微波诱变乳酸高产菌选育及淀粉废水发酵制乳酸工艺研究
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摘要
针对目前淀粉工业废水处理与资源化以及微生物诱变育种技术中存在的问题,基于分子生物学和微波辐照技术,本文进行了用于淀粉工业废水资源化处理的乳酸高产菌微波诱变育种技术及相关基础研究。建立微波诱变选育高产乳酸菌的技术方法,并设计研制了相关实验装置,进行乳酸高产菌株的选育;采用生理生化和分子生物学方法对突变菌种进行表征,深入探讨微波诱变乳酸菌的过程和机制,考查微波场中影响高产乳酸菌选育的主要因素及影响规律,同时,对玉米淀粉工业废水用于发酵制备乳酸工艺进行了研究,并将活性炭纤维(ACF)作为固定乳酸菌载体引入发酵体系,建立了ACF的HNO3-Fe(Ⅲ)改性方法,选择315型阴离子交换树脂对发酵液中的乳酸进行分离提取。
本研究采用干酪乳杆菌鼠李糖亚种(Lactobacillus casei subsp.)X1-12作为原始出发菌种,自行设计一种功率连续可调水循环冷却式微波辐照育种装置,用以抵消微波辐照时产生的热效应,在低功率微波条件下,对干酪乳杆菌X1-12进行诱变处理,在微波功率400W,辐照时间3min的诱变条件下,筛选得到一株突变株W4-3-9,L-乳酸产量为115.8g/L,比出发菌株提高了58.0%,连续遗传10代,产酸性状稳定。通过对微波辐照前后乳酸菌细胞表面形态和遗传物质DNA形貌的AFM观察对比可知,微波辐照前乳酸菌表面有明显凸起,而辐照后乳酸菌表面光滑,且遗传物质DNA有明显的开环和断裂情况。进一步对出发菌株X1-12和突变株W4-3-9进行了AFLP DNA指纹图谱分析,聚丙烯酰胺凝胶电泳结果显示出差异条带,并对d21差异带进行测序,在GenBank中进行BLAST序列类似性检索,得出该差异序列与乳酸脱氢酶序列相似,说明微波辐照后乳酸脱氢酶的酶切位点发生了变化。
将选育的高产L-乳酸菌种W4-3-9应用于淀粉工业废水为原料制备乳酸工艺中,其结果表明:玉米浸泡废水与工艺废水体积配比为3:7、葡萄糖添加量70g/L、初始pH 6.8、接种量5%、在温度37℃下厌氧发酵72h,L-乳酸产量可达62.28 g/L,并且发酵过程对废水的COD有45%的去除,NH4+-N去除率75%。
对淀粉废水中的微生物进行平板稀释法分离,初步鉴定废水中存在酵母菌、霉菌、葡萄球菌、芽孢杆菌四类主要微生物。研究微波辐照对淀粉废水培养基杀菌条件,达到培养基细菌总数为0cfu/mL时,微波功率为500W,辐照时间100s。废水培养基中总糖、还原糖、蛋白质营养成分灭菌后损失率为:11.76%、18.77%和7.42%,优于高压蒸汽灭菌的营养成分损失。
实验选择315型阴离子交换树脂对发酵液中的乳酸进行分离提取,通过吸附等温线实验及上柱条件与洗脱条件优化,最终确定最佳上柱流速为1.5BV/h、上柱pH1.9、洗脱剂为去离子水、洗脱流速为1BV/h。在实现乳酸与乙酸、柠檬酸、丙酮酸及葡萄糖等良好的分离基础上,乳酸纯度从原发酵液中的72.4%提高到85.3%,从吸附到洗脱,全过程中乳酸提取率为79.2%。
采用固定化菌体发酵法,研究ACF载体影响乳酸菌固定化的影响因素。实验用浓HCl、H2SO4、HNO3及FeCl3对ACF表面进行改性,并对改性前后的ACF进行XPS射线衍射表征和表面菌膜厚度扫描电镜观察,HNO3-Fe(Ⅲ)协同改性后单丝菌膜厚度为40μm。ACF表面酸性含氧官能团增加,表面正电荷也增加,改性后ACF表面特性能很好地促进乳酸菌的吸附固定,固定乳酸菌的ACF载体用于发酵过程,发酵时间缩短到48h,L-乳酸产量68.6g/L较无载体发酵的L-乳酸量增加了6.4 g/L,同时为后序乳酸发酵液分离提供了良好条件。
实验表明:玉米淀粉生产废水作为发酵培养基,改性ACF作乳酸菌载体,采用阴离子交换树脂吸分离提取发酵液中乳酸,整个工艺可以实现淀粉废水的资源化、无害化,具有经济与环境的双重效益。
To solve the problems in the treatment and resource recovery of the starch industry wastewater and the mutation breeding of microogranism, this study focuses on the microwave mutation breeding technology of lactic acid bacteria with high L-lactic acid yielding ability, based on molecular biology and microwave irradiation technology. The technology of breeding lactic acid bacteria with high lactic acid yielding ability by microwave mutation was developed, the ralted experimental device was designed and developed, breeding of strain with high lactica acid yielding ability was developed; the mutant strain was characterized by physiological, biochemical and molecular biological methods, the process and mechanism of mutation of lactic acid yielding baceria by microwave was discussed, major influencing factors and rule in microwave field for the strain with high lactic acid yielding ability was investigated. Meanwhile, the technology of fermentation production of L-lactic acid by using wastewater of starch industry was studied, activated carbon fibers (ACF) was introduced into the frementation system as carrier immobilizing lactic acid bacteria, modifying method of HNO3-Fe(Ⅲ) of ACF was consructed. 315 anion exchange resin was used to separate the lactic acid form the fermentation broth.
