Patent Publication Number: US-2022220016-A1

Title: Manganese-oxidizing fungus and uses thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the priority benefit of China Application No. 202110048920.0, filed on Jan. 14, 2021. The entirety of the above-mentioned patent application is hereby incorporated by reference herein and made a part of this specification. 
     BACKGROUND 
     Technical Field 
     The present disclosure relates to the technical field of environmental microorganisms, and in particular to a manganese-oxidizing fungus and uses thereof. 
     Description of Related Art 
     In recent years, biological manganese oxides have good adsorption and oxidation capacity; compared with chemosynthetic manganese oxides, biological manganese oxides have weak crystallization, small grain size, high valence state of Mn, and have many octahedral cavities in the structure thereof. Therefore, biological manganese oxides have stronger adsorption capacity, oxidation capacity and other surface activity. Moreover, biological manganese oxides are produced in eco-friendly conditions without the excessive use of energy sources. Therefore, the biological manganese oxide may be regarded as a cost-effective and environment-friendly material, and used for the remediation of the polluted environment. 
     The formation mechanism of manganese oxides may be divided into a chemical origin and a biogenic origin. Comparatively speaking, the biogenic origin believes that microorganisms are mainly mediated by Mn oxidization process in natural world; and the biologically mediated Mn oxidization rate is several times faster than the chemo-catalysis rate, and even tens of thousands times above. Up to now, numerous types of microorganisms have been found to have Mn oxidation capacity; and these manganese oxides are widely distributed in the wild, and manganese-oxidizing bacteria in the continental ecosystem mainly exist in soils, deposits, water pipes, desert varnish, caves and ores. Manganese-oxidizing bacteria in freshwater system mainly exist in hot springs of fresh water, rivers, streams, ponds, Mn-enriched surface films in shallow lakes, iron-manganese concretion, deposits in fresh water, aerobiotic/anoxic interfaces of bays. 
     Bacteria and fungi are regarded as important Mn transforming agents to oxidize Mn 2+  into Mn (III, and IV) and to produce nanoscale biological manganese oxides having poor crystallization. Currently, Mn-related microbial oxidization is mainly focused on bacteria, including the removal of heavy metals in a polluted environment by a manganese-oxidizing bacterium, structure features of a product formed by manganese oxidization via a manganese-oxidizing bacterium, a manganese oxidization mechanism of a manganese-oxidizing bacterium, and the like. For example, a patent CN112094766 A discloses a manganese oxidizing bacterium; while researchers have scarcely known about the contribution of manganese-oxidizing fungi to the transformation of manganese oxides in the environment and influences thereof on the fate of inorganic substances in the environment. However, compared with bacteria, filamentous fungi are more superior to oxidizing manganese in the environment: (1) the growing ability of fungi within a wide pH range; (2) the resistance of fungi to high-concentration toxic metals and organic pollutants. Many fungi capable of oxidizing manganese (II) can be separated from soils, rock surfaces, sediments, sludge, fresh water and other environment, including Acridine,  Alternaria, Cladosporium , Anapiculatisporites,  Curvularia, Penicillium  and the like. The studies on manganese-oxidizing fungi are mainly focused on  Acremonium  sp. and  Phanerochaete chrysosporium . By biosorption and oxidization of these manganese-oxidizing fungi, manganese ions Mn (II) in water are allowed to form biological manganese oxides. These findings bring a brand-new idea for the biological removal of manganese, oxidization and adsorption of pollutants in water (such as, heavy metals, and trace organic pollutants). Therefore, the process is a novel biological treatment method of manganese-containing waste water, and has a broad application prospect. Nutritional deficiency and competitive effects of microorganisms are present in natural hydrological conditions, which will influence the survival and manganese oxidation activity of strains. Therefore, it is very important to seek manganese-oxidizing strains suitable for natural hydrological conditions. 
     SUMMARY 
     The objective of the present disclosure is to provide a manganese-oxidizing fungus and uses thereof, thus solving the above problems. The manganese-oxidizing fungus in this present disclosure belongs to  Cladosporium  sp., and can be suitable for complex natural water bodies and exert manganese-oxidizing effects, and has very good application prospects. 
