定向驯化对异养蛋白核小球藻硒的耐受性及富集能力的影响

硒(Se)是生物体必不可少的微量元素,硒缺乏会导致人产生克山病、大骨节病等疾病,缺硒也会给畜牧业带来巨大损失。目前的补硒产品存在硒含量和生物利用度低、安全性差等问题,而通过小球藻培养可获得生物利用度高、安全性好的有机硒,因此是非常有应用前景的补硒产品。首先,为了获得对硒的耐受性和富集能力更强的藻种,研究通过定向驯化的方式逐步提高培养基中Na₂SeO₃浓度来驯化蛋白核小球藻,并对驯化时间和驯化过程中Na₂SeO₃的浓度梯度进行了优化。结果表明,驯化后的藻种对硒的耐受性和富集能力明显提高。在5L发酵罐中,驯化后的藻株可以耐受40mg/L的Na₂SeO₃,胞内有机硒合成速率提高了175.6%。之后,在5L发酵罐中进一步优化了硒的补加方式,在异养培养过程中分批补料添加40mg/LNa₂SeO₃时,最终获得的蛋白核小球藻细胞干重达106.4g/L,有机硒含量为1227mg/kg,有机硒合成速率为1.36mg/(L·h)。研究结果与已有蛋白核小球藻异养富硒文献报道的最高细胞密度75g/L和最高有机硒含量560mg/kg相比分别提高了41.9%和119.1%。上述结果表明,通过定向驯化的方法,可大大提高蛋白核小球藻对硒的耐受性和富集能力。     

蛋白核小球藻在光协迫下的超补偿现象

2000．3－2000．7以蛋白核小球藻（Chlorella pyrenoidosa）为实验材料,测定了其在光胁迫条件下及胁迫解除后细胞的生长密度及生长率。结果表明：光照条件恢复后,遮光处理组较对照组在短期内有更高的产量及生长速率。藻类中存在的这种特性很可能也是形成赤潮爆发的一个生物内源性因素。     

蛋白核小球藻在光胁迫下的超补偿现象

2000.3-2000.7以蛋白核小球藻(Chlorella pyrenoidosa)为实验材料,测定了其在光胁迫条件下及胁迫解除后细胞的生长密度及生长率。结果表明:光照条件恢复后,遮光处理组较对照组在短期内有更高的产量及生长速率。藻类中存在的这种特性很可能也是形成赤潮爆发的一个生物内源性因素。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoidosa No.2。研究了培养基组成及培养条件对其细胞生长和油脂积累的影响。结果表明, 最适培养基组成为(g/L)：葡萄糖 20, 甘氨酸 0.08, MgSO4·7H2O 0.4, K2HPO4 1.0, FeSO4·7H2O 0.004; 适宜的培养温度、初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和 650 Lux。在上述优化条件下培养7 d, Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L 和 40.15%提高到6.56 g/L和59.90%, 油脂产量提高了162%。Chlorella pyrenoidosa No.2能以木糖为碳源产油脂, 可望用于以木质纤维素等可再生生物质资源为原料生产油脂。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 不饱和脂肪酸含量达71％左右, 可作为生产生物柴油的原料。     

蛋白核小球藻高效同化硝态氮联产微藻蛋白

本研究旨在建立利用微藻去除高浓度废水中硝酸根并转化为藻蛋白的创新技术。先在摇瓶中研究了培养模式和光照模式对于混养蛋白核小球藻的生物量产量、硝酸根同化速率和藻蛋白产量的影响,随后在5 L光发酵罐中成功进行了放大验证。结果表明,在摇瓶培养中,不回流培养基的补料分批培养是最佳培养模式,可获得最高生物量产量为35.95 g/L,硝酸根平均同化速率为2.06 g/(L·d),藻蛋白含量可高达42.44%干重;采用阶梯式增加光强的光照模式,能显著提高细胞比生长速率,最高达到0.65 d–1。在5 L光发酵罐中连续培养128 h,最高生物量产量和硝酸根平均同化速率分别达到66.22 g/L和4.38 g/(L·d),最高藻蛋白含量可达干重的47.13%。本研究能为高效处理工业废硝酸或高浓度硝酸盐废水提供微藻光发酵技术,基于微藻的生物转化过程可联产高蛋白微藻生物质,有利于实现这类废水资源化利用、变废为宝。     

