Isolation,Identification And Coal Degradation Of Schizophyllum Commune From Coalbed Sediment Down To 1924 M Below The Ocean Floor

Subseafloor sediments are the largest carbon sink on Earth and play an important role in ocean and atmospheric chemistry,and global climate.In recent years,studies of fungal groups in subseafloor sediments gradually increased,but the depth is limited(<1626mbsf,meters below seafloor),and has not yet been involved nutrient sources and functions of deeper fungal life.Here we isolated and identified the culturable fungi and studied its ability to degrade the coal from lignite-associated sediment sample obtained by the drilling vessel Chikyu from 1924 meters below seafloor(mbsf)at Site C0020(41°10'35"N,142°12'01"E)off the Shimokita Peninsula,Japan during the Integrated Ocean Drilling Program(IODP)Expedition 337.A white filamentous fungal strain was successfully isolated based on culture-dependent method(Isolation medium,IM)under anaerobic conditions from the core sample C0020A-15R-5,and identified to be Schizophyllum commune belonging to phylum of Basidiomycota,Agaricales,Schizophyllaceae,Schizophyllum based on morphological observation and rRNA sequence analysis.The growth characteristics of strain 15R-5-F01 were compared with that of the ocean originated strain MCCC 3A00233 and the terrestrial strains CFCC 7252 under different conditions.The optimal condition for S.commune 15R-5-F01 growth is 30?,2.92%salinity and anaerobic.The growth curve of strain 15R-5-F01 is similar to the terrestrial strains of S.commune CFCC 7252.This result indicated that S.commune 15R-5-F01 could adapt to the in situ environment and might be alive in the subseafloor environment.Meanwile,the strain 15R-5-FO1 might be derived from the terrestrial environment or deposited with plant residue in the ancient time.Coal liquefaction was observed by spreading coal powders on the mycelia which were cultured in the medium for 3 days.Detection using light microscopy(400×)showed that the lignite powders were closely attached on the mycelia of strain 15R-5-F01.These results suggest that the strain 15R-5-F01 has potential to degrade lignite.Degradation of Xinjiang lignite by S.commune 15R-5-F01 was determined by using flask shaking method under different conditions for 20 days.Glucose whether adding to the media or not as well as the culture condition whther aerobic or anaerobic did not significantly affect lignite degradation,the average degradation rate is 15%.However,glucose did affect the biomass of the fungus in the experiments,0.15g in the media with glucose but 0.01 g in the media without glucose if the fungus were cultured under anaerobic condition.The changes of the surface structure in the process of lignite degradation were detected using scanning electron microscopy(SEM),elemental analysis and Fourier transform infrared spectral analysis(FTIR).The fungus could package the lignite on the surface and corrode it,resulting in the decrease of C and H concents by 1.68%-3.31%and 0.64%-0.69%respectively,and reduction of aromatic compounds in lignite surface.GC-MS analysis showed that the intermediates of lignite degradation mainly were 1,4-Anthracenedione,2,3-dihydro-9,10-dihydroxy,n-Hexadecanoic acid and 2,6-Dimethylbenzaldehy.Moreover,the lignite could be completely minerized to the final product of CH4(2.84mg/m3)based on GC analysis.The degradation of lignite by S.commune 15R-5-F01 varied under aerobic and anaerobic conditions.At aerobic condition,the degraded lignite showed the following phenomena:C content decreased by 3.31%,free hydroxyl(3650cm-1)increased a little bit,and 3 intermediate compounds observed in the metabolites based on GC-MS analysis.However,at anaerobic condition,the lignite showed the following phenomena:C content significantly decreased by 1.68%hydroxyl with hydrogen bonds(3200-3500cm-1)reduced and more than 10 intermediate compounds were detected.The same methods were used to test if the fungus can degrade the higher rank coal-Wannan bitumite.Results showed that the given bitumite could be degraded by the fungus.Glucose in the media and oxygen in the culture condition did not affect the degradation capacity(6.5%-9%)of S.commune 15R-5-F01.However,glucose had a great influence on the biomass of S.commune 15R-5-F01 in the process of degradation of bitumite.The biomass was 0.01 g in the media without addition of glucose,which was significantly lower than the biomass of 0.08-0.12g in the media supplended with glucose.After degradation under aerobic and anaerobic conditions,the content of C and H in bitumite decreased by 0.67%-1.45%and 0.13%-0.25%,respectively,and hydroxyl(3200-3500cm-1)increased based FTIR analysis.The activities of ligninolytic enzymes have been screened and measured at the end of degradation process of coal.Results showed that the coal degradation by S.commune 15R-5-F01 mainly depend on the production of MnP and Lip,and weak Laccase.Moreover,the metabolism of coal by the fungus is nothing to do with addition of glucose in the media.Creactivity:(1)Fungus was firstly observed in the coalbed below ocean floor down to 1924m,which was the new record of fungal distribution in the deep biosphere in the world.(2)It was the first report about S.commune,which inhabited in the sediment below the ocean floor.(3)This was the first report about S.commune having the potential in coal degradation under both aerobic and anaerobic conditions.To be summarized,we believe that fungi are the important components in the deep biosphere and play key roles in carbon cycle of in the subseafloor envionments.