     The manganese-oxidizing fungus disclosed in the present disclosure has high Mn 2+  oxidization efficiency within a scope of temperature (15-30° C.) and a scope of neutral pH (6.0-7.5). 
     The objective of the present disclosure is achieved by the following technical solution: 
     A manganese-oxidizing fungus is provided, and the manganese-oxidizing fungus is  Cladosporium  sp. XM01, and has been preserved in China General Microbiological Culture Collection Center (accession number: CGMCC NO. 21083). 
     The manganese-oxidizing fungus of this present disclosure is obtained by domesticating, separating and purifying the soil derived from the nearby Xiangtan Manganese Mine of Hunan Province. 
     ITS rRNA sequencing results of XM01 in the present disclosure is led to GenBank database of NCBI for homology comparison. The results indicate that the maximum similar strain is  Cladosporium  sp. having a similarity of 99%. Therefore, the strain can be judged to be  Cladosporium  sp. and named  Cladosporium  sp. XM01. 
     The manganese-oxidizing fungus in this present disclosure grows and has Mn 2+  oxidation activity under the conditions that a temperature range is 15-30° C., a pH range is 6.0-7.5, and preferably 7, and a Mn 2+  concentration is not higher than 800 μM. Mn 2+  removal rate is up to 99.9% and manganese oxidation rate is up to over 80%. 
     The manganese-oxidizing fungus in this present disclosure lives in a non-sterilized water body or a solid matrix and exerts Mn 2+  oxidization activity. 
     The manganese-oxidizing fungus in this present disclosure is used in the treatment of toxic metallic elements or trace organic pollutants to remove Mn 2+  in a water body or a solid matrix, or used in a preparation of a microbial agent, or used in a preparation of an adsorbent for metallic elements. The water body includes industrial wastewater, domestic wastewater, underground water and tap water; and the solid matrix includes a soil and a deposit. 
     A gene sequence of a manganese-oxidizing fungus, is shown in SEQ ID NO.1. 
     A microbial agent, includes a  Cladosporium  sp. XM01 strain with the accession number of CGMCC NO. 21083 as an active ingredient. 
     A method for removing Mn 2+  from a water body or a solid matrix, including the following steps of: inoculating the manganese-oxidizing fungus or a microbial agent including the manganese-oxidizing fungus onto a water body or a solid matrix for culturing for a proper period under the conditions of 15-30° C. and pH=6.0-7.5, where the water body includes industrial wastewater, domestic wastewater, underground water and tap water; and the solid matrix includes a soil and a deposit. 
     Compared with the prior art, the present disclosure has the following advantages: 
     (1) Low manganese oxidization cost: during the overall oxidization, a conventional fungal PYG medium is used to achieve high-density activization of a strain; moreover, in later oxidizing process, trace trophic factors can achieve better oxidization effect without complex and expensive drugs and apparatus. 
     (2) Great application potential: the independently screened manganese-oxidizing strain has better Mn 2+  oxidation capacity; the high-valent insoluble manganese oxides produced have a higher value of research in the aspects, such as catalytic oxidation, degradation, adsorption of water-borne pollutants (e.g., heavy metals, and organic pollutants); therefore, the strain is considered to be used in remediation and remediation of water environment and soils. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a phylogenetic dendrogram of  Cladosporium  sp. XM01 in this present disclosure based on ITS rRNA genes. 
         FIG. 2  shows electrophoretogram of  Cladosporium  sp. XM01 in this present disclosure (DL2000 band distribution: 2000, 1000, 750, 500, 250, and 100 bp). 
         FIGS. 3 a -3 c    shows a SEM graph (2kX) of biological manganese oxides produced by 10  Cladosporium  sp. XM01 in this present disclosure and energy spectrum analysis corresponding to markers, where the box portion denotes the markers ( FIGS. 3 b  and 3 c   ,  FIG. 3 b    denotes marker 1 and  FIG. 3 c    denotes marker 2). 
         FIG. 4  shows Mn 2+  adsorption and oxidization properties (initial concentration is 200 μM) of  Cladosporium  sp. XM01 in this present disclosure. 
         FIG. 5  shows a Mn 2+  oxidization curve of  Cladosporium  sp. XM01 in this present disclosure at different concentrations of manganese. 
         FIG. 6  shows Mn 2+  oxidation activity of  Cladosporium  sp. XM01 in this present disclosure at different temperature. 
         FIG. 7  shows Mn 2+  oxidation activity of  Cladosporium  sp. XM01 in this present disclosure at different pH values. 
     
