【作者】 张永杰；

【导师】 杨生玉；

【作者基本信息】 河南大学 ， 微生物学， 2017， 硕士

【摘要】 聚羟基丁酸是一种生物可降解的高分子材料,可应用于医学、环境保护、农业等领域,其用途广泛,具有广阔的工业应用价值。本文研究了从化工厂污水中筛选出1株能够以甘油作为碳源来合成PHB的菌株。首先采用16S rDNA基因序列分析,确定该菌株属于假单胞菌科（Pseudomonadaceae）假单胞菌属（Pseudomonas）,然后依据与其相关的生理生化特性试验,确定该菌为恶臭假单胞菌（Pseudomonas putida）,并将其命名为PP25。进行初步发酵试验,结果表明该菌以2%甘油作为主要碳源时,PHB产量为2.456 g/L。本文分别建立紫外分光光度法及高效液相色谱法测定假单胞菌胞内的PHB含量。在紫外分光光度法的建立中以超声波破壁作为细胞破壁的预处理方法,考察了超声时间、三氯甲烷的蒸发时间、水浴时间、硫酸用量等因素,确定最优试验条件。紫外分光光度法于1.0³.5μg/mL内呈线性关系。采用该方法对样品测定时的回收率是95.93%¹04.79%,相对标准偏差是1.13%⁴.27%。高效液相色谱法采用Aminex HPX-87C分析柱,采用紫外检测器对胞内的PHB进行分离测定。经过色谱条件优化,最终确定5 mmol/L硫酸作为流动相,流速选择0.7 mL/min,柱温选择50℃。采用高效液相色谱法检测样品时回收率是98.91%¹01.97%,相对标准偏差是0.763%².014%。本文研究了温度、摇床转速、初始pH、接种量、装液量、碳源、氮源、无机盐、金属离子等因素对PP25生长及PHB积累的影响。然后依据Placket-Burman试验,筛选对PP25菌株合成PHB有显著影响的因素。结果表明:甘油,硫酸铵及初始pH 3个因子对PP25菌株合成PHB有显著的影响。利用最陡爬坡试验及响应面分析法探究3个显著因素对PP25菌株积累PHB的关系。结果表明:当甘油为26.892 g/L,（NH₄）₂SO₄为3.043g/L,初始pH为7.004时,聚羟基丁酸最大估计值为8.062 g/L。经过对模型验证,PHB实际合成为7.881 g/L,与理论产量相似,比优化前提高了32.52%。最终确定最优的条件为是甘油26.892 g/L,柠檬酸2 g/L,（NH₄）₂SO₄ 3.043 g/L,Na₂HPO₄·12H₂O 15 g/L,NaH₂PO₂·H₂O 4 g/L,FeSO₄·7H₂O 0.4 g/L,MgSO₄·7H₂O 1.2 g/L,初始pH 7.004。本文探究了通气量、温度、pH对PP25菌株在5 L发酵罐中积累PHB的影响。结果表明:通气量选择1.2 vvm、温度选择28℃和pH选择6.5时,菌体生物量为18.04 g/L,PHB产量为10.617 g/L。比优化前分别提高了46.19%和78.95%。在此基础上,进行不同时间段补加甘油及补加不同C/N比的复合碳氮源补料工艺研究,确定在发酵24 h后补加C/N比为5:1的复合碳氮源（甘油60 g,硫酸铵12 g）时,比只补加甘油时分别提高了19.42%和19.11%。本文研究了珠磨时间、珠磨质量、氯仿提取时间、温度、细胞浓度、醇沉时间及乙醇和氯仿相得比例对胞内PHB提取率的影响。结果表明珠磨时间3 min,珠磨量100 mg、氯仿提取时间78.3 min、温度54.5℃、细胞浓度20.5 mg/mL、氯仿相和乙醇比例1:4及醇沉时间70 min是胞内的PHB提取率最高为84.20%。经过纯化后PHB的提取率为73.51%,PHB的纯度为98.97%。更多还原

