水母雪莲细胞悬浮培养黄酮合成的调控及生物反应器放大培养

水母雪莲（Sausswea medusa Maxim）为菊科风毛菊属植物，其主要的活性成份为黄酮类化台物。为进一步提高雪莲细胞培养物的黄酮含量和实现雪莲细胞培养生产黄酮类化合物的工业化，本文开展了水母雪莲细胞悬浮培养黄酮生物合成的调控及雪莲细胞生物反应器放大培养的可行性研究。 在水母雪莲细胞悬浮体系中加入苯丙氨酸和乙酸钠两种前体，结果显示，它们均能促进细胞内黄酮的生物合成，但它们对细胞的生长也有一定的抑制作用。实验表明，前体的添加时间均以第6天为宜，它们的最佳添加浓度都是0.1 mmol/L。苯丙氨酸、乙酸钠两种前体协同添加，黄酮产量是对照的1.94倍，比它们单独加入更能促进细胞的黄酮合成。 两种非生物诱导子硝酸银和谷胱甘肚都能诱导水母雪莲悬浮培养细胞黄酮的生物合成，诱导子的作用效果与诱导于的浓度和添加时间有关。二者协同诱导，获得的黄酮产量达是对照的1.72倍，高于它们单独加入时的黄酮产量。 应用2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响，发现在75 r/min、700-1000 L/min和4.0-5.0 9 DW/L接种量下细胞生长和黄酮合成比较好。经过12 d培养细胞干重达13.8 9 DW/L,黄酮产量416 mg/L。水母雪莲细胞生长及黄酮合成的进程表明，黄酮积累与细胞生长呈正相关。对细胞聚集体分布的研究发现，剪切力等因素使细胞聚集体分裂，使反应器中细胞生长受到影响，黄酮产量较摇瓶中降低。

Effects of water movement and aeration system on the survival and growth of hatchery bred sugpo (Penaeus monodon Fabricius) in earthen nursery ponds

An experiment was conducted to determine the effects of water movement and airlift aeration on the survival and growth of P. monodon fry reared from P sub(4)P sub(5) to P sub(32)P sub(33) in earthen brackishwater ponds. The high survival rates obtained justify the need for aeration when using the earliest stages of fry (P sub(4)P sub(5)) at higher stocking densities. For older stages regardless of source and at lower stocking densities, nursery operations based on traditional methods could also achieve better survival rates.

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d(-1)) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell(-1)) compared with cells grown by other culture methods (0.27-0.49 pg cell(-1)). The highest cell density and cellular toxin content were 17190 cells mL(-1) and 1.26 pg cell(-1) respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.

A perspective on fish gonad manipulation for biotechnical applications

The gonad is an essential organ for generating sperm and ova in vertebrates. This review describes several pilot studies on gonad gene manipulation and development in fish. With antisense RNA techniques, we suppressed the gonad development, and thus the fertility, of an antisense gonadotropin-releasing hormone (sGnRH) transgenic common carp. Then, using a tissue-specific exogenous gene excision strategy with sexual compensation, we knocked out the gonad-specific transgene. Under the control of the rainbow trout protamine promoter, the transgenic fish expressed the reporter gene eGFP specifically in the spermary. These results indicate that the fish gonad is a new model organ that can improve contemporary biotechnology experiments. Herein we discuss the potential of fish gonad manipulation for resolving important biosafety problems regarding transgenic fish generation and producing the new transgenic animal bioreactor.

