CN115011484A - 一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法 - Google Patents
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Abstract
一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,涉及一种去除重金属六价铬和促进微藻产油脂的方法。本发明是要解决六价铬容易溶解,并具有很强的氧化能力,损害生物的遗传物质以及不可再生能源的供应已经难以满足日益增长的能源需求的技术问题。本发明首先将微藻接种到含有重金属Cr(Ⅵ)的酸性培养基中,培养基的初始pH为3.5,培养温度为15℃;培养微藻至对数生长后期离心收集藻细胞,冻干称重,利用超声破碎结合有机溶剂提取干藻粉中的油脂。酸性条件与实际含有重金属的废水接近,且低温环境适宜一些寒冷地区废水处理。本发明方法能够有效的去除重金属Cr(Ⅵ),并促进微藻油脂积累。
Description
技术领域
本发明涉及一种去除重金属六价铬和促进微藻产油脂的方法。
背景技术
工业化生产和人类活动向环境中释放了大量的金属,但是这些金属中只有一部分是环境必不可少的,而其他非必需的重金属对生态则构成了威胁。铬被认为是一种致癌物质,特别是六价铬,其毒性是三价铬的100倍以上,六价铬容易溶解,并具有很强的氧化能力,会损害生物的遗传物质,铬(Ⅵ)的生物积累对生态***来说是一个严重的问题。此外,一些微生物如微藻,在培养过程中有很强的去除重金属的能力,采用微藻去除废水中的六价铬是一种经济、环保和可持续的方法。
目前,随着经济全球化和工业化进行的不断发展,不可再生能源的供应已经难以满足日益增长的能源需求,因此,需要寻求一种替代传统化石燃料的新型能源。生物柴油由于清洁、环保、十六烷值高等优点而成为传统化石能源良好的替代品。而微藻作为生产生物柴油的候选生产原料,由于生长周期快、油脂含量高、不占用耕地等优势而备受关注。利用微藻生产生物柴油,同时利用微藻生物吸附重金属,可同时实现生产可再生绿色能源和减少环境污染。
发明内容
本发明是要解决六价铬容易溶解,并具有很强的氧化能力,损害生物的遗传物质以及不可再生能源的供应已经难以满足日益增长的能源需求的技术问题,而提供一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法。
本发明的低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法是按以下步骤进行的:
一、种子液的制备:将微藻接种至含有10g/L~12g/L葡萄糖的BG-11培养基中培养至对数生长期,得到微藻培养的种子液;
二、藻液的培养:将步骤一中所获得的微藻培养的种子液接种至含有0.5mg/L~40mg/L的Cr(Ⅵ)的BG-11培养基,调整pH为3.5~4,培养温度为14℃~16℃,光暗比为光12h:暗12h,摇床转速为150r/min~160r/min,培养至对数生长后期,然后离心收集藻细胞;
三、微藻油脂的提取:采用超声破碎结合有机溶剂提取藻细胞内的油脂;
所述的采用超声破碎结合有机溶剂提取藻细胞内的油脂的方法是:将步骤二中收集的藻细胞用蒸馏水反复洗涤2次~3次,冻干,随后加入氯仿-甲醇溶液,在功率为200W~220W下超声破碎至藻体发白,离心收集有机相干燥获得油脂;
所述的氯仿-甲醇溶液中氯仿和甲醇的体积比为2:1;
所述的冻干后的藻粉的质量与氯仿-甲醇溶液的体积比为0.1g:(10mL~12mL)。
本发明在步骤二中选择酸性环境培养(pH为3.5~4)的原因:有色金属行业在金属矿山开采、选矿、冶炼等过程中会产生pH较低且含有重金属的酸性废水,如不经处理会腐蚀下水管道、水工构筑物等基础设施,且其中的重金属会严重污染地表水、地下水或土壤,影响动植物的生长繁殖和农作物生长,对环境质量和人体健康造成巨大的危害。
本发明在步骤二中选择低温培养(14℃~16℃)的原因:低温环境根据目前北方环境经常处于较为低温的环境,因此采用低温酸性条件下处理重金属。
