热带普通小球藻培养模式的筛选及其培养基的优化

【背景】从海南热带海区中分离得到一株微藻,其生长速度快、适应力强,经鉴定该微藻为普通小球藻。【目的】提高热带普通小球藻的生长速率。【方法】以"宁波大学3#微藻培养液配方"为基础培养液,分别添加有机碳(C6H12O6和CH3COONa)对热带普通小球藻进行自养、兼养及异养培养,获得促进热带普通小球藻快速生长的培养方式。在"宁波大学3#微藻培养液配方"的基础上对热带普通小球藻的兼养培养基配方进行优化,并用优化兼养培养基与"宁波大学3#微藻培养基"对比培养热带普通小球藻。【结果】添加6 g/L CH3COONa的兼养模式促进热带普通小球藻生长效果最好;优化的兼养培养基配方为:6 g/L CH3COONa,20 mg/L(NH4)2SO4-N,5 mg/L Na H2PO4-P,3 mg/L Fe SO4-Fe,1 mg/L Vitamin B1和0.000 5 mg/L Vitamin B12。对比培养实验结果显示,培养第6天,兼养培养液收获的生物量(细胞密度)达4.20×107 cells/m L,是"宁波大学3#配方微藻培养液"的2.30倍。【结论】兼养培养模式为热带普通小球藻的最佳培养模式,优化的兼养培养基极显著地提高了热带普通小球藻的生物量(P0.01)。     

自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。     

葡萄糖对小球藻(Chloralla sp. HN08)光合作用和生长的影响

【目的】探讨葡萄糖作为外加碳源对热带海洋小球藻(Chloralla sp.HN08)生物质生产和脂、光合色素、碳水化合物及可溶性蛋白等细胞主要成份含量的影响。【方法】分析比较小球藻HN08在光合自养和兼养(添加10 g/L葡萄糖)2种营养方式下的生长速率、细胞密度、光合放氧速率、油脂相对含量,以及可溶性总糖、淀粉和可溶性蛋白的含量。【结果】结果表明,在光照条件下葡萄糖(10 g/L)能促进小球藻(Chloralla sp.HN08)生长,提高细胞终密度,而异养条件下藻细胞逐渐衰亡。兼养条件下,细胞相对生长速率及细胞终密度分别是自养条件下的6.8倍和1.3倍。兼养藻细胞中可溶性糖、淀粉、油脂含量显著高于(P0.05)光合自养细胞,然而可溶性蛋白质和光合色素含量显著低于(P0.05)光合自养细胞。添加葡萄糖的小球藻液的光饱和点和呼吸速率均高于光自养条件下的细胞,但2种培养条件下藻液的净光合速率无显著差异(P0.05)。【结论】光照条件下,添加葡萄糖可显著提高小球藻HN08相对生长速率和细胞终密度,促进油脂与淀粉的积累。     

氮源对雨生红球藻绿色生长期细胞生长的影响

【背景】雨生红球藻(Haematococcus pluvialis)细胞能合成积累具有超强抗氧化活性的虾青素,是生产高值天然虾青素的优异藻种。【目的】解析不同氮源对雨生红球藻生长的效应,以期建立优化氮素营养提高雨生红球藻生物量和虾青素产量的技术体系。【方法】选用Na NO3和NH_4Cl为氮源、辅以pH缓冲液Hepes,培养雨生红球藻藻株797,测试2种不同氮源对雨生红球藻藻液pH、营养生长期(绿色生长期)藻细胞生长、叶绿素含量和生物量等的影响。【结果】以Na NO3为氮源培养的雨生红球藻细胞的比生长速率、生物量、叶绿素a和叶绿素b含量均高于以NH_4Cl为氮源培养的藻细胞。不同氮源对雨生红球藻培养液的pH值有显著影响,NH_4Cl氮源导致培养液pH值降低,然而Na NO3氮源则导致培养液pH值上升。添加pH缓冲液Hepes能有效稳定培养液的pH值,并促进雨生红球藻的生长,尤以NH_4Cl为氮源添加Hepes的效果更显著。不同氮源导致雨生红球藻营养生长阶段细胞的生长和生物量等差异主要源于不同氮源引起藻液pH的变化。【结论】添加pH缓冲液Hepes可有效控制藻液pH,进而显著促进以Na NO3和NH_4Cl为氮源的雨生红球藻营养生长阶段细胞的生长和生物量积累。     

