摘要
螺旋藻为原核蓝藻,小球藻为真核绿藻,本文以这两类不同的微藻为生物载体,即螺旋藻(包括极大螺旋藻S.maxima和钝顶螺旋藻S.platensis)和小球藻(包括蛋白核小球藻C.pyrenoidosa和普通小球藻C.vulgaris),研究其对硒的同族重元素碲(Te)的吸收转化,及其生理生化响应。取得了一些有重要意义的结果:
1.在螺旋藻接种第1天加入不同剂量的碲,结果表明Te(Ⅳ)对两种螺旋藻的生长表现出不同的效应。低剂量时(χ_(Te)≤0.028,即C_(Te)≤2 mg·L~(-1)),Te(Ⅳ)对S.platensis生长有促进作用,而对S.maxima观察不到促进作用。高剂量时(对于S.platensis,χ_(Te)≥4.4,即C_(Te)≥320 mg·L~(-1);对于S.maxima,χ_(Te)≥11.5,即C_(Te)≥560 mg·L~(-1)),两种螺旋藻几乎全部死亡,但通过比较两种螺旋藻的半数有效浓度96hEC_(50)和碲剂量χ_(Te),发现两种螺旋藻对Te(Ⅳ)的敏感性表现出明显差异,S.maxima对高剂量Te(Ⅳ)的耐受性较S.platensis强。
2.在小球藻接种第工天加入不同剂量的碲,结果表明蛋白核小球藻和普通小球藻对碲的生理生化响应与螺旋藻明显不同。低剂量的碲处理(对于C.pyrenoidosa,χ_(Te)≤0.33,即C_(Te)≤10 mg·L~(-1);对于C.vulgaris,χ_(Te)≤0.20,即_(Te)≤10 mg·L~(-1))对小球藻生长基本无影响;而高剂量(对于C.pyrenoidosa,χ_(Te)≥3.33,即C_(Te)≥100 mg·L~(-1);对于C.vulgaris,χ_(Te)≥2.00,即C_(Te)≥100 mg·L~(-1))的碲处理时,小球藻的生长不但没有受到抑制,反而得到促进。
3.分别在螺旋藻的生长初期(即接种后第3～5天分3次添加Na_2TeO_3 650、750、850和950 mg·L~(-1))和稳定生长期(即7～9天分3次添加Na_2TeO_3 700、800、900和1000 mg·L~(-1))添加碲。生长初期实验组,最大碲剂量出现在第5天(4个碲处理组分别为1.91,2.14,2.43,2.88);稳定生长期实验组,最大碲剂量出现在第9天(4个碲处理组分别为0.90,0.99,1.14,1.26)。尽管从浓度上稳定生长期实验组的碲浓度(1000 mg·L~(-1))是最大的,但其最大碲剂量(1.26)却小于生长初期实验组的最小碲剂量(1.91)。生长初期实验组的藻细胞一直处于较大的碲剂量下,碲表现为毒性因子,致使4个碲处理组螺旋藻的最终生物量、水溶性蛋白、藻蓝蛋白和别藻蓝蛋白,以及色素的含量均低于对照组。而在藻的稳定生长期,由于每个藻细胞所承受的Te(Ⅳ)的量并不抑制藻的生长,此时,碲表现为营养因子,螺旋藻的蛋白、色素等均表现不同程度的增加,而且谷胱甘肽过氧化物酶(GPX)活性也随着碲剂量的增加而增大。
4.进行恒定剂量碲处理实验,维持微藻在整个生长期的碲剂量χ_(Te)为恒定水平。对于螺旋藻的研究结果表明,χ_(Te)≤0.2时,Te(Ⅳ)对S.platensis的生长有促进作用,水溶性蛋白和光合色素含量高于对照组;χ_(Te)=0.4时,Te(Ⅳ)对S.platensis产生了抑制作用,水溶性蛋白含量下降,MDA含量上升。实验结果提示χ_(Te)≤0.2时,Te(Ⅳ)对S.platensis是营养因子;χ_(Te)≥0.4时,Te(Ⅳ)对S.platensis是毒性因子。对于小球藻的研究结果表明,恒定剂量碲处理对小球藻的生理生化性质的影响比较复杂,在实验所设定的碲剂量范围内,不能很明显的区分营养因子和毒性因子。当碲剂量为0.2时,小球藻的生物量和水溶性蛋白含量均最大,但此时除叶绿素a与对照接近外,其它色素含量降低。
5.碲处理方式影响微藻对Te(Ⅳ)的吸收代谢行为。对于螺旋藻,在恒定剂量碲处理的第1天加Te(Ⅳ),螺旋藻中的有机碲产率并不是很高,添加的Te(Ⅳ)大部分被转化为气态碲(86.7%～91.0%),培养液和藻体中的碲含量都很少,这提示了螺旋藻在碲污染的生物修复中具有巨大的应用前景。而在螺旋藻稳定生长期添加Te(Ⅳ),藻体中的有机碲比率较高,对富碲螺旋藻的生产有很好的参考价值。在恒定剂量碲处理的第1天加Te(Ⅳ),小球藻有机碲比率均达到80%左右。而且在较高剂量时,小球藻可以把更多的无机碲转化为气态碲挥发出去。实验结果表明在适当的条件下,螺旋藻和小球藻都是无机碲生物有机化的良好载体,而且在碲污染的生物修复中都可发挥作用。
