Enhanced anthocyanin production by repeated-batch culture of strawberry cells with medium shift

Repeated-batch cultures of strawberry cells (Fragaria ananassa cv. Shikinari) subjected to four medium-shift procedures (constant LS medium, constant B5 medium, alternation between LS and B5 starting from LS and alternation between LS and B5 starting from B5) were investigated for the enhanced anthocyanin productivity. To determine the optimum period for repeated batch cultures, two medium-shift periods of 9 and 14 days were studied, which represent the end of the exponential growth phase and the stationary phase. By comparison with the corresponding batch cultures, higher anthocyanin productivity was achieved for all the repeated-batch cultures at a 9-day medium-shift period. The average anthocyanin productivity was enhanced 1.7-and 1.76-fold by repeated-batch cultures in constant LS and constant B5 medium at a 9-day shift period for 45 days, respectively. No further improvement was observed when the medium was alternated between LS (the growth medium) and B5 (the production medium). Anthocyanin production was unstable at a 14-day shift period regardless of the medium-shift procedures. The results show that it is feasible to improve anthocyanin production by a repeated-batch culture of strawberry cells.

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.

A simple and low-cost airlift photobioreactor for microalgal mass culture

A simple, low-cost, and efficient airlift photobioreactor for microalgal mass culture was designed and developed. The reactor was made of Plexiglas, and composed of three major parts: outer tube, draft tube and air duct. The fluid-dynamic characteristics of the airlift reactor were studied. The system proved to be well suited to the mass cultivation of a marine microalga, Chlorella sp. In batch culture, the biomass volumetric output rate of 0.21 g l(-1) d(-1) was obtained at the superficial gas velocity of 4 mm s(-1) in the draft tube.

Effects of gibberellin A(3) on growth and microcystin production in Microcystis aeruginosa (cyanophyta)

Environmental. factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A(3) (GA(3)) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA3 on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001-25mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA3 treatment. GA3 increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA3 was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-Linked immunosorbent assay (ELISA) increased in GA3 treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA3 increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin. (C) 2007 Elsevier GmbH. All rights reserved.

Calibration of alkenone unsaturation index with growth temperature for a lacustrine species, Chrysotila lamellosa (Haptophyceae)

Although long chain alkenones (LCKs) occur widely in lacustrine sediments, their origin is not clear. Here, we report a lacustrine source, the non-calcifying species Chrysotila lamellosa Anand (Haptophyceae), collected and isolated from an inland saline water body, Lake Xiarinur (Inner Mongolia, China). Its alketione pattern is similar to those of coastal marine strains of C lamellosa,but the relationship between U-37(K') index and culture temperature for the lacustrine species is quite different from that of the coastal species. A significant feature of the alkenones in this strain of C lamellosa is a lack of C-38 methyl alkenones, which might be used to distinguish the species from the marine haptophyte species Emiliania huxleyi and Gephyrocapsa oceanica. The higher C-38 tetraunsaturated compound abundance might be another important feature for distinguishing the C lamellosa alkenone producer from the coastal species Isochrysis galbana. This alkenone distribution pattern has been detected in many lakes, which suggests that C lamellosa or a closely related species might be a very common alkenone precursor in lacustrine systems. We examined U-37(K') and U-37(K) values for C lamellosa as a function of culture temperature in a batch culture experiment. The calibration for U-37(K') vs. culture temperature (T) was U-37(K') = 0.0011 x T-2 - 0.0157 x T + 0.1057(n = 14, r(2) = 0.99) from 10 degrees C to 22 degrees C or U-37(K') = 0.0257 x T - 0.2608(n = 9, r(2) = 0.97) from 14 degrees C to 22 degrees C. U-37(K) vs. culture temperature was U-37(K) = 0 0377 x T - 0.5992(n = 14, r(2) = 0.98) from 10 degrees C to 22 degrees C. Our experiments show that the alkenone unsaturation index (U-37(K')) is strongly controlled by culture temperature and can be used for palaeoclimate reconstruction. (C) 2007 Elsevier Ltd. All rights reserved.