Lactobacillus casei subsp, X1-12, was used as the starter strain for mutation, a microwave irradiation breeding device with continuous power and adjustable water circulating cooling was designed to counteract the heating effect produced by the microwave irradiation. Under a microwave power of 400W and irradiation length of 3min, a mutated strain W4-3-9 with high-yielding L-lactic acid was obtained by screening. Compared with the starting strain X1-12,the L-lactic acid production of W4-3-9 was increased by 58.0% to a concentration of 115.8g/L.The strain maintained the ability to produce a high L-lactic acid level after 10 generations. Cell surface morphology and DNA structures of parental and mutated strains were observed by atomic force microscopy (AFM). Amplified fragment length polymorphism (AFLP) DNA fingerprint experiments were conducted on X1-12 and W4-3-9, polyacrylamide gel electrophoresis suggested difference in AFLP band pattern between the mutated and non-mutated strains, and the order of different band of d21 were checked, BLAST sequence similarity index in GenBank was conducted, the results revealed that catalytic site of lactate dehydrogenase (DHL) was changed due to the microwave induced mutation.
Mutated Strain W4-3-9, which had high L-lactic acid yield, was applied in the technology of producing L-lactic acid from starch industry wastewater. The results showed that when the ratio of corn steep water and waste water was 3:7, glucose was 70g/L, initial pH was 6.8, inoculation was 5%, after anaerobic fermentation at 37℃for 72h, the yield of L-lactic acid was 62.28 g/L, and 45% of the COD of wastewater was removed, the removal of NH4+-N was 75%, B/C was increased, biodegradability.was also improved.
After separating microorganism in starch wastewater with plate dilution method, four main kinds of microbe including coccus, bacillus, molds, yeast were found by the preliminary identification. Sterilization condition of microwave irradiation for starch wastewater culture medium was studied. When microwave irradiation power reached 500W, microwave irradiation time got 100s, micro-organisms were killed totally, the total number of micro-organisms was 0 cfu/mL. The loss ratio of total sugar, reducing sugar,and protein nutrition in starch wastewater culture medium are 11.76%、18.77%、7.42% , is better than the result of high-pressure steam sterilization.
315 anion exchange resin was used to separate the lactic acid form the fermentation broth, after adsorption isotherm experiment and the optimization experiment, the optimal conditions were determined as follows: flow rate was 1.5BV/h, pH was 1.9, eluent was deionized water, elution rate was 1BV/h. based on the realization of the separation of lactic acid from acetic acid, citric acid, pyruvic acid and glucose, the purity was improved from72.4% to 85.3%. From adsorption to elution, the extract rate of lactic acid was 79.2%.
Effect of activated carbon fibers (ACF) carrier on immobilization of lactic acid was studied by biomass immobilization fermentation. ACF surface was modified by HCl, H2SO4, HNO3 and Fe3+ with high concentration, and ACF before and after modification was characterized by XPS and the thickness of the biofilm was observed by scanning electron microscope (SEM). After HNO3-Fe(Ⅲ) co-modification, the thickness of single biofilm was 40μm. The acidic oxygen-containing functional group increased on the surface of ACF, the positive charge also increased, after modification, the surface property of ACF improved the immobilization of lactic acid bacteria. When the ACF carrier immobilizing lactic acid was used to ferment, the fermentation time reduced to 48h, compared with non-carrier, the yield of L-lactic acid increased from 62.2g/L to 68.6 g/L, providing good conditions for the separation of later lactic acid broth.