    
    
     DESCRIPTION OF THE EMBODIMENTS 
     The present disclosure will be further described in details with reference to detailed examples; and those skilled in the art should understand that the present disclosure is not limited to these detailed examples. 
     Unless otherwise specified, methods in the following examples should be conventional methods; and the reagents used therein are conventional reagents available in the market. 
     Example 1 
     Isolation and Purification of a Manganese-Oxidizing Fungus  Cladosporium  sp. XM01 
       Cladosporium  sp. XM01 was obtained by sampling, domesticating, separating and purifying the soil derived from the manganese ore stacked in Xiangtan Manganese Mine of Hunan Province. Specific steps were as follows: 
     (1) Preliminary screening: black brown soil was collected from the nearby Xiangtan Manganese Mine of Hunan Province; and 2 g soil was taken and added to an HAY medium sterilized by high temperature (0.246 g/L sodium acetate; 0.15 g/L yeast powder; 0.05 g/L magnesium sulfate heptahydrate; 5 mg/L dipotassium phosphate; 2 mL/L mineral salts; where per liter of mineral salts included the ingredients below: 3.7 g calcium chloride dihydrate; 0.44 g zinc sulfate heptahydrate; 0.29 g sodium molybdate dihydrate; 2.5 g boric acid; 5 mg copper sulfate pentahydrate; and 1.0 g ferric chloride hexahydrate) in a laboratory to make the content of MnCl 2  being 200 μM; a buffer solution in the medium was HEPES having a final concentration of 20 mM and pH of 7.0; where the MnCl 2  solution and HEPES buffer solution were filtered by a 0.22 μm filter membrane and sterilized for addition. The above soil was domesticated for 7 d every time on a 170 rpm table in dark place at 25° C., after the domestication, 5 mL domesticated fungal solution was taken and added onto a freshly-prepared 45 mL HAY medium for continuous domestication for 4 times in total. 
     (2) Secondary screening: after the domestication, the fungal solution was diluted 10 −1  to 10 −7  folds by sterilized tap water; 0.1 mL diluent was respectively taken and coated on a solid medium containing 100 μM Mn 2+  (prepared by adding 2% agar to the above liquid HAY culture medium) for isolation, and cultured in the dark at 25° C. After obvious colonies grew on the medium, a single colony forming brown substances thereon was picked and marked out for isolation and purification. A strain having stronger manganese oxidation capacity was determined by surveying the growth rate and Mn 2+  oxidation rate of these fungi. 
     (3) Liquid culture conditions of the  Cladosporium  sp. XM01 strain: a 150 mL HAY medium was added to a 250 mL conical flask, and an HEPES buffer solution having a final concentration of 20 mM and a pH value of 6.0-8.0 was added; and the table was configured at 170 rpm and 15-30° C. The strain cells were collected by centrifugation for 10 min at 3000-5000 r/min. 
     Example 2 
     Molecular Biological Identification of  Cladosporium  sp. XM01 
       Cladosporium  sp. XM01 was extracted by a fungal genome kit (purchased from Omega, Code no. D3390-02), and subjected to PCR amplification by using universal primers ITS1(TCCGTAGGT GAACCTGCGG) and ITS4(TCCTCCGCTTATTGATATGC) for fungi ITS rRNA genes. PCR reaction system: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 10 × Ex Taq buffer 
                 2.0 
                 μl 
               
               
                   
                 5 u Ex Taq 
                 0.2 
                 μl 
               
               
                   
                 2.5 mM dNTP Mix 
                 1.6 
                 μl 
               
               
                   
                 5 p Primer 1 
                 1 
                 μl 
               
               
                   
                 5 p Primer 2 
                 1 
                 μl 
               
               
                   
                 DNA 
                 0.5 
                 μl 
               
               
                   
                 ddH 2 O 
                 13.7 
                 μl 
               
               
                   
                 Total volume 
                 20 
                 μl 
               
               
                   
                   
               
            
           
         
       