本发明公开了一种低温酸性条件下去除重金属Cr(Ⅵ)及促进微藻产油脂的方法,主要包括:首先将微藻接种到含有重金属Cr(Ⅵ)的酸性培养基中,培养基中Cr(Ⅵ)的浓度为0.5~40mg/L,培养基的初始pH为3.5,培养温度为15℃;培养微藻至对数生长后期离心收集藻细胞,冻干称重,利用超声破碎结合有机溶剂提取干藻粉中的油脂。酸性条件与实际含有重金属的废水接近,且低温环境适宜一些寒冷地区废水处理。本发明方法操作简单,能够有效的去除重金属Cr(Ⅵ),去除率可达到91%;并促进微藻油脂积累,油脂含量最高达到61.03%,油脂产率最高达到131.79mg/L·d。本发明为利用微藻处理废水同时促进微藻产油等问题提供了一种有效的技术手段。
附图说明
图1为Cr(Ⅵ)的去除效率图。
具体实施方式
具体实施方式一:本实施方式为一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,具体是按以下步骤进行的:
一、种子液的制备:将微藻接种至含有10g/L~12g/L葡萄糖的BG-11培养基中培养至对数生长期,得到微藻培养的种子液;
二、藻液的培养:将步骤一中所获得的微藻培养的种子液接种至含有0.5mg/L~40mg/L的Cr(Ⅵ)的BG-11培养基,调整pH为3.5~4,培养温度为14℃~16℃,光暗比为光12h:暗12h,摇床转速为150r/min~160r/min,培养至对数生长后期,然后离心收集藻细胞;
三、微藻油脂的提取:采用超声破碎结合有机溶剂提取藻细胞内的油脂;
所述的采用超声破碎结合有机溶剂提取藻细胞内的油脂的方法是:将步骤二中收集的藻细胞用蒸馏水反复洗涤2次~3次,冻干,随后加入氯仿-甲醇溶液,在功率为200W~220W下超声破碎至藻体发白,离心收集有机相干燥获得油脂;
所述的氯仿-甲醇溶液中氯仿和甲醇的体积比为2:1;
所述的冻干后的藻粉的质量与氯仿-甲醇溶液的体积比为0.1g:(10mL~12mL)。
具体实施方式二:本实施方式与具体实施方式一不同的是:步骤一中所述的微藻为栅藻。其他与具体实施方式一相同。
具体实施方式三:本实施方式与具体实施方式一或二不同的是:步骤一中将微藻接种至含有10g/L葡萄糖的BG-11培养基中培养至对数生长期,得到微藻培养的种子液。其他与具体实施方式一或二相同。
具体实施方式四:本实施方式与具体实施方式一至三之一不同的是:步骤二中调整pH为3.5。其他与具体实施方式一至三之一相同。
具体实施方式五:本实施方式与具体实施方式四不同的是:步骤二中调整pH的方法是通过添加1mol/L的盐酸实现的。其他与具体实施方式四相同。
具体实施方式六:本实施方式与具体实施方式五不同的是:步骤二中对数生长后期为进入对数生长期7天~9天。其他与具体实施方式五相同。
具体实施方式七:本实施方式与具体实施方式六不同的是:步骤二中摇床转速为150r/min。其他与具体实施方式六相同。
具体实施方式八:本实施方式与具体实施方式七不同的是:步骤二中离心转速为8000r/min,离心时间为5min。其他与具体实施方式七相同。
具体实施方式九:本实施方式与具体实施方式八不同的是:步骤三中在功率为200W下超声破碎至藻体发白。其他与具体实施方式八相同。
具体实施方式十:本实施方式与具体实施方式九不同的是:步骤三中所述的冻干后的产物的质量与氯仿-甲醇溶液的体积比为0.1g:10mL。其他与具体实施方式九相同。
用以下试验对本发明进行验证:
试验一:本试验为一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,具体是按以下步骤进行的:
一、种子液的制备:将微藻接种至含有10g/L葡萄糖的BG-11培养基中培养至对数生长期,得到微藻培养的种子液;步骤一中所述的微藻为栅藻(ParachlorellakessleriR-3),采用已公开中国发明专利《一种富油微藻的高通量筛选方法》(申请号:CN201310096030.2)中的方法筛选获得;
二、藻液的培养:将步骤一中所获得的微藻培养的种子液接种至含有0.5mg/L的Cr(Ⅵ)的BG-11培养基,调整pH为3.5,培养温度为15℃,光暗比为光12h:暗12h,摇床转速为150r/min,培养至对数生长后期,然后离心收集藻细胞;调整pH的方法是通过添加1mol/L的盐酸实现的;步骤二中对数生长后期为进入对数生长期7天~9天;步骤二中离心转速为8000r/min,离心时间为5min;