厌氧产氢菌的分离筛选、诱变选育及其产氢特性的研究

本文从四川绵竹酒厂、成都市龙泉长安垃圾填埋场以及四川大学荷花池底的厌氧污泥中先后分离得到63株厌氧产氢菌，其中H-8、H-61、HC-10等16株产氢细菌产氢能力较高，HC-10的产氢能力最高，最大产氢量和最大产氢速率分别达到2840 ml H2/L培养基和25.39 mmol H2/g drycell·h，对HC-10进行生理生化鉴定和分子生物学鉴定，判定其为clostridium sp.，对HC-10的产氢条件进行了研究，结果表明，该菌的最适生长温度为35 ℃，最适生长初始pH为7，以葡萄糖为最佳碳源，以蛋白胨为最佳氮源，不利用无机氮源，其产氢发酵液相产物以乙醇和乙酸为主，其发酵类型属于乙醇型发酵。此外，以酒糟废液作为底物，进行了菌株HC-10的生物强化试验，研究表明，投加了HC-10的强化系统其产氢量比对照高出40.32%。 同时为了获得厌氧产氢菌的高效突变株，分别以产氢菌H-8和H-61为原始菌株进行微波诱变处理，对微波诱变参数进行了优化，考察了突变株的遗传稳定性、产氢特性及耐酸性。菌株H-8经过微波诱变得到5株高产氢突变株HW7、HW33、HW181、HW184、HW195，经多次传代表明HW195是稳定的高产突变株。突变株HW195具有较好的耐酸性，在pH值为2.8时仍能生长。通过间歇发酵实验，其最大产氢量和最大产氢速率分别达到2460 mL/L培养基和27.97 mmol H2/g drycell·h，比原始菌分别提高了50.75%和41.7%。菌株H-61经过微波诱变后选育得到的突变株HW-18，其最大产氢量和最大产氢速率分别达到2190 mL/L培养基和25.86 mmol H2/g drycell·h，比原始菌分别提高了23.03%和31.00%。 为了对比各种诱变方式对产氢菌产氢能力的影响，以厌氧产氢菌H-61为原始菌株，先后经亚硝基胍(NTG)、紫外(UV)诱变，选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下，其产氢量为3024 mL/L培养基，比原始菌株提高了69.89%；其最大产氢速率为33.19 mmol H2/g drycell·h，比原始菌株提高了68.14%。经过多次传代实验，稳定性良好。其发酵末端产物以乙醇和乙酸为主，属于典型乙醇型发酵。其最适产氢初始pH为6.5，最适生长温度为36 ℃，以蔗糖为最佳碳源。与原始菌株相比，突变株HCM-23的产氢特性发生了改变，如生长延滞期延长，可利用无机氮源等。 From anaerobic activated sludge, 16 strains of hydrogen producing bacteria were newly isolated. One of them named as HC-10 had the highest hydrogen producing capability, under the batch fermentative hydrogen production condition, the maximal hydrogen yield and hydrogen production rate was 2840 mL/L culture and 25.39 mmol H2/g drycell·h. It was identified as clostridium sp.HC-10 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen producing of strain HC-10 were achieved as: initial pH 7.0, temperature 35 ℃, glucose as the favorite substrate, Moreover, using distiller's solubles wastewater as substrate, HC-10 strain was added in the biohydrogen producing system to research the bioaugmentation effection. The results showed that the hydrogen production of bioaugmentation system was 40.32% higher than the noaugmentation system. An anaerobic, hydrogen producing strain H-8 was irradiated by microwave to optimize the microwave mutagenesis condition, and to test the heredity, hydrogen-producing potential and aciduric of the mutants. An aciduric mutant named as HW195 with steady hydrogen-producing capability was obtained, which can grow at pH 2.8. Its capability of hydrogen production was tested in the batch culture experiments. The maximum hydrogen yield and hydrogen production rate was 2460 mL/L culture and 29.97 mmol H2/g drycell·h, which was 50.7% and 41.7% higher than those of the initial strain, respectively. When used the strain H-61 as original strain, a mutant named as HW18 was obtained. The maximum hydrogen yield and hydrogen production rate was 2190 mL/L culture and 25.86 mmol H2/g drycell·h, which was 23.03% and 31.00% higher than those of the initial strain, respectively. The results demonstrated that microwave mutagenesis could be used in the field of hydrogen producing microorganism. The hydrogen producing strain H-61 was used as an original strain which was induced by NTG and UV for increasing and the hydrogen production capability. One of the highest efficient H2-producing mutants was named as HCM-23 with its stable hydrogen production capability. which was tested in the batch culture experiments. With the condition of 10 g/L glucose, its cumulative hydrogen yield and hydrogen production rate was 3024 mL/L culture and 33.19 mmol H2/g drycell·h, 69.89％and 68.14％ higher than that of the original strain, respectively. The terminal liquid product compositions showed that the mutant HCM-23 fermentation was ethanol type, while the original strain H-61 fermentation was butyric acid type. Varieties of parameters of hydrogen production fermentation were studied, including time, carbon source, nitrogen source, glucose concentration, glucose utilization, initial pH and incubation temperature had been studied, indicated the optimum condition of hydrogen production for the mutant HCM-23 as initial pH6.5, temperature 36 ℃, and the favorite substrate was sucrose. The hydrogen production characters of the mutant and the original strain were different, such as, the growth lag phase and the utilization of inorganic nitrogen source, etc. This work shows a good application potential of NTG-UV combined mutation in the biohydrogen production. And the hydrogen production mechanism and metabolic pathway should be explored furthermore.

