利用固定化技术探究光质对小球藻的影响

采用海藻酸钙凝胶包埋法对小球藻进行固定化,探究不同光质对小球藻生物量和溶解氧产生的影响。结果表明,白光对小球藻生物量的影响大于黄红光,但差异不显著,小球藻可能主要吸收利用黄红光;固定化小球藻制约了小球藻生物量的高速增长,但固定化能显著提高小球藻的产氧速率。通过让学生参与完整的科学探究活动,落实STS教育,培养学生的创新精神和实践能力,达成生物学学科核心素养的培养。     

铀对两种小球藻生长和光合作用的影响

为了探究铀对藻类生长及光合作用的影响,筛选新的基于光合作用的水体铀污染生态风险评价指标,本试验采用不同浓度铀（0、0.5、1、5、10、20mg U·L-1）分别处理普通小球藻(Cholorella vulgaris)和黄龙普通小球藻两种来自不同生境的微藻,在处理后的第3、5、7、10、14d进行相对生长速率、光合放氧速率、叶绿素含量和叶绿素荧光动力学参数等指标的测定。结果表明：（1）0.5mg·L-1低浓度铀处理显著促进两种小球藻的生长和光合作用效率,表现为两种微藻的相对生长速率、光合放氧速率、光系统II最大光化学量子产量Fv/Fm、实际光化学量子产量Y（Ⅱ）、相对电子传递速率rETR等叶绿素荧光参数等指标均显著高于对照, 而5-20 mg·L-1高浓度铀处理则显著抑制两种小球藻的生长和光合作用;（2）黄龙普通小球藻比普通小球藻对铀处理更敏感,在1mg·L-1处理浓度下生长与光合作用就受到显著抑制,可以用来作为水体铀污染生物监测的指示生物;（3）回归分析表明,不同浓度铀处理下,叶绿素荧光参数Y(II)和rETR的响应速度快于相对生长速率、光合放氧速率、叶绿素含量和Fv/Fm等指标的变化,可以作为水体铀污染生态风险评价的敏感指标。     

小球藻生物富集锌、镉机制的研究

藻类对许多重金属具有较强的生物富集能力,小球藻（Chlorella spp.)分别在不同系列Zn^2 ,Cd^2 浓度下培养8d间隔2d,测定小球藻的生物量及Zn和Cd的含量。结果表明,小球藻随水体中Zn^2 和Cd^2 的浓度不同,在重重金属水体中暴露时间不同,具有不同的富集速率和能力。     

优化滩涂养殖水体生态结构和调节水质的研究

采用在滩涂鱼塘和虾池中接种小球藻和有益微生物的方法,探讨了调控滩涂养殖水体生态结构的浮游生物种群、数量、生物量及化学环境的可行性。结果表明,虾池和鱼塘中接种小球藻后,小球藻数量均大幅度增加,变为优势种群,分别是其对照的16.92倍、4.76倍;浮游动物生物量为4.32mg·L^-1和2.84mg·L^-1,分别比对照增加19.3%、2.5%,同样,光合细菌、酵母菌及硝化细菌显著地改变了水体藻类和浮游动物种群的组成、数量、比例及生物量等,“酵母菌＋硝化细菌”的处理,使水体氨态氮浓度下降最明显,为对照的44％,虾池生化耗氧和化学耗氧,为对照的56.5%和38.4%,增加了水体溶解氧和初级生产力。     

氮、锰、硫缺乏对蛋白核小球藻Chlorella pyrenoidosa光合产氢及其生长的影响

本实验通过研究缺氮、缺锰和缺硫对蛋白核小球藻Chlorella pyrenoidosa产氢的影响,发现缺氮、缺锰及缺硫条件下该藻均能产氢,但在缺氮条件下产氢量最高,约为88.613μL H2/mgChla,分别是对照组、缺锰和缺硫实验组产氢量的4.61倍、1.92倍和3.63倍。通过对光合、呼吸及生长的比较研究,发现缺锰对该藻光合、呼吸及生长的影响要小于缺氮和缺硫;与正常培养条件相比,缺锰、缺硫抑制藻细胞的光合放氧和生长,对呼吸影响小,而缺氮不仅最大程度抑制光合放氧和生长,同时使呼吸作用增强,这为进一步优化该藻产氢条件及研究其产氢机制提供了线索。     

