CN105039437A - 一种利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法 - Google Patents
一种利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法 Download PDFInfo
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Abstract
本发明公开了一种利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,包括以下步骤:将废醪液稀释,作为产油单针藻的基本培养基;在基本培养基中加入无机盐,用NaOH调节pH值,配置成培养基;在500mL三角瓶内装入250mL最终制备得到的培养基,灭菌后接入产油单针藻进行培养;用离心分离法收集细胞,用氯仿/甲醇提取液提取油脂,上述提取油脂加2mL3%硫酸-甲醇进行甲酯化制备得到生物柴油。本发明简单易行、能大幅缩短产油单针藻的培养时间、降低生产成本,并显著增加油脂产量,从而大大提高了微藻生产生物柴油的效率。
Description
技术领域
本发明属于生物柴油技术领域,具体涉及一种利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法。
背景技术
生物柴油是指以植物油脂、动物油脂、微生物油脂、废餐饮油等为原料油通过酯交换工艺制成的甲酯或乙酯燃料。与常规柴油相比,它具有可再生、易降解、燃烧后污染物排放低、温室气体排放低等特点。
微藻类作为生产生物柴油原料具有许多优点,具有简单的细胞结构;光合作用效率高、生物产量高、生长繁殖快、生长周期短;且油脂含量较高;对生长条件的要求不高,能够在极端的条件下生存;且微藻中不含有硫、无毒且可降解。因此,利用微藻生产生物柴油具有广阔的发展前景。
糖蜜酒精废醪液是甘蔗或甜菜糖厂在精制糖产品过程中,所得的副产物糖蜜经过发酵及蒸馏后所排放的废醪液,其化学耗氧量(COD)达40000~50000mg/L,生物需氧量(BOD)达20000~30000mg/L,氨氮1500~2500mg/L,其中还含有许多氨基酸、蛋白质、糖类以及Na、K、Ca、Mg、P等矿物元素,其固形物含量为8%~10%。
云南省是全国主要蔗糖产地之一,全省拥有80多家制糖企业,遍布全省14个地州,涉及40多个县。这些糖厂都附设有酒精生产车间,但其生产过程中会产生大量的酸性高浓度有机废水,具有潜在的危害性。一个配有日产量为20t酒精生产能力的糖厂,其排放的酒精生产废水污染负荷,相当于一个40万人口的城市排放生活污水的污染负荷,会造成水体富营养化、缺氧、鱼虾绝迹、水质恶化、发臭,严重污染地表地下水。如酒精生产排放的废水污染负荷是云南最主要的水污染源,占云南废水排放污染负荷总量约40%。
如何对酒精废醪液进行经济有效的治理,至今仍是糖厂污染治理的一个难题。目前国内使用的一些技术和方法,如氧化塘、农灌法、厌氧制沼气、蒸发浓缩、制取单细胞蛋白饲料等,有的投资太高、经济运行性差、效果不显著,有的条件苛刻、使用范围受限制,难以进一步推广运用。而用废醪液作为基本培养基培养微藻生产生物柴油,不仅可以解决环境污染处理问题,还能为企业带来经济效益,完全可做到“变废为宝”,将会为云南省制糖企业带来很好的前景。
发明内容
本发明的目的是提供一种利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,该方法过程简单,生产成本低。
本发明所采用的技术方案是,一种利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,包括以下步骤:
步骤1、制备培养基:将废醪液稀释,作为产油微藻的基本培养基,然后向基础培养基中加入无机盐,调整pH值,制备得到培养基;
步骤2、产油微藻培养:将上述培养基高压高温灭菌20min,接入产油单针藻,藻细胞密度控制在106个/ml,进行光照摇瓶培养;
步骤3、制备生物柴油:将培养到稳定期的微藻富集,提取油脂,制备得到生物柴油。
本发明的特点还在于:
步骤1中废醪液梯度稀释80~110倍,制备得到的培养基的pH值为6.8~7.1。