影响沼气甲基产孢弧菌生长及其甲烷单加氧酶活性的若干因素研究

本文叙述了影响甲烷氧化细菌沼气甲基产孢弧菌81Z菌株生长和甲烷单加氧酶(MMO)活性的若干因素。沼气甲基产孢弧菌81Z菌株细胞生长被高浓度PO43-(>8mM),NH4+([NH4cl]>500mg/l)抑制；[CuSO4·5H2O]在0～4mg/l范围内。生长随[Cu2+]升高而加强，低[Cu2+](0.1mg/l CuSO4·5H2O)培养基中，添加Cocl2·6H2O(0.238mg/l)；促进菌体细胞生长。发酵罐分批培养过程中，生长延迟期过后，沼气甲基产孢弧菌81Z菌株细胞MMO比活很快达到最高，并稳定至对数生长中后期，随即急剧下降至初始水平。发现沼气甲基产孢弧菌81Z细胞中存在一种MMO活性，它不同于已报道过的两种MMO,MMOL最适PH6.2～6.4，4℃相对稳定，其产生不受培养基中[Cu2+]调控能与甲醇-甲醇脱氢酶系统相偶联，在无细胞抽提液中其活性被400μM[Cu2+]抑制。在低[Cu2+]发酵罐培养条件下，沼气甲基产孢弧菌81Z菌株产生可溶性MMC，其最适PH7.0，4℃不稳定，可被DE-52分离为三组分：A、B、C。为了获得沼气甲基产孢弧菌81Z细胞MMO的最佳催化活性，①采用高[Cu2+]培养基进行发酵罐培养，收集对数生长中期的细胞；②选择反应缓冲液PH6.3；③反应体系中添加5mM甲醇或甲酸是有效的方法。在本研究所采取过的最佳条件下，测得MMO活性为15.9nmol/min·mg干细胞，是以前报道的该菌株活性0.97nmol/min·mg干细胞的十六倍。Some factors which influence growth and MMO activity of Methylosporovibrio methanica 81Z were described. The growth of Methylosporovibrio methanica 81Z is inhibited by high concentration of PO43-(8mM)or NH4+(500mg/lNH4cl). The growth of Methylosporovibrio methanica 81Z increased with rising of copper concentration up to 4mg/l CuSO4·5H2O. At low copper concentration(0.1mg/lCuSO4·5H2O),adding Cocl2·6H2O(0.238mg/l)could enhance the growth of Methylosporovibrio methanica 81Z.With batch culture of Methylosporovibrio methanica 81Z in a fermentor, after lag phase, the activity of MMO reached the highest level rapidly and steady until later log phase, then falled to initial level.MMOL activity differenct from that of two types of MMO reported before was found from Methylosporovibrio methanica 81Z with optimum PH value from 6.2 to 6.4 and relative stabilty at 4℃. Synthsis of the MMOL was not regulated by copper concentaration in medium. Its activity could couple with methane-l-methanoldehydrogenase system, and in cell-free extract, were inhibited by 400μm copper ion. At low copper concentration(0.1mg/lCuSO4·5H2O) and in a fermentor, Methylosporovibrio methanica 81Z could syntheis soluble MMO similar to solble MMO reported before by Palton and Patel. Its optimum PH value was 7.0. It was unstable at 4℃. It could be resoluted into three components: A, B, and C. It was effentive for obtaining the maxtmum MMO with Methylosporovibrio methanica 81Z that (1) to keep high copper concentration(4mg/lCuSO4·5H2O) in a fermentor and harvest cell at middlel lag phase;(2) to choose 6.3 as the PH value of reaction buffer;(3)and to add 5mM methanol or formate into reaction system. In this dy, the MMO activity of cells of Methylosporovibrio methanica 81Z was reached 15.9 nmol/min.mg, dry weight, sixteen times as high as the value(0.97nmol/min.mg, dry weight) reported with the same strain.