     
     PCR experiments were performed using the following program: initial denaturation at 95° C. for 5 min, followed by 25 cycles of denaturation at 95° C. for 30 s, primer annealing at 56° C. for 30 s and extension at 72° C. for 30 s. A final long extension was at 72° C. for 10 min. 
     Samples were sequenced via the amplified PCR product by Majorbio. Sequences obtained by sequencing were as shown in SEQ ID NO.1, and were compared to the sequences in database by BLAST on line; sequences having greater than 97% similarity were selected as reference sequences; and a phylogenetic tree of fungal system was constructed by Neighbor-Joining with Mega4.0 software ( FIG. 1 ). 
     Example 3 
     Scanning Electron Microscope (SEM) and Energy Dispersive X-Ray Detector (EDX) of  Cladosporium  sp. XM01 
     The isolated  Cladosporium  sp. XM01 was inoculated into a Mn 2 -containing liquid HAY culture medium (inoculum size was 1×10 5  conidia/mL); and cultured in the dark for 3 d at 25° C. and 170 rpm to collect biological manganese oxides; the biological manganese oxides were subjected to SEM and EDX. The results of SEM combined with EDX were as shown in  FIG. 3  and table 1; marker 1 denoted an aggregate containing manganese oxides; marker 2 denoted manganese oxide-free mycelium surface; higher content of manganese oxides was detected in the marker 1 of the  Cladosporium  sp. XM01 strain cells, being up to 30.81%; compared with marker 2, the manganese content increased by 25.68%. Thus, it can be seen that the strain XM01 could form manganese oxide aggregates on the epispore, and may participate in the manganese oxidation process via protein factors distributed on the epispore. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 EDX results 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Mass percentage 
                   
                 Atom percentage 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Element 
                 Marker 1 
                 Marker 2 
                 Marker 1 
                 Marker 2 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 C K a   
                 29.90 
                 52.52 
                 45.89 
                 61.59 
               
               
                   
                 N K a   
                 3.23 
                 7.16 
                 4.26 
                 7.20 
               
               
                   
                 O K a   
                 32.19 
                 32.51 
                 37.09 
                 28.62 
               
               
                   
                 Na K a   
                 0.39 
                 0.19 
                 0.31 
                 0.12 
               
               
                   
                 Mg K a   
                 0.41 
                 0.22 
                 0.31 
                 0.13 
               
               
                   
                 Al K a   
                 0.39 
                 0.22 
                 0.26 
                 0.11 
               
               
                   
                 Si K a   
                 0.25 
                 0.13 
                 0.16 
                 0.06 
               
               
                   
                 P K a   
                 1.57 
                 1.32 
                 0.93 
                 0.60 
               
               
                   
                 S K a   
                 0.45 
                 0.37 
                 0.26 
                 0.16 
               
               
                   
                 K K a   
                 0.41 
                 0.23 
                 0.19 
                 0.08 
               
               
                   
                 Mn K a   
                 30.81 
                 5.13 
                 10.34 
                 1.31 
               
               
                   
                   
               
               
                   
                 Note: 
               
               
                   
                 K a  referred to energy excited by an atomic layer K. 
               
            
           
         
       