步骤1中培养基的制备方法为:在基本培养基中加入下述无机盐及用量:NaNO3:600-800mg/L;K2HPO4.3H2O:30-50mg/L;MgSO4.7H2O:65-85mg/L;CaCl2.2H2O:30-45mg/L;Na2CO3:10-30mg/L;MnCl2.4H2O:1.0-2.5mg/L;ZnSO4.7H2O:0.1-0.3mg/L;CuSO4:0.05-0.1mg/L;(NH4)6Mo7O24:2-6mg/L;Co(NO3)2.6H2O:0.02-0.06mg/L;Na2.EDTA:0.5-2mg/L;H3BO3:2-6mg/L;柠檬酸:4-8mg/L;柠檬酸铁:4-8mg/L;调整PH值至6.8~7.1,配置成培养基。
步骤2中培养条件为:光照培养温度为23~26℃,光照强度为2500~4000lux,摇床转速为130~160r/min,制备得到单针藻培养液。
步骤3中制备生物柴油具体为:将培养液离心富集,离心条件为3000r/min转速条件下离心6min,并以蒸馏水反复洗涤2次后冻干;添加冻干藻粉2倍体积石英砂研磨20min后用体积比为2:1的氯仿-甲醇进行油脂提取,有机溶剂重复提取3次后收集有机相浓缩,上述提取油脂加2mL3%硫酸-甲醇进行甲酯化制备得到生物油脂。
本发明的有益效果是:
(1)本发明能节省碳源和氮源,提高了糖的利用率和转化率,降低了生产成本;废醪液实现零排放,有利于保护环境;将环境污染的处理和微藻的培养结合在一起,变废为宝。
(2)本发明与一般光照自养培养微藻相比,生长周期缩短了67~75%,到达稳定期时候的最大生物量较一般光照自养培养微藻提高了5~22%,达到4.2~4.88mg/L,油脂含量提高了8~17%,达到54~58.5%。
(3)本发明中产油微藻能很好地在废醪液培养基中生长,不但解决了环境污染问题,而且将污染物转化为可再生的生物能源。微藻在废醪液中不但能很好地生长,缩短了生长周期,而且还能促进产油微藻油脂含量的增加。可能存在的作用机制是,废醪液里边的氮源、碳源能被微藻很好地吸收并利用,废醪液里边也可能存在促进微藻生长的作用因子。
具体实施方式
下面结合具体实施方式对本发明进行详细说明。
实施例1
将废醪液梯度稀释80倍,作为产油微藻的基本培养基,然后向基础培养基中加入下述无机盐及用量(mg/L):NaNO3:700mg/L;K2HPO4.3H2O:40mg/L;MgSO4.7H2O:75mg/L;CaCl2.2H2O:40mg/L;Na2CO3:20mg/L;MnCl2.4H2O:1mg/L;ZnSO4.7H2O:0.2mg/L;CuSO4:0.075mg/L;(NH4)6Mo7O24:4mg/L;Co(NO3)2.6H2O:0.04mg/L;Na2.EDTA:1.25mg/L;H3BO3:4mg/L;柠檬酸:6mg/L;柠檬酸铁:7mg/L;用1.0M的NaOH调节pH值调至6.8;高压高温灭菌20min,接入产油单针藻,藻细胞密度控制在106个/mL,光照培养温度为23℃,光照强度为2500lux,摇床转速为130r/min,进行光照摇瓶培养。
实施例2
将废醪液梯度稀释90倍,作为产油微藻的基本培养基,然后向基础培养基中加入下述无机盐及用量:NaNO3:600mg/L;K2HPO4.3H2O:50mg/L;MgSO4.7H2O:65mg/L;CaCl2.2H2O:45mg/L;Na2CO3:10mg/L;MnCl2.4H2O:2.5mg/L;ZnSO4.7H2O:0.1mg/L;CuSO4:0.1mg/L;(NH4)6Mo7O24:2mg/L;Co(NO3)2.6H2O:0.06mg/L;Na2.EDTA:0.5mg/L;H3BO3:6mg/L;柠檬酸:4mg/L;柠檬酸铁:8mg/L,调整pH值为6.9。高压高温灭菌20min,接入产油单针藻,藻细胞密度控制在106个/mL,光照培养温度为24℃,光照强度为3000lux,摇床转速为140r/min,进行光照摇瓶培养。
实施例3
将废醪液梯度稀释100倍,作为产油微藻的基本培养基,然后向基础培养基中加入下述无机盐及用量:NaNO3:800mg/L;K2HPO4.3H2O:30mg/L;MgSO4.7H2O:85mg/L;CaCl2.2H2O:30mg/L;Na2CO3:30mg/L;MnCl2.4H2O:1.0mg/L;ZnSO4.7H2O:0.3mg/L;CuSO4:0.05mg/L;(NH4)6Mo7O24:2mg/L;Co(NO3)2.6H2O:0.02mg/L;Na2.EDTA:2mg/L;H3BO3:2mg/L;柠檬酸:8mg/L;柠檬酸铁:4mg/L;调整pH值为7;高压高温灭菌20min,接入产油单针藻,藻细胞密度控制在106个/mL,光照培养温度为25℃,光照强度为3500lux,摇床转速为150r/min,进行光照摇瓶培养。