吲哚美辛调控小球藻生长及代谢的初步研究

本研究通过在小球藻培养液中添加不同浓度的吲哚美辛(0mg/L,50mg/L,75mg/L,100mg/L,112.5mg/L,125 mg/L,150 mg/L)来研究其对小球藻生长及代谢的影响,并探索其影响机制。研究表明:吲哚美辛对小球藻生长和代谢的影响与其浓度有关,低浓度吲哚美辛(≤75 mg/L)可促进小球藻的生长,而高浓度(≥100 mg/L)则会抑制其生长,且在75 mg/L处理组中,吲哚美辛对小球藻生长的促进作用最明显,培养液p H值的变化最小,小球藻可溶性糖含量最高(3.12μg/106 cells)。而在高浓度(150 mg/L)处理组中,吲哚美辛可显著促进小球藻细胞内叶绿素a和可溶性蛋白的积累,可分别达到0.38μg/10~6 cells和7.09μg/106 cells,油脂含量也相对于对照组提高了60%。研究将为探索吲哚美辛调控小球藻生长及代谢的作用机制提供理论基础和技术资料,也将为小球藻的产业化培养及其代谢产物的调控提供思路。     

不同营养方式对普通小球藻生长代谢及生化组分的影响

摘要:【目的】系统研究自养、混养和异养3种营养方式对真核模式微藻———普通小球藻(Chlorella vulgaris)生长特性、细胞生化组分和碳代谢途径关键酶活性的影响。【方法】以C．vulgaris为研究对象,通过设置光合自养、混养和异养3 种营养方式,采用光谱学、色谱学方法,研究不同营养方式对C．vulgaris从生长特性、细胞组分合成和碳代谢等方面的影响。【结果】C．vulgaris依次经自养至混养和异养的培养方式转变中,藻细胞的可溶性糖和油脂含量显著提高,油脂中C16、C18不饱和脂肪酸的相对含量降低,而饱和脂肪酸的含量升高;蛋白质含量、光合色素含量显著下降,18种氨基酸的相对含量也呈下降趋势;葡萄糖的添加可抑制藻细胞吸收和积累除碳元素以外的其他测试元素。在添加葡萄糖的前提下,光照可促进藻细胞的生长量、不饱和脂肪酸和氨基酸,以及除碳元素以外的其他测试参数增加。对微藻胞外碳酸酐酶和核酮糖-1,5-二磷酸 羧化酶的活性分析结果表明,异养和混养直接影响C．vulgaris的碳代谢途径。【结论】光源和葡萄糖的供给与否直接影响C．vulgaris的生长代谢和生化组分合成,葡萄糖的添加在显著促进藻细胞生物量积累的同时,刺激碳素(糖类和油脂)生化成分的合成,而抑制氮素成分(蛋白质和光合色素)的合成;在光照条件下培养基质中葡萄糖的浓度和消耗水平直接决定藻细胞主营自养或异养生长。添加有机碳源葡萄糖的混养(光照)和异养(暗处理)培养可促进藻细胞的生长,异养和自养的生物量之和接近于混养,表明混养是最佳的藻细胞营养生长方式。     

有机碳源和氮源对三角褐指藻生长的影响

研究了8种有机碳源和4种氮源对三角褐指藻(Phaeodactylum tricornutum)生长的影响.结果表明,三角褐指藻具有兼养生长的能力,碳浓度为5mmol/L和50mmol/L时,葡萄糖、果糖、乙酸钠和甘油对其生长有明显的促进作用,乳酸钠和乙醇抑制藻细胞生长,半乳糖和柠檬酸钠对其生长随有机碳浓度而异.甘油、葡萄糖和乙酸钠的适宜浓度范围分别为5 -800mmol/L、100 -400mmol/L和50 -300mmol/L.培养液中分别加入50mmol/L甘油、400mmol/L葡萄糖和150mmol/L乙酸钠,培养第14天,三角褐指藻的最大生物量分别为对照的1.45倍、1.25倍和1.11倍.甘油兼养生长的最优氮源是尿素,适宜浓度范围为0.88-8.8mmol/L.当尿素浓度为4.4mmol/L时,最高生物量可达1.31g/L.     

粗甘油优化污泥脱水液培养小球藻过程的研究

污泥脱水液为污泥压缩过程产生的污水,因其含有N、P等营养物,可用于微藻的培养。但污泥脱水液碳氮比低,可利用碳源有限,影响微藻生长。本研究考察外加不同浓度(1 g/L,2 g/L,4 g/L,6 g/L)生物柴油副产物-粗甘油对污泥脱水液培养小球藻过程的影响。结果表明:1 g/L、2 g/L粗甘油浓度能促进小球藻生长,藻生物量为1.29 g/L、1.45 g/L;2 g/L粗甘油浓度下氨氮、总氮去除率达99.32%和97.52%。粗甘油被分解后易使培养体系pH降至7以下,使总磷去除率比对照组略低。外加1 g/L、2 g/L粗甘油组的COD去除量分别为553.00 mg/L和405.00 mg/L。藻细胞元素和傅里叶红外光谱分析表明补加粗甘油后藻细胞中C元素和H元素相对含量均明显增加,C元素含量约为对照组的1.5倍;2 g/L粗甘油组的蛋白质与脂类物质含量均高于对照组。MPBR半连续培养小球藻过程中,HRT为5 d时藻生物量维持在1.99~2.21 g/L,大约为批次生物量的1.50倍;氨氮、总氮、总磷、COD的去除率分别在96.26%~99.20%、92.44%~94.04%、53.63%~58.58%、59.44%~65.57%。     

小球藻高密度培养及油脂提取条件的优化

【目的】高密度培养小球藻及优化油脂提取条件。【方法】通过进行单因素实验研究不同培养基组成及环境因子对其细胞生长影响,并采用超声波提取法进行正交实验对藻粉油脂提取条件进行研究。【结果】对椭圆小球藻Y4进行异养培养,最适培养条件为:葡萄糖50 g/L,硝酸钾2 g/L,适宜的培养温度、摇床转速和接种量分别为29°C、180 r/min和20%。在此基础上,进行了1 L发酵罐培养实验,获得了干重18.25 g/L的生物量。通过对油脂提取条件进行优化,Y4的油脂提取率由优化前的25.0%提高到60.2%,提高了35.2%。【结论】优化了小球藻的培养条件及油脂提取条件,促进了小球藻的开发和利用。     

一株高蛋白含量小球藻TX的分离鉴定及其诱变育种

【背景】小球藻由于蛋白含量高、营养丰富,在水产养殖上可直接作为鱼、虾、贝类的优质饵料。【目的】对从养殖环境中分离的小球藻进行诱变,选育生长快、蛋白含量高的突变株,为水产养殖天然饵料生产提供优良藻种资源。【方法】以从养殖环境中筛选的生长相对较快且蛋白含量较高的TX作为出发藻株,对该藻株进行分子鉴定,并对该藻株进行紫外诱变、甲基磺酸乙脂(ethyl methyl sulfonate,EMS)诱变和复合诱变,采用96孔板高通量筛选技术和递进式重复筛选方法选育高生物量、高蛋白突变株。【结果】经18SrRNA基因序列分析,TX鉴定为Chlorella sorokiniana,从540个可能的突变株中筛选到8个遗传稳定且生长较快的突变株,其中H10的总蛋白含量达64.2%,可溶性蛋白含量达0.44g/L,干重达0.72g/L,分别较出发藻株提高3.4%、15.8%和26.2%。【结论】突变株H10蛋白含量高且生长较快,可用于天然饵料生产。     

Two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production

A two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production was studied, wherein high density heterotrophic cultures of Chlorellasorokiniana serve as seed for subsequent phototrophic growth. The data showed growth rate, cell density and productivity of heterotrophic C.sorokiniana were 3.0, 3.3 and 7.4 times higher than phototrophic counterpart, respectively. Hetero- and phototrophic algal seeds had similar biomass/lipid production and fatty acid profile when inoculated into phototrophic culture system. To expand the application, food waste and wastewater were tested as feedstock for heterotrophic growth, and supported cell growth successfully. These results demonstrated the advantages of using heterotrophic algae cells as seeds for open algae culture system. Additionally, high inoculation rate of heterotrophic algal seed can be utilized as an effective method for contamination control. This two-stage heterotrophic phototrophic process is promising to provide a more efficient way for large scale production of algal biomass and biofuels.     