     
     Example 4 
     Studies on Biological Oxidation Properties of the  Cladosporium  sp. XM01 Strain to Mn 2+   
     The Mn 2+  oxidation and adsorption capacity of the manganese-oxidizing fungus ( Cladosporium  sp. XM01) were surveyed in the Mn 2+ -adding HAY medium; it was found that the fungus in this present disclosure had good oxidation and adsorption capacity to Mn 2+  and could remove soluble Mn 2+  in the matrix completely within a short time, so that most of the Mn 2+  were transformed into water-insoluble manganese oxides. 
     Specific steps were as follows: 
     (1) Manganese Adsorption and Oxidation Properties of the Fungus in this Present Disclosure at Different Periods of Time 
     The isolated  Cladosporium  sp. XM01 was inoculated into sterilized liquid HAY culture medium (inoculum size was 1×10 5  conidia ml −1 ) containing 200 μM Mn 2+ , and cultured at 25° C. and 170 rpm for 3 days in the dark. Samples were taken at specific intervals to measure the concentration of Mn 2+  remaining in a culture solution and to collect biological manganese oxides; and then adsorption and oxidation capacity of the biological manganese oxides to Mn 2+  were measured by a two-step extraction method. 
     Samples of the culture solution were filtered by a 0.45 μm filter membrane, and then the concentration of Mn 2+  remaining in the culture solution was measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) (Agilent, 5110 series). 
     Two-step extraction method: a medium was centrifuged for 10 min at 3000 r/min to collect precipitates (including mycelia and solid-phase manganese oxides); afterwards, the precipitates were washed by deionized water and resuspended by 20 mM copper sulfate for 16 h to extract extracellularly adsorbed Mn 2+ ; and then the precipitates were washed by deionized water to thoroughly disrupt cells in the precipitates by a cell disruption method (working for 3 s at 2 h interval for 15 acoustic wave cycles in total), and the disrupted samples were treated by an extracellular adsorption method to extract Mn 2  absorbed intracellularly, and finally, treated by 50 mM hydroxylamine hydrochloride to extract the oxidized Mn. The concentration of manganese in the extractive was measured by ICP-AES (Agilent, 5110 series). 
     As shown in  FIG. 4 , the strain was sensitive to the environment and had lower oxidation capacity to manganese after being cultured for 0-24 h; and Mn (II) was mainly removed by extracellular adsorption. After being cultured for 24-48 h, the strain was up to the increased logarithmic phase and achieved a rapid increase in quantity, which was beneficial to adsorption; the adsorption rate was 13.64% at 48 h; and the strain adapted to the environment and had an ability to oxidize manganese. After being cultured for 48-66 h, the adsorption rate increased slowly, and the cell adsorption rate was up to 21.18% at 66 h; while after being cultured for 72 h, Mn (II) in the solution was basically removed, and the adsorption capacity suffered a decrease; and the adsorption rate decreased to 18.63%; this was probably because the strain rapidly increased in the logarithmic phase in quantity, and accordingly the adsorption rate increased rapidly. In a stabilization stage, the adsorption capacity tended to be stable and even decline, resulting in the slow growth of the adsorption rate. After the grain was cultured for 24 h, Mn (II) oxidation enhanced rapidly, and the manganese oxidation rate was up to 81.18% at 72 h. This finding provided that the  Cladosporium  sp. XM01 strain had high manganese oxidation capacity. The adsorption capacity tended to decline at 72 h, while the oxidation capacity was tending to increase. This was the result of oxidizing the adsorbed manganese. In a word, Mn (II) oxidation of the manganese-oxidizing fungus played a leading role in the removal of soluble Mn (II). 
     EXISTING LITERATURE 
     
         
         1. Zhang Y, Tang Y K, et al. A novel manganese oxidizing bacterium- Aeromonas hydrophila  strain DS02: Mn(II) oxidization and biogenic Mn oxides generation. Journal of Hazardous Materials, 2019, 367: 539-545. 
         2. Tang W W, Gong J M, Wu L J, et al. DGGE diversity of manganese mine samples and isolation of a  Lysinibacillus  sp. efficient in removal of high Mn (II) concentrations. Chemosphere, 2016, 165: 277-283. 
       
    
     As shown in Table 2, compared with the manganese oxidizing bacterium reported in the above existing literature, the manganese-oxidizing fungus in this present patent has a manganese removal rate of 99.9%; and the manganese oxidation rate is up to 81.18%; therefore, the manganese-oxidizing fungus of this present disclosure has significant advantages. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Comparison in manganese removal effect and oxidation 
               
               
                 effect of different manganese-oxidizing bacteria 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Manga- 
                 Manga- 
                   
               
               
                   
                   
                 nese 
                 nese 
               
               
                   
                 Manganese-oxidizing 
                 removal 
                 oxidation 
               
               
                 No. 
                 bacteria 
                 rate 
                 rate 
                 Literature 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                   Aeromonas  sp. DS02 
                 89.6% 
                 49.6% 
                 Zhang et al., 2019 
               
               
                 2 
                   Lysinibacillus  sp. 
                 94.7% 
                 55.9% 
                 Tang et al., 2016 
               
               
                 3 
                   Cladosporium  sp. 
                 99.9% 
                 81.18% 
                 The present patent 
               
               
                   
               
            
           
         
       
     