Results showed that starch industry wastewater resource recovery and harmlessness can be realized by using wastewater from corn starch as fermentation medium, ACF as lactic acid bacteria carrier, separating lactic acid from fermentation broth by anion exchange resin adsorption, which brought both good economic and environmental benefits.
本研究采用干酪乳杆菌鼠李糖亚种(Lactobacillus casei subsp.)X1-12作为原始出发菌种,自行设计一种功率连续可调水循环冷却式微波辐照育种装置,用以抵消微波辐照时产生的热效应,在低功率微波条件下,对干酪乳杆菌X1-12进行诱变处理,在微波功率400W,辐照时间3min的诱变条件下,筛选得到一株突变株W4-3-9,L-乳酸产量为115.8g/L,比出发菌株提高了58.0%,连续遗传10代,产酸性状稳定。通过对微波辐照前后乳酸菌细胞表面形态和遗传物质DNA形貌的AFM观察对比可知,微波辐照前乳酸菌表面有明显凸起,而辐照后乳酸菌表面光滑,且遗传物质DNA有明显的开环和断裂情况。进一步对出发菌株X1-12和突变株W4-3-9进行了AFLP DNA指纹图谱分析,聚丙烯酰胺凝胶电泳结果显示出差异条带,并对d21差异带进行测序,在GenBank中进行BLAST序列类似性检索,得出该差异序列与乳酸脱氢酶序列相似,说明微波辐照后乳酸脱氢酶的酶切位点发生了变化。
将选育的高产L-乳酸菌种W4-3-9应用于淀粉工业废水为原料制备乳酸工艺中,其结果表明:玉米浸泡废水与工艺废水体积配比为3:7、葡萄糖添加量70g/L、初始pH 6.8、接种量5%、在温度37℃下厌氧发酵72h,L-乳酸产量可达62.28 g/L,并且发酵过程对废水的COD有45%的去除,NH4+-N去除率75%。
对淀粉废水中的微生物进行平板稀释法分离,初步鉴定废水中存在酵母菌、霉菌、葡萄球菌、芽孢杆菌四类主要微生物。研究微波辐照对淀粉废水培养基杀菌条件,达到培养基细菌总数为0cfu/mL时,微波功率为500W,辐照时间100s。废水培养基中总糖、还原糖、蛋白质营养成分灭菌后损失率为:11.76%、18.77%和7.42%,优于高压蒸汽灭菌的营养成分损失。
实验选择315型阴离子交换树脂对发酵液中的乳酸进行分离提取,通过吸附等温线实验及上柱条件与洗脱条件优化,最终确定最佳上柱流速为1.5BV/h、上柱pH1.9、洗脱剂为去离子水、洗脱流速为1BV/h。在实现乳酸与乙酸、柠檬酸、丙酮酸及葡萄糖等良好的分离基础上,乳酸纯度从原发酵液中的72.4%提高到85.3%,从吸附到洗脱,全过程中乳酸提取率为79.2%。
采用固定化菌体发酵法,研究ACF载体影响乳酸菌固定化的影响因素。实验用浓HCl、H2SO4、HNO3及FeCl3对ACF表面进行改性,并对改性前后的ACF进行XPS射线衍射表征和表面菌膜厚度扫描电镜观察,HNO3-Fe(Ⅲ)协同改性后单丝菌膜厚度为40μm。ACF表面酸性含氧官能团增加,表面正电荷也增加,改性后ACF表面特性能很好地促进乳酸菌的吸附固定,固定乳酸菌的ACF载体用于发酵过程,发酵时间缩短到48h,L-乳酸产量68.6g/L较无载体发酵的L-乳酸量增加了6.4 g/L,同时为后序乳酸发酵液分离提供了良好条件。
实验表明:玉米淀粉生产废水作为发酵培养基,改性ACF作乳酸菌载体,采用阴离子交换树脂吸分离提取发酵液中乳酸,整个工艺可以实现淀粉废水的资源化、无害化,具有经济与环境的双重效益。
To solve the problems in the treatment and resource recovery of the starch industry wastewater and the mutation breeding of microogranism, this study focuses on the microwave mutation breeding technology of lactic acid bacteria with high L-lactic acid yielding ability, based on molecular biology and microwave irradiation technology. The technology of breeding lactic acid bacteria with high lactic acid yielding ability by microwave mutation was developed, the ralted experimental device was designed and developed, breeding of strain with high lactica acid yielding ability was developed; the mutant strain was characterized by physiological, biochemical and molecular biological methods, the process and mechanism of mutation of lactic acid yielding baceria by microwave was discussed, major influencing factors and rule in microwave field for the strain with high lactic acid yielding ability was investigated. Meanwhile, the technology of fermentation production of L-lactic acid by using wastewater of starch industry was studied, activated carbon fibers (ACF) was introduced into the frementation system as carrier immobilizing lactic acid bacteria, modifying method of HNO3-Fe(Ⅲ) of ACF was consructed. 315 anion exchange resin was used to separate the lactic acid form the fermentation broth.
