Fungi-bacteria composite microecologics and methods for preparing and using the same

Abstract

A fungi-bacteria composite microecologies, including: ester-degrading fungi including Trichoderma sp. LW-1 which has been deposited in China Center for Type Culture Collection (CCTCC) with an accession number: CCTCC NO. M2014176 and Aspergillus sp. HD-2 which has been deposited in CCTCC with an accession number: CCTCC NO. M2014175; an alkene-degrading fungus including Ophiostoma sp. LLC which has been deposited in CCTCC with an accession number CCTCC NO. M2014531; a BTEX-degrading bacterium including Zoogloea sp. HJ1 which has been deposited in CCTCC with an accession number: CCTCC NO. M2012235; and a chlorinated hydrocarbon-degrading bacterium including Pandoraea sp. FLX-1 which has been deposited in CCTCC with an accession number: CCTCC NO. M2011242.

The invention claimed is: 
     
         1 . A fungi-bacteria composite microecologies, comprising:
 a) ester-degrading fungi comprising  Trichoderma  sp. LW-1 which has been deposited in China Center for Type Culture Collection (CCTCC) with an accession number: CCTCC NO. M2014176 and has a DNA sequence represented by SEQ. ID. NO. 1 and  Aspergillus  sp. HD-2 which has been deposited in CCTCC with an accession number: CCTCC NO. M2014175 and has a DNA sequence represented by SEQ. ID. NO. 2;   b) an alkene-degrading fungus comprising  Ophiostoma  sp. LLC which has been deposited in CCTCC with an accession number CCTCC NO. M2014531 and has a DNA sequence represented by SEQ. ID. NO. 3;   c) a BTEX-degrading bacterium comprising  Zoogloea  sp. HJ1 which has been deposited in CCTCC with an accession number: CCTCC NO. M2012235 and has a DNA sequence represented by SEQ. ID. NO. 4; and   d) a chlorinated hydrocarbon-degrading bacterium comprising  Pandoraea  sp. FIX-1 which has been deposited in CCTCC with an accession number: CCTCC NO. M2011242 and has a DNA sequence represented by SEQ. ID. NO. 5;   wherein
 both  Zoogloea  sp. HJ1 and  Pandoraea  sp. FLX-1 are bacteria; and 
   Ophiostoma  sp. LLC,  Trichoderma  sp. LW-1, and  Aspergillus  sp. HD-2 are fungi. 
   
     
     
         2 . The composite microecologies of  claim 1 , wherein the composite microecologies is in the form of a solid powder, and a number of live bacteria/fungi per gram of the composite microecologies reaches between 10 8  and 10 9 . 
     
     
         3 . A method for preparing the fungi-bacteria composite microecologies of  claim 1 , the method comprising:
 1) inoculating liquid culture media containing inorganic salts using seed culture tube slants of  Zoogloea  sp. HJ1 and  Pandoraea  sp. FLX-1, respectively, for activation, in which, toluene and dichloromethane are provided as sole carbon sources, respectively; and conducting high-density fermentation in fermenters after activation;   2) inoculating a potato dextrose agar (PDA) culture medium using a seed culture plate of  Ophiostoma  sp. LLC for activation, conducting high-density fermentation in a fermenter, in which, α-pinene is provided as a sole carbon source;   3) mixings strains obtained from the high-density fermentations of 1) and 2), sterilizing a solid-state fermentation (SSF) culture medium, inoculating the SSF culture medium with mixed strains for solid-state fermentation, controlling a fermentation temperature of between 30 and 40° C. and a fermentation time of between 24 and 60 hrs;   4) vacuum drying a product obtained from the solid-state fermentation of 3), controlling a drying temperature at 40° C. and a drying time at between 24 and 48 hrs; and grinding a resulting product into a powder after drying;   5) inoculating PDA culture media with seed culture plates of  Aspergillus  sp. HD-2 and  Trichoderma  sp. LW-1 for activation, respectively; inoculating an improved Czapek Dox culture plate containing butyl acetate and an improved Czapek Dox culture plate containing ethyl acetate with activated  Aspergillus  sp. HD-2 and  Trichoderma  sp. LW-1, respectively, and acquiring a large amounts of spores respectively from the Czapek Dox culture plates after between 3 and 5 d cultivation; and   6) evenly mixing the powder obtained from 4) and the spores obtained from 5) at a weight ratio of (3-5):1, whereby obtaining a composite microecologies.   
     
