Bioconversion Of Steam Exploded Eucalyptus Urophylla Wood To Single Cell Protein

Bioconversion of steam exploded wood to single cell protein is important for using the wood cutting and machining residues and settling the feed protein lacking situations.In order to make clear the techniques of bioconversion of Eucallptus urophylla wood biomass to single cell protein,this research took triennial Eucalyptus urophylla wood pretreated by steam explosion as materials,the general chemical analytic method and the advanced analytic techniques including SEM,X-ray diffraction spectrum and IR spectrum were applied to investigate the effects of steam explosion on chemical components,appearance and structure of steam exploded Eucallptus urophylla wood.The bioconversion process including cellulase fermentation,hydrolyzing by cellulase enzymes,decolorizing of hydrolysate and single cell protein fermentation were also studied,the amino acids and mineral elements in single cell protein product were analyzed.The results were as follows:1) The steam explosion pretreatment caused the relaxation between wood fiber and the breakage of pit membrane,which consequently enhanced the available surface area of enzymes,and improved the performance of biochemical reaction.The mensuration to chemical components and analysis by IR spectrum showed that steam explosion pretreatment also made part of hemicellulose and lignin dissolve, cellulose was basically not losing except little decomposing in amorphous area,it was favorable for improving enzymic hydrolysis.If pretreated by 0.2%sulphuric acid before steam explosion,Eucallptus urophylla wood fiber became smaller and thinner,the available surface area became larger,at the same time,the deligninification the breakage degree of pit membrane increased,thus could benefit the cellulase enzymes action.2) The optimized technical conditions of cellulase and xylanase fermentation using Trichoderma viride and Aspergillus niger mixed strains in exploded Eucallptus urophylla wood substrate were: inoculum dosage 6%,inoculum propotion(T.v/A.n) 1:1,substrate concentration 1.0%,temperature 28℃,initial pH 4.5,for 4d fermentation.3) The pretreatment before steam explosion to Eucallptus urophylla wood material and the steam pressure had prominent effects to cellulase and xylanase fermentation.The CMC cellulase(endo -β-1, 4 glucanase),FPase(Filter Paper cellulase) and xylanase activities improved evidently if pretreated with 0.2%sulphuric acid before explosion in the same steam pressure and the results by single factor analysis indicated that enzymatic activities differences were notable;However,if pretreated with ammonia,these three enzymatic activities reduced and the difference between treated and untreated was also notable. 4) The best cellulose hydrolyzing conditions of steam exploded Eucallptus urophylla wood with cellulase enzyme were:48 h of hydrolyzing at temperature 45～50℃,pH 4.8,substrate concentration 2 %,cellulase dosage 25 FPIU/g substrate;The best decoloration condition with active carbon were: temperature 70℃,pH 2,90 min,active carbon dosage 3.5%,and the decoloration rate and the total reducing sugar regaining was 77.02±0.52%and 80.81±0.23%respectively in best conditions.5) The pretreatment before steam explosion to Eucallptus urophylla wood material and the steam pressure had prominent effects to its cellulase hydrolysis competence.Saccharification of exploded Eucallptus urophylla wood improved evidently if pretreated with 0.2%sulphuric acid before explosion in the same steam pressure,however it reduced if pretreated with ammonia.The reducing sugar concentration in liquid hydrolysate and the saccharification rate was 12.72±0.26 g/L and 82.43%±0.017 respectively,if the material which exploded at 2.2 MPa steam pressure and pretreated with 0.2% sulphuric acid before explosion was hydrolyzed in best hydrolysis conditions.6) The analysis in high performance liquid chromatography showed that glucose,xylose, arabinose and disaccharide contents were 69.39±0.67%,27.53±0.40%,0.65±0.05%,2.44±0.88% respectively in cellulase liquid hydrolysate of exploded Eucallptus urophylla wood.Acetic acid concentration in liquid hydrolysate did not change with the reducing sugar concentration diversification, but furfural concentration enhanced with the sugar concentration increasing.There was a fall of furfural concentration if the liquid hydrolysate was decolored,but the acetic acid concentration did not reduce.7) The optimal culture medium ingredients of single cell protein fermentation were:4.0 g/L (NH₄)₂HPO₄ as the nitrogen source,0.5 g/L yeast lixiviating matter,1.0 g/L KH₂PO₄ and 0.3 g/L MgSO₄·7H₂O.The optimal inoculation fashion in shake flask were:10⁷ cell/mL inoculum concentration, the Sacchromyces cerevisive and Candida utilis 2.587 mixed(S.c:C.u = 1:3) strains fermentation and 10%inoculum dosage.The optimal technical conditions were:31～34℃temperature,initial pH4.5, 11.33～17.52g/L initial reducing sugar concentration,25mL fermentation liquid in 250mL shake flask.8) There were multiple single sugars in cellulase liquid hydrolysate of exploded Eucallptus urophylla wood,a diauxic growth and sequential subatrate utilization were observed in Sacchromyces cerevisive and Candida utilis 2.587 mixed fermentation.D-glucosse was utilized first(within 9 h after inoculation) by two strains,D-xylose and L-arabinose utilization by Candida utilis 2.587 commenced shortly before the depiction of D-glucose.D-glucose was consumed with that inoculation,the fast fall of its concentration appeared in 6～9 h after inoculation,the glucose was almostly depleted in 9 h.D-xylose reduced after glucose depletion,its complete utilization appeared 15 h after inoculation,L-arabinose utilization as well as came forth 9 h after inoculation.Furthermore,the acetic acid could be assimilated by yeast in fermentation.9) Biomass yield coefficient(Y_x/s) and Specific growth rate(μ) of discolored hydrolyzate single cell protein fermentation were higher and specific rate of substrate consumption(Ï…) was lower than undiscolored hydrolyzate,which indicated that the inhibiting substances in liquid hydrolysate were removed,it was favorable to yeast growth. 10) Kinetic parameters of maximum biomass concentration(X_max),rate of biomass formation(Q_x) and specific growth rate(μ) enhanced with the substrate concentration increasing in initial substrate concentration range of 6～40 g/L,the biomass yield coefficients reduced instead.The values of maximum specific growth rate(μ_max) and substrate saturation constant(K_S) were 0.093 h^-1 and 0.88 g/L respectively by estimate.11) The dry matter,crude protein and ash content were 92.82%,43.59%and 7.68%separately in single cell protein products of this research,there were balancing various aminao acids for animal growth in protein,especially there was a higher lysine content,which occupied 7.27%of the total protein;Also there were higher trace elements in single cell protein products,for example,the iron,zinc and manganese contents reached 216,353,and 16.70 mg/kg separately.