蛋白核小球藻凝集素的分离纯化及部分性质研究

蛋白核小球藻藻粉的PBS抽提液经硫酸铵二步分级沉淀 ,再经DEAE Sepharose和SephadexG 10 0层析 ,从中分离纯化得到蛋白核小球藻凝集素 (CPL)。经测定 ,该凝集素为单个亚基的蛋白质 ,相对亚基分子量为 1 4× 10 4 — 1 5× 10 4 ,分子中不含糖。在氨基酸组成中 ,苯丙氨酸 (Phe)的含量最高 ,其次是天冬氨酸 (Asp)和谷氨酸 (Glu) ,不含组氨酸 (His)。CPL能够凝集兔、绵羊及鸽子红细胞 ,其中对兔红细胞的凝集活性最大 ,最低浓度为 6 88μg/mL ,对鸡、鸭及人红细胞 (A型、O型及B型 )无凝集活性。卵黏蛋白和 7种单糖对CPL的凝血活性具有抑制作用。CPL具有很好的热稳定性 ,在 90℃处理 10min不失活。     

增强UV-B对蛋白核小球藻中NO释放的影响

研究了蛋白核小球藻在增强UV—B辐射下NO信号的产生。结果表明：NO释放量与UV—B的强度相关。在藻类细胞培养液中加入NOS的竞争性抑制剂硝基精氨酸(LNNA),不能抑制NO的释放。加入NO清除剂乙酰半胱氨酸(NAC),能明显减少NO的释放。在无氮和有氮培养基中NO同样的释放。这些结果说明NO生成途径不经过依赖L-精氨酸的NOS和依赖NO3^-和硝酸还原酶,NO的底物不是L-精氨酸和NO3^-在无氮和有氮培养基中同样有NO3^-的形成,NAC减少NO2^-的释放,说明生成了NO2^-有可能是NO释放后生成的。     

蛋白核小球藻脂溶性化合物的抑菌活性及成分分析

利用纸片法对蛋白核小球藻（Chlorella pyrenoidosa Chick.）脂溶性化合物的粗脂以及经硅胶柱层析分离后的不同组分进行了抑菌实验。结果表明,蛋白核小球藻的粗脂有极强的抑菌活性,其石油醚洗脱组分占总脂的63.9％,抑菌活性最弱;乙醇洗脱级分占总脂的32.2％,抑菌活性最强;苯洗脱组分只占总脂的3.9％,对玉米大斑病菌（Helminthosporium turcicum Pass）有极强的抑制活性。各洗脱组分及粗脂对革兰氏阴性菌均我抑制活性。成分分析结果表明,石油醚洗脱组分以烷烃类化合物为主,其中1-十九烯经烃含量最高;苯洗脱组分以6,10,14-三甲基-2-十五酮、十六碳酮和2-十一烷酮这3种酮类化合物为主;而乙醇洗脱组分含有大量的脂肪酸,其中γ-亚麻酸含量最高（56.673％）。蛋白核小球藻脂溶怀化合物的抑菌活性是其各种化学成分相互综合作用的结果。     

药用植物浸提液抑制蛋白核小球藻生长的化感效应

研究了11种药用植物浸提液对水华藻种蛋白核小球藻的影响,结果显示:黄连、重楼、贯众、防己4种药用植物的浸提液均有抑藻效应。当药用植物浸提液的相对浓度为1g/L处理时,其半抑制效应时间LT50的排序为:防己重楼黄连贯众。进一步研究防己的相对浓度梯度抑藻效应,结果表明:在相对浓度为2g/L时,实验3d后的藻细胞几乎全部死亡;防己的抑藻效应受贮藏时间和贮藏温度的影响不显著。在所研究的药用植物中,防己的抑藻效果最好,在抑藻方面有较大应用前景,其它3种药用植物对轻度藻类爆发的控制也有潜在应用价值。     