固定化红酵母细胞合成L-苯丙氨酸研究

In this paper, bioconversion of trans-cinnamic acid(t-Ca)to L-phenylalanine (L-phe) has been investigated by using immobilized yeast cells with induced L-phe Ammonia-lyase(PAL, EC.4.3.1.5) as biocatalysts. The contents are the following. (1) Thirty strains of yeasts, including two genera (Rhodotorula, Sporobolomyces), six species (R. glutinis R. minuta,R.rubra,R.sineses,R.roseus and S.salmonicolor)were screened for their ability to converse the substrates, t-Ca and ammonia, to the product, L-phe, by using yeast cells as biocatalyst, and primary evaluation for PAL activity of the selected strains was investigated. From the results of the screening experiments, it was found that 22 strains were able to produce L-phe from t-Ca with the range of conversion yield from 2% to 67%. Studies on PAL formation time course during cultivation show that the maximum PAL activity of several different strains ranges from 2.3 to 14.4×10-3U/mg cell dry weight. The biomass of tested strains at their maximum enzyme activity is also greatly varied. (2)One of the selected strains, R. rubra as 2.166, was used for immobilized cells as biocatalysts to produce L-phe. The optimum conversion conditions and effective stablization agents were investigated. The results shown that polyacrylamide gel was chosen as a suitable matrix for immobilization of the yeast cells, and it can retain 88% of the PAL activity in the reverse direction at the following reactive conditions: [t-Ca]: 34mM. [NH4OH]: 6.OM.PH10.00, temperature: 30℃. (3) The effects of various kinds of effectors on the production of L-phe were also examined. Membrane permeabilizing agents can stimulate L-phe synthesis, but make the stability of PAL decline greatly. Polyalchoholic agents and glutamic acid were very effective for the stabilization of PAL. At the presence of glutamic acid (5%), the half life of L-phe productivity with the immobilized cells was extended to 192 hours, which was much higher than most of that having been reproted, while the half life of resting cells was only about 15 hours. (4) Use of initial velocity studies on the kinetics of enzyme-catalized reaction indicated that the apparent Km value was 13.0mM for the immobilized cells, and 4.8mM for the resting cells. Thermostability of the immobilized cells was better than the resting cells. Fluid bed bioreactor is more effective than batch bioreator in prolonging the thermostability of the biocatalysts. (5) CGA- 688 resin column chromatographic procedure was employed in the isolation and purification of L-phe, t-Ca and other substances from the reactire mixture. (6) Preparative-scale production of L-phe on a level of gram amount by immobilized cells from the culture broth of R. rubra AS2.166 allowed for the conversion yield with 30%. The characteristic physico-chemical criteria (including melting point, optical activity, elements analysis, IR, NMR) are the same with the standard L-phe. 本文报告了利用诱导的苯丙氨酸解氨酶 (PAL.EC.4.3.1.5)催化反式肉桂酸(t-Ca)氨加 成制备L-苯丙氨酸(L-phe)的研究，主要内容为：(1) 我们搜集了三十株酵母菌株，利用全细胞转化t-Ca生成L-phe的能力进行了直 接筛选，并对其PAL活性水平进行了初步评估研究。研究结果表明，其中22株酵母具有转化t-Ca生产L-phe的能力，它们包括 Rhodotorula glutinis,R.rubra, R.sineses 和Sporobolomyces roseus 的菌株，转化率在2-67%。细胞生长和PAL形成过程的研究 表明，不同菌株PAL最大活力在2.3-14.4×10-3U/mg 细胞干重，达到最大PAL活性时各株酵母的生长情况也极不一致。(2) 利用筛 选出的一株深红酵母R.rubra AS2.166 作为供试菌株，研究了细胞固定化条件下生物转化的最适条件及PAL在固定化条件下的稳定 性。结果表明以聚丙烯酰胺凝胶包埋法较为理想，能使细胞合成L-phe活力保持88%，最适t-Ca浓度为34mM，最适NH4OH浓度为6M,最 适PH10.0，最适温度45℃。(3) 多种效应物对L-phe 合成的影响研究表明：表面活性剂能刺激L-phe的合成，但使PAL稳定性下降。 多羟基化合物及Glu对PAL的稳定十分有效在有Glu存在下，能使固定化细胞合成L-phe的半寿期达192小时左右，高于大部分现已报 导的固定化结果。(4) 用初速度法研究了深红酵母AS2.166中PAL的酶促反应特征，测得固定化细胞对t-Ca的表观米氏常数Km为 13.0mM，全细胞为4.8mM，细胞固定后热稳定性提高。(5) 建立了适合低浓度分离纯化产物与底物的聚苯乙烯大孔树脂柱层析技术 ，能使L-phe与t-Ca及产物混合物中其它成分有效分开。(6) 利用固定化的R.rubra AS2.166细胞所做的制备实验能够使L-phe的产 率达到30%左右，其主要的理化指标(包括熔点、比旋光度、元素分析、IR、NMR等)与标准L-phe一致。