三、微藻油脂的提取:采用超声破碎结合有机溶剂提取藻细胞内的油脂;
所述的采用超声破碎结合有机溶剂提取藻细胞内的油脂的方法是:将步骤二中收集的藻细胞用蒸馏水反复洗涤3次,冻干,随后加入氯仿-甲醇溶液,在功率为200W下超声破碎至藻体发白,离心收集有机相干燥获得油脂;
所述的氯仿-甲醇溶液中氯仿和甲醇的体积比为2:1;
所述的冻干后的藻粉的质量与氯仿-甲醇溶液的体积比为0.1g:10mL。
重金属去除效率的测定:将步骤二中离心收集的上清液过0.22μm的水系滤膜后,用ICP-OES测定上清液中的Cr(Ⅵ)含量。
对步骤三中冻干后的产物进行生物量测试。
结果表明,低温酸性条件下0.5mg/L的Cr(Ⅵ)的实验组,微藻的生物量为2.05g/L,步骤三中获得的油脂中油脂含量53.84%,油脂产率为116.73mg/L·d。
试验二:本试验与试验一不同的是:步骤二中将步骤一中所获得的微藻培养的种子液接种至含有1mg/L的Cr(Ⅵ)的BG-11培养基。其它与试验一相同。
结果表明,低温酸性条件下添加1mg/L的Cr(Ⅵ)的实验组,微藻的生物量为2.09g/L,获得的油脂中油脂含量53.11%,油脂产率为117.29mg/L·d。
试验三:本试验与试验一不同的是:步骤二中将步骤一中所获得的微藻培养的种子液接种至含有0mg/L的Cr(Ⅵ)的BG-11培养基(即没有加六价铬)。其它与试验一相同。
结果表明,低温酸性条件下添加0mg/L的Cr(Ⅵ)的对照组,微藻的生物量为1.93g/L,获得的油脂中油脂含量51.89%和油脂产率为105.22mg/L·d。
试验四:本试验与试验一不同的是:步骤二中将步骤一中所获得的微藻培养的种子液接种至含有2mg/L的Cr(Ⅵ)的BG-11培养基。其它与试验一相同。
结果表明,低温酸性条件下添加2mg/L的Cr(Ⅵ)的实验组,微藻的生物量为2.40g/L,获得的油脂中油脂含量51.65%,油脂产率为131.79mg/L·d。
试验五:本试验与试验一不同的是:步骤二中将步骤一中所获得的微藻培养的种子液接种至含有5mg/L的Cr(Ⅵ)的BG-11培养基。其它与试验一相同。
结果表明,低温酸性条件下添加5mg/L的Cr(Ⅵ)的实验组,微藻的生物量为1.39g/L,获得的油脂中油脂含量61.03%,油脂产率为89.30mg/L·d。
试验六:本试验与试验一不同的是:步骤二中将步骤一中所获得的微藻培养的种子液接种至含有10mg/L的Cr(Ⅵ)的BG-11培养基。其它与试验一相同。
结果表明,低温酸性条件下添加10mg/L的Cr(Ⅵ)的实验组,微藻的生物量为1.35g/L,获得的油脂中油脂含量25.31%,油脂产率为35.12mg/L·d。
试验七:本试验与试验一不同的是:步骤二中将步骤一中所获得的微藻培养的种子液接种至含有20mg/L的Cr(Ⅵ)的BG-11培养基。其它与试验一相同。
结果表明,低温酸性条件下添加20mg/L的Cr(Ⅵ)的实验组,微藻的生物量为1.05g/L,获得的油脂中油脂含量18.55%,油脂产率为19.63mg/L·d。
试验八:本试验与试验一不同的是:步骤二中将步骤一中所获得的微藻培养的种子液接种至含有40mg/L的Cr(Ⅵ)的BG-11培养基。其它与试验一相同。
结果表明,低温酸性条件下添加40mg/L的Cr(Ⅵ)的实验组,微藻的生物量为0.69g/L,获得的油脂中油脂含量18.05%,油脂产率为11.84mg/L·d。
结果分析:表1汇总了试验一至试验八中低温酸性条件下不同浓度的Cr(Ⅵ)浓度下对微藻生物量、油脂含量及油脂产率的影响。
表1低温酸性条件下不同浓度的Cr(Ⅵ)浓度下对微藻生物量、油脂含量和油脂产率的影响
上述结果表明,低温酸性条件下,较试验三中添加0mg/L Cr(Ⅵ),试验一、试验二和试验四中微藻生物量均有不同的程度的提升,这说明适当浓度的重金属Cr(Ⅵ)能促进微藻的生物量积累,但是当Cr(Ⅵ)浓度超过5mg/L时,微藻生物量由于重金属Cr(Ⅵ)浓度过高,生物量急剧下降。对于油脂含量,试验一、试验二和试验四较试验三没有显著性不同,而当Cr(Ⅵ)浓度为5mg/L(试验五)时,油脂含量达到最高,达到61.03%。而试验四中当Cr(Ⅵ)浓度为2mg/L时,油脂产率最高,达到131.79mg/L·d。