pH对小球藻Chlorella sp. XQ-200419光合作用、生长和产油的影响

以一种生长快、油脂含量高的小球藻(Chlorella sp. XQ-200419)为实验材料, 利用测定净光合放氧速率的方法研究了pH对其光合作用的影响; 使用改良的BG-11培养基在微藻环形培养池模拟系统中进行分批培养, 培养周期为8d, 培养过程中使用 pH控制仪在线监测藻液的pH, 根据pH变化, 自动接通、关闭CO2通气管道, 将藻液pH分别控制在5.06.0, 7.08.0, 8.09.0, 9.010.0, 10.011.0内, 研究pH对生长速率、生物质面积产率、总脂含量和总脂面积产率的影响。主要结果如下: 藻液pH对小球藻Chlorella sp. XQ-200419光合放氧、生长速率、生物质产率、总脂含量和产率都有显著影响, 适宜的pH范围是7.09.0, 在此范围内, 光合放氧、生长速率、生物质产率、总脂含量和产率均保持较高水平, 且pH的影响不显著; pH低于7.0, 高于9.0, 其光合放氧、生长速率、生物质产率、总脂含量和产率都显著降低。这表明pH对小球藻Chlorella sp. XQ-200419光合作用的影响和对生长、产油的影响是一致的。pH 7.08.0, 小球藻的生物质平均面积产率和总脂平均面积产率都达到最大值, 分别是8.9 g/(m2d)和2269.5 mg/(m2d); 当藻液pH超过10.0, 生物质平均面积产率和总脂平均面积产率分别降低42.1%和60.0%。适合于小球藻生长的pH也有利于其积累油脂, 所以, pH对小球藻产油的影响是一种适宜模式, 而非胁迫模式。规模化培养小球藻Chlorella sp. XQ-200419, 通过补充CO2将藻液pH控制在7.09.0内, 可以获得高生物质产率和总脂产率。研究结果反映出pH对小球藻光合作用、生长和产油影响的规律, 也为规模化培养小球藻生产微藻油脂过程中合理控制藻液pH提供了依据。       

固定化技术在促进光合细菌产氢中的应用

氢气是一种新型的清洁高效能源,制氢技术的创新是目前研究的热点。将新型的技术及材料应用到生物制氢工艺中,从而促进生物制氢技术的产氢效率和工程应用是研究的重点之一。该文阐述了光合细菌在固定化生长条件下发酵产氢的最新研究进展,从固定化技术的原理、固定化方法的应用进展及影响因素几个方面进行了综述,详细阐述了包括包埋、悬浮载体附着生长及固定生物膜法等几种固定化方法对光发酵产氢的作用,介绍了国内外用于固定化的新型材料,并对今后的研究重点及方向进行了展望。     

光合生物色素-蛋白质复合物的多样性

通过对近年来放氧型光合生物有关研究结果的概括分析,提出放氧型光合生物光系统Ⅰ、光系统Ⅱ及捕光色素-蛋白质复合物具有多样性,并根据其PSⅠ复合物的77 K荧光发射特点,指出放氧型光合生物光合系统中的能量传递方式也可能存在多样性.     

温度变化对藻类光合电子传递与光合放氧关系的影响

由于直接测定藻类的光合速率耗时且不方便,研究者们常通过测定藻类光合电子传递速率的方式来间接反映其光合速率,理论上,以氧气产生来度量的总光合速率（PGross）与电子传递速率(ETR)之间应该存在很好的线性关系。然而,由于温度的变化会影响藻类的光呼吸等耗氧的生理过程从而影响光合作用中的氧气释放,因此温度可能会对PGross与ETR之间的线性关系产生影响。研究了温度变化对蛋白核小球藻（Cholorella pyrenoidosa）、菱形藻（Nitzschia sp.）和水生集胞藻（Synechocystis aquetilis Sauv.）的总光合放氧速率（PGross）与电子传递速率(ETR)之间比率的影响,结果表明PGross/ETR随温度的升高而降低,低温条件下PGross/ETR比值较高,说明在相同的电子传递速率的情况下水的光裂解产生的氧有更多的可以释放出来;在高温条件下PGross/ETR比值相对较低,说明高温条件下可能有相对更多的水光裂解产生的氧被用于耗氧的生理过程而没有释放出来。研究表明当温度发生变化时,光合放氧与电子传递之间并不呈线性关系,这说明将ETR作为实际光合生产的评价指标时要谨慎,不能不加分析地直接应用。     