垃圾渗滤液场外硝化－原位反硝化脱氮协同填埋场温室气体减排

垃圾卫生填埋是国内外城市垃圾的主要处置方法。垃圾渗滤液是渗入填埋场垃圾的降水混合垃圾降解过程中产生的物质而形成的混合物，是垃圾填埋场向环境排放的主要污染物。渗滤液由于其所含高浓度有机和无机污染物，且其中很多物质有生物毒性或难生物降解，难于治理。特别是到填埋晚期，渗滤液中高浓度的氨氮更是增加了治理的难度。渗滤液场外硝化－原位反硝化是填埋场氮管理的新途径。本文利用从环境中筛选出优势硝化功能菌对渗滤液中的高浓度氨氮进行生物硝化，经硝化后的渗滤液回灌至以垃圾柱模拟的生物反应器填埋场，在填埋场内实现原位反硝化。 上述目标通过以下两部分来实现： 第一部分：渗滤液场外硝化。首先从污水厂的硝化污泥中富集并筛选出硝化功能菌，在模拟氨氮废水中优化。将驯化的硝化功能菌接种于连续式完全混合反应器（CSTR）进行高氨氮渗滤液硝化研究。在200余天的连续运行中，反应器硝化和有机物去除效果良好。在最大氨氮负荷和有机物负荷分别为0.65 g N l-1 d-1 和3.84 g COD l-1 d-1时，氨氮和COD去除率分别高于99%和57%。实验过程中发现，游离氨（FA）和溶解氧（DO）浓度对反应器中亚硝酸盐的积累影响很大。 第二部分：渗滤液原位反硝化。本文利用一个垃圾填充柱模拟生物反应器填埋场，研究了硝化渗滤液回灌对垃圾降解的影响，和回灌的硝化渗滤液中TON（总氧化态氮）对填埋场生物反应器产甲烷作用的影响。最后利用变性梯度凝胶电泳（DGGE）分析了硝化渗滤液回灌对垃圾填埋场菌群结构的影响。结果表明：回灌的TON被完全还原，反硝化为主要反应，最大TON负荷为28.6 mg N kg-1 TS d-1。当垃圾柱TON负荷大于11.4 mg N kg-1 TS d-1时，出现了产甲烷抑制，抑制作用随TON负荷的增加而加强。在此过程中，反硝化逐渐代替产甲烷作用成为填埋场内垃圾降解的主要反应，且更多产生的是清洁的氮气，而非温室气体甲烷。直到实验结束时，回灌硝化渗滤液的垃圾柱的甲烷产量仅相当于对照的2.5%，并且回灌的硝化渗滤液还加速了填埋场垃圾的降解与稳定。通过DGGE进行菌群结构分析发现，由于TON对填埋场的长期作用，反硝化菌增多而产甲烷菌减少。 Landfill still remains the chief method for MSW management around the world. Leachate is a mixture of rainfall permeating through landfill and organic and inorganic matters generated during decomposition of the wastes in the landfills, characterized as highly complicated and refractory wastewater. Ex-situ nitrification and sequential in-situ denitrification represents a novel approach to nitrogen management at landfills. In the present paper, nitrification was carried out in a continuous stirred tank reactor (CSTR) inoculated with nitrifying bacteria which were isolated from municipal WWTP of Chengdu city. The nitrified leachate from CSTR was recirculated to a lab-scale municipal solid waste (MSW) column where in-situ denitrification took place. The above object was achived through two parts as following: First, ex-situ nitification of leachate. After acclimated in simulated wastewater for 3 month, nitrifying bacteria isolated from WWTP nitrifying sludge were added to the CSTR for nitrification. The results over 200 days showed that the maximum nitrogen loading rate (NLR) and the maximum organic loading rate (OLR) was 0.65 g N l-1 d-1 and 3.84 g COD l-1 d-1, respectively. The ammonia and COD removal was over 99% and 57%, respectively. Moreover, the effects of free ammonia (FA) and dissolved oxygen (DO) on nitrification were investigated. Second, in-situ denitrification was studied in a municipal solid waste (MSW) column. Variation of nitrified leachate and its effects on the decomposition of municipal solid waste (MSW) were studied in a lab-scale MSW column to which nitrified leachate was recirculated. Additionally, DGGE was employed to investigate the microbial community of both MSW columns. The results suggested: complete reduction of total oxidized nitrogen (TON) was obtained with maximum TON load of 28.6 mg N kg-1 TS d-1 and denitrification was the main reaction responsible. Methanogenesis inhibition was observed while TON load was over 11.4 mg N kg-1 TS d-1 and the inhibition was enhanced with the increase of TON load. Denitrification gradually took over methanogenesis to become the main reaction responsible for decomposition of MSW while nitrogen gas, a clean byproduct, was generated instead. Till the end of the experiment, the average weekly methane production in the denitrification column was as low as 2.5% of that of the control, and the rate of decompition and stability of MSW was accelerated by the recirculation of the nitrified leachate.Owing to long term exposure of nitrified leachate to landfill, denitrifying bacteria increased and methanogen decreased.