Lactobacillus casei subsp, X1-12, was used as the starter strain for mutation, a microwave irradiation breeding device with continuous power and adjustable water circulating cooling was designed to counteract the heating effect produced by the microwave irradiation. Under a microwave power of 400W and irradiation length of 3min, a mutated strain W4-3-9 with high-yielding L-lactic acid was obtained by screening. Compared with the starting strain X1-12,the L-lactic acid production of W4-3-9 was increased by 58.0% to a concentration of 115.8g/L.The strain maintained the ability to produce a high L-lactic acid level after 10 generations. Cell surface morphology and DNA structures of parental and mutated strains were observed by atomic force microscopy (AFM). Amplified fragment length polymorphism (AFLP) DNA fingerprint experiments were conducted on X1-12 and W4-3-9, polyacrylamide gel electrophoresis suggested difference in AFLP band pattern between the mutated and non-mutated strains, and the order of different band of d21 were checked, BLAST sequence similarity index in GenBank was conducted, the results revealed that catalytic site of lactate dehydrogenase (DHL) was changed due to the microwave induced mutation.
Mutated Strain W4-3-9, which had high L-lactic acid yield, was applied in the technology of producing L-lactic acid from starch industry wastewater. The results showed that when the ratio of corn steep water and waste water was 3:7, glucose was 70g/L, initial pH was 6.8, inoculation was 5%, after anaerobic fermentation at 37℃for 72h, the yield of L-lactic acid was 62.28 g/L, and 45% of the COD of wastewater was removed, the removal of NH4+-N was 75%, B/C was increased, biodegradability.was also improved.
After separating microorganism in starch wastewater with plate dilution method, four main kinds of microbe including coccus, bacillus, molds, yeast were found by the preliminary identification. Sterilization condition of microwave irradiation for starch wastewater culture medium was studied. When microwave irradiation power reached 500W, microwave irradiation time got 100s, micro-organisms were killed totally, the total number of micro-organisms was 0 cfu/mL. The loss ratio of total sugar, reducing sugar,and protein nutrition in starch wastewater culture medium are 11.76%、18.77%、7.42% , is better than the result of high-pressure steam sterilization.
315 anion exchange resin was used to separate the lactic acid form the fermentation broth, after adsorption isotherm experiment and the optimization experiment, the optimal conditions were determined as follows: flow rate was 1.5BV/h, pH was 1.9, eluent was deionized water, elution rate was 1BV/h. based on the realization of the separation of lactic acid from acetic acid, citric acid, pyruvic acid and glucose, the purity was improved from72.4% to 85.3%. From adsorption to elution, the extract rate of lactic acid was 79.2%.
Effect of activated carbon fibers (ACF) carrier on immobilization of lactic acid was studied by biomass immobilization fermentation. ACF surface was modified by HCl, H2SO4, HNO3 and Fe3+ with high concentration, and ACF before and after modification was characterized by XPS and the thickness of the biofilm was observed by scanning electron microscope (SEM). After HNO3-Fe(Ⅲ) co-modification, the thickness of single biofilm was 40μm. The acidic oxygen-containing functional group increased on the surface of ACF, the positive charge also increased, after modification, the surface property of ACF improved the immobilization of lactic acid bacteria. When the ACF carrier immobilizing lactic acid was used to ferment, the fermentation time reduced to 48h, compared with non-carrier, the yield of L-lactic acid increased from 62.2g/L to 68.6 g/L, providing good conditions for the separation of later lactic acid broth.
Results showed that starch industry wastewater resource recovery and harmlessness can be realized by using wastewater from corn starch as fermentation medium, ACF as lactic acid bacteria carrier, separating lactic acid from fermentation broth by anion exchange resin adsorption, which brought both good economic and environmental benefits.
引文
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