Screening And Application Of High-yielding Iogo-cellulase Stains In Producing Single Cell Protein(SCP)Fodder

Lignocelluloses are abundant source in the world and can be converted to makeethanol, methane, single cell protein and so on. This method can not only make fulluse of this natural resource, but also solve the environmental problems which arecaused by abandoning this material at random. The conversion of Lignocelluloses isthe most important step to realize reasonable use of them which includes pretreatmentand enzymolysis of Lignocelluloses. This paper mainly studies these two aspects.The corn straws were pretreated in high pressure cooker. The conditions ofpretreatment were investigated and the optimum conditions were determined: dosageof NaOH is14%, solid-to-liquid ratio is1:7and holding temperature is130℃1h＋140℃1h. Under this condition, the kappa number of corn straws reached the lowestlevel. the residual alkali in black liquid and the yield of corn straw were reasonable.The kappa number was12.43and the yield is56.7%. Then the pretreated cornstraws were hydrolyzed by cellulase and xylanase. The hydrolysis processconfirmed the results of pretreatment. Then the conditions of hydrolysis wereinvestigated and the optimum conditions were determined.100mL pretreated cornstraws were put into250mL flask, its concentration is3%, dosage of cellulase is100U/g, dosage of xylanase is600U/g and pH is4.8. The pretreated corn strawswere hydrolyzed for72h at48℃. Under this condition, the concentration of reducingsugar of enzymatic liquid reached25.6mg/mL. The cellulase production strain T.viride8140, which was stored at lab of Enzymeengineering of OUC, was treated with UV and DES. Taking CMC-Na as the solecarbon source, one mutant strain was screened on Congo red medium. Itstransparent zone was larger then the residual and its Hc reached2.1. After manytimes further screens, one mutant strain was obtained which grow well with highenzyme activity, numbered as T.viride8140UEUE4-80. After several passages, itsCMC-ase reached22.79U/mL, twice more then the original strain. FPA reached2.56U/mL,1.5times more then original strain. The xylanase reached38.09U/mL,1.3times more then original strain. The stability of T.viride8140UEUE4-80wasstudied. The results showed that there is a little declination in its enzyme activity,which indicated that T.viride8140UEUE4-80can be used for future research.Under liquid cultivation, the culture medium, which includes carbon sources,nitrogen sources, pH, metal ion and surface-active agent, was studied and the optimalwas decided.1000mL nutritive salt((NH4)SO42.0g/L, KH2PO42.0g/L, Urea0.3g/L,Peptone0.75g/L, CaCl20.3g/L, MgSO4·7H2O0.3g/L, FeSO4·7H2O5.0mg/L,MnSO4·H2O1.6mg/L, ZnSO4·7H2O1.4mg/L, CoCl22.0mg/L), Avicel10g/L, bran10g/L, Twain-802mL/L, pH6. After the optimum medium was determined, thedetermination of the optimum conditions of liquid fermentation was followed. Thebest condition was decided as: culture temperature is28℃, liquid medium volume is50mL/300mL, shaking speed is180r/min, inoculated with spores, inoculum dose is5%. Under this condition, the CMC-ase, xylanase and FPA of the mutantrespectively reached24.91U/mL,2.81U/mL and105.5U/mL.Under solid fermentation, the enzyme activity of the mutant strain was studied.At first, the optimum solid medium was obtained through investigating thecarbon resources, nitrogen resources and metal ion. The ingredients of the mostproper medium are: carbon ratio is6:3:1(bran/pretreated corn straw/soybean mealw/w/w), KH2PO40.3%,(NH4)SO40.45%, Urea0.15%, CaCl20.15%, MgSO4·7H2O0.2%.Secondly, through a series of experimental studies, which include moisturecontent, loading volume, fermentation temperature and inculum dose. The best solid conditions for enzyme production were as follows: solid/liquid is1:2,20g solidmedium was put into500mL triangular flask, inoculated with10%inculum dose,cultivated for4days under32℃. Under this condition, the CMC-ase, xylanase andFPA of the mutant respectively reached47.04U/g,10.07U/g and1015U/g.Finally, basing on the conditions of solid fermentation in triangular flask,extensive cultivation was performed in a box which was made by our lab. Themutant strain grew well. After4days cultivation, the CMC-ase reached43.3U/g,FPA reached8.2U/g, xylanase reached994U/g. The results indicated that followingtraining can be performed to realize mass production.