     (2) Manganese Oxidation Properties of the Fungus in this Present Disclosure Under Different Concentrations of Mn 2+   
     The Mn 2+  oxidation capacity of the manganese-oxidizing fungus ( Cladosporium  sp. XM01) in this present disclosure was surveyed in a Mn 2+ -adding medium. It was found that the fungus in this present disclosure had very good oxidation capacity to Mn 2+ , and could completely remove the soluble Mn 2+  in the matrix within a short time; and most of the Mn 2+  were transformed into water-insoluble manganese oxides. Through the culture experiments under different initial concentrations of Mn 2+ , it was found that the fungus in this present disclosure could completely remove 800 μM Mn 2+  and lower; moreover, with the increase of the Mn 2+  initial concentration, the manganese oxidation activity of the fungus tended to be lower slightly. Specific steps were as follows: 
     The isolated  Cladosporium  sp. XM01 was inoculated into sterilized liquid HAY culture medium (inoculum size was 1×10 5  conidia/mL) containing different concentrations of Mn 2+ , and cultured at 25° C. and 170 rpm in the dark. Samples of the culture solution were taken at specific intervals, and filtered by a 0.45 μm filter membrane, and then the concentration of Mn 2+  remaining in the culture solution was measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) (Agilent, 5110 series). 
     As shown in  FIG. 5 , the fungus could thoroughly remove 800 μM Mn 2+  and lower; and with the increase of the Mn 2+  concentration, the manganese oxidation activity of the fungus to Mn 2+  tended to be lower slightly; but 800 μM concentration of Mn 2+  has been much greater than the concentration of Mn 2+  in common rivers or underground water. The results indicate that the fungal flora can thoroughly oxidize the Mn 2+  within a concentration range in a common water body. 
     (3) Manganese Oxidation Properties of the Fungus in the Present Disclosure Under Different Temperature Conditions 
     Based on the studies on the manganese oxidation properties of the fungus under different culture temperature, it was found that the fungus in the present disclosure had Mn 2+  oxidation activity within a temperature range of 15−30° C.; and from the angle of practical application, it was relatively appropriate to choose 20-30° C. Specific steps were as follows: 
     The isolated  Cladosporium  sp. XM01 was inoculated into sterilized liquid HAY culture medium (inoculum size was 1×10 5  conidia/mL), and Mn 2+  having a final concentration of 200 μM and a 20 mM HEPES buffer solution having a pH value of 7.0 were added in a filtering way; and then respectively placed onto tables in the dark at 170 rpm and 15° C., 20° C., 25° C., and 30° C. Samples of the culture solution were taken at specific intervals, and filtered by a 0.45 μm filter membrane, and then the concentration of Mn 2+  remaining in the culture solution was measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) (Agilent, 5110 series). 
     As shown in  FIG. 6 , the manganese oxidation activity of the fungus decreased with the decrease of the culture temperature; and the oxidation rate was the slowest at 15° C., and faster slightly at 20° C., and up to the maximum at 25-30° C. It indicates that the most suitable oxidizing temperature of the fungus was in a range from 25 to 30° C. The fungus can exert manganese oxidation activity at a low temperature of 15° C., showing its stronger adaptability to the temperature. 
     (4) Manganese Oxidation Properties of the Fungus in the Present Disclosure Under Different pH Conditions 
     Through the oxidation experiments at different initial pH values, it was found that the most suitable pH for manganese oxidation of the fungus was 7; and the specific steps were as follows: 
     The isolated  Cladosporium  sp. XM01 was inoculated into sterilized liquid HAY culture medium (inoculum size was 1×10 5  conidia/mL), and Mn 2+  having a final concentration of 200 μM was added in a filtering way; and then pH was respectively adjusted to 6 and 6.5 by an MES buffer solution; then the pH was respectively adjusted to 7.0 and 7.5 by an HEPES buffer solution; and cultured on a table at 170 rpm in the dark. Samples of the culture solution were taken at specific intervals, and filtered by a 0.45 μm filter membrane, and then the concentration of Mn 2+  remaining in the culture solution was measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) (Agilent, 5110 series). 
     As shown in  FIG. 7 , the manganese oxidation rate was up to the maximum at pH=7; the fungus could basically oxidize Mn 2+  within a pH range of 6.0-7.5; moreover, the pH range is consistent with the pH of most of the water bodies in natural world, indicating that the fungus can exert manganese oxidation activity under pH conditions of the natural water body. 
     To sum up, researchers found that the  Cladosporium  sp. XM01 had Mn 2+  oxidation activity within a pH range of 6.0-7.5 and a temperature range of 15−30° C., and had Mn 2+  oxidation activity under the condition that Mn 2  concentration was not higher than 800 μM. 
     The above description made to the examples is convenient for those skilled in the art to understand and use the present disclosure. Apparently, those skilled in the art can readily make various amendments to these examples, and apply the general principles described herein to other examples without any inventive labor. Therefore, the present disclosure is not limited to the above examples; based on the present disclosure, improvements and amendments made by those skilled in the art without departing from the spirit and scope of the present disclosure shall fall within the protection scope of the present disclosure.