Independent exponential feeding of glycerol and methanol for fed-batch culture of recombinant Hansenula polymorpha DL-1

As a novel feeding strategy for aptomizing human epidermal growth factor (hEGF) production with a recombinant Hansenula polymorpha DL-1 using the methanol oxidase (MOX) promoter in H. polymorpha DL-1, independent exponential feeding of two substrates was used. A simple kinetic model considering the cell growth on two substrates was established and used to calculate the respective feeding rates of glycerol and methanol. In the fedbatch culture with methanol-only feeding, the optimal set point of specific growth rate on methanol was found to be 0.10 h⁻¹. When the fed-batch cultures were conducted by the independent feeding of glycerol and methanol, the actual specific growth rate on glycerol and methanol was slightly lower than the set point of specific growth rate. By the uncoupled feeding of glycerol and methanol the volumetric productivity of hEGF increased from 6.4 to 8.0 mg/(L·h), compared with methanol-only feeding.     

Effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans in an airlift reactor

The effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans P56 in an airlift reactor was investigated. A maximum polysaccharide concentration (18.5 g/L), biomass dry weight (14.0 g/L), polysaccharide yield (38.5%), and sugar utilization (96%) was achieved at a shear rate of 42 s⁻¹. A. pullulans grown on beet molasses produced a mixture of pullulan and other polysaccharides. The highest value of pullulan proportion (30% of total polysaccharide) was obtained at a low shear rate (42 s⁻¹). The apparent viscosity of the fermentation broth increased as the shear rate increased up to 42 s⁻¹ and then decreased. On the other hand, the dissolved oxygen concentration and the volumetric mass transfer coefficient increased with the increase of the shear rate from 21 to 84 s⁻¹. The external addition of L-glutamic acid, olive oil, and Tween-80 improved significantly the production of crude polysaccharide (27.0 g/L), but the pullulan content of the polysaccharide was low (20%).     

微流控芯片技术在生命科学研究中的应用

微流控芯片最初起源于分析化学领域,是一种采用精细加工技术,在数平方厘米的基片,制作出微通道网络结构及其它功能单元,以实现集微量样品制备、进样、反应、分离及检测于一体的快速、高效、低耗的微型分析实验装置.随着微电子及微机械制作技术的不断进步,近年来微流控芯片技术发展迅猛,并开始在化学、生命科学及医学器件等领域发挥重要作用.本文首先简单介绍了微流控芯片制作材料和工艺,然后主要阐述了其在蛋白质分离、免疫分析、DNA分析和测序、细胞培养及检测等方面的应用进展.     

Concavine, an unusual diterpenic alkaloid produced by the fungus Clitocybe concava

A novel alkaloid with an unprecedented ring system consisting of a dodecahydro-7-oxa-9a-aza-benzo[a]azulene ring (1), has been isolated from cultures of Clitocybe concava (Basidiomycetae). Its structure and relative stereochemistry were elucidated on the basis of spectroscopic data. Concavine presents a weak antibacterial activity.     

A stereotypic,transplantable liver tissue-culture system

A method of coculturing adult rat hepatic parenchymal cells (PC) and stromal cells in a three-dimensional framework of nylon filtration screens or biodegradable polymer meshes was developed in our laboratory. Rat liver stroma, which includes vascular and bile duct endothelial cells, fat-storing cells, fibroblasts, and Kupffer cells, were isolated by gradient centrifugation afterin situ liver perfusion and expanded in monolayer culture prior to seeding onto nylon screens or bioresorbable polyglycolic acid (PGA) polymers oriented into a felt-like construct. A second inoculum of freshly isolated PC was applied after the stromal cells became established. Histological analyses revealed that PC proliferation occurred until all available space for expansion within the template was exhausted. These cells retained their rounded morphology, and after 4–5 wk 7–9 “layers” of PC filled the 140-μm deep template. Dioxin-inducible cytochrome P450 activity was detected for up to 58 d in culture, and albumin, fibrinogen, transferrin, and soluble fibