基于常压室温等离子体技术的金藻诱变筛选方法

以高生长速率或高油脂含量藻株为目标,以湛江等鞭金藻为例,报道了一种基于常压室温等离子体技术的微藻诱变及快速分级筛选方法。即以叶绿素荧光动力学参数F_v/F_m大于0.68的金藻为出发藻株、以致死率90%为阈值确定最适诱变电流为1.4~1.5A,诱变时间为24~30s。分别在室温常光及胁迫条件(高温常光和高光室温)下,按照孔板至摇瓶至反应器三级培养进行筛选。在孔板培养过程中以比生长速度结合尼罗红荧光强度变化实现高通量初筛,最终在反应器培养中进行提取验证,并以候选藻株为出发进行二次诱变筛选。结果表明,室温常光条件诱变株筛出率为0.7%,胁迫条件筛出率为0.9%;一次诱变诱变株筛出率为0.6%,二次诱变筛出率为1.2%;二次诱变和胁迫条件筛出率更高,更易得到性状变化的诱变株。     

低场核磁共振技术快速检测小球藻油脂含量及其在高通量选育中的应用

为了建立一个高效的高产油微藻诱变育种流程,微藻中油脂含量快速和准确的测定在其中具有重要作用。在本研究中,首先利用低场核磁共振技术,建立了直接检测干藻粉和培养液中小球藻油脂含量的方法,其信号强度与细胞中油脂含量存在特异的线性关系,干藻粉和藻液中油脂含量与信号值拟合的R2均高于0.99,说明该方法用于小球藻油脂含量的检测是准确和可行的。同时该方法与传统油脂测量方法相比,具有快速、简便和准确等优点。但其通量不及尼罗红染色法,因此,我们开发了将尼罗红染色法用于初筛,低场核磁共振技术用于复筛的新型高通量藻种复合筛选方法,并将此筛选方法应用于一种异养高产油原壳小球藻的诱变育种过程中。首先从3 098株诱变藻种中初筛得到108株具有较高油脂含量的藻株,然后利用低场核磁共振技术复筛得到9株高产油性能的藻株,其中一株甘油三酯含量超过20%,比原始藻株提高1倍,培养168 h后培养液油脂浓度达到5 g/L,证明此诱变育种流程不仅提高了筛选的效率还可靠且可行。     

盐度和pH对平凡舟形藻生长和油脂积累的影响

为研究盐度和pH对淡水硅藻生长和油脂含量的影响,对一株分离自野外采集水样中的平凡舟形藻(Navicula trivialis)进行研究,通过设置不同盐度0、0.03、0.06、0.12、0.18和0.24 mol/L和不同pH 4.5、6.0、7.5、8.5、9.5和10.5进行胁迫,测定各处理对平凡舟形藻的生长、叶绿素a含量、叶绿素荧光参数和油脂含量的影响。结果表明:盐浓度为0.12 mol/L、pH 7.5时,平凡舟形藻细胞密度和叶绿素a含量最高;盐浓度为0.24 mol/L、pH 7.5时,总脂含量最高,分别为34.93%和33.5%。结果表明,平凡舟形藻对不同盐度和pH的适应性不同,这在一定程度上影响其生长和油脂含量。     

微拟球藻富油藻株筛选及柱状光生物反应器培养评价研究

研究以亲脂性荧光染料BODIPY505/515和流式细胞仪为基础, 从多株诱变海洋微拟球藻(Nannochloropsis oceanica)中筛选到4株候选富油藻株(MT-1,2,3,4), 并利用柱状光生物反应器对诱变株的产油能力进行了综合评价。结果表明, 藻株筛选时最佳BODIPY505/515使用浓度为0.87 μg/mL, 染色时间为10min; 4株诱变株产油性能较野生株有较大提高, 其中MT-4油脂积累达到了干重的66%, 油脂产率比野生型藻株提高了45%, 达到了27.32 mg/(L·d)。4株诱变株的脂肪酸组成合适, 其中C16和C18之和占78%以上, 且主要以饱和脂肪酸和单不饱和脂肪酸为主; 多不饱和脂肪酸只占总脂肪酸的6%—8%, 非常适合生物柴油生产。研究提供了一种针对海洋微拟球藻富油藻株快速、有效的筛选方法, 并以此为基础筛选得到4株极具生物柴油生产潜力的候选藻株, 有望用于规模化生产。     

