转兔防御素基因小球藻生物反应器异养培养工艺研究

根据5L生物反应器中转兔防御素(NP1)基因小球藻培养过程特征,建立了基于在补糖、补KNO3、pH和溶氧(DO)控制的5L生物反应器中进行转NP1基因小球藻异养培养的工艺,同时将此培养工艺放大到15L生物反应器中。转NP1基因小球藻在15L反应器中培养133h的细胞密度可达34.2g/L,平均生长速率为0.257g/(L·h),比5L生物反应器中培养131h的细胞密度27.8g/L和平均生长速率0.21g/(L·h)分别提高了23%和22%。研究结果证实了转NP1基因小球藻放大培养的可行性,为实现高密度、高表达的大规模培养奠定了基础。     

小球藻光生物反应器脱除空气中二氧化碳的研究

采用自制的光生物反应器,研究了不同条件对小球藻脱除空气中CO2效果的影响.光生物反应器为圆柱形有机玻璃容器,底面积0.0154m2,高0.2m,容积为3L,反应器内小球藻藻体干重约为7.2g.实验结果表明,用小球藻来固定CO2不仅能脱除通入气体中的CO2,而且可以促进小球藻生长.通入气体的流量和CO2浓度对CO2的脱除率有很大影响.在适宜的实验条件下(通入气体流速为0.6L/min、CO2浓度约1%、温度25~30℃、光强3500lux、pH9.5~8.5),入口气体中CO2约有50%被脱除,该光生物反应器脱除CO2的能力约为0.118g/(L.h).可见利用小球藻光生物反应器固定CO2具有较好的脱除效果,值得进一步研究.     

膜光生物反应器污水养藻技术及其膜污染研究

在膜光生物反应器中利用市政污水培养螺旋藻,对微藻生物量、膜污染物和膜清洗方法进行了考察分析,为膜技术应用于微藻培养体系提供参考.实验结果表明,连续培养过程中,反应器中藻密度范围为1.79~2.05g/L,产率为0.10g/(L·d),NH_4~+-N、TP去除率分别为93.54%~98.97%、48.37%~66.91%.较高藻密度、碱性环境下,藻细胞残体及其分泌的EOM等有机物和无机沉淀物会共同引起膜污染.SEM、EEM和EDX结果显示,进水膜面主要是由藻细胞残体、蛋白质和SMP等有机物形成的滤饼层污染,出水膜面附着含Ca为主的无机沉淀物,部分膜孔堵塞.先后用pH为2的盐酸和200mg/L的NaClO溶液浸泡污染膜片2h,清洗后膜通量恢复了94.29%.     

AnMBR处理活性黑KN-B印染废水

实验对厌氧膜生物反应器(AnMBR)处理活性黑KN-B印染废水进行了探讨.研究了厌氧反应器内活性污泥的驯化培养状况,考察了厌氧反应器与管式超滤膜对印染废水BOD5,COD的去除作用,并对厌氧反应器产气量、出水的碳酸氢盐碱度和VFA进行了分析,通过UV-Vis光谱图分析了AnMBR对活性黑KN-B的脱色效果.结果表明,液流较低的低负荷启动方式使该AnMBR活性污泥的驯化培养顺利进行,厌氧反应器污泥床区域的污泥浓度培养至35 g/L;AnMBR对废水BOD5和COD的去除率分别达到90%和85%以上;该厌氧反应器的活性污泥体系和缓冲体系相对稳定,出水的碳酸氢盐碱度和VFA分别在16~18mmol/L与1.0~3.5 mmol/L的范围内波动,pH稳定在7.70~8.00之间;AnMBR对500mg/L的活性黑KN-B废水的脱色率能达到90%以上.     

