本文關鍵詞： 葉綠素a測定 熒光檢測法 藻體標準品 懸浮穩(wěn)定劑 真空冷凍干燥法　出處：《河北科技大學》2016年碩士論文　論文類型：學位論文

【摘要】：葉綠素a含量是水體富營養(yǎng)化指標,熒光法因測定時間短、可以實現現場在線測定等優(yōu)勢在水體葉綠a含量測定中得到廣泛應用,但在測定過程,存在著藻細胞在水體分散不均勻、藻體懸浮體系不穩(wěn)定、易沉降,無藻體標準品等問題,影響測量結果的準確度。論文研究了利用水體懸浮劑制備藻體標準品用于葉綠素熒光現場測定的制備技術,技術研究對水體葉綠素熒光法測定的方法標準化有重要意義。文章簡述了熒光檢測法對葉綠素a測定的國內外研究進展,微生物懸浮穩(wěn)定技術、保存技術的研究進展。以葉綠素a的實驗室熒光法測定方法為基礎,得到如下結論:1)基于蛋白核小球藻的自身優(yōu)勢及實驗優(yōu)勢,確定蛋白核小球藻作為目標藻體,建立了蛋白核小球藻的吸光度值與熒光強度值的標準曲線y=74.831x+16.94(R2=0.9945),最優(yōu)D(663 nm)范圍為0.219~0.777,對應的熒光強度值為31.606~74.722。2)藻細胞沉降,導致其熒光值受靜置時間影響較大,靜置1 min時,熒光值的衰減率達△F/F0=-9.96%,無法獲得準確的藻濃度。3)藻體培養(yǎng)過程中向培養(yǎng)基中添加瓊脂粉0.8~1.0 g·L-1培養(yǎng)蛋白核小球藻,藻液的懸浮性、均勻性、穩(wěn)定性都明顯增強。4)在熒光法測定水樣葉綠素過程中,提出一種藻體懸浮穩(wěn)定技術,在藻液熒光測定環(huán)節(jié)添加復合懸浮劑,即瓊脂0.06%、黃原膠0.20%、卡拉膠0.40%,T=1h時熒光值的變化率為-1.16%,并對實際水樣測定,T=1 h時熒光值的變化率小于±3.5%、精密度較高小于1.5%。5)對長勢較好的蛋白核小球藻在最佳離心條件下,即離心轉速為4 500 r.min-1,離心時間為為10min濃縮100倍;保護劑:15%脫脂奶粉,體積比1:1混合;預凍方式:4℃預凍12 h+液氮(-196℃)冷凍12 h;凍干保存溫度:-20℃。保存時間:40 d(存活率高于40%);100 d(存活率高于30%)。
[Abstract]:Chlorophyll a content is a eutrophication index of water body. Because of the short measuring time, fluorescence method can be used widely in the determination of leaf green a content in water body, but it can be used in the determination process. There are some problems such as uneven dispersion of algal cells in the water body, unstable suspension system of algae body, easy settling, and no algal body standard, etc. In this paper, the preparation technology of algae standard for chlorophyll fluorescence in situ was studied. The technical research is of great significance to the standardization of the methods for the determination of chlorophyll a in water body. This paper briefly introduces the domestic and foreign research progress of fluorescence detection for the determination of chlorophyll a and the technique of microorganism suspension stabilization. Based on the fluorescence assay of chlorophyll a in laboratory, the following conclusions are obtained: 1) based on the advantages of Chlorella protein and its experimental advantages, Chlorella Proteinuca is identified as the target alga. The standard curve of absorbance value and fluorescence intensity value of Chlorella pyrenoidosa was established. The optimum range of DX 663nm was 0.2196nm and the corresponding fluorescence intensity value was 31.606n 74.722.2) the fluorescence value of Chlorella globosa was significantly influenced by standing time, and the fluorescence value was statically affected by 1 min. The attenuation rate of fluorescence value was as high as F / F _ 0 ~ (-9.96). It was impossible to obtain accurate algal concentration. 3) adding Agar powder (0.81.0 g 路L ~ (-1)) to the culture medium of Chlorella vulgaris, the suspension and homogeneity of algal fluid, were obtained by adding Agar powder (0.81.0 g 路L ~ (-1)) to the culture medium. In the process of determination of chlorophyll in water samples by fluorescence method, a suspension stabilization technique for algae was put forward, in which compound suspensions were added in algal liquid fluorescence determination. That is, Agar 0.06, Xanthan 0.20, carrageenan 0.40T + 1 h fluorescence change rate is -1.16, and the change rate of fluorescence value is less than 鹵3.5 at 1 h and higher precision is less than 1.5. 5) under the best centrifugation conditions. The centrifugal speed is 4 500 r.min-1, the centrifugation time is 10 min, the concentration is 100 times, the protective agent is 1: 15% skim milk powder, the volume ratio is 1: 1; The pre-freezing method was pre-frozen at 4 鈩，

本文編號：1516896