微生物学通报 MAR 20, 2009, 36(3): 383~388 Microbiology tongbao@im.ac.cn 2009 by Institute of Microbiology, CAS 研究报告 生物油脂高产菌株筛选方法研究 宋安东 1* 冯冲 1,2 王风芹 1 谢慧 1 (1. 450002) (2. 450044) 摘要 : 本文采用脂肪粒计数法 苏丹 III 菌泥染色法 碳饥饿检出法等对生物油脂高产菌株进行了筛选比较研究 通过试验表明, 苏丹 III 菌泥染色法简便快捷, 其结果与菌体的油脂含量有较好的相关性, 此法是筛选产脂菌株的较理想的初筛方法 碳饥饿检出法准确性较高, 但过于繁琐 ; 相比之下, 脂肪粒计数法虽简便, 但缺乏准确性, 使用时必须同时考虑脂肪粒的大小 通过筛选, 获得了一株生物油脂高产菌株 IFFI01368, 其菌体中油脂含量为 54.25% 关键词 : 生物油脂, 筛选方法, 脂肪粒, 油脂含量 Research of Screening Method of the High Bio-oil Production Microorganisms SONG An-Dong 1* FENG Chong 1,2 WANG Feng-Qin 1 XIE Hui 1 (1. College of Life Science, Henan Agricultural University, Zhengzhou, Henan 450002, China) (2. Experimental Management Centre, Zhongzhou University, Zhengzhou, Henan 450044, China) Abstract: Fat Particles Counting(FPC), Yeast Mud Dying by Sudan III(YMDSIII), Culture of Lacking Carbon(CLC) were compared and approached in the essay. The results show that YMDSIII is simple and direct, its results are better interrelated with oil-producing quantity of yeast. This method is a relatively ideal screening method on choosing oil-producing microorganisms. CLC is relatively accurate, but it is too tedious. By comparison, FPC is simple and convenient, but it is short of accuracy. When it is used, it must consider the size of fat particles. By screening, cellular oil content reached 54.25% (W/W). Keywords: Bio-oil, Screening methed, Fat particles, Oil contents,,,,,, [1,2] (microbial oils)(single cell oil, SCO), (Bio-oil),,, [3 5], 基金项目 :(No. 70741032); (No. 2006D00070597); (No. 20060400797) * 通讯作者 :Tel: 86-371-63555153; : song1666@126.com 收稿日期 :2008-08-07; 接受日期 :2008-10-17
384 微生物学通报 2009, Vol.36, No.3 [6], 70% 60%, [7,8] 65% [9], ;,,,, ;, ; [3], ;, [10 12],,,,, ;,,, Ⅲ ; 1 材料与方法 1.1 菌种 ( 1) 1.2 培养基及试剂 1.2.1 麦芽汁培养基 : 12 Bé~13 Bé, ph, : 0.1 MPa, 20 min 1.2.2 酵母产脂培养基 :0.8%, 0.3%, 10%, ph 5.8, : 0.1 MPa, 20 min 1.2.3 霉菌产脂培养基 : 10%, 0.15%, 0.05%, 0.05%, 0.1%, 0.7%, 0.25%, FeSO 4 CaCl 2 CuSO 4 ZnSO 4, ph 7.4, : 0.1 MPa, 20 min 1.2.4 缺碳培养基 :0.2%, 0.3%, ph 5.8, : 0.1 MPa, 20 min 0.15%, 0.05%, 0.1%, 0.5%, 0.25%, FeSO 4 CaCl 2 CuSO 4 ZnSO 4, ph 7.4, : 0.1 MPa, 20 min 1.2.5 苏丹黑 B 染色液 [13] : B 0.25 g, 70% 100 ml 1.2.6 苏丹 染色液 : 95%, 0.2%(M/V) III 1.3 试验方法 1.3.1 产脂发酵 10%, 28 C 150 r/min 1.3.2 生物量测定 : 4500 r/min 10 min,,, 1 Table 1 Experimental strains Strain class Yeast Mould Chinese name Latin No. Resource Trichosporon fermentans IFFI01368 Trichosporon cutaneum IFFI01367 Trichosporon capitatum As2.1385 Lipomyces starkeyi As2.1390 Lipomyces konoenkoae IFFI01714 Mortierella vinacea As3.3414 Mortierella isabellina As3.3410 Phycomyces blakesleeanus As3.2061