Microprojectile bombardment of Laminaria japonica gametophytes and rapid propagation of transgenic lines within a bubble-column bioreactor

A human acidic fibroblast growth factor gene, hafgf, was successfully transferred into Laminaria japonica (kelp) gametophytes via microprojectile bombardment using the biolistic PDS-1000/He gene gun. Following phosphinothricin screening, PCR detection and Southern blot analysis, transgenic L. japonica gametophytes were cultivated in an illuminated bubble-column bioreactor to optimize growth conditions. A maximal final dry cell density of 1,695 mg l(-1) was obtained in a batch culture having an initial dry cell density of 129.75 mg l(-1). This was achieved using an aeration rate of 1.08 l air min(-1) l(-1) culture in a medium containing 1.5 mM inorganic nitrate and 0.15 mM phosphate. In addition, the relationship between different nitrogen sources and growth of transgenic gametophytes indicated that both urea and sodium nitrate were effective nitrogen sources for cell growth, while ammonium ions inhibited growth of these gametophytes.

Pilot-scale fermentation and purification of the recombinant allophycocyanin over-expressed in Escherichia coli

A recombinant allophycocyanin (rAPC), used for treatment of tumors, has been expressed in E. coli which was grown in glucose fed-batch culture in a 30 l fermentor. Recombinant allophycocyanin was purified from soluble E. coli cell lysate using hydrophobic interaction chromatography followed by chromatography using amylose affinity column. The purity of product was greater than 98% and yielded an average of 5.5 g kg(-1) dry cells. Recombinant allophycocyanin significantly inhibited H-22 hepatoma (p (0.01) in mice with inhibition rates ranging from 36% to 62% with doses from 6.25 to 50 mg kg(-1) d(-1).

Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation

The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by anaerobic mixed culture fermentation was investigated using batch experiments at 37 degrees C. Seven varieties of typical individual components of OFMSW including rice, potato, lettuce, lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production potential. Experimental results showed that the boiling treated anaerobic sludge was effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is the most optimal substrate for fermentative hydrogen production compared with proteins, lipids and lignocelluloses. This conclusion was also substantiated by experimental results of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas produced from rice, potato and lettuce were 57-70%, 41-55% and 37-67%. 2008 International Association for Hydrogen Energy.