硫和pH对蛋白核小球藻光照产氢的影响

在有无硫及pH5.0-8.0下对蛋白核小球藻(Chlorella pyrenoidosa)光照产氢的影响进行了研究。结果表明,在持续光照(165μmolm-2s-1)条件下,从有硫培养液(TAP培养液)内叶绿素a含量、Fv/Fm值及ΦPSII值的变化表明蛋白核小球藻在pH6.0-7.0时生长最佳,生长旺盛易形成暂时的无氧环境而利于藻产氢。最高的产氢速率和总产氢量出现在pH7.0,分别是0.10mlmg-1chlh-1和1.39ml。从无硫培养液(TAP-S培养液)内叶绿素a含量、Fv/Fm值及ΦPSII值的变化表明蛋白核小球藻生长明显受抑制,形成的无氧环境持久,故产氢持久,总产氢量比有硫培养液内高。蛋白核小球藻在pH5.5培养液内的Fv/Fm值后期高于其他4种pH值的,表明潜在的PSII光化学效率高,在光照条件下产氢电子主要来源于PSII,故pH5.5的无硫培养液内藻的产氢速率和总产氢量最大,分别是0.58mlmg-1chlh-1和10.98ml。说明pH为5.5的无硫培养液是蛋白核小球藻产氢的最佳条件。     

Rapid light-response curves of chlorophyll fluorescence in microalgae: relationship to steady-state light curves and non-photochemical quenching in benthic diatom-dominated assemblages

Rapid light-response curves (RLC) of variable chlorophyll fluorescence were measured on estuarine benthic microalgae with the purpose of characterising its response to changes in ambient light, and of investigating the relationship to steady-state light-response curves (LC). The response of RLCs to changes in ambient light (E, defined as the irradiance level to which a sample is acclimated to prior to the start of the RLC) was characterised by constructing light-response curves for the RLC parameters α _RLC, the initial slope, ETR_m,RLC, the maximum relative electron transport rate, and E _k,RLC, the light-saturation parameter. Measurements were carried out on diatom-dominated suspensions of benthic microalgae and RLC and LC parameters were compared for a wide range of ambient light conditions, time of day, season and sample taxonomic composition. The photoresponse of RLC parameters was typically bi-phasic, consisting of an initial increase of all parameters under low ambient light (E < 21–181 μmol m⁻² s⁻¹), and of a phase during which α _RLC decreased significantly with E, and the increase of ETR_m,RLC and E _k,RLC was attenuated. The relationship between RLC and LC parameters was dependent on ambient irradiance, with significant correlations being found between α _RLC and α, and between ETR_m,RLC and ETR_m, for samples acclimated to low and to high ambient irradiances, respectively. The decline of α _RLC under high light (Δα _RLC) was strongly correlated (P < 0.001 in all cases) with the level of non-photochemical quenching (NPQ) measured before each RLC. These results indicate the possibility of using RLCs to characterise the steady-state photoacclimation status of a sample, by estimating the LC parameter E _k, and to trace short-term changes in NPQ levels without dark incubation.     

Relationship of rapid light curves of variable fluorescence to photoacclimation and non-photochemical quenching in a benthic diatom

