5-0.8 v) comparing to others indicates its more application potential in lignin degradation.
Numerous of studies have been conducted in laccase production by fungi fermentation. Simultaneously, there were signi cant differences in laccase activity among different strains, screening the potential producer is still relevant for customization of laccase production.
Therefore, this study aimed to screen a kind of fungus with high yield of laccase for lignin degradation, to make full use of waste agricultural straws. Then a strain with lignin degradation ability was isolated and identi ed as Cerrena unicolor GC.u01 by 18S rDNA gene-sequencing technology, and its related enzyme production performances under different processes were studied.

Screening and identi cation of GC.u01
A puri ed strain designed as GC.u01 was selected for further study. There is high identity between the phylogenetic tree of 18S rDNA sequences from GenBank and GC.u01 constructed using the N-J method (Fig 1). According to the phylogenetic tree, GC.u01 showed high degree of genetic similarity to Cerrena unicolor (FN907915.1), which suggested that GC.u01 belongs to the same species of Cerrena unicolor, therefore it was named as Cerrena unicolor GC.u01. Fig 2(a) and (b), the colony of Cerrena unicolor GC.u01 on PDA plate was snow-white circle, and mycelium was short and messy as catkin uffy, with the incubation time of Cerrena unicolor GC.u01 increased in liquid medium, many small white pellets of even size grew from the original pellets. The hyphae morphology of Cerrena unicolor GC.u01 was affected signi cantly by culture conditions , Fig 2 (d) showed more branched and thinner hyphae than that in Fig 2 (c). It is believed that the enzyme production by fungi is correlated with hyphal tips (Krull et al. 2013). Therefore, submerged and solid-state fermentation by Cerrena unicolor GC.u01 were conducted for lignin-degrading enzymes production.

As shown in
Ligninolytic enzymes from Cerrena unicolor GC.u01 via submerged fermentation  Table 1. The activities of these three ligninolytic enzymes were signi cantly different. Lac activity was generally increased rstly and then decreased. The maximum activity of Lac was 1605.28 U·L -1 at 8 th day, following decreased sharply because of the substrate gradually exhausted.
The activities of LiP and MnP were unstable, their maximum activities were 17.63 U·L -1 at 8 th day and 28.31 U·L -1 at 9 th day, respectively. The results showed that Cerrena unicolor GC.u01 could secrete Lac mainly to degrade lignin, and this performance is similar to Pycnoporus cinnabarinus (Eggert et al. 1996a;Eggert et al. 1996b).
The pH of the reaction system was maintained between 5 and 6. The lowest pH 5.50 was observed at 8 th day, which was the time of maximum Lac activity performed. However, the variation tendency of pH was generally contrary to Lac activity, indicating that pH had crucial in uence on Lac activity. MnP were detected, they were both low and unstable comparing with that of Lac under the same conditions. Therefore, Cerrena unicolor GC.u01 is a potential strain for Lac production via solid-state fermentation and corn stalk is the suitable fermentation substrate. In addition, solid-liquid ratio is the most signi cant difference between the submerged and solid-state fermentation, the change of moisture content (M) in solid-state fermentation may exert an in uence on the enzyme activity. As shown in Table 3, in the solid-state fermentation, the moisture contents with different substrates were all around 80%, and the highest moisture content was obtained at the 6 th day, which was 81.53%, 81.54% and 82.46%, respectively. The pH of solid-state fermentation was basically maintained and there was no signi cant difference compared with that of submerged fermentation with corn stalk as substrate. Nevertheless, the pH values with different substrates were various, in which the pH of rice straw fermentation rstly decreased and then increased. The fermentation pH of wheat straw basically showed a trend of continuous decrease, from 5.86 at the 4 th day to 5.24 at the end. On the contrary, the fermentation pH of corn straw increased from 5.58 on the 8 th day to 5.91 at the 10 th day, after that, it decreased slightly but remained stable. Compared with the initial value, the nal pH of rice straw and corn straw increased respectively, while that of wheat straw decreased. Lignin degradation is the bottlenecks of e cient utilization of biomass. The cellulose components of three stalks through solid fermentation by newly isolated fungus Cerrena unicolor GC.u01 were analyzed, and the results were shown in Table 4 and Fig 4. The lignin degradation ratios of wheat, rice and corn stalks were up to 24.3%, 34.3% and 26.2%, respectively. Although the hemicellulose and cellulose contents decreased slightly, but the soluble component increased markedly. Solid-state fermentation is in favor of Lac activity by Cerrena unicolor GC.u01 comparing to submerged fermentation both with corn stalks as substrate, which mimics the natural environment of most fungi (Hölker et al. 2004; Barrios-Gonzalez 2012), while corn stalks contains appropriate nutrients and available chemical components, and its relatively loose structure and low lignin content make it much easy for biological application. Solid-liquid ratio is the most signi cant difference between the submerged and solid-state fermentation. Moisture content is a key factor in solid-state fermentation that in uences the laccase production, Patel et al. found that 80% moisture content was most suitable for the Lac production by Tricholoma giganteum AGHP (Patel and Gupte 2016), which was approach to our results. Noteworthily, optimum moisture level varies among different species, and the optimal water content for fungal laccase production by solid-state is within a narrow range of 80% -90% (Krishna 2005;Xin and Geng 2011;Xu et al. 2020). In addition to moisture content, mycelia morphology and energy&mass transfer also are responsible for the differences in enzyme activity between the two fermentation processes (Xin and Geng 2011; Krull et al. 2013). However, there is no universal regularity applies to all fungi or bacteria. On the other hand, the optimum initial pH of fungal laccase activity is around 5 and kept stable at about 6 (Patel and Gupte 2016). The pH change during Lac production of Cerrena unicolor GC.u01 basically followed this characteristic.
Xylose is the main monomer of straw hemicellulose, cellulose can also be further , and the maximum lignin degradation ratio was 34.3% in the present study, which was not proportional to its high enzyme activity for the following reasons: the complex structure of lignin, the complex enzymatic system involved in its degradation, and the low activities of MnP and Lip. However, the high laccase activity demonstrated the potential application of Cerrena unicolor GC.u01 in laccase production, Cerrena unicolor GC.u01 could collaborate with other bacteria or fungi to improve the lignin degradation rate.
As shown in Table 5 Mostly, solid-state fermentation has more advantages in enzymes production than submerged processes using agro-industrial residues as carbon source. Moreover, enzymes produced by solid-state fermentation are less inhibited by the substrate, temperature and pH (Holker and Lenz 2005; Barrios-Gonzalez 2012). In brief, the types of stalk and the fermentation method showed signi cant in uence on the lignocellulosic activity of fungi. And Cerrena unicolor GC.u01 could be employed as an potential producer of the industrially enzyme laccase in solid-state fermentation bioreactor. In summary, the end-product of laccase catalyzed oxidation is H 2 O without other toxic byproducts, thus laccase is considered as "green catalyst ", which brings it broad application prospects. Up to now, some achievements have been made in laccase research, but no organism can be really used in large-scale industrial production. In this work, the Cerrena unicolor GC.U01 can secrete a higher yield of laccase when using abundant corn stalk as carbon source for solid fermentation, the reaction conditions are mild, which provides a reference for the industrialized production of laccase.