The toxicity of phenolic compounds to soil algal population and toChlorella vulgaris andNostoc linckia

Summary p-Nitrophenol (PNP),m-nitrophenol (MNP), 2,4-dinitrophenol (DNP) and catechol were tested for their effects on algal population in a soil and on pure cultures of two algae isolated from soil. Both PNP and MNP, even at 0.5 kg ha⁻¹ level were toxic to the soil algae; high doses effected increase in toxicity. Inhibition of algae was relatively more with PNP compared to the other two nitrophenols. Catechol treatment up to 1.0 kg ha⁻¹ led to a significant initial enhancement of algae with a subsequent far less toxic effect. The toxicity of the phenolic compounds towardChlorella vulgaris, a green alga andNostoc linckia, a blue-green alga, decreased in the order: MNP≧PNP>DNP>Catechol. However, algicidal or algistatic effect of the test chemicals was fairly more againstC. vulgaris, suggesting that the eukaryotic alga is highly sensitive to such soil pollutants compared to the prokaryotic alga.     

O-Dealkylation of coumarin and resorufin ethers by unicellular green algae: kinetic properties of Chlorella fusca and Chlorella sorokiniana

O-Dealkylations of resorufin and coumarin ethers, mediated by microsomal cytochrome P450 mono-oxygenases from animals, plants and microorganisms, are shown here to be performed also by intact cells of the unicellular green algaeChlorella fusca andChlorella sorokiniana. The activity of theO-dealkylation of these ethers was up to tenfold higher withChlorella sorokiniana. Both algae dealkylated methyl-, ethyl-, and pentylethers of resorufin and coumarin. Dealkylation in vivo indicated efficient absorption of methoxy- and ethoxyresorufin, confirmed by the respective absorption kinetics. Piperonylbutoxide and 1-aminobenzotriazole, known inhibitors of plant and mammalian cytochrome P450s, significantly inhibited theO-dealkylase activity of both algal strains. The use of synchronized cultures of both algae revealed that efficiency ofO-dealkylation depends on the stage of the cell cycle: during the growth phase, theO-dealkylase activities increased more than proportional, and the distinct drop in activity during the last hours of the light period indicated the appearance of an endogenous substrate.     

一株可增强莱茵衣藻耐盐能力细菌MEZX29的分离与鉴定

【背景】水体中的藻类、细菌及这些微生物之间的相互作用对水体生态系统的功能有着重要作用。近年来,一些河流、湖泊等淡水资源的盐渍化不断加重,对水体生态系统造成严重影响。然而,高盐胁迫条件如何影响藻类与其他细菌的相互作用,以及是否存在能够促进藻类耐盐能力的有益细菌等问题尚未得到深入研究。【目的】分离和鉴定可以促进淡水藻类莱茵衣藻抗盐能力的细菌,并开展相关机制分析。【方法】通过富集培养、筛选和共接种实验,获得可以促进衣藻耐盐的细菌;基于活细胞浓度、叶绿素含量等参数评价衣藻在不同条件下的生长能力;对菌株进行16S rRNA基因序列分析和基因组分析,预测其可能的菌藻相互作用机制。【结果】获得一株在250-290 mmol/L NaCl条件下可以显著增强衣藻耐盐能力的菌株MEZX29,16S rRNA基因序列分析表明,该菌可能属于Rhodococcus qingshengii;基因组分析结果表明,该细菌含有参与糖代谢、乙烯合成、生物膜形成等途径的基因,这些基因可能在促进衣藻抗盐过程中起到重要作用。【结论】Rhodococcus qingshengiiMEZX29可以增强莱茵衣藻21gr抵抗高盐胁迫的能力,为研究藻类与其他微生物之间的有益相互作用提供了新的材料。     

Microbial load in mass cultures of green algaeScenedesmus acutus and its processed powder

Microbial contamination in cultures of the alga,Scenedesmus acutus raised in outdoor open tanks and also in the processed powder of the alga was monitored; The total bacterial population increased with time during the growth period of six days. When a combination of molasses and carbondioxide was employed as carbon source for this alga, the bacterial load increased to 10 colony forming units/ml. Yeast, molds and also coliforms were quantitated. Drum-drying the algae drastically reduced the bacterial load and storing the algal powder for a period of over 3 months did not increase the bacterial load. Pathogens likeSalmonella andStaphylococcus were not detectable either in the open cultures or in the drumdried algal powder. Although there are not set standards available in literature on the permissible level of the microbial contamination in algal biomass for use in foods, the microbial load appears to be within the limits of permissible levels stipulated by Indian Standard Institution standards for baby foods.     

