SCREENING OF USEFUL MICROORGANISMS WITH CELL SORTER

通过流式细胞术筛选特定特征的微生物(在本研究中为高产量)，并对这些微生物进行流式细胞术分选。所使用的模型系统是：(1)荧光类胡萝卜素虾青素(ASX)和微藻雨生球藻，它在包囊时产生ASX；(2)维生素B1(B1；或硫胺素)，它不是荧光的，但被化学转化为荧光衍生物硫色素；以及清酒酵母(酿酒酵母)，它产生大量的B1，以及(3)辅酶吡咯喹啉醌(PQQ)和两种产生PQQ的乳酸菌，主要以全酶(奎诺蛋白)的形式产生。荧光信号越强，包囊大小越大(显微镜观察)，说明…分选更多的雨生红曲霉用于ASX-超高产。这些包囊被再次分析，显示出与诱变前相似的细胞学图谱，表明获得了不稳定的高产特性。清酒酵母在不同条件下培养，细胞被包裹在凝胶微滴(GMD)中，典型的和成功的用多孔玻璃过滤器来产生微集落。在一定条件下，特别是反应时间较短的条件下，通过快速中和反应混合物，可以测定出GMDs中的B1含量，与整个培养物中的B1含量有很好的相关性(用硫代铬法测定)，并有一定比例的GMDs能使活细胞再生。在整个培养过程中获得了多样性的细胞学，但与PQQ含量没有很好的相关性。紫外光(625 Nm)不适合用于PQQ，因为有其他化合物干扰PQQ。在PQQ的进一步研究中，可以考虑使用弧灯和氢灯。较少

Microorganisms were screened by flow cytometry for specific characteristics (hyperproduction, in this study) and such organisms were flow cytometrically sorted. Model systems used were :(1) a fluorescent carotenoid astaxanthin (ASX) and a microalga Heamatococcus pluvialis, which produces ASX when encysting,(2) vitamin B1 (B1 ; or thiamin), which is not fluorescent but is chemically transformed to a fluorescent derivative thiochrome, and a sake yeast (Saccharomyces cerevisiae), which produces large amounts of B1, and(3) a coenzyme pyrroloquinoline quinone (PQQ) and two lactic-acid bacteria Acetobacter acetiand Gluconobacter oxidans, which produce PQQ, mainly as holoenzymes (quinoproteins).Hyperproduction of these compounds was induced by treatment with mutagenic compounds and hyperproducers were sorted.Cysts of H.pluvialis were analysed by flow cytometry. The stronger the fluorescence signal was, the larger the size of the cysts was (as microscopic observation), demonstrating sorting of … More H.pluvialis for ASX-hyperproduction. Such cysts were encysted and analysed again to show similar cytographic pattems as those before mutagenesis, suggesting the unstable hyperproducing characteristic obtained.The sake yeast was cultured under various conditions and cells were encapsulated in gel microdroplets (GMDs), typically and successfully with porous glass filter, to produce microcolonies. After thiochrome reaction under certain conditions, epecially with shorter reaction periods and by quick neutralisation of the reaction mixtures, GMDs could be measured for B1 contents with good correlation with the B1 content of the whole cultue (assayed by thiochrome method) and some percentages of GMDs gave regeneration of living cells.The lactic-acid bacteria were cultured under various conditions and cytometrically analysed. Variety of cytogrammes was obtained but without well correlation to the PQQ contents in the whole culture. The UV laser used (625 nm) was not suitable for PQQ because of other compounds which interfered PQQ.Use of arc and deuterium lamps might be considered in further study with PQQ. Less