两株钝顶螺旋藻紫外诱变株的特征

采用紫外诱变的方法筛选获得了两株优良的稳定钝顶螺旋藻突变株M1-3和M5-1,与出发株相比,M5-1较粗大,M1-3较细,但长很长,藻体螺旋数超过40;两株突变株的生长速度和光合放氧速率均有显著提高;M1-3的藻蓝蛋白含量高于出发藻株20.2%;突变株的长碳连不饱和脂肪酸含量高于出发藻株,总脂中M1-3含花生四烯酸（20：4）4.93%、M5-1含EPA（20：5）2.49%。两株突变株对NH4^ 和Zn^2 的抗性也发生了改变。     

富油脂微藻育种技术研究进展

微藻因生长迅速、光合作用效率高、分布范围广和油脂积累能力强等优势,已被认为是生产生物柴油的理想原料。诱变育种可改善野生型微藻生长缓慢、油脂含量低和环境适应能力弱等缺陷,提高了以微藻生产生物柴油的可行性。概述了物理、化学和基因工程三类诱变育种方法的研究现状,介绍了低场核磁共振和荧光激活细胞分选两种富油脂微藻筛选技术以及一种测定诱变藻株致死率的方法,讨论了三种诱变方法的应用前景,供相关研究人员参考。     

大庆湖藻的分离、鉴定和高油脂含量筛选

目的:对大庆6个典型湖泊的湖藻进行分离和鉴定,筛选出高油脂含量的大庆湖藻。方法:采用吸管分离法、稀释平板分离法和平板划线法共分离出8株硅藻和5株绿藻,并利用化学方法对所分离的湖藻进行油脂含量测定。结果:所分离13株藻种名称和油脂含量分别为GF-1四尾栅藻9.3%、GF-2鼓藻2.7%、GF-3布朗葡萄藻20.9%、GF-4布纹藻32.6%、HY-1卵囊藻5.5%、HY-2舟形藻37.1%、HY-3二形栅藻6.4%、YL-1舟形藻32.2%、YL-2刀形布纹藻38.3%、YL-3舟形藻34.5%、HQ-1脆杆藻36.6%、QY-1盒形藻33.8%、CY-1脆杆藻29.4%。结论:筛选出YL-2、HY-2和HQ-1三株高油脂含量的大庆湖藻。     

一株高脂土壤小球藻的分离鉴定及脂质分析

对采自山西省庞泉沟国家自然保护区的土壤中的藻种进行分离鉴定, 获得了一株优良的高脂绿藻。经显微形态观察鉴定, 该藻株的形态特征属于小球藻属Chlorella (Chlorellasp. PQG67)。进一步对其rbcL和18S rDNA基因序列进行分析并构建系统树, 结果表明基因序列与普通小球藻Ch. vulgaris同源并聚为一支, 确定其为一株普通小球藻Ch. vulgaris PQG67。在不同光照强度下连续培养后测定其油脂含量稳定在30%左右, 在不同NaCl浓度胁迫条件下可达40%以上, 并通过叶绿素荧光值测量探索该藻株生长趋势。通过傅立叶变换红外光谱图对其油脂积累过程分析, 显示该藻株脂类成分在1634/cm附近, 有vC=O伸缩振动谱带, 随着培养时间的延长, 脂质含量的相对强度也在增加。可见该藻株具有较高的生长速率及产油能力, 是一株具产业化应用潜力的优良产油藻株。     

生长速度快且油脂产率高的湛江等鞭金藻诱变株的筛选

【目的】筛选生长速度快的高产油湛江等鞭金藻诱变株。【方法】利用常压室温等离子体射流诱变技术对湛江等鞭金藻进行诱变,通过96孔板、摇瓶和反应器培养对诱变株进行筛选。【结果】传至第13代,600 m L反应器培养7 d,诱变株IM110020最大比生长速率、细胞密度和油脂产率可分别达0.72 d-1、16 750×104 cells/m L、109.8 mg/(L·d),分别比野生株提高12.5%、20.8%和17.9%。【结论】获得了一株生长速度快且油脂产率高的诱变株IM110020,且经过多次传代性质稳定。     

倍频Nd：YAG激光对钝顶螺旋藻的诱变效应

利用倍频 Nd:YAG激光 (波长 53 2 nm,功率 50 0 m W,功率密度 1 60 m W/cm2 )诱变钝顶螺旋藻 ,辐照时间为 1 5min、1 0 min、5min通过测定藻丝形态参数、叶绿素 a、β 胡萝卜素、生长速度 ,比较倍频 Nd:YAG激光对钝顶螺旋藻生长的影响。实验结果表明 :与出发株相比 ,经倍频 Nd:YAG激光辐照后 ,藻丝形态发生变化 ,藻丝长、螺旋数、螺旋长变小 ;1 5min,1 0 min辐照组出现螺旋变松驰 ;1 0 min,5min辐照组促进藻的生长和叶绿素 a含量提高 ,使生长速度提高。三个诱变时间剂量都有利于 β 胡萝卡素积累 ,含量增幅最高达 2 2 .3 %。     