: 385 1.3.3 油脂提取 [14] :, 40 ml, 20 min, 10 min, - (2:1, V/V), 30 min, 4500 r/min l5 min,,,, 1.4 筛选方法 1.4.1 脂肪粒计数法 :,, B l5 min,,, 2 min,, [15] 1.4.2 缺碳培养基检出法 :, 28 C~30 C, 48 h 1.4.3 苏丹 菌泥染色法 :,, 50 C 20 min, 20 min, 95% 3 min, 2 结果与分析 2.1 菌体生长情况观察 2, IFFI 01368 IFFI01367 IFFI01714 2 d, 2.2 脂肪粒计数法,, 2.2.1 显微观察的脂肪粒 1~5,, 01368, 01367,,, 2.2.2 同一菌株中不同抽样细胞间脂肪粒数目的差异 : 5,, 3 3, 01367, 表 2 斜面菌体生长情况 Table 2 Incline mycelium growth situation Culture time Strain 48 h 65 h 60 h IFFI01368 + + + + + + + + + + + + IFFI01367 + + + + + + + + + + + + IFFI01714 + + + + + + + + + + + + As2.1390 + + + + + + + + + + + As2.1385 + + + + + + + + + + As3.3414 + + + + + + + + + As3.3410 + + + + + + + + + As3.2061 + + + + + : ; + + + + + + + + + +:. Note: : Not growth; +,+ +, + + +,+ + + +: High level of colony growth. 表 3 不同菌株细胞内脂肪粒数目 Table 3 Number of fat particles in different yeast / Number of fat particles/cell Strain 1 2 3 4 5 01368 8 6 10 7 10 8.2 01367 12 8 11 8 9 9.6 01714 5 3 4 2 4 3.6 2.1390 4 1 5 2 4 3.2 2.1385 2 5 3 7 3 4 3.3414 2 3 1 4 2 2.4 3.3410 4 3 3 1 5 3.2 3.2061 1 4 2 1 2 2
386 微生物学通报 2009, Vol.36, No.3 Fig. 1 图 1 01368 苏丹黑染色 01368 stained with Sudan Black B 图 2 Fig. 2 01367 苏丹黑染色 01367 stained with Sudan Black B 图 5 Fig. 5 3.3410 苏丹黑染色 3.3410 stained with Sudan Black B 图 3 3.2061 苏丹黑染色 Fig. 3 3.2061 stained with Sudan Black B 图 4 2.1390 苏丹黑染色 Fig. 4 2.1390 stained with Sudan Black B 1, 01368,, 01368 01367,,,, ;, 2.3 碳饥饿检出法,,,,,,,, [16] 4, 01368 5, 3.3414 3.3410 3.2061 1%,, 2.4 苏丹 菌泥染色法,
: 387,,, 表 4 产脂菌株在缺碳培养基上的生长情况 Table 4 Growth of the oil-product microorganisms with CLC Strain (%) Oil content in body (d) Appear time of colony 01368 54.25 5 01367 48.2 7 01714 14.7 10 2.1390 8.3 11 2.1385 5.8 12 3.3414 2.1 13 3.3410 1.03 13 3.2061 1.9 13 5, 2,, III ( ),, 3, 01368, 01367, 01368, III 01368, 01368, IFFI01368, 54.25% 3 结论及讨论 3.1 结论 III, ;, ;,,,, III IFFI01368, 54.25% 3.2 讨论,,,, III 3 01368,,,,,, Table 5 表 5 苏丹 Ⅲ 染色后菌泥颜色与菌体含油率的相关性 The interrelation of the strain mud color after sudan III stain to oil content Strain Sense determination Ⅲ YMDSⅢ Sequence (%) Oil content Oil extraction Sequence 01368 + + + 1 54.25 1 01367 + + 2 48.2 2 01714 + 3 14.7 3 2.1385 + 4 5.8 5 2.1390 5 8.3 4 3.3414 6 2.1 6 3.3410 7 1.03 8 3.2061 8 1.9 7 + :. Note: +, : That were close to the high-fat, low-fat level of the cell color.
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