一种新型广谱的原核型分泌表达载体的建立及人表皮生长因子的表达

通过Cyanobase藻类学数据库查询,从集胞藻6803转译后加工过程中的某些蛋白的分泌信号序列得到了可以用于分泌型表达的信号序列,进一步通过SignalP 3.0 Server分析软件预验证,从集胞藻6803得到4种不同蛋白的信号序列A、B、C、D,将这些信号序列插入pET-His-EGF表达载体hegf基因上游,得到了带有分泌型信号的pS-X系列分泌型表达载体,人表皮生长因子(hEGF)在其中的pS-A载体中实现了分泌型表达,hEGF分泌到周质空间的相对表达量约为1%,在其它3个载体中未见表达.获得的pS-A分泌型表达载体是一种新型分泌型表达载体,它利用蓝藻信号序列作为分泌表达的信号肽,实现了hEGF多肽在大肠杆菌中分泌表达,通过融合白亚细胞定位法处理得到的hEGF多肽在胞质、周质空间和培养基中表达的比例为85:33:25(density intensity/mm2),利用渗透休克处理方法得到的比例为50:37:25.由于A信号序列本身是集胞藻6803膜蛋白(ID:s110172/NCBI-GI:1001433)的分泌信号肽,说明pS-A载体是一种广谱的原核型分泌表达载体.这一结果为实现hEGF在螺旋藻中的分泌型表达奠定了基础.     

微藻在TiO_2纳米管阵列界面的培养及应用

通过阳极氧化法在金属Ti箔表面制备了TiO_2纳米管阵列。以集胞藻(synechocystis sp)、鱼腥藻(anabaena sp)和LKG 3种微藻作为实验藻种,通过控制接种量和培养条件等因素,实现了3种微藻在TiO_2纳米管阵列表面的附着生长。将3种微藻附着的TiO_2纳米管阵列制备成光电极(简称负载电极),与阴极材料和电解液组装成太阳能电池,测定负载电极的光电流响应。结果显示由于微藻细胞的附着,TiO_2负载电极的光电流值均有所增大,最大达3倍以上。     

中心组合优化重组毕赤酵母生产S-腺苷甲硫氨酸用发酵培养基

利用统计学实验设计方法优化了重组毕赤酵母生产S-腺苷甲硫氨酸(SAM)的发酵培养基.采用中心组合设计和响应面分析对诱导相培养基中硫酸铵、甲醇和L-甲硫氨酸三个关键因素的浓度进行了优化,使SAM在全合成培养基上的摇瓶产量提高了60%,达到1.9g/L,并考察了胞内SAM合成酶活性的变化与SAM积累的相关性.     

海洋鱼类弧菌病疫苗的制备--新型简易鳗弧菌(Vibrio anguillarum)培养基优化①

以一株从我国山东海域爆发弧菌病鲈鱼体内分离得到的病原菌鳗弧菌（Vibrio anguillarum MV425 O1型）为研究对象,对其实现高密度生产海洋鱼类弧菌病疫苗的培养基进行了优化研究。研究表明,碳源的NaCl造成的渗透压以及由发酵碳源和氮源导致的pH值变化是影响细胞正常生长的重要因素。海水中微量元素（除铁元素）对菌体生长的作用可被酵母浸出物补偿,高铁浓度有助于菌体生长。在确定显著影响因子的基础上,采用可旋转中心复合设计方案和响应面法进行了统计模型优化,优化设计有效地提高了细胞培养密度,实现在摇瓶条件下细胞干重5．8g/L和30L规模反应器中26．2g/L的较好的初步放大结果,并可替代海水培养基。     

类产碱假单胞菌对不同型号PVA的降解特性研究

对类产碱假单胞菌降解不同型号PVA(0588,1788,2088,1750,1799)的降解效果及降解特性进行了研究。采用UV2900紫外分光光度计测吸光度法测定了各型号PVA的降解率,同时研究了PVA的分子量和醇解度、PVA浓度、接种菌种浓度、摇瓶转速等对PVA降解率的影响。结果表明:醇解度一定时,高聚合度PVA的降解效率远低于低聚合度PVA;而聚合度一定时,醇解度对降解率的影响不明显。在培养温度为30℃,培养时间为72 h,摇瓶转速为180 rad/min,PVA的质量浓度为0.5 g/L,接种菌的体积分数为4.0%时,PVA的降解率可达90.15%。     

