Methyl Red And Malachite Green Degradation By A Strain Schizophyllum Commune 15R-5-F01 Isolated From A Coalbed At 1.9Km Below The Seafloor

Azo and triphenylmethane dyes are widely used in textile industry.Discharge of the dye-wasted water could cause serious water pollution resulting in death of aquatic plants and animals,and balance disturbance of natural ecosystem,which directly or indirectly threaten the safety of human beings.Most of the dyes are difficult to degrade.The conventional physicochemical treatments used for dyes degradation are not only expensive and inefficient,but also easy to cause secondary pollution.However,the biological methods used for dye degradation have attracted more and more attentions since they are low cost,high efficiency and eco-friendly.The application of Integrated Ocean Drilling Program(IODP)made it possible to obtain microorganisms from deep subseafloor biosphere using advanced drilling technology.These microbes may have special application due to the unique biological and physiological traits they have.The drilling depth of IODP Expedition 337 ranged from 1200 to 2466 mbsf(meters below seafloor).Many pure cultures of fungal species have been obtaed from the sediment samples in our lab.In order to explore microbial resources in subseafloor sediments,some of the previously isolated fungi were evaluated for their degradation potential against various types of dyes.One strain named as Schizophyllum commune 15R-5-F01 showed broad and strong degradation activities against several dyes,such as methyl red(MR),neutral red(NR),reactive blue 21(RB21),malachite green(MG),and trypan blue(TB).Compared with other dyes,MR belonging to azo dye and MG of triphenylmethane dye are degraded more esily by the strain 15R-5-F01.Thus,in this study,we mainly focus on the factors that may affect the biodegradation efficiency of MR and MG by the strain,the composition of the degraded products and their biological toxicity,as well as the enzymes responsible for the degradation of the dyes.Results are as followings:1.Study on the optimal conditions of oxgen,temperature,pH,carbon and nitrogen resources on MR and MG degradation by strain 15R-5-F01 in shaking flasks using step by step optimization technique of singal factors.Results showed:(1)Oxygen did not affect the biodegradation of MR and MG by the strain with degradation rate(DR)>96%.(2)The optimal pH for MR and MG degradation was 6.0,DR>96%.(3)The optimal temperature for MR and MG degradation was 30?,DR>97%.(4)The optimal carbon substrat for MR degradation was lactose at concentration of 15.0 g/L;for MG was maltodextrin at concentration of 10.0 g/L.At these conditions,the DR of was>98%.(5)The optimal nitrogen substrat for MR degradation was bovine brain extract at concentration of 5.0 g/L;for MG was yeast extract at concentration of 2.5 g/L.At these conditions,the DR of was>97%.In summary,the optimization culture condition for MR degradation by the strain is:15.0 g/L lactose,5.0 g/L bovine brain extract,pH 6.0,30?;while for MG is:10.0 g/L maltodextrin,2.5 g/L yeast extract,pH 6.0,30?.2.Effects of salinities,metal ions and concentrations,and contents of dyes on MR or MG degradation by strain 15R-5-FO1 in in the optimal culture conditions.Results showed:(1)The concentration of salt in the culture severely affect the biodegradation capacity.At 0-9%of NaCl,the DR of MR could reach as high as 87%,while at 0-12%of NaCl,the DR of MG could reach as high as 81%.However,the DR would be reduced by increasing the salinities in the cultures.(2)The test 13 metal ions exhibited different effects on MR and MG degradation at 0.1 mM,however,Zn2+and Cu2+did not affect the DR of MR and MG,respectively.Except Zn2+and Cu2+,other ions displayed inhibitory effect on the DR of MR or MG,in particular,Cd2+strongly reduced the DR of MR and MG by 20.28%and 53.44%respectively.Further experiment revealed that Zn2+rarely influenced the DR of MR at the given culture condition at the concentration as high as 2.0 mM.Similarly,the highest tolerance of strain 15R-5-F01 to Cu2+was l.0 mM for MG degradation at the same culture condition.(3)The concentration of dyes in the culture severely affect the biodegradation capacity.At 50-500 mg/L of MR and ?250 mg/L of MG,the DR could reach as high as 91%.However,the DR would be reduced by increasing the concentration of MR or MG in the cultures.For example,at 400 mg/L of MG,the DR reduced to 43.64%,which was about 55.27%less than the DR at 100 mg/L.3.Continuous degradation ability on MR or MG by strain 15R-5-F01 in the optimal culture conditions.Results showed:(1)At the batch number of 1-3,the DR of MR could reach as high as 85%,but the DR began to decline significantly at the batch number 4,which was only 32.05%at the batch number 6.(2)At the batch number of 1-7,the DR of MG could reach as high as 98%,but the DR began to decline significantly at the batch number 8,which was only 49.69%at the batch number 10.4.Enzymatic activity test showed that MnP was mainly responsible for MR degradation,followed by Lac and LiP;while,for MG degradation,MnP and Lac played the major roles.5.Data of GC-MS analysis showed that the strain 15R-5-F01 could degrade MR into N,N-dimethyl-p-phenylenediamine,2-aminobenzoic acid,aniline and N,N-dimethylaniline;MG into 4-(dimethylamino)benzophenone and 4-dimethylaminophenol.6.Phytotoxic data indicated that both MR and MG exhibited strong inhibition against seed germination and seedling growth of Oryza sativa,Triticum aestivum and Vigna umbellata at 100 mg/L,while their degraded producton hardly had inhibition at the same experimental condition.Moreover,compoud MG also showed growth inhibition against both gram positive bacteria(Staphylococcus aureus,Bacillus subtilis)and gram negative bacterium(Pseudomonas aeruginosa),but lost its toxicity after degradation.Based on the above results,we believe that the subseafloor sediment S.commune 15R-5-F01 possess efficient degradation ability on MR and MG,which can be used as a potential biodegradation resource to treat dyes with efficient and environmentally friendly features.