6.通过谱学方法观察微藻对碲处理的响应,结果表明,碲处理后基本不影响谱峰位置。对于螺旋藻,吸收光谱显示,较高剂量的碲对藻蓝蛋白、叶绿素a的损伤较大;荧光光谱显示较高的碲剂量下,各荧光峰强度最弱。对于小球藻,在实验的碲剂量下对其UV-Vis、荧光、红外光谱影响不大,而且UW-Vis和荧光光谱仍可观察到较高剂量(χ_(Te)=0.4时)的碲处理组各谱峰强度基本最大。这与前面的结果一致,即一定的高剂量的碲,有利于小球藻的生长。因此,对于真核小球藻和原核螺旋藻,两种藻确实表现出对碲不同的响应。
Spirulina,a blue green alga,is a prokaryote.Chlorella,a single-celled green alga,is a eukaryote.Spirulina(S.maxima and S.platensis) and Chlorella(C.pyrenoidosa and C.vulgaris) are used in this paper to compare their different responses to tellurium,which is in the same group as selenium in the periodic table of elements.The physiological and biochemical characteristics of Spirulina and Chlorella,and the absorption and distribution of tellurium in those algae after the addition of tellurium are studied in this research.Some important results are as follows:
1.Two species of Spirulina responded differently to the different tellurium dose(χ_(Te)) when Te(Ⅳ) was added on the first day of inoculation.At the low tellurium dose(χ_(Te)≤0.028,C_(Te)≤2 mg·L~(-1)) Te(Ⅳ) promoted the growth of S.platensis.However,no effect was observed on S.maxima.At high tellurium dose(for S.platensis,χ_(Te)≥4.4,C_(Te)≥320 mg·L~(-1);for S.maxima,χ_(Te)≥11.5,C_(Te)≥560 mg·L~(-1)),both Spirulina died after the addition of tellurium.S.maxima can endure more Te(Ⅳ) than S.platensis by comparing their 96hEC_(50) and tellurium dose.
2.Two species of Chlorella had the same responses to tellurium dose(χ_(Te)) when Te(Ⅳ) was added on the first day of inoculation.These responses,however,were totally different from Spirulina.At low tellurium dose(for C.pyrenoidosa,χ_(Te)≤0.33,C_(Te)≤10 mg·L~(-1);for C.vulgaris,χ_(Te)≤0.20,C_(Te)≤10 mg·L~(-1)),there was no effect to the growth of Chlorella. But at high tellurium dose(for C.pyrenoidosa,χ_(Te)≥3.33,C_(Te)≥100 mg·L~(-1);for C.vulgaris,χ_(Te)≥2.00,C_(Te)≥100 mg·L~(-1)),the growth of Chlorella was promoted instead of being stressed as with Spirulina.