图1为Cr(Ⅵ)的去除效率图,横坐标为步骤二中的培养时间,■为试验一,●为试验二,▲为试验四、为试验五,◆为试验六,为试验七,为试验八,可以看出,重金属的去除效率也与Cr(Ⅵ)的添加浓度相关,当Cr(Ⅵ)添加浓度为0.5mg/L~10mg/L时去除效率较高,其中0.5mg/L时(试验一)达到最高,可达到91%;但当Cr(Ⅵ)浓度为20mg/L~40mg/L时,去除效率最高仅达到60.1%。
因此,通过表1和图1中,微藻的生物量、油脂含量、油脂产率和Cr(Ⅵ)的去除效率的结果看来,这些结果为低温酸性条件下微藻去除重金属Cr(Ⅵ)和油脂积累提供了数据支持及理论支持。可以看出,低温酸性条件下,微藻能够高效的积累油脂和去除重金属Cr(Ⅵ),重金属Cr(Ⅵ)的添加范围为0.5~40mg/L,获得最佳的油脂含量和Cr(Ⅵ)的去除效率,由此可见,在低温酸性条件下微藻R-3能够高效的积累油脂和去除重金属Cr(Ⅵ),这是一种可行的、经济的、有效的方法,满足处理重金属废水和微藻生产生物柴油的基本需求。
Claims (10)
1.一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法是按以下步骤进行的:
一、种子液的制备:将微藻接种至含有10g/L~12g/L葡萄糖的BG-11培养基中培养至对数生长期,得到微藻培养的种子液;
二、藻液的培养:将步骤一中所获得的微藻培养的种子液接种至含有0.5mg/L~40mg/L的Cr(Ⅵ)的BG-11培养基,调整pH为3.5~4,培养温度为14℃~16℃,光暗比为光12h:暗12h,摇床转速为150r/min~160r/min,培养至对数生长后期,然后离心收集藻细胞;
三、微藻油脂的提取:采用超声破碎结合有机溶剂提取藻细胞内的油脂;
所述的采用超声破碎结合有机溶剂提取藻细胞内的油脂的方法是:将步骤二中收集的藻细胞用蒸馏水反复洗涤2次~3次,冻干,随后加入氯仿-甲醇溶液,在功率为200W~220W下超声破碎至藻体发白,离心收集有机相干燥获得油脂;
所述的氯仿-甲醇溶液中氯仿和甲醇的体积比为2:1;
所述的冻干后藻粉的质量与氯仿-甲醇溶液的体积比为0.1g:(10mL~12mL)。
2.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤一中所述的微藻为栅藻。
3.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤一中将微藻接种至含有10g/L葡萄糖的BG-11培养基中培养至对数生长期,得到微藻培养的种子液。
4.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤二中调整pH为3.5。
5.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤二中调整pH的方法是通过添加1mol/L的盐酸实现的。
6.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤二中对数生长后期为进入对数生长期7天~9天。
7.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤二中摇床转速为150r/min。
8.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤二中离心转速为8000r/min,离心时间为5min。
9.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤三中在功率为200W下超声破碎至藻体发白。
10.根据权利要求1所述的一种低温酸性条件下去除重金属六价铬和促进微藻产油脂的方法,其特征在于步骤三中所述的冻干后的产物的质量与氯仿-甲醇溶液的体积比为0.1g:10mL。
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