实施例4
将废醪液梯度稀释110倍,作为产油微藻的基本培养基,然后向基础培养基中加入无机盐,调整pH值为7.1。然后向基础培养基中加入下述无机盐及用量:NaNO3:700mg/L;K2HPO4.3H2O:40mg/L;MgSO4.7H2O:75mg/L;CaCl2.2H2O:40mg/L;Na2CO3:20mg/L;MnCl2.4H2O:1mg/L;ZnSO4.7H2O:0.2mg/L;CuSO4:0.075mg/L;(NH4)6Mo7O24:4mg/L;Co(NO3)2.6H2O:0.04mg/L;Na2.EDTA:1.25mg/L;H3BO3:4mg/L;柠檬酸:6mg/L;柠檬酸铁:7mg/L;调整pH值为7.1,高压高温灭菌20min,接入产油单针藻,藻细胞密度控制在106个/mL,光照培养温度为26℃,光照强度为4000lux,摇床转速为160r/min,进行光照摇瓶培养。
将实施例1至4中得到的培养液离心富集(3000r/min,6min),并以蒸馏水反复洗涤2次后冻干称重。添加冻干藻粉2倍质量石英砂研磨20min后用氯仿-甲醇(2:1,v/v)进行油脂提取,有机溶剂重复提取3次后收集有机相浓缩称重,上述提取油脂加2mL3%硫酸-甲醇进行甲酯化制备得到生物柴油。
结果表明:本发明与一般光照自养培养微藻相比,生长周期缩短了67~75%,到达稳定期时候的最大生物量较空白对照提高了5~22%,达到4.2~4.88mg/L,油脂含量提高了8~17%,达到54~58.5%。
Claims (5)
1.一种利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,其特征在于,包括以下步骤:
步骤1、制备培养基:将废醪液梯度稀释,作为产油微藻的基本培养基,然后向基础培养基中加入无机盐,调整pH值,制备得到培养基;
步骤2、产油微藻培养:将上述培养基高压高温灭菌20min,接入产油单针藻,藻细胞密度控制在106个/ml,进行光照摇瓶培养;
步骤3、制备生物柴油:将培养到稳定期的微藻富集,提取油脂,制备得到生物柴油。
2.根据权利要求1所述的利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,其特征在于,所述步骤1中废醪液梯度稀释倍数为80~110倍,制备得到的培养基的pH值为6.8~7.1。
3.根据权利要求1所述的利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,其特征在于,所述步骤1中培养基的制备方法为:在基本培养基中加入下述无机盐及用量:NaNO3:600-800mg/L;K2HPO4.3H2O:30-50mg/L;MgSO4.7H2O:65-85mg/L;CaCl2.2H2O:30-45mg/L;Na2CO3:10-30mg/L;MnCl2.4H2O:1.0-2.5mg/L;ZnSO4.7H2O:0.1-0.3mg/L;CuSO4:0.05-0.1mg/L;(NH4)6Mo7O24:2-6mg/L;Co(NO3)2.6H2O:0.02-0.06mg/L;Na2.EDTA:0.5-2mg/L;H3BO3:2-6mg/L;柠檬酸:4-8mg/L;柠檬酸铁:4-8mg/L;调整pH值至6.8~7.1,配置成培养基。
4.根据权利要求1所述的利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,其特征在于,所述步骤2中培养条件为:光照培养温度为23~26℃,光照强度为2500~4000lux,摇床转速为130~160r/min,制备得到单针藻培养液。
5.根据权利要求1所述的利用糖蜜酒精废醪液培养单针藻生产生物柴油的方法,其特征在于,所述步骤3中制备生物柴油具体为:将培养液离心富集,离心条件为3000r/min转速条件下离心6min,并以蒸馏水反复洗涤2次后冻干;添加冻干藻粉2倍体积石英砂研磨20min后用体积比为2:1的氯仿-甲醇进行油脂提取,有机溶剂重复提取3次后收集有机相浓缩,上述提取油脂加2mL3%硫酸-甲醇进行甲酯化制备得到生物柴油。
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