【作者】 张晓燕；

【导师】 赵广杰；

【作者基本信息】 北京林业大学 ， 木材科学与技术， 2008， 博士

【摘要】 用蒸汽爆破预处理的木材进行生物转化单细胞蛋白的研究对于解决我国木材采伐、加工剩余物的利用和饲料蛋白缺乏的现状有重要意义。为了弄清楚蒸汽爆破尾叶桉木材生物转化单细胞蛋白的工艺,解决我国饲料蛋白缺乏的现状,本研究以三年生尾叶桉（Eucalyptus urophylla）为原料,采用蒸汽爆破方法进行预处理,通过常规化学分析方法及扫描电镜、X射线衍射、红外光谱等现代分析手段,对蒸汽爆破尾叶桉木材的微观形貌和化学成分进行了分析,并研究了蒸汽爆破尾叶桉木材生物转化单细胞蛋白的工艺过程,包括发酵产酶、酶水解、水解液脱色及水解液发酵单细胞蛋白,并对单细胞蛋白产品进行了氨基酸和矿质元素营养分析,得到的主要结论归纳如下:1.蒸汽爆破处理能使尾叶桉木材纤维之间的结合变得松散,纹孔膜破损,从而提高生物酶对纤维素的可及度,改善其生物化学反应性能。对化学成分的测定和红外光谱的分析结果表明,蒸汽爆破能溶解出一定量的半纤维素和木质素,纤维素除无定形区少量降解外基本不受损失,有利于提高酶解率。蒸汽爆破前用0.2%硫酸预处理,尾叶桉木材纤维变得更细碎,表面积更大,同时木质素脱除率提高,纹孔膜破损比例增加,更适合于纤维素酶的作用。2.用绿色木霉（Trichoderma viride）和黑曲霉（Aspergillus niger）混合菌种发酵,蒸汽爆破尾叶桉木材发酵产纤维素酶和木聚糖酶的最佳工艺条件为:接种量6%、菌种配比（绿色木霉/黑曲霉）为1∶1、底物浓度1.0%,在温度28℃、初始pH值4.5条件下发酵4d。3.原料在蒸汽爆破前的预处理及蒸汽压力对其发酵产酶能力有显著的影响,爆破前用0.2%硫酸预处理可使相同蒸汽压力下CMC酶活力（β-1,4-葡聚糖内切酶活力,羧甲基纤维素酶活力）、FPA酶活力（滤纸酶活力）和木聚糖酶活力明显提高,且通过单因素方差分析表明酶活力差异极显著;然而氨水预处理却降低了这三种酶的活力,且处理与未处理间差异也极显著。4.蒸汽爆破尾叶桉木材纤维素酶水解的最佳工艺条件是:温度45～50℃、pH 4.8、底物浓度2%,酶用量25 FPIU/g底物,酶解时间48 h;水解液用活性炭脱色的最佳条件是温度70℃、pH 2、脱色时间90 min、活性炭用量3.5%,该条件下水解液的脱色率达到77.02±0.52%,总还原糖回收率为80.81±0.23%。5.原料在蒸汽爆破前的预处理及蒸汽压力对其用纤维素酶水解能力有显著的影响,爆破前用0.2%硫酸预处理可使相同蒸汽压力下爆破尾叶桉木材的纤维素酶水解糖化率明显提高,而氨水预处理却使水解率降低。在2.2 Mpa的蒸汽压力下,0.2%硫酸预处理的爆破材料,用最佳工艺条件（即底物2.0%,酶用量20 FPIU/g底物,50℃,pH 4.8,水解48 h）进行纤维素酶水解,其水解液中还原糖浓度为12.72±0.26 g/L,水解糖化率达82.43%±0.017。6.蒸汽爆破尾叶桉木材纤维素酶水解液用高效液相色谱分析表明,其中葡萄糖、木糖、阿拉伯糖、二糖的百分含量分别为69.39±0.67%、27.53±0.40%、0.65±0.05%、2.44±0.88%,乙酸含量随糖浓度的高低没有明显变化,而糠醛含量会随糖浓度的提高而增加。水解液经脱色处理会降低糠醛的含量,但不会降低乙酸含量。7.蒸汽爆破尾叶桉木材纤维素酶水解液发酵单细胞蛋白的最适培养基组成:磷酸氢二铵为氮源,加入量为4.0g/L,酵母浸粉,磷酸二氢钾和硫酸镁的加入量分别为0.5、1.0和0.3g/L;最佳接种方式为:种子浓度10⁷ cell/mL,接种酿酒酵母（Sacchromycescerevisive）和假丝酵母（Candida utilis 2.587）混合菌种,配比为S.c:C.u=1∶3,接种量为10%;最佳工艺条件为:发酵温度31～34℃,初始pH为4.5,初始还原糖浓度11.33～17.52g/L,250mL摇瓶发酵的最适体积为25mL。8.蒸汽爆破尾叶桉木材纤维素酶水解液中含有多种单糖,酿酒酵母和假丝酵母混合接种发酵对单糖的利用是有顺序的,且会出现二次生长现象。在有葡萄糖的时候,两个菌种优先利用葡萄糖,葡萄糖即将被消耗完时,假丝酵母也可以利用木糖,阿拉伯糖等五碳糖作为其生长的碳源继续生长。葡萄糖在接种后随即被消耗,6～9 h时间内葡萄糖浓度降低最快,到发酵9 h时几乎被消耗完毕;而木糖浓度的变化是在发酵开始9 h之内浓度几乎不降低,到9 h葡萄糖被消耗完毕后,木糖才开始减少,发酵15 h时木糖也完全被利用;阿拉伯糖浓度的降低也是出现在发酵9 h之后。此外,乙酸在发酵的过程中也可以被酵母利用。9.尾叶桉木材纤维素酶水解液经脱色后发酵单细胞蛋白,菌体得率系数(Y_x/s)和菌体比生长速率（μ）比未脱色液高,基质比消耗速率（ν）比未脱色液低,表明脱色过程脱去了水解液中对发酵有抑制作用的物质,对酵母菌体的生长繁殖是有利的。10.在6～40g/L的初始基质浓度范围内,随基质浓度的升高,得到的最高菌体浓度(X_max)、菌体生产率（Q_x）和菌体比生长速率（μ）也随之升高,但菌体得率系数(Y_x/s),却逐渐降低,最大比生长速率(μ_max)和基质饱和常数（K_S）的值分别为0.093 h^-1和0.88 g/L。11.本研究制备的单细胞蛋白产品的干物质、粗蛋白含量、灰分分别达到92.82%、43.59%和7.68%,蛋白质中含动物生长发育所需的各种氨基酸,且氨基酸组分平衡,尤其是赖氨酸含量高,占到总蛋白质的7.27%;微量元素含量高,铁、锌、锰分别达到216、353、16.70 mg/kg。更多还原

【Abstract】 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（υ） 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.更多还原