铜锈环棱螺对水华中常见藻类的抑制效应

藻类水华频发已成为全球性的水环境问题,近年来,生物控藻法因具有环境友好的特点而备受关注。本研究以我国富营养化湖泊中常见大型底栖动物铜锈环棱螺为操纵生物,通过室内培养试验,研究了其对水华水体中常见蓝藻铜绿微囊藻、绿藻普通小球藻和斜生栅藻生长及光合活性的影响,以期阐明螺-藻间相互作用关系,探究铜锈环棱螺作为生物操纵物种的可行性。结果表明: 铜锈环棱螺能在短时间内大量摄食藻细胞,其对铜绿微囊藻产毒株和不产毒株以及斜生栅藻的去除率均在12 h内达到最大值,分别为73.7%、73.2%和51.1%;其对普通小球藻的摄食强于其他藻类,至试验结束时去除率达到99.2%。产毒铜绿微囊藻产生的微囊藻毒素会在铜锈环棱螺体内累积并促发其肝脏病理学变化,进而阻碍铜锈环棱螺的摄食。试验后期各处理组藻细胞光合活性均显著低于对照组,表明铜锈环棱螺的摄食作用对藻细胞造成了损伤,抑制了其大量增殖。此外,当不产毒铜绿微囊藻与斜生栅藻混合时,铜锈环棱螺的选择摄食性导致微囊藻的优势地位被斜生栅藻所取代。因此,铜锈环棱螺可以通过摄食作用抑制藻类的光合活性并降低其生物量,从而在一定程度上抑制或减缓水华的形成。     

固定化过程对聚球藻生理生化特性的影响

【背景】聚球藻(Synechococcus)是一类生长于海水中的单细胞蓝细菌,因生长迅速被用来净化污水。为了降低成本,生产上采用了固定化措施,但聚球藻固定化后,细胞内的生理生化变化尚未见报道。【目的】研究聚球藻固定化后生理生化及净化能力的变化,为促进聚球藻的应用提供科学依据。【方法】以海藻酸钠和氯化钙为主要原料固定聚球藻;利用显微观察法计算聚球藻的生长速率;利用溶氧仪检测聚球藻的净光合效率;利用荧光法检测一氧化氮(NO)含量;利用分光光度计法检测叶绿素含量、蛋白含量、硝酸还原酶(NR)活性、Rubisco羧化酶活性及水质指标。【结果】固定化过程使聚球藻的最大比生长速率降低24.30%。固定化后聚球藻的净光合速率降低范围为9.10%-29.10%,但固定化过程对叶绿素含量没有明显的影响。固定化使聚球藻Rubisco羧化酶活性、NO含量和NR活性分别降低25.70%、32.10%和40.00%,使聚球藻去除红鳍东方鲀养殖废水中总氮、总磷、氨氮和亚硝酸盐的能力分别降低30.00%、17.70%、20.20%和21.20%,但对去除硝酸盐及化学需氧量(Chemical oxygen demand,COD)的能力没有影响。【结论】固定化过程抑制了聚球藻NR的活性,使其NO产量减少,NO通过转录后修饰的方式降低了光合作用关键酶Rubisco的活性。Rubisco的活性降低使光合效率降低,导致固定化聚球藻的比生长速率和净化污水的能力降低。     

Use of surface-immobilized Trichoderma in batch and fed-batch fermentations

Needle-punch polyester was shown to be an effective support material for the immobilization of Trichoderma reesei Rut C30. When used as a resident inoculum for a batch process, the immobilized Trichoderma was very stable and resulted in a reduced rate of biomass generation in the bulk liquid phase as compared to cultures inoculated with free mycelium. Fed-batch fermentations with the immobilized Trichoderma produced ca. 80% of the activity of those using free cells; however, the activity was more stable and the crude enzyme broth produced had a greatly reduced biomass concentration.     