     
         4 . The method of  claim 3 , wherein
 the liquid culture medium containing inorganic salts and culture media in the fermenters of 1) comprise: 0.376 g/L of KH 2 PO 4 , 0,456 g/L of K 2 HPO 4 , 0.48 g/L of (NH 4 ) 2 SO 4 , 0.68 g/L of NaNO 3 , 0.25 g/L of Mg(NO 3 ) 2 , 0.011 g/L of CaCl 2 .2H 2 O, trace elements (0.06 g/L of MnCl 2 .H 2 O, 0.088 g/L of ZnCL 2 , 0.01 g/L of KI, 0.1 g/l of NaMoO 4 .2H 2 O, and 0.05 g/I, of H 3 BO 3 , and pH values thereof are between 7.0 and 7.2;   the liquid culture medium containing the inorganic salts and the culture media in the fermenters are performed with moist heat sterilization at a temperature of 121° C. for between 30 and 40 min;   toluene and dichloromethane are supplied for  Zoogloea  sp. HJ1 and  Pandoraea  sp. FLX-1 as the carbon sources in activation and high-density fermentation, respectively; and   temperatures for the activation and fermentation cultivation of strains in 1) are controlled at between 30 and 35° C. and dissolved oxygen contents are controlled at between 2 and 3 mg/L.   
     
     
         5 . The method of  claim 3 , wherein
 the PDA culture medium in 2) comprises: 200 g/L of a potato, 20 g/L of glucose (or sucrose), and 20 g/L of an agar, and a pH value thereof is 6.5;   a culture medium in the fermenter in 2) comprises: 2.0 g/L of NH 4 Cl, 0.47 g/L of Na 2 HPO 4 , 0.45 g/L of KH 2 PO 4 , 0.5 g/L of MgSO 4 , 0.01 g/L of anhydrous CaCl 2 , and trace elements (0.001 g/L of Mn 2+ , Fe 2+ , Cu 2+ , and Zn 2+ , respectively), a pH value thereof is between 4.2 and 4.6, and a carbon source thereof is α-pinene;   both the PDA culture medium and the culture medium of the fermenter in 2) are performed with moist heat sterilization at a temperature of 121° C. for between 30 and 40 min;   the activation and the fermentation cultivation of  Ophiostoma  sp. LLC 2) are conducted at temperatures of between 30 and 35° C.; and   a dissolved oxygen concentration during the fermentation cultivation is controlled at between 2 and 3 mg/L.   
     
     
         6 . The method of  claim 3 , wherein
 the SSF culture medium of step 3) comprises a solid state composite comprising between 45 and 50 wt. of wheat bran, between 25and30wt. % of a sawdust, and between 25 and 30 wt. % of and a powdered activated carbon;   an aqueous solution having a volume of between and 2 times of that of the solid state composite is added to yield a mixture;   the aqueous solution comprises: 20 g/L of a yeast extract, 20 g/L of a potato, and 5 g/L, of NaCl;   a pH value of the mixture is regulated to be between 6.8 and 7.2; and   the mixture is conducted with moist heat sterilization at a temperature of 121° C. for between 30 and 40 mm and then cooled to obtain the SSF culture medium.   
     
     
         7 . The method of  claim 3 , wherein an inoculum of the mixed strains in 3) is between 5 and 20%. 
     
     
         8 . The method of  claim 3 , wherein
 in step 5), the improved Czapek Dox culture plates comprises: 3 g/L of NaNO 3 , 0.5 g/L of MgSO 4 , 0.5 g/L of KCl, 0.01 g/L of FeSO 4 , and 20 g of an agar, and pH values thereof is between 6.0 and 6.5;   butyl acetate and ethyl acetate are supplied as carbon sources for  Aspergillus  sp. HD-2 and  Trichoderma  sp. LW-1, respectively; and   the improved Czapek Dox culture plates are conducted with moist heat sterilization at a temperature of 121° C. for between 30 and 40 min.   
     
     
         9 . The method of  claim 3 , wherein the composite microecologies Obtained from 6) is solid powder state and is adapted to maintain viabilities thereof after preservation on at room temperature or a temperature of 4° C. for more than 45 d. 
     
     
         10 . A method for treating waste gas comprising chlorinated hydrocarbons, alkenes, aromatic hydrocarbons, and esters, the method comprising applying the fungi-bacteria composite microeeologics of  claim 1 , wherein
 the fungi-bacteria composite microecologies is directly added to an inoculation sludge in a reactor; and   an addition of the fungi-bacteria composite microecologies in controlled to be between 0.5 and 2 kg per cubic meter of a filler.