氮、磷浓度对灌注搅拌式光生物反应器连续培养海带配子体细胞的影响

用搅拌式灌注光生物反应器对海带（Laminaria japonica）配子体细胞进行了连续培养,并研究了氮、磷两种营养元素对反应器培养海带配子体细胞的影响,首次定量描述了不同的恒定氮、磷浓度对配子体细胞生长的长期影响。研究结果表明,在培养液氮、磷摩尔浓度比固定在16：1,NO3^-维持浓度达到0．412mmol／L时,细胞在氮、磷稳定期的比生长速率达到最大值0．118d^-1,而NO3^-稳定浓度达到约2．50mmol／L时,细胞的生长开始明显受到抑制;在抑制阀值下,建立的Monod数学模型较精确地描述了细胞生长速率和氮、磷稳定浓度之间的关系。实验结果为大型海藻细胞或组织的光生物反应器培养提供了理论依据。     

节旋藻FACHB341 Rubisco基因部分序列的克隆和分析

以节旋藻FACHB34l为材料，对所克隆Rubisco基因进行了核苷酸序列测定和分析，由此推导出相应的氨基酸序列，并与部分其他蓝藻的同源基因进行了同源性分析。结果表明：所克隆DNA片段包含Rubisco大小亚基基因部分序列及rbcX基因序列，长度为2073bp，其中rbcL和rbcX基因之间存在两个转录茎环结构；大小亚基酸性氨基酸和碱性氨基酸的比例分别为13．14％和14．51％，疏水氨基酸的比例为42．16％；rbcL核苷酸序列与集胞藻PCC6803、Prochlorothrix hallandica、聚球藻PCC630l、Agmenellum quadruplicatum和鱼腥藻PCC7120同源序列的相似性分别为91．7％、79．9％、74．8％、77．2％和76．1％；rbcS核苷酸序列与鱼腥藻PCC7120同源序列的相似性为67．6％，而与PCC630l和集胞藻PCC7002同源序列的相似性分别为30．1％和63．8％。     

Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time.

In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.     

Statistical factor-screening and optimization in slurry phase bioremediation of 2,4,6-trinitrotoluene contaminated soil

Since slurry phase bioremediation is a promising treatment for recalcitrant compounds such as 2,4,6-trinitrotoluene (TNT), a statistical study was conducted for the first time to optimize TNT removal (TR) in slurry phase. Fractional factorial design method, 2(IV)(7-3), was firstly adopted and four out of the seven examined factors were screened as effective. Subsequently, central composite design and response surface methodology were employed to model and optimize TR within 15 days. A quadratic model (R(2) = 0.9415) was obtained, by which the optimal values of 6.25 g/L glucose, 4.92 g/L Tween 80, 20.23% (w/v) slurry concentration and 5.75% (v/v) inoculum size were estimated. Validation experiments at optimal factor levels resulted in 95.2% TR, showing a good agreement with model prediction of 96.1%. Additionally, the effect of aeration rate (0-4 vvm) on TR was investigated in a 1-liter bioreactor. Maximum TR of 95% was achieved at 3 vvm within 9 days, while reaching the same removal level in flasks needed 15 days. This reveals that improved oxygen supply in bioreactor significantly reduces bioremediation time in comparison with shake flasks.     

Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.     

Batch growth kinetics of an indigenous mixed microbial culture utilizing m-cresol as the sole carbon source

An indigenous mixed microbial culture, isolated from a sewage treatment plant located in Guwahati was used to study biodegradation of m-cresol in batch shake flasks. m-Cresol concentration in the growth media was varied from 100mg/L to 900mg/L. The degradation kinetics was found to follow a three-half-order model at all initial m-cresol concentrations with regression values greater than 0.97. A maximum observed specific degradation rate of 0.585h(-1) was observed at 200mg/L m-cresol concentration in the medium. In the range of m-cresol concentrations used in the study, specific growth rate of the culture and specific degradation rates were observed to follow substrate inhibition kinetics. These two rates were fitted to kinetic models of Edward, Haldane, Luong, Han-Levenspiel, and Yano-Koga that are used to explain substrate inhibition on growth of microbial culture. Out of these models Luong and Han-Levenspiel models fitted the experimental data best with lowest root mean square error values. Biokinetic constants estimated from these two models showed good potential of the indigenous mixed culture in degrading m-cresol in wastewaters.     

