CN111378254B - 盐单胞菌Halomonas sp ZY-1利用秸秆和地沟油廉价合成生物可降解地膜 - Google Patents
盐单胞菌Halomonas sp ZY-1利用秸秆和地沟油廉价合成生物可降解地膜 Download PDFInfo
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Abstract
本发明公开了一种盐单胞菌Halomonas sp.ZY‑1在开放不灭菌条件下,利用秸秆与地沟油合成生物可降解地膜的方法。秸秆经处理得到秸秆上清液,配制秸杆上清液与地沟油体积比为35:1‑40:1的MS培养基。在序批式反应器(SBR)内Halomonas sp.ZY‑1利用不灭菌的秸杆上清液与地沟油MS培养基好氧培养获得菌体,提取胞内聚酯类物质。将聚酯类物质进一步改性获得拉伸强度16‑18MPa的地膜,将地膜深埋20‑30cm耕地土壤中,35d减重率40%‑50%,而置于干燥空气中35d减重率0.3%‑0.5%。以嗜盐菌不灭菌条件下利用废弃物合成生物地膜最大限度降低生产成本,亦为实现绿色生态循环、农业可持续发展提供技术支持。
Description
技术领域
本发明涉及盐单胞菌Halomonas sp.ZY-1在不灭菌、开放条件下,利用秸秆与地沟油廉价合成生物可降解地膜的方法,属于农用物资领域。
背景技术
干旱和低温是我国北方农业可持续发展的主要障碍之一,严重影响了东北地区农业经济的发展。地膜的推广大大改善了地方性干旱现象,也改善了现代农业的生产局限。地膜覆盖技术虽然给我国干旱地区农作物的生产带来了很大改善和提升,但也伴随着许多问题,现在广泛使用的地膜主要由石油基材料制成,化学结构稳定,短时间内不能降解。而且会污染土壤,影响农作物种植。目前微生物生产可降解塑料主要利用精致碳源如葡萄糖、鼠李糖、糖蜜等作为发酵底物,而能够以秸秆、地沟油等粗制碳源的较少。在微生物生产可降解塑料时会利用温性菌种,需要高温高压灭菌,而很少利用极端微生物进行可降解塑料的生产。盐单胞菌Halomonas sp.ZY-1的优势主要体现在可以在高盐高碱环境(60g/L NaCl,pH 10)的条件下利用秸秆上清液和处理过的地沟油为碳源生产可降解塑料PHA。生产PHA的其他菌株中利用的主要是废水、废油、活性污泥,而少见利用秸秆碳源,因此菌种在底物选择和废弃物利用上有较大优势。且生物可降解生物地膜在干燥的空气中35d减重率0.3%-0.5%,深埋20-30 cm耕地土壤中,35d减重率40%-50%,并随光照增强,地表温度升高,降解速率也会加快,生物可降解地膜易被分解转化为土壤腐殖质,缓解土壤板结问题。
盐单胞菌Halomonas sp.ZY-1在高盐高碱(pH 9.0-10,NaCl 60g/L)特定环境条件下生存,而非嗜盐嗜碱微生物不能在这种特殊环境中存活,因此可有效的避免杂菌污染,节约高压、蒸汽灭菌能源和封闭发酵设备的投资。此外,利用秸秆和地沟油代替精致碳源,降低生产成本的同时节约资源,因此利用嗜盐菌在高盐高碱环境生长可抑制杂菌的特性,降低生产中高能耗灭菌和发酵罐的投资,廉价低能耗的利用秸秆和地沟油合成的生物可降解地膜代替石油基塑料,不仅可以避免白色污染、保护环境,亦加速石化经济向绿色循环经济的转变合成廉价、环保、可降解材料提供技术保障。
发明内容
本发明针对石化塑料耗费石化能源,以及引发白色污染等系列问题,提出Halomonas sp. ZY-1利用秸秆与地沟油在续批式反应器(SBR)内开放条件下合成生物可降解地膜生产方法,进一步优化其材料学性能制成弹性和透光度符合条件的生物可降解地膜。该方法合成生物可降解地膜绿色环保、无污染还能增加土壤肥力,可用做合成地膜。
为实现上述目的,本发明的技术方案为:
(1)将秸秆用自来水冲洗至无土,50-60℃烘干,经粉碎机粉碎,过100-200目细筛,获得秸秆粉。将50-75g秸秆粉置于1L的2mol/L NaOH溶液中,105-115℃高温处理1-2h,冷却后4000-6000r/min离心10min,收集秸秆上清液。