【Abstract】 PHB is a kind of biodegradable polymer materials,which can be used in medicine,environmental protection,agriculture and other fields,it has wide applications and wide industrial application value.The strain was identified as Pseudomonas by the 16 S r DNA gene sequence analysis,Then,the bacterium was identified as Pseudomonas putida according to the physiological and biochemical characteristics test.The strain was named PP25.The results of preliminary fermentation test showed the yield of PHB was about 2.456 g/L when 2% glycerol was used as the main carbon source.In this paper,UV method and HPLC method were established to determine the content of PHB in Pseudomonas sp.In the establishment of UV spectrophotometry,the method of ultrasonic wall breaking was as the pretreatment of cells,the optimum conditions were determined by the factors such as the ultrasonic time,the evaporation time,the water bath time and the dosage of sulfuric acid.The result was a linear relationship within 1.0 ~ 3.5 g/m L with UV spectrophotometric method,the recovery rate was95.93%~104.79% and the relative standard deviation was 1.13% ~ 4.27%.HPLC method was used to analyze the intracellular PHB by Aminex HPX-87 C column.The final results were obtained after optimizing the chromatographic conditions,5 mmol/L sulfuric acid was as the mobile phase,the flow rate was 0.7 m L/min,column temperature was 50 o C,the recovery rate of the sample was 98.91% ~ 101.97%,and the relative standard deviation was 0.763% ~ 2.014%.In the paper,the factors affecting the growth of PP25 and the accumulation of PHB were studied,including temperature,shaking speed,initial p H,inoculation amount,liquid volume,carbon source,nitrogen source,inorganic salt,metal ion and so on.The factors were screened by the Placket-Burman experiment,which had significant influence on the PHB synthesis of PP25.The results showed that glycerol,ammonium sulfate and initial p H had significant effects on the synthesis of PHB of PP25 strain.The relationship between 3 significant factors and PHB accumulation of PP25 were studied by steepest ascent test and response surface methodology.The results showed that the optimum conditions were glycerol at 26.892 g/L,ammonium sulphate at 3.043 g/L,and initial p H at 7.004,and the maximum estimated value of poly(hydroxybutyrate)was about 8.062 g/L.The actual PHB synthesis was 7.881 g/Lafter verification of the model,which was similar to the theoretical yield,which increased 32.52% at optimized condition.The optimal conditions were determined that Glycerol was 26.892 g/L,citric acid was2 g/L,ammonium sulfate was 3.043 g/L,two sodium hydrogen phosphate was 15 g/L,sodium dihydrogen phosphate was 4 g/L,seven water ferrous sulfate was 0.4 g/L,magnesium Sulfate was1.2 g/L,and initial p H was 7.004.In this paper,the effects of aeration rate,temperature and p H on the accumulation PHB of PP25 in 5 L fermentor were investigated.The results showed that the optimal fermentation conditions were aeration rate at 1.2 vvm,temperature at 28°C,and p H at 6.5,the biomass was 18.04 g/L and the yield of PHB was about 10.617 g/L under these conditions,which was about respectively 46.19%、78.95% greater than those obtained with initial fermentation conditions.On this basis,the feeding process of different time periods was studied,feeding composition was glycerol or different ratio of carbon and nitrogen sources.The results showed that the compound carbon and nitrogen source(glycerol 60 g,ammonium sulfate 12 g)was added after the fermentation 24 h,the C/N ratio was 5:1,finally,the dry weight and yield of PHB were further increased by 71.40% and by 54.95%,respectively.In this paper,the effects of some factors on the extraction rate of intracellular PHB were studied,including the time of bead milling,the quality of bead grinding,the time of chloroform extraction,temperature,cell concentration,alcohol precipitation time and the ratio of ethanol to chloroform.The results showed that the milling time was 3 min,bead grinding capacity of 100 mg,chloroform extraction time was 78.3 min,the temperature was 54.5o C,cell concentration was 20.5 mg/m L,the ratio of chloroform and alcohol was 1:4,and the alcohol precipitation time was 70 min,the intracellular PHB extraction rate was 84.20% with these condition.After purification,the extraction rate of PHB was73.51%,and the purity of PHB was 98.97%.更多还原