Improvement of Process Stability of Microbiological Quinoline Degradation in a Three‐Phase Fluidized Bed Reactor

The biological degradation of quinoline by suspended and immobilized Comamonas acidovorans was studied under continuous and discontinuous operating conditions in a three‐phase fluidized bed reactor. C. acidovorans degrades quinoline into biomass and carbon dioxide. Quinoline and the intermediates of its metabolic pathway are found only by quinoline shockloads. The continuous degradation of quinoline by suspended biomass was only possible, if the dilution rate was less than the growth rate (μ_max =0.42 h^–1) and the concentration of a shockload was less than 1 kg/m³. A concentration greater than 1 kg/m³ led to an irreversible damage of the cells. Hence, two different carrier materials were used for immobilization by attachment, to increase the stability of the process. Using immobilization of biomass on carriers decouples the hydrodynamic retention time and the growth rate of the microorganisms. A comparison of the carrier material showed no differences with respect of activity and stability of the biofilm. The process stability of a three‐phase fluidized bed reactor was increased by immobilized biomass. The degradation of toxic shockloads was only possible with immobilized biomass. A dynamic model has been developed to describe the concentration profile of quinoline, 2‐hydroxyquinoline as metabolite and the suspended biomass. A comparison of the measured and calculated values showed good agreement.     

Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans

The effect of in situ immobilization of Gluconobacter oxydans on a novel carrier material in a repeated-fed-batch operated packed-bed bubble-column bioreactor for the production of the fine chemical dihydroxyacetone was investigated experimentally. The carrier material were biocompatible, durable, coated Ralu-rings. The coating was a porous silicone matrix with satisfactory wetting characteristics. Settling of cells was relatively rapid. The cells were protected from abrasion caused by shear forces. A sufficiently high oxygen supply rate to the immobilized cells was provided due to the high oxygen permeability of the silicone matrix. The immobilized biomass was estimated to be about 65% of the total biomass contained in the bioreactor after 18 days of operation. The observed space-time yield was approx. 76% higher compared to a similar process which was performed without an optimized fermentation medium. Compared to previous experiments with a trickle-bed bioreactor, the space-time yield was approx. 3.7 times higher. A typical time course of the immobilization process was observed: after an induction phase, a transition phase followed which later on gave way to a nearly linear accumulation phase. A stationary phase with regard to the amount of immobilized active cells, however, was not reached. Hence, a higher bioreactor performance than observed could be expected at longer operation times.     

米曲霉F-81产中性蛋白酶的固定化条件及其特性

以海藻酸钠为载体,戊二醛为交联剂固定化米曲霉F-81产中性蛋白酶,研究了固定化条件及固定化酶的性质。结果表明,固定化的最佳条件为:固定化时间1 h、海澡酸钠浓度4%、戊二醛浓度9%、CaCl2浓度0.7 mol/L。在此条件下固定化的中性蛋白酶活力为游离酶活力的68%。固定化酶的最适作用温度为65℃,最适作用pH值为7.0。60℃下酶稳定性较好,80℃下处理60 min,粗酶中几乎检测不到酶活力;中性蛋白酶pH稳定范围为6.5-9.5。Km值为24.83 mg/mL,最大反应速率Vmax为0.043 12 mg/min。     

Efficient immobilization technique for enhancement of cellobiose dehydrogenase activity on silica gel

In this study, cellobiose dehydrogenase (CDH) of Phanerochaete chrysosporium ATCC 32629 was immobilized on silica gel for the further application of CDH in the saccharification process of biomass. To prevent the loss of enzyme activity during enzyme immobilization, the pretreatment of CDH was performed by various pretreatment materials before immobilization. When pretreated enzymes were used in immobilization, the activities of immobilized CDH were higher than non-pretreated CDH even in same amounts of immobilized protein. The specific activity of pretreated immobilized CDH with lactose was about two times higher than that of non-pretreated immobilized CDH. Moreover, the pretreated immobilized CDH showed better reusability than non-pretreated immobilized CDH, with 67.3% of its original activity being retained after 9 reuses.     