(2)配制MS培养基,其成分为秸秆上清液750-800mL/L、地沟油17-20mL/L、酵母浸提物5-10 g/L、Na2HPO4·12H2O 10-15g/L、KH2PO4 1-2g/L、NH4Cl 1-2g/L、MgSO4 0.1-1.5g/L、Fe(III)-NH4-Citrate 0.05-0.1g/L、CaCl2·2H2O 0.02-0.1g/L、ZnSO4·7H2O 0.1-0.5g/L、NaCI 60-70g/L,pH调至9.5-11;
(3)取1-5%的Halomonas sp.ZY-1种子液接于含秸秆上清液和地沟油为碳源的MS培养基的SBR 反应器中,搅拌器转速为200-240r/min,25-30℃,溶解氧约爆气12h/周期,每天2周期,连续培养100-140 h收集发酵液;
(4)将发酵液4000-6000r/min离心10min收集菌体,置于-20℃预冷12-14h,在冻干机内冻干12-24h,依次经蒸馏水洗、离心、丙酮洗、离心、再水洗和离心(4000-6000r/min离心 10min,上同);将菌体放入锥形瓶中,加入氯仿:次氯酸钠3:1(v/v)的混合溶液,混匀后4000-6000r/min离心10min,吸取下层氯仿相平铺于洁净的容器中,加入预冷的95%乙醇10-15ml,析出聚酯类物质。
(5)将聚酯类物质60-80份硫、PBAT 30-50份、碳酸钙0.5-1份、二氧化钛0.2-0.5份均匀混合。放入高速混合机中以800-1000r/min的转速搅拌15-20min,混合均匀后送入双螺杆挤出、造粒获得的混合物粒子;
(6)将获得的混合物粒子在吹膜机组上吹塑成膜,制成生物可降解地膜。
本发明提供的秸秆与地沟油合成生物可降解地膜的方法优势在于:
(1)利用秸秆与地沟油在SBR反应器中开放、不灭菌条件下合成生物可降解地膜,从原来到发酵过程都在降低生物可降解地膜生产成本。
(2)该方法合成的生物可降解地膜强拉伸强度为16-18MPa。该方法合成的生物可降解地膜具有良好的生物可降解性,在20-25℃环境下的耕地土壤深埋20-30cm耕地土壤中,35d 减重率40%-50%,且生物可降解生物地膜在干燥的空气中35d减重率0.3%-0.5%,可在农业生产应用于地膜生产。
本发明所用盐单胞菌Halomonas sp.ZY-1,样品取自大庆市大同区盐碱土壤经筛选分离得到,Genebank登录号为MH428215,经系统发育分析鉴定上述嗜盐菌与Halomonas.elongata相似度为99%,命名为Halomonas sp.ZY-1,见图1。于2018年11月22日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),地址位于北京市朝阳区北辰西路1号院3号,保藏编号CGMCC No.16773。
附图说明
图1为Halomonas sp.ZY-1系统发育树
图2为生物可降解地膜深埋20-30cm耕地土壤中的减重
图3Halomonas sp.ZY-1以不同体积地沟油和秸秆培养菌体干重与聚酯类物质差异
图4为透射电镜观察Halomonas sp.ZY-1胞内积累聚酯类物质(细胞内白色物质是本专利用以合成地膜的原料)
图5为Halomonas sp.ZY-1利用秸秆与地沟油合成的聚酯类物质
图6扫描电镜(SEM)扩大500×和10000×观察合成的聚酯类物质
图7为秸秆与地沟油合成聚酯类物质与PHB标准品的红外光谱对比分析(FTIR)
图8为不同聚己二酸/对苯二甲酸丁二酯含量合成地膜拉伸强度
图9为聚酯类物质改性前后合成膜对比
图10为秸秆与地沟油发酵和改性后合成生物地膜图
具体实施方式
实施例1、生物可降解地膜的自然降解情况
本研究为模拟生物可降解地膜在自然条件下降解的情况,以生物可降解地膜埋藏在土壤中减重率为指标考察其降解能力。
(1)取3份制备好的生物可降解地膜样品,每份20-30mg,记为初始质量M0。
(2)将生物可降解地膜样品分别置于20-25℃环境下,15-20cm深的耕地中,每隔7d将样品取出去除表面土壤,置于40-55℃的烘箱中进行干燥处理后称重记为Mn。