Glucose-6P dehydrogenase in Chlorella sorokiniana (211/8k): an enzyme with unusual characteristics

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH—EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki_NADPH is 103 μM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki_NADPH decreased to 48 μM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.     

Flocculation of algae using chitosan

Flocculation of three freshwater algae, Spirulina,Oscillatoria and Chlorella, and onebrackish alga, Synechocystis, using chitosan was studiedinthe pH range 4 to 9, and chlorophyll-a concentrations inthe range 80 to 800 mg m^–3, which produces aturbidity of 10 to 100 nephelometric turbidity units (NTU) in water. Chitosanreduced the algal content effectively by flocculation and settling. Theflocculation efficiency is very sensitive to pH, and reached a maximum at pH7.0for the freshwater species, but lower for the marine species. The optimalchitosan concentration that is required to effect maximum flocculation dependedon the concentration of alga. Flocculation and settling were faster whenconcentrations of chitosan higher than optimal are used. The settled algalcellsare intact and live, but will not be redispersed by mechanical agitation. Thede-algated water may be reused to produce fresh cultures of algae.     

Genomics of green algal hydrogen research

This article summarizes knowledge on genes and their respective proteins in the field of green algal hydrogen research. Emphasis is placed on recently cloned genes from the unicellular green alga Chlamydomonas reinhardtii, including HydA1 and HydA2, which encode homologous [Fe]-hydrogenases, Tla1, which encodes a chlorophyll antenna size regulatory gene, SulP, which encodes a chloroplast sulfate permease, and Sta7, which encodes an isoamylase. Analysis of the structure and function of these genes and of their respective proteins in C. reinhardtii, and related unicellular green algae, is presented in light of the role they play in the hydrogen metabolism in these organisms. A discussion is offered as to the potential application of these genes in the field of hydrogen photoproduction.     

Screening of algal strains for metal removal capabilities

Eight algal species were tested for their ability to remove five toxic metalsduring 30-min exposures to single-metal (1 mg L^-1) solutions at pH7. Efficacy of metal bioremoval varied according to algal species and metal. Al⁺³ was best removed by the thermophilic blue-green alga(cyanobacterium) Mastigocladus laminosus, Hg⁺² and Zn⁺² by the thermophilic and acidophilic red alga Cyanidiumcaldarium, and Cd⁺² by C. caldarium and the green alga Scenedesmus quadricauda. All of these alga/metal combinations resultedin >90% metal removal. However, none of the eight algal speciesremoved more than 10% of Cr⁺⁶. Results indicate that some toxicmetals are more readily removed than others are by algae and that selectionof appropriate strains could potentially enhance bioremoval of specificmetals from wastewater at neutral pH.     

Stable isotope labelling method for studies of saccharide metabolism in Agardhiella subulata

Stable isotopes are preferable in many ways to radioactive isotopes for metabolic studies designed to elucidate biosynthetic pathways. We have developed the methodology to utilize ¹³C-labelled compounds in tracer studies of saccharide metabolism in the red algae. Cultures of Agardhiella subulata were pulse-chase labelled with ¹³C0₂ and ¹²C0₂. Gas chromatography/mass spectrometry (GC-MS) and ¹³C-NMR provided for positive identification of labelled carbohydrate metabolites. In addition, GC-MS can be used to profile the monosaccharide composition of algal species and combined GC-MS and ¹³C-NMR can disclose which carbon(s) is (are) labelled and the extent of labelling. In ¹³C0₂ incubated plants, the label is clearly detected in floridoside and floridean starch. After chasing the labelled alga with ¹²CO₂ for three days or storing the pulse-chase labelled alga in darkness for 6 days, labels disappeared from both floridoside and starch and the contents of these two carbohydrates became very low. More detailed biochemical analysis is being continued to identify labelled cell wall polysaccharides and/or their precursors.