Effect of Cold Rolling Ratio and Heating Rate of Batch Annealing on Textures of Micro-Carbon Steel Sheet

在四辊冷轧试验机和Gleeble-1500试验机上进行了热轧微碳钢板的冷轧和退火试验。用D/max-RC衍射仪测量了试样的,/”层织构,并用Roe软件进行了ODF分析。研究表明,所研究的热轧微碳深冲板压下率约为75%,退火升温速度为20-40℃/h时,试样为{111}织构特征；压下率较大(80%)时,退火织构为较弱的{111}组分。无论{111}织构还是非{111}织构都是在形核阶段开始形成,在晶粒长大优先长大,受到定向形核和选择生长双重机制的作用。

Study of Growth Mechanism of Lysozyme Crystal by Batch Crystallization Method

The lysozyme crystals were made by batch crystallization method and the distribution of aggregate in solution were measured by dynamic light scattering. The results showed that the dimension of aggregate increased with the increase of the concentration of lysozyme and NaCl, lysozyme molecules aggregated gradually in solution and finally arrived at balance each other. The higher the concentrations of lysozyme and NaCl were, the faster the growth rate of (I 10) face was. The growth rates of lysozyme crystal were obtained by a Zeiss microscope, and the effective surface energy (a) of growing steps were calculated about 4.01 X 10(-8) J.cm(-2) according to the model of multiple two-dimensional nucleation mechanism.

A Novel Counter Sheet-Flow Sandwich Cell Culture Device For Mammalian Cell Growth In Space

Cell culture and growth in space is crucial to understand the cellular responses under microgravity. The effects of microgravity were coupled with such environment restrictions as medium perfusion, in which the underlying mechanism has been poorly understood. In the present work, a customer-made counter sheet-flow sandwich cell culture device was developed upon a biomechanical concept from fish gill breathing. The sandwich culture unit consists of two side chambers where the medium flow is counter-directional, a central chamber where the cells are cultured, and two porous polycarbonate membranes between side and central chambers. Flow dynamics analysis revealed the symmetrical velocity profile and uniform low shear rate distribution of flowing medium inside the central culture chamber, which promotes sufficient mass transport and nutrient supply for mammalian cell growth. An on-orbit experiment performed on a recovery satellite was used to validate the availability of the device.

A Trabecular Bone Explant Model of Osteocyte–Osteoblast Co-Culture for Bone Mechanobiology

The osteocyte network is recognized as the major mechanical sensor in the bone remodeling process, and osteocyte-osteoblast communication acts as an important mediator in the coordination of bone formation and turnover. In this study, we developed a novel 3D trabecular bone explant co-culture model that allows live osteocytes situated in their native extracellular matrix environment to be interconnected with seeded osteoblasts on the bone surface. Using a low-level medium perfusion system, the viability of in situ osteocytes in bone explants was maintained for up to 4 weeks, and functional gap junction intercellular communication (GJIC) was successfully established between osteocytes and seeded primary osteoblasts. Using this novel co-culture model, the effects of dynamic deformational loading, GJIC, and prostaglandin E-2 (PGE(2)) release on functional bone adaptation were further investigated. The results showed that dynamical deformational loading can significantly increase the PGE(2) release by bone cells, bone formation, and the apparent elastic modulus of bone explants. However, the inhibition of gap junctions or the PGE(2) pathway dramatically attenuated the effects of mechanical loading. This 3D trabecular bone explant co-culture model has great potential to fill in the critical gap in knowledge regarding the role of osteocytes as a mechano-sensor and how osteocytes transmit signals to regulate osteoblasts function and skeletal integrity as reflected in its mechanical properties.