(3)生物可降解地膜的减重率计算如下:
降解情况如图2,在干燥的空气中35d减重率为0.5%在20-25℃将可降解地膜深埋于 15-20cm耕地土壤中,35d减重率达40-50%,分解后能增加土壤腐殖质,缓解土壤板结问题,最后转化为二氧化碳和水。
实施例2、Halomonas sp.ZY-1在SBR反应器中不灭菌合成聚酯类物质
(1)粉碎的秸秆过100-200目细筛获得秸秆粉,每1L的1-2mol/L NaOH溶液浸泡75-140g 秸秆粉,高温高压处理2-4h,4000-6000r/min离心10min收集秸秆上清液,主要成分为纤维素 2.5-6%、木糖15-30%、木质素16-25%、酸溶木质素1-5%;
(2)MS培养基成分为:秸秆上清液750-800mL/L、地沟油17-20mL/L、酵母浸提物5-10g/L、 Na2HPO4·12H2O 10-15g/L、KH2PO4 1-2g/L、NH4Cl 1-2g/L、MgSO4 0.1-1.5g/L、Fe(III)-NH4-Citrate 0.05-0.1 g/L、CaCl2·2H2O 0.02-0.1g/L、ZnSO4·7H2O 0.1-0.5g/L、NaCI 60-70g/L,pH调至9.5-11,不灭菌直接用于培养Halomonas sp.ZY-1;
(3)在排水比为10/13的SBR反应器中,接种1-5%的Halomonas sp.ZY-1种子液接于含秸秆上清液和地沟油为碳源的MS培养基的SBR反应器中,搅拌器转速为200-240r/min,25-30℃,溶解氧大于2mg/L 供氧12h/周期,每天2周期,连续培养100-140h收集发酵液;
(4)采用氯仿次氯酸钠法提取聚酯类物质,观察发酵不同时期产量变化(如图3),取适量聚脂类物质,利用扫描电镜观察材料的表面形态;红外光谱分析合成的生物膜与PHB标准品 (Sigma)在4000-500cm-1处进行比对。
产量分析结果如图3所示,在固定的秸秆上清液条件下,随地沟油含量的增加聚酯类物质产量也逐渐增加,浓度为20mL/L的地沟油,即秸秆上清液与地沟油比值为40:1时,聚酯类物质产量最高为1.4-1.6g/L,Halomonas sp.ZY-1胞内积累的物质提取后呈现透明的膜(图 5)。透射电镜观察Halomonas sp.ZY-1细胞内积累白色粉末物质(图4)。
实施例3、Halomonas sp.ZY-1利用水稻秸秆与地沟油合成聚酯类物质
根据实施例2中聚酯类物质产量分析结果,秸秆合成聚酯类物质产量低,且聚酯类物质的成膜性较差。添加地沟油(成分90-92%油酸,3-4%甘油三酯,2-3%植物油,2-3%动物油) 进行改善,将秸秆与地沟油作为碳源配制MS培养基。
(1)秸秆和地沟油梯度MS培养基配置:800mL/L秸秆上清液、酵母浸提物5g/L、Na2HPO4 12H2O 10-15g/L、KH2PO4 1-2g/L、NH4Cl 1-2g/L、MgSO4 0.1-1.5g/L、Fe(III)-NH4-Citrate 0.05-0.1g/L、CaCl2·2H2O 0.02-0.1g/L、ZnSO4·7H2O 0.1-0.5g/L,地沟油分别为5mL/L、10mL/L、15mL/L、20mL/L、25mL/L、30ml/L;
(2)将LB培养基过夜活化的Halomonas sp.ZY-1种子液以1-5%接种量接入MS培养基,在36-38℃震荡培养箱内转速为140-160r/min培养80-86h;
(3)采用氯仿次氯酸钠法提取聚酯类物质,分析不同含量地沟油对其产量变化如图4所示,在秸秆上清液体积固定条件下,随地沟油含量的增加聚酯类物质产量也逐渐增加,浓度为20mL/L的地沟油时,即秸秆上清液与地沟油比值为40:1时,聚酯类物质最高产量为 1.5-1.7g/L。
实施例4扫描电镜分析(SEM)和红外光谱分析(FTIR)
(1)取适量秸秆与地沟油合成聚脂类物质,通过扫描电镜观察材料的表面形态;