金钟藤和葛藤在干旱与复水条件下的生理比较

对外来入侵种金钟藤和本地种葛藤进行干旱胁迫和水分恢复处理,比较了两种植物抗氧化酶(SOD、CAT)活性、丙二醛(MDA)含量及叶绿素荧光参数的变化。研究表明,干旱胁迫下两种植物SOD、CAT活性均高于复水后,金钟藤高于相同处理下的葛藤;金钟藤在干旱与复水条件下的MDA含量无差异,而葛藤干旱下高于复水后,不同处理下葛藤均高于金钟藤;干旱条件下金钟藤比葛藤有更强的抗氧化能力,膜脂过氧化水平明显较低,MDA与SOD、CAT呈明显的负相关。两种植物在复水后Fv/Fm、ETR与ΦPSⅡ值均显著高于干旱时,葛藤变化程度略大于金钟藤。复水后金钟藤的NPQ值增加而葛藤降低,且干旱时葛藤大于金钟藤,复水后金钟藤大于葛藤。综合比较,金钟藤比葛藤对干旱的生理适应能力强,这可能是其入侵性的重要特征。     

深黄被孢霉利用不同碳源产油脂比较

本研究主要探讨深黄被孢霉M2菌株对生物质全糖的利用,考察其碳源同化能力、不同碳源下产脂情况以及对玉米皮渣的利用能力。研究结果表明,M2菌株能够利用葡萄糖、木糖、阿拉伯糖和甘露糖进行生长和油脂积累。M2菌株以6%糖浓度的玉米皮渣水解液为底物发酵培养,油脂微生物生物量达18.2g/L,干菌体油脂含量45.7%,单位体积发酵液油脂产量为8.3g/L。     

A comparative analysis of wild and laboratory grown Laminaria abyssalis (Phaeophyta) using modulated fluorescence

Laminaria abyssalis fronds were either collected at the Brazilian costal area - 40 meters below sea level - or grown in the laboratory. The photochemical yield as defined by the Fv/Fm and the Fo - the dark fluorescence level when all PSII centers are open - varied with the distance from the stipe to the tip of the blade in wild grown fronds while it stayed constant in the laboratory grown plants. The chlorophyll a/c ratio levels decreased in the wild fronds from 12 (near the stipe) to 6 near the top. The chlorophyll c content increased from 0.8 to near 1.7 mg cm^–2 in the wild fronds. The laboratory fronds did not show variations in their chlorophyll contents. The wild fronds pattern changed after 2 months kept in the laboratory, producing similar results to those grown in the laboratory. The results indicate that the levels of the antenna complex in the wild fronds increase from the stipe to the top of the blade, in a fashion similar of the sun/shade leaves. Also, results show, that this alga is able to adapt itself to new light conditions, possibly increasing its level of antenna complex and photosynthetic units.Abbreviations PSII Photosystem II - Fo Chlorophyll fluorescence when all PSII are opened - Fm Chlorophyll Fluorescence when all PSII are closed - Fv Variable Fluorescence (Fm-Fo) - Fv/Fm Quantum Yield for Photochemistry     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

Development of microalga Scenedesmus dimorphus mutant with higher lipid content by radiation breeding

In this study, a high lipid-accumulating mutant strain of the microalgae Scenedesmus dimorphus was developed via radiation breeding. To induce mutant strain, S. dimorphus was gamma-irradiated at doses from 100 to 800 Gy, and then a mutant (Sd-Pm210) with 25 % increased lipid content was selected using Nile red staining methodology. Sd-Pm210 showed morphological changes and had higher growth rate compared to the wild type. From random amplified polymorphic DNA analysis, partial genetic modifications were also observed in Sd-Pm210. In comparisons of lipid content between wild type and Sd-Pm210 using thin-layer chromatography, the content of triacylglycerol was markedly higher in the Sd-Pm210 strain. The total peak area of fatty acid methyl ester was shown to have about 1.4-fold increase in Sd-Pm210, and major fatty acids were identified as palmitic acid, oleic acid, linoleic acid, and linolenic acid. To define the metabolic changes in the mutant strain, 2-dimensional electrophoresis was conducted. Several proteins related to lipid synthesis and energy metabolisms were overexpressed in the mutant strain. These results showed that radiation breeding can be utilized for the development of efficient microalgae strains for biofuel production.     