Primmorphs from archaeocytes-dominant cell population of the sponge Hymeniacidon perleve: Improved cell proliferation and spiculogenesis

Marine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development. In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge-derived bioactive metabolites; however, it is still a challenging task. The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed-cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte-dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed-cell population (MCP). Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types. A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation. Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved. Furthermore, a time-dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP. This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge-derived drugs. (C) 2003 Wiley Periodicals, Inc.

Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l(-1) was obtained in batch culture with an initial dry cell weight of 126 mg l(-1) and with aeration rate of 1.2 l air min(-1) l(-1) culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 mu g g(-1) dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.

Comparison of photobioreactors for cultivation of Undaria pinnatifida gametophytes

Undaria pinnatifida gametophytes were grown in 2.5 l bubble column and airlift reactor at 25 degreesC and light intensity of 40 mumol m(-2) s(-1) for 6 days. With aeration at 1 l min(-1), the airlift reactor yielded higher growth rate (0.12 mg DW ml(-1) d(-1)) than a bubble column (0.08 mg DW ml(-1) d(-1)). The advantages were related to the more homogeneous fluid dynamic characteristics of the airlift reactor.

Expression, purification, and characterization of recombinant Chinese shrimp crustin-like protein (CruFc) in Pichia pastoris

A crustin-like protein (CruFc) from Fenneropenaeus chinensis was expressed in Pichia pastoris and then purified to electrophoretic homogeneity on a Sephacryl S-100 column with a band corresponding to the expected one (13 kDa) shown by 15% SDS-PAGE. Western blot indicated that the rCruFc specifically reacted with polyclonal rabbit anti-Fenneropenaeus chinensis CruFc. Production in a 5 l bioreactor gave 237 mg rCruFc/l. Antimicrobial assay revealed that 4 mu M rCruFc inhibited growth of Staphylococcus aureus.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Synthesis of a silica-bonded bovine serum albumin s-triazine chiral stationary phase for high-performance liquid chromatographic resolution of enantiomers

A novel method of synthesizing protein chiral stationary phase (protein-CSP) is proposed with 2,4,6-trichloro-1,3,5-triazine as the activator. The bovine serum albumin (BSA) based chiral columns (150x4.6 mm I.D.) were prepared successfully within 8 h. With tryptophan as the probe solute, it was observed that the BSA immobilized by this method had a better ability to distinguish enantiomers than that activated by glutaric dialdehyde. This may be due to the well-maintained BSA conformation and the larger amount of BSA immobilized on the silica gel. The BSA-CSP prepared by this method was relatively stable under experimental conditions, and the resolution of 13 chiral compounds was achieved. The coupling reaction in this method is mild, reliable and reproducible; it is also suitable for the immobilization of various biopolymers in the preparation of bioreactor, biosensor and affinity chromatography columns. (C) 2000 Elsevier Science B.V. All rights reserved.

Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage.

Ohlsen K, Ternes T, Werner G, Wallner U, Löffler D, Ziebuhr W, Witte W, Hacker J. Institute for Molecular Biology of Infectious Diseases, The University of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. knut.ohlsen@mail.uni-wuerzburg.de The growing rate of microbial pathogens becoming resistant to standard antibiotics is an important threat to public health. In order to assess the role of antibiotics in the environment on the spread of resistance factors, the impact of subinhibitory concentrations of antibiotics in sewage on gene transfer was investigated using conjugative gentamicin resistance (aacA-aphD) plasmids of Staphylococcus aureus. Furthermore, the concentration of antibiotics in hospital sewage was measured by high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry. Several antibiotics were found to be present in sewage, e.g. ciprofloxacin up to 0.051 mgl(-1) and erythromycin up to 0.027 mgl(-1). Resistance plasmid transfer occurred both on solidified (dewatered) sewage and in liquid sewage in a bioreactor with a frequency of 1.1x10(-5)-5.0x10(-8). However, low-level concentrations of antibiotics measured in sewage are below concentrations that can increase plasmid transfer frequencies of gentamicin resistance plasmids of staphylococci.

Lovastatin production by Aspergillus terreus using lignocellulose biomass in large scale packed bed reactor

The effect of superficial air velocity on lovastatin production by Aspergillus terreus PL10 using wheat bran and wheat straw was investigated in a 7 l and a 1200 l packed bed reactor. Mass transfer and reaction limitations on bioconversion in the 1200 l reactor was studied based on a central composite design of experiments constructed using the superficial air velocity and solid substrate composition as variables and lovastatin production as response.
The surface response prediction showed a maximum lovastatin production of 1.86 mg g-1 dry substrate on day 5 of the bioconversion process when the reactor was operated using 0.19 vvm airflow rate (23.37 cm min-1 superficial air velocity) and 54% substrate composition (wC). Lovastatin production did not increase significantly with superficial air velocity in the 7 l reactor. Variation in temperature and exit CO2 composition was recorded, and the Damköhler number was calculated for lovastatin production at these two scales. The results showed that in larger reactors mass transfer limitation controlled bioconversion while in smaller reactors bioconversion was controlled by reaction rate limitations. In addition, mass transfer limitations in larger reactors reduced the rate of metabolic heat removal, resulting in hot spots within the substrate bed.

Lovastatin biosynthesis depends on the carbon-nitrogen proportion: model development and controller design

Lovastatin biosynthesis depends on the relative concentrations of dissolved oxygen and the carbon and nitrogen resources. An elucidation of the underlying relationship would facilitate the derivation of a controller for the improvement of lovastatin yield in bioprocesses. To achieve this goal, batch submerged cultivation experiments of lovastatin production by Aspergillus flavipus BICC 5174, using both lactose and glucose as carbon sources, were performed in a 7 liter bioreactor and the data used to determine how the relative concentrations of lactose, glucose, glutamine and oxygen affected lovastatin yield. A model was developed based on these results and its prediction was validated using an independent set of batch data obtained from a 15-liter bioreactor using five statistical measures, including the Willmott index of agreement. A nonlinear controller was designed considering that dissolved oxygen and lactose concentrations could be measured online, and using the lactose feed rate and airflow rate as process inputs. Simulation experiments were performed to demonstrate that a practical implementation of the nonlinear controller would result in satisfactory outcomes. This is the first model that correlates lovastatin biosynthesis to carbon-nitrogen proportion and possesses a structure suitable for implementing a strategy for controlling lovastatin production.