The response of rapid light–response curves (RLCs) of variable fluorescence to changes in short- and long-term photoacclimation status was studied in an estuarine benthic diatom. The diatom Nitzschia palea was grown under low- (LL, 20 μmol m⁻² s⁻¹) and high-light (HL, 400 μmol m⁻² s⁻¹) conditions, with the purpose of characterising the effects of long-term photoacclimation on (i) steady-state light–response curves (LC) of relative electron transport rate, rETR, (ii) the response of RLCs to changes in ambient irradiance (E, the irradiance to which the sample is acclimated to immediately before the RLCs), (iii) the relationship of RLCs to LC parameters and non-photochemical quenching (NPQ). Photoacclimation to LL and HL conditions induced distinct light–response patterns of rETR and NPQ. Higher growth light resulted in rETR vs. E curves with lower initial slopes (α, 0.591 μmol⁻¹ m² s vs. 0.661 μmol⁻¹ m² s, for HL and LL, respectively) and markedly higher maximum rates (rETR_m, 95.9 vs. 29.3), reached under higher E levels (higher light-saturation coefficient, E_k: 162.4 μmol m⁻² s⁻¹ vs. 44.3 μmol m⁻² s⁻¹). Acclimation to HL induced bi-phasic NPQ vs. E curves, with minimum values reached under low E levels (15–25 μmol m⁻² s⁻¹) and not on dark-acclimated samples. The response of RLCs to changes in ambient irradiance varied with the long-term photoacclimation status of the samples. The initial slope, α_RLC, decreased monotonically with E in LL cultures, from 0.68 to 0.25 μmol⁻¹ m² s, while varied bi-phasically in HL-acclimated samples. Typically, α_RLC of HL cultures increased under low E, reaching a maximum of 0.61 μmol⁻¹ m² s under 25–55 μmol m⁻² s⁻¹, and decreased gradually under higher E levels to 0.25 μmol⁻¹ m² s. RLC maximum rETR, rETR_m,RLC, and saturation coefficient E_k,RLC, increased with E following a saturation-like pattern, with the HL cultures presenting markedly higher values for all the E range (maximum rETR_m,RLC values were 108.6 and 33.4 for HL and LL cultures, respectively). An inverse relationship was consistently found between α_RLC and NPQ, both on LL and HL cultures, causing strong correlations (P < 0.001 in all cases) between NPQ and the high light-induced decrease of α_RLC, Δα_RLC. RLCs were confirmed to also provide information on the long-term photoacclimation status, as significant correlations (P < 0.001 both for HL and LL cultures) were verified between E_k and an index based on RLC parameters, Ê_k, both for LL and HL cultures. These results reinforce the usefulness of RLCs as a tool for inferring on the short- and long-term photoacclimation status of samples with different long-term light histories, through the estimation of LC parameters and the monitoring of NPQ levels.     

蛋白核小球藻生长和无机碳利用对不同光照强度和CO2浓度的响应

为了探讨光照强度和CO₂浓度对蛋白核小球藻(Chlorella pyrenoidosa)生长、无机碳利用的复合效应, 丰富绿藻中无机碳浓缩机制的资料, 该文设置两种光照强度(40和120 µmol photons•m^-2•s^-1)和两种CO₂浓度(0.04%和0.16%)组合成4种条件, 比较了蛋白核小球藻生长、无机碳浓度、pH补偿点、光合放氧速率、碳酸酐酶(CA)活性和α-CA基因转录表达对这4种培养条件的响应。结果发现: 蛋白核小球藻在高光强高CO₂浓度组生长最快; 低光强高CO₂浓度组培养体系中总无机碳浓度为1163.3 µmol•L^-1, 显著高于其他3组; 高光强低CO₂浓度组藻的pH补偿点最高(9.8), 而低光强高CO₂浓度组藻的pH补偿点最低(8.6); 低光强高CO₂浓度组藻的最大光合速率(V_max)和最大光合速率一半时的无机碳浓度(K_0.5)最高, 分别是其他3组的1.28-1.91倍和1.61-2.00倍; 高光强低CO₂浓度组藻的胞外CA活性最高; 而低光强低CO₂浓度组藻的胞外α-CA基因表达量显著高于其他3组。以上结果表明低CO₂浓度可促进蛋白核小球藻的pH补偿点和无机碳亲和力的提高, 诱导胞外CA活性及α-CA基因的表达; 该藻主要以HCO₃^-为无机碳源, 其对无机碳的利用受光照的调节。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoi-dosa No.2.研究了培养基组成及培养条件对其细胞生长和油脂积累的影响.结果表明,最适培养基组成为(g/L):葡萄糖20,甘氨酸0.08,MgSO4·7H2O 0.4,K2HPO4 1.0,FeSO4·7H2O 0.004;适宜的培养温度,初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和650 Lux.在上述优化条件下培养7 d,Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L和40.15%提高到6.56 g/L和59.90%,油脂产量提高了162%.Chlorella pyrenoidosa No.2能以木糖为碳源产油脂,可望用于以木质纤维素等可再生生物质资源为原料生产油脂.气相色谱分析表明该油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达71%左右,可作为生产生物柴油的原料.     