Effect of glyphosate toxicity on growth, pigment and alkaline phosphatase activity in cyanobacterium Anabaena doliolum: a role of inorganic phosphate in glyphosate tolerance

Response of glyphosate toxicity on photoautotrophic cyanobacterium A. doliolum and its mutant strain was investigated. Chlorophyll a content of both the wild type and mutant strain in the presence of glyphosate (N-phosphonomethyl glycine) initially showed an increasing trend when supplemented with Pi and a declining tendency under the Pi-starved condition. The results suggested that both the wild type and mutant strains were more sensitive to glyphosate in the absence of phosphate. Alkaline phosphatase activity of wild type strain in the presence of Pi, enhanced in response to addition of glyphosate (40 microg/ml), but the activity remained unaltered by addition of glyphosate in the Pi-starved cells, whereas the alkaline phosphatase activity in the mutant strain under both Pi-starved as well as unstarved conditions was stimulated (approximately 5.4 and 3.1-fold, respectively) by addition of glyphosate. The results on alkaline phosphatase activity indicated a glyphosate-induced depletion in the phosphate content of the cells, particularly in the mutant strain, as evident from the stimulated activity of alkaline phosphatase. It is suggested that enzyme activity in the Pi-starved wild type cells may not be influenced any further by glyphosate, as cellular phosphate reserve might not be available for further depletion.     

Effect of Cadmium Ions on Antioxidant Defense System in Sunflower Cotyledons

Sunflower (Helianthus annuus L.) seeds were germinated and grown in the presence of 50, 100 and 200 μM CdCl₂. The lower concentration (50 μM) of Cd² ions produced slight decrease in reduced glutathione (GSH) content and overall increase (except superoxide dismutase) in antioxidant enzyme activities, and in H₂O₂ concentration. Chlorophyll content, lipid peroxidation and protein oxidation were not affected under 50 μM CdCl₂. GSH content was diminished under 100 and 200 μM CdCl₂, and except for superoxide dismutase, which activity remained unaltered, overall decreases in the antioxidant enzyme activities (catalase, ascorbate peroxidase, dehydroascorbate peroxidase, glutathione reductase) and in guaiacol peroxidase were observed. These Cd² concentrations caused a decrease in chlorophyll content as well as an increase in lipid peroxidation, protein oxidation and H₂O₂ concentration. All the observed effects were more evident with the highest concentration of cadmium chloride used. This revised version was published online in July 2006 with corrections to the Cover Date.     

培养条件对产油微生物生长的影响

为了筛选出高产油菌株, 首先采用细胞形态学方法与细胞化学方法(苏丹III染色法)对4株高产油脂菌株进行初筛, 并通过索氏提取法对初筛菌株油脂含量进行分析, 确定M2菌株为实验菌株, 其油脂含量达53.09%。为了增加产油微生物油脂产量, 本试验考察了不同发酵条件对其细胞生长和油脂积累的影响。优化工艺参数为: 10° Bx玉米皮渣水解液为培养基质, 0.2% NaNO3为氮源, pH 6.0、28oC下发酵培养6 d, 微生物油脂含量75.21%, 菌体生物量30.40 g/L, 油脂产量22.86 g/L。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 主要含有16碳和18碳系脂肪酸, 可作为生物柴油的原料, 不饱和脂肪酸含量达68%, 可应用于医药化工领域。     

Enhanced biomass and gamma-linolenic acid production of mutant strain Arthrospira platensis

A mutant of Arthrospira platensis PCC 9108, strain M9108, obtained by mutagenesis with UV treatment, was able to mixotrophically grow in an SOT medium containing 40 g of glucose/l. The biomass and specific growth rate of strain M9108 (4.10 g/l and 0.70/d) were 1.9-fold and 1.4-fold higher, respectively, than those of the wild type (2.21 g/l and 0.58/d) under mixotrophic culture condition. In addition, when compared with the wild type, the content of gamma- linolenic acid (GLA) in the mutant was increased when glucose concentration was increased. Compared with the wild type, the GLA content of the mutant was 2-fold higher in autotrophic culture and about 3-fold higher in mixotrophic culture. Thus, the mutant appears to possess more efficient facility to assimilate and metabolize glucose and to produce more GLA than its wild-type strain.     

Phase separation in chlorophyll a containing dipalmitoyllecithin vesicles. A fluorescence and photoacoustic study

Chlorophyll a was incorporated into dipalmitoyllecithin vesicles in different concentrations. Depending on the physical state of the lipid, the chlorophyll can aggregate into domains. This phase separation was demonstrated by fluorescence as well as by photoacoustic measurements.