Increased production of extracellular polysaccharide by Porphyridium cruentum immobilized in foam sheets

Large‐size plate bioreactors were used to compare the production of extracellular polysaccharide by the red microalga Porphyridium cruentum when grown in suspension and in a foam sheet. A well‐defined illuminated area and unidirectional light propagation allowed us to generate information that is better quantified when expressed in terms of illuminated area. This is essential for meaningful comparison of data, especially considering that for a well‐designed and managed bioreactor, the culture production rates are believed to be light limited. At the same level of illumination, the culture immobilized in foam showed double the production rate of extracellular polysaccharide compared with the culture in suspension. The saturation level of biomass density per unit of illuminated area was eight times higher for the immobilized culture compared with the culture in suspension. Despite the increased biomass density for the immobilized culture, an increase in the light level above the optimum found for the culture in suspension reduced the extracellular polysaccharide production, suggesting that the photoinhibition light level was surpassed.     

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB = 5,5′-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the APTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability.     

The Microscopic Examination of Phytophthora cinnamomi in Plant Tissues Using Fluorescent In Situ Hybridization

The microscopic examination of Phytophthora cinnamomi in plant tissues is often difficult as structures such as hyphae, chlamydospores and oospores are frequently indistinguishable from those of other fungi and oomycetes, with histological stains not enabling species differentiation. This lack of staining specificity makes the localization of P. cinnamomi hyphae and reproductive structures within plant tissues difficult, especially in woody tissues. This study demonstrates that with the use of a species‐specific fluorescently labelled DNA probe, P. cinnamomi can be specifically detected and visualized directly using fluorescent in situ hybridization (FISH) without damage to plant or pathogen cell integrity or the need for subculturing. This approach provides a new application for FISH with potential use in the detailed study of plant–pathogen interactions in plants.     

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor

Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.     

Consensus Brain-derived Protein,Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.     

Acquisition of Cu, Zn, Mn and Fe by mycorrhizal maize (Zea mays L.) grown in soil at different P and micronutrient levels

Sustainability of soil-plant systems requires, among other things, good development and function of mycorrhizal symbioses. The effects of P and micronutrient levels on development of an arbuscular mycorrhizal fungus (AMF) and uptake of Zn, Cu, Mn and Fe by maize (Zea mays L.) were studied. A pot experiment with maize either inoculated or not with Glomus intraradices was conducted in a sand:soil (3 :1) mix (pH 6.5) in a greenhouse. Our goal was to evaluate the contribution of mycorrhizae to uptake of Cu, Zn, Mn and Fe by maize as influenced by soil P and micronutrient levels. Two levels of P (10 and 40 mg kg⁻¹ soil) and three levels of a micronutrient mixture: 0, 1X and 2X (1X contained, in mg kg⁻¹ soil, 4.2 Fe, 1.2 Mn, 0.24 Zn, 0.06 Cu, 0.78 B and 0.036 Mo), were applied to pots. There were more extraradical hyphae at the low P level than at the high P level when no micronutrients were added to the soil. Root inoculation with mycorrhiza and application of micronutrients increased shoot biomass. Total Zn content in shoots was higher in mycorrhizal than non-mycorrhizal plants grown in soils with low P and low or no micronutrient addition. Total Cu content in shoots was increased by mycorrhizal colonization when no micronutrients were added. Mycorrhizal plants had lower Mn contents than non-mycorrhizal plants only at the highest soil micronutrient level. AMF increased total shoot Fe content when no micronutrients were added, but decreased shoot Fe when plants were grown at the high level of micronutrient addition. The effects of G. intraradices on Zn, Cu, Mn, and Fe uptake varied with micronutrient and P levels added to soil. Accepted: 27 December 1999     

固定化微生物在生物膜式生化需氧量传感器中的应用现状

生化需氧量(Biochemical oxygen demand,BOD)微生物传感器是一种快速检测水样中有机污染物含量的设备,固定化微生物是其核心部件之一,对其稳定性、响应时间、使用寿命及实际应用范围等性能有着重要影响。生物膜式BOD传感器较其他类型的BOD微生物传感器具有结构简单、灵敏度高、响应时间短等优点,受到广泛的研究和应用。本文主要针对固定化微生物在生物膜式BOD传感器中的应用情况,概述较典型的微生物固定化方式的原理、特点及应用;总结几类应用较多或具有较好前景的载体材料,并讨论载体特性与传感器性能之间的关系;综述微生物在该领域的应用现状;简要介绍生物膜式BOD传感器的实际应用及商业化现状,比较其与另外几种BOD微生物传感器的优缺点;分析生物膜式BOD传感器中固定化微生物现存的一些问题及其发展趋势。     

应用固定化里氏木霉糖化玉米秆纤维素的研究

采用多孔聚酯材料固定里氏木霉（TrichodermareeseiRutC30）菌丝细胞,将固定化细胞在生长限制条件下重复分批培养,使纤维酶的合成与玉米秆纤维原料的酶解糖化耦合在一个反应器中同时进行。在30℃、初始pH4.8、摇瓶转速150r／min的条件下,连续重复进行12次分批培养试验。每批玉米秆用量为60g／L,培养周期4．5d,共54d。培养液中含滤纸酶活力平均为0.70IU／ml,还原糖26．41g／L,糖化率达到理论值的89.11％。固定化菌丝形态正常,菌量保持在10g／L左右。在间歇添料条件下,玉米秆原料的总量可提高到120g／L,7d后还原糖浓度达52.81g／L,糖化率为89．20％。利用固定化里氏木霉同时产酶和糖化植物纤维原料,工艺简便、成本低廉、易于连续自动化操作,是一条有效利用可再生纤维素资源的新途径。     

利用固定化黑曲霉单宁酶制备没食子酸的研究

用海藻酸钙载体包埋单宁酶,制备出转化五倍子单宁成没食子酸能力较好的固定化酶。研究了固定化条件和固定化单宁酶的部分性质,结果表明：最佳固定化条件为海藻酸钠90mg包埋单宁酶546u(3mL,182u/Ml),在1%～2%CaCl₂中作硬化处理;固定化单宁酶的最适温度为45℃,在10～50℃范围内稳定;其最适Ph值为6.5,在Ph5～7之间基本稳定;在此基础上,进行了没食子酸实验室克量级酶法制备实验,3次实验没食子酸产品的平均产率达到61%。和目前所用工业生产没食子酸的硫酸水解法相当,具有潜在的工业开发价值。     

Degradation of chlorophenols catalyzed by laccase

The degradations of 2,4-dichlorophenol (2,4-DCP), 4-chlorophenol (4-CP) and 2-chlorophenol (2-CP) catalyzed by laccase were carried out. The optimal condition regarding degradation efficiency was also discussed, which included reaction time, pH value, temperature, concentration series of chlorophenols and laccase. Results showed that the capability of laccase was the best, while to oxidize 2,4-DCP among the above-mentioned chlorophenols. Within 10 h, the removal efficiency of 2,4-DCP, 2-CP and 4-CP could reach 94%, 75% and 69%, respectively. The optimal pH for laccase to degrade chlorophenols was around 5.5. The increase of laccase concentration or temperature might result in the degradation promotion. The trends of degradation percentage were various among these three chlorophenols with the concentration increase of chlorophenols. Degradation of 2,4-DCP is a first-order reaction and the reaction activation energy is about 44.8 kJ mol⁻¹. When laccase was immobilized on chitosan, crosslinked with glutaraldehyde, the activity of immobilized laccase was lower than that of free laccase, but the stability improved significantly. The removal efficiency of immobilized laccase to 2,4-DCP still remained over 65% after six cycles of operation.