Cultivation of Chlorella pyrenoidosa in soybean processing wastewater

Chlorella pyrenoidosa was cultivated in soybean processing wastewater (SPW) in batch and fed-batch cultures without a supply of additional nutrients. The alga was able to remove 77.8 ± 5.7%, 88.8 ± 1.0%, 89.1 ± 0.6% and 70.3 ± 11.4% of soluble chemical oxygen demand (SCOD_Cr), total nitrogen (TN), NH₄⁺-N and total phosphate (TP), respectively, after 120 h in fed-batch culture. C. pyrenoidosa attained an average biomass productivity of 0.64 g L⁻¹ d⁻¹, an average lipid content of 37.00 ± 9.34%, and a high lipid productivity of 0.40 g L⁻¹ d⁻¹. Therefore, cultivation of C. pyrenoidosa in SPW could yield cleaner water and useful biomass.     

小球藻对U(VI)的生物吸附特性

试验研究了小球藻吸附U(VI)的过程, 探讨了吸附机理、吸附热力学和动力学。考查了pH值、时间、U(VI)的起始浓度和温度等对吸附的影响。研究表明, pH值对小球藻的吸附效果影响较大, 小球藻吸附U(VI)的最佳pH值为6, 最大吸附量为2.7 mg/g, 吸附在5 min内基本达到平衡。小球藻对U(VI)的吸附量与其浓度的正相关; 温度在20℃~30℃时, 对铀的吸附影响不大。实验结果还表明, 吸附过程符合准二级动力学方程, 其相关系数达0.99, 该吸附为多种反应同时作用的复杂过程。U(VI)在小球藻上的吸附行为可以很好地用Langmuir等温方程来描述。     

小球藻对U(VI)的生物吸附特性

试验研究了小球藻吸附U(VI)的过程,探讨了吸附机理、吸附热力学和动力学.考查了pH值、时间、U(VI)的起始浓度和温度等对吸附的影响.研究表明,pH值对小球藻的吸附效果影响较大,小球藻吸附U(VI)的最佳pH值为6,最大吸附量为2.7mg/g,吸附在5min内基本达到平衡.小球藻对U(VI)的吸附量与其浓度的正相关;温度在20℃-30℃时,对铀的吸附影响不大.实验结果还表明,吸附过程符合准二级动力学方程,其相关系数达0.99,该吸附为多种反应同时作用的复杂过程.U(VI)在小球藻上的吸附行为可以很好地用Langmuir等温方程来描述.     

不同光质对小球藻光自养培养积累油脂的影响

研究了5种光质对小球藻(Chlorella vulgaris)M209256生长和产油的影响。结果表明:蓝光为小球藻的最适生长和产油光质;与其他光质相比,蓝光培养的小球藻生物量和油脂含量均较高,为2.40×107个/mL和28%;红光培养的小球藻生长最慢且油脂含量最低,为1.32×107个/mL和15.13%,表现出明显的"红降"现象。在GCMS分析的基础上,对油脂甲酯化后的十六烷值进行评估,结果发现:蓝光的十六烷值最高;5种光质培养的小球藻所产油脂,甲酯十六烷值均在47以上。因此,小球藻油脂所制备的生物柴油具有较好的燃烧性能。