Conclusion
Cerrena unicolor GC.u01 is a newly screened white rot fungus in the present work, which is capable of degrading ligninby highly activity laccase. The activity of laccase produced by this strain was in uenced by the culture methods and straw types, and the lignin degradation rate was up to 34.3%. More studies on conditions optimization and enzymatic properties are still necessary for application of Cerrena unicolor GC.u01 in the future to degrade agricultural waste and dyestuff, treat sewage, produce laccase as an industrial microorganism and so on.

Materials And Methods
Enrichment and identi cation of fungus strain 1 g solid-sample collected from birch forest was dissolved with 10 mL sterilized redistilled water and leached for 24 h, leaching solution was diluted 10, 100 and 1000 times before cultured on PDA plate. When the fungi appeared, different strains were isolated, and repeated plate streaking was carried out at 28℃ for about 3 days until puri ed isolates were obtained (Wang et al., 2018). The puri ed colony were selected for further studies.
Genomic DNA of a puri ed strain GC.u01 was extracted and the ampli cation of its 18S rDNA gene conducted with the universal primers (Forward-GTAGTCATATGCTTGTCTC; Reverse-TCCGCAGGTTCACCTACGGA). The polymerase chain reaction (PCR) was performed with PCR mix kit (2×pfu Master mix, GK8008, Shanghai Generay Biotech Co., Ltd). PCR product were examined by electrophoresis with 1% (w/v) agarose gel including 0.5 mg·mL -1 ethidium bromide, then puri ed and sent to sequence (Sangon Biotech Co., Ltd). The partial 18S rDNA sequence data was submitted to GenBank database for blasting (Access number: MW150799). Several similar sequences were obtained to construct phylogenetic tree using the neighbor-joining method (N-J) by MEGA 5.0 (Tamura et al. 2011).

Determination of cellulose components in straw
Dried stalks collected from farmlands around Ji'nan was ground and passed through 20 meshes to obtain the powder sample. The contents of hemicellulose, cellulose and lignin were determined according to previous report ).

Measurement of ligninolytic enzymes and data analysis
For the submerged fermentation experiment, the puri ed isolation GC.u01 was inoculated into 250 mL conical ask with 50 mL liquid fermentation medium and 2.15 g stalks. The fermentation was carried out at 28℃ in a rotary shaker (150 rpm). For the solid-state fermentation experiment, the reaction system with 18 mL medium and 5 g stalks was placed under the same condition with that of submerged fermentation. Three parallel samples were set for each test.
For enzyme activity assay, culturing samples were centrifuged at 6000 rpm for 10 min to remove The enzyme activity for submerged fermentation was calculated as Eq (1) , and Eq (2) was employed for the solid-fermentation.
Where the Vt and Vs represent the total volume of measurement system (3 mL) and volume of supernatant added to the reaction (0.15 mL), respectively. The extinction coefficient ε for Lac, MnP and LiP are 3.6×10⁴, 9.3×10³ and 6.5×10³ L·mol -1 ·cm -1 , and reaction time t are 1, 2 and 2 min, respectively. n = 1. M means moisture content.
The carboxymethyl cellulase (CMCase) and xylanase activity were determined according to the reports (Wood and Bhat 1988; Tabka et al. 2006).
The lignin degradation rate was calculated by Eq (3)