磁珠固定化结核分枝杆菌二氢叶酸还原酶及其表征

比较Ni~(2+)-NTA磁珠和羧基磁珠固定结核分枝杆菌二氢叶酸还原酶(Mycobacteriumtuberculosis dihydrofolate reductase,Mt DHFR),探索适合小分子配体混合物库筛选的Mt DHFR固定化方法。重组表达带6×His标签Mt DHFR,纯化后表征酶学性质,比较用Ni~(2+)-NTA磁珠和羧基磁珠固定化时相应固定化容量、保留活性、稳定性及对抑制剂响应。结果表明,Ni~(2+)-NTA磁珠对Mt DHFR固定化容量为(93±12)mg/g磁珠(n=3),但酶比活保留不超过32%,Ni~(2+)明显抑制酶活性,EDTA与Ni~(2+)呈协同抑制效应,Fe~(3+)无显著干扰。羧基磁珠活化固定Mt DHFR的容量(8.6±0.6) mg/g磁珠(n=3),固定化酶比活保留(87±4)%(n=3)。在含50 mmol/L KCl的100 mmol/L HEPES (pH 7.0)中,游离和固定化Mt DHFR在0℃保存16 h活性都无显著改变,但在25℃保存16 h,游离酶活性下降近60%而羧基磁珠固定化Mt DHFR活性下降仅35%。甲氨喋呤对游离Mt DHFR和固定化Mt DHFR的IC50无显著差异(P0.05)。综上,Ni~(2+)-NTA磁珠不适合固定化Mt DHFR;羧基磁珠固定化Mt DHFR能保留活性、热稳定性及对抑制剂的响应,该固定化方法有望用于快速筛选其配体混合物库。     

Optimization of covalent immobilization of pectinase on sodium alginate support

Pectinase was immobilized on a sodium alginate support using glutaraldehyde and retained 66% activity. The optimal pH for activity shifted from 3.0 to 3.5 after immobilization; however, the optimum temperature remained unchanged at 40°C. The immobilized enzyme also had a higher thermal stability and reusability than the free enzyme, and retained 80% of initial activity after 11 batch reactions.     

A fast bioassay for phytotoxicity measurements using immobilized photosynthetic membranes

The potential of thylakoid membranes immobilized in an albumin-glutaraldehyde crosslinked matrix in a fast bioassay for phytotoxicity measurements in aqueous samples is studied. Free and immobilized preparations are compared for their electron transport activity measured as the initial rate of oxygen evolution with 2,5-cichlorobenzoquinone as the artificial electron acceptor. Immobilized thylakoids were much stable under storage conditions; in the dark, at 4 degrees C, they were fully stable in terms of photosynthetic activity for a period of 200 h. The immobilized membranes were as sensitive as the free thylakoids for the detection of most of the compounds tested (metal cations, sulfite, nitrite, and herbicides), all known as inhibitors of photosynthetic electron transport. In some instances, the immobilized preparations were even more sensitive than the free counterparts. The sensitivity could be further increased by lowering chlorophyll concentration in the assay. The short incubation period required ( approximately 10 to 15 min) and the small volume of the assay (3 mL) suggest that this type of material should be useful in the detection of locations or effluents with phytotoxic character. (c) 1994 John Wiley & Sons, Inc.     

用固定化弗劳地柠檬酸杆菌XP05从溶液中回收铂

比较了5种固定弗劳地柠檬酸杆菌XP05菌体的方法,其中明胶海藻酸钠包埋法为固定菌体的最佳方法。扫描电子显微镜观察表明,XP05菌体较均匀地分布于包埋基质中。固定化XP05菌体吸附Pt^4＋受吸附时间、固定化菌体浓度、溶液的pH值和Pt^4＋起始浓度的影响。吸附作用是一个快速的过程;吸附Pt^4＋的最适pH值为1.5;在50～250 mg P^4＋/L范围内,吸附量与Pt^4＋起始浓度成线性关系,吸附过程符合Langmuir和Freundlich吸附等温模型。在Pt^4＋起始浓度250 mg/L、固定化菌体2.0 g/L、pH 1.5和30℃条件下,振荡吸附60 min, 吸附量为35.3 mg/g。0.5 mol/L HCl能使吸附在固定化菌体上的Pt解吸98.7%。从废铂催化剂处理液回收铂的结果表明,在Pt^4＋起始浓度111.8 mg/L、固定化菌体4.0 g/L、pH 1.5和30℃条件下,振荡吸附60 min, 吸附量为20.9 mg/g。在填充床反应器中,在Pt^4＋起始浓度50 mg/L、流速1.2 ml/min、固定化菌体1.86 g的条件下,饱和吸附量达24.7 mg/g; 固定化XP05菌体经4次吸附解吸循环后吸附率仍达78％。