3.Te(Ⅳ) was added to Spirulina at the different stages of their growth.At the initial stage, Te(Ⅳ) was added on the 3rd to the 5th day after inoculation with the concentration of 650, 750,850 and 950 mg·L~(-1).The maximum tellurium dose on the 5th day was 1.91,2.14, 2.43 and 2.88 for different test sets.At the stable stage,Te(Ⅳ) was added on the 7th to the 9th day after inoculation with the concentration of 700,800,900 and 1000 mg·L~(-1),and the maximum tellurium dose on the 9th day was 0.90,0.99,1.14 and 1.26 respectively. Though the Te(Ⅳ) concentration of 1000 mg·L~(-1) is the maximum in its test sets,itsχ_(Te) is 1.26.This is smaller than the minimumχ_(Te) 1.91 in the initial stage test sets.When Te(Ⅳ) was added to Spirulina at the initial stage,the algae cells were in higher tellurium dose, and Te(Ⅳ) had toxicity to Spirulina.So the final biomass,the content of soluble protein, phycocyanin,allophycocyanin and pigments of Spirulina were all lower than that of the control group.While at the stable stage of Spirulina,Te(Ⅳ) was nutrient to Spirulina.So the protein and pigments of Spirulina were increased,and the activicty of GPX was also increased with the increase of tellurium dose.
4.We set different Te dose(χ_(Te)) and keptχ_(Te) invariable during the growth of S.platensis and C.pyrenoidosa.It indicated that the growth of S.platensis was promoted and the content of protein and pigments were higher than that of the control group whenχ_(Te) was less than 0.2.However,its growth was stressed,the content of protein being decreased, and MDA was increased whenχ_(Te) was 0.4.This implied that atχ_(Te)≤0.2,Te was nutrient to S.platensis.But atχ_(Te)≥0.4,Te was toxic to it.The results for C.pyrenoidosa were complicated,and it was difficult to distinguish when Te was nutrient or toxic to C. pyrenoidosa.Whenχ_(Te) was 0.2,the biomass and content of soluble protein were the maximum,but the content of all the pigments reduced except chlorophyll a,which was close to that of control.
5.The absorption and distribution of tellurium in the algae were affected by the different methods of adding tellurium.For S.platensis,when Te(Ⅳ) was added on the first day of inoculation andχ_(Te) was kept invariable,it was found that most of the Te(Ⅳ) was released from algal cells in the form of volatile Te,at about 86.7%～91.0%.The content of organic Te in S.platens& was not high,while the organic Te was higher in S.platensis when Te was added at the stable stage in S.platensis.For C.pyrenoidosa,the organic Te in it was about 80%when Te(Ⅳ) was added on the first day of inoculation andχ_(Te) was kept invariable.More Te(Ⅳ) could be transformed into volatile Te by C.pyrenoidosa at higher Te dose.The results indicated that both Spirulina and Chlorella were good biological carriers for Te(Ⅳ),and both of them could be used to restore tellurium pollution.
6.The effects of Te to different algae were observed by different methods of spectra,and the results indicated that all the peaks didn't change their positions after the addtion of Te to S.platensis and C.vulgaris.For S.platensis,the UV-Vis spectrum demonstrated that more harm was done to phycocyanin and chlorophyll a at high Te dose than other pigments;the fluorescence spectra showed that the peak intensities reduced greatly at high Te dose.For C.vulgaris,the UV-Vis,fluorescence and FT-IR spectra didn't change significantly.Moreover,when Te dose was 0.4,the peak intensities were almost the highest.This was consistent with the results we got before,that is,at a certain amount of high Te dose,the growth of C.vulgaris was promoted.In conclusion,two different species of algae had different responses to Te.
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