Physiological Responses of Synechocystis sp. PCC 6803 under Clinorotation

Photosystem efficiency and the characteristic on oxidative stress were examined to elucidate the metabolic responses of Synechocystis sp. PCC 6803 to short-term clinorotation. Results compiled when using clinostat to simulate microgravity for 60?h, showed that clinorotation clearly prohibited the photochemical quantum yield, but promoted the synthesis of chlorophyll and total protein. This may be a compensatory mechanism for the algal cell to maintain its normal metabolism. An increased malondialdehyde (MDA) content of algal cell upon clinorotation, together with an enhanced catalase (CAT) activity was observed during the whole period of clinorotation. One conclusion is that short-term clinorotation acts as a kind of stress, and that these physiological responses may be a special way for an algal cell to adapt itself to a different environment other than earth gravity.     

2008年夏季广州大气消光系数与细粒子的关系

为了解夏季广州大气中细粒子消光特性,利用浊度仪、黑碳仪和自动气象站获得散射系数μsp、吸收系数μap以及气象要素等观测数据,并利用采样器采集大气悬浮颗粒物PM2.5和PM1.0样品获取其质量浓度。结果表明,散射系数μsp、吸收系数μap和大气消光系数(μext=μsp+μap)的平均值分别为(226±111)、(53±20)、(280±125)Mm-1;大气悬浮颗粒PM2.5和PM1.0的质量浓度分别为(53.7±23.2)、(46.1±21.3)μg/m3;消光系数μext与PM2.5和PM1.0的相关性系数分别达到0.96和0.93;PM2.5和PM1.0的平均质量浓度消光效率分别为到5.2、5.5 m2/g,表明大气中小粒径的粒子对消光系数的影响更为明显。     

槽边循环电解法回收电镀废水中Ni的研究

对槽边循环电解法回收电镀废水中Ni进行了分阶段实验研究,包括小型试验、扩大试验以及系统扩大试验。试验结果表明,适合工业生产中使用的操作条件如下:Ni离子浓度可在0.5～2.5g/L变化,电流密度40～80 A/m2,槽电压3～6 V,pH=4.5～5.5;当Ni离子浓度保持在1.0～2.5g/L,若连续操作电流密度采用100～150 A/m2,则电流效率仍大于40%;若从高浓度操作到低浓度,每次回收到0.5 g/L,电流密度应取40～80 A/m2,操作10次后,可回收到使浓度小于0.1 g/L,弃掉,则回收槽的回收率大于99%;由3类不同规模试验的对比中可以看出,在相同条件下,电流效率几乎相等。研究表明,采用槽边循环电解法回收电镀废水中的Ni是完全可行的,且可保证较高回收效率。     

Biotreatment of an effluent from a wood laminate industry using Lentinula edodes UEC 2019

The aim of this work was to evaluate the performance of the fungus Lentinula edodes UEC 2019 in the treatment of an effluent derived from a wood laminate manufacturing process. The factorial design methodology was employed to verify the influence of the source of nitrogen, co-substratum and treatment time variables on the color and total phenols reduction. Assays were conducted in Erlenmeyer flasks and bench bioreactor and the obtained results showed that the best conditions for the effluent remediation were observed when using the effluent itself as the nitrogen source (1g/L) and glucose (79 g/L) as co-substratum (glucose) during a treatment period of 30 days. It was possible the remove 92% phenol, 97% color and reduce the COD in the bioreactor by 99% while controlling the temperature, pH and feeding. These results demonstrate that the fungus was able to use the effluent components as substrate and that it has potential for use in wood lamination effluent remediation.