(2)分别称取一定量PHB标准品(Sigma)、秸秆与地沟油合成聚脂类物质,采用溴化钾压片法制样,在测试4000-500cm-1处进行红外光谱分析;
扫描电镜观察材料的表面形态如图6,在500倍电镜观察下对比,秸秆合成聚脂类物质表面较粗糙,而秸秆地沟油合成的聚脂类物质表面较平整;10000倍电镜下,秸秆合成的聚脂类物质有明显的带状裂痕,而秸秆地沟油合成的材料相对裂痕少。能谱分析显示:秸秆与地沟油合成聚脂类物质的碳原子含量为72.5-73%。
红外光谱分析如图7,两种聚脂类物质在1450、2980和1720cm-1处,对应CH2、CH、 C=O基团分别有吸附带。与PHB标准品的红外图谱相对应,因此该聚合物为聚羟基脂肪酸酯类物质。
实施例5生物可降解地膜材料学性质的优化
将Halomonas sp.ZY-1利用秸秆与地沟油合成聚酯类物质制作成生物可降解地膜。
(3)设置聚己二酸/对苯二甲酸丁二酯比例为20、30、40、50、60不同质量份,其他成分为:聚酯类物质80质量份、碳酸钙0.5质量份,二氧化钛0.2质量份,放入高速混合机中,以800-1000r/min的转速搅拌15-20min,混合均匀。
(4)得到混合物送入双螺杆挤出机,螺杆转速160r/min,喂料速度20r/min,160-180℃下挤出造粒,得到混合物粒子。
(5)将获得的混合物粒子在吹膜机组上吹塑成膜,制成薄膜见图10。
(6)按GB2568测试薄膜的拉伸强度。
测定拉伸力结果图9所示,聚己二酸/对苯二甲酸丁二酯为50质量份时,获得的生物可降解地膜强度最高,拉伸强度可达16MPa。
为实现上述目的,本发明的技术方案为:
(1)将秸秆用自来水冲洗至无土,50-60℃烘干,经粉碎机粉碎,过100-200目细筛,获得秸秆粉。将50-75g秸秆粉置于1L的2mol/L NaOH溶液中,105-115℃高温处理1-2h,冷却后4000-6000r/min离心10min,收集秸秆上清液。
(2)配制MS培养基,其成分为秸秆上清液750-800mL/L、地沟油17-20mL/L、酵母浸提物5g/L、Na2HPO4·12H2O 10-15g/L、KH2PO4 1-2g/L、NH4Cl 1-2g/L、MgSO4 0.1-1.5g/L、Fe(III)-NH4-Citrate 0.05-0.1g/L、CaCl2·2H2O 0.02-0.1g/L、ZnSO4·7H2O 0.1-0.5g/L、NaCI 60 g/L,pH调至8.0-10。
(3)将1-5%的Halomonas sp.ZY-1菌种接种到LB培养基(pH 8.0-10,NaCI 60g/L)中,在 36-38℃震荡培养箱内转速为140-160r/min培养12-14h,取1-5%的种子液接于MS培养基中,在36-38℃震荡培养箱内转速为140-160r/min培养70-90h。
(4)收集菌体,置于-20℃预冷12-14h,在冻干机内冻干12-24h。依次经蒸馏水洗、离心、丙酮洗、离心、再水洗、离心,4000-6000r/min离心10min,将菌体放入锥形瓶中,加入氯仿:次氯酸钠体积比为3:1的混合溶液,混匀后4000-6000r/min离心10min,吸取下层氯仿相平铺于洁净的容器中,加入预冷的95%乙醇10-15ml,析出聚酯类物质。
(5)将聚酯类物质60-80份、聚己二酸/对苯二甲酸丁二酯30-50份、碳酸钙0.5-1份、二氧化钛0.2-0.5份放入高速混合机中,以800-1000r/min的转速搅拌15-20min,混合均匀。
(6)得到混合物送入双螺杆挤出机,螺杆转速160r/min,喂料速度20r/min,160-180℃下挤出造粒,得到混合物粒子,将混合物粒子在吹膜机组上吹塑成膜,制成生物可降解地膜。
Claims (4)
1.一种盐单胞菌(Halomonas sp.) ZY-1在开放、不灭菌条件下利用秸秆和地沟油合成生物可降解地膜的方法,其特征是,生物可降解地膜的合成步骤如下:
步骤1:将秸秆粉碎过100-200目细筛获得秸秆粉,每1L的1-2 mol/L NaOH溶液浸泡75-140 g秸秆粉,高温高压处理2-4 h,4000-6000 r/min离心10 min收集秸秆上清液,上清液成分为纤维素2.5-6%、木糖15-30%、木质素16-25%、酸溶木质素1-5%;上清液添加地沟油使得秸秆上清液与地沟油体积比为35-40:1,配制MS培养基;
步骤2:在序批式反应器(SBR)中配制秸秆上清液和地沟油为碳源的MS培养基,直接取1-5%盐单胞菌(Halomonas sp.) ZY-1种子液,接种于SBR反应器含秸秆上清液和地沟油为碳源不灭菌的MS培养基中,搅拌器转速为200-240 r/min,25-30℃,溶解氧大于2mg/L供氧12h/周期,每天2周期,连续培养100-140 h收集发酵液;所述盐单胞菌(Halomonas sp.)ZY-1与Halomonas.elongata相似度为99%,Genebank登录号为MH428215,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,于2018年11月22日保藏,编号为CGMCC No.16773;
步骤3:采用氯仿-次氯酸钠法破壁提取胞内聚酯类物质:(1) 将发酵液离心收集菌体,置于-20℃预冷12-14h,在冻干机内冻干12-24h,依次经蒸馏水洗、4000-6000r/min离心10min、丙酮洗、离心、再水洗和离心;(2) 将菌体放入锥形瓶中,加入氯仿:次氯酸钠为3:1(v/v)的混合溶液,混匀后4000-6000r/min离心10min,吸取下层氯仿相平铺于洁净的容器中,加入预冷的95%乙醇10 -15mL析出聚酯类物质,将聚酯类物质、聚己二酸/对苯二甲酸丁二酯、碳酸钙、二氧化钛按一定质量比混匀后熔融,挤粒吹塑成生物可降解地膜;
步骤4:经材料学性质分析,地膜具有生物可降解性、韧性、透明度,能够作为合成农用地膜的材料;
步骤1中所述地沟油,其特征是,成分为90-92%油酸、3-4%甘油三酯、2-3%植物油、2-3%动物油;
步骤1中所述秸秆上清液和地沟油为碳源的MS培养基,其特征是,秸秆上清液750-800mL/L、地沟油 17-20 mL/L、酵母浸提物 5-10 g/L、Na2HPO4•12H2O 10-15 g/L、KH2PO4 1-2g/L、NH4Cl 1-2 g/L、MgSO4 0.1-1.5 g/L、Fe(III)-NH4-Citrate 0.05-0.1 g/L、CaCl2•2H2O0.02-0.1 g/L、ZnSO4•7H2O 0.1-0.5 g/L、NaCI 60 -70g/L,pH调至9.5-11,不灭菌直接用于培养盐单胞菌(Halomonas sp.)ZY-1;
步骤2中所述SBR,其特征是,SBR反应器的主罐体为玻璃,滗水器、电磁阀运行所需的零件均为耐酸碱的304钢,反应器总容积为15 L,有效容积13 L,每周期进水10 L,排水比10/13。
2.根据权利要求1的方法,其特征在于,步骤3中所述生物可降解地膜以质量份数计,包括聚酯类物质60-80份、PBAT 30-50份、碳酸钙0.5-1份、二氧化钛0.2-0.5份,放入高速混合机中以800-1000 r/min的转速搅拌15-20 min,混合均匀后送入双螺杆挤出、造粒获得的混合物粒子在吹膜机组上吹塑成生物可降解地膜。
3.根据权利要求1的方法,其特征在于,步骤3中所述生物可降解地膜,其特征是,经红外光谱与PHB标准品分析比对发现在1450、2980和1720 cm-1处,对应CH2、CH、C=O基团分别有吸附带,与PHB标准品的红外图谱相对应,主要有碳、氢、氧三种元素组成,彻底分解后产物为二氧化碳和水。
4.根据权利要求1的方法,其特征在于,步骤3中所述生物可降解地膜,其特征是,在干燥的空气中35d减重率0.3%-0.5%,深埋20-30 cm耕地土壤中,35d减重率40%-50%,分解产物为水和二氧化碳不污染环境,适用于农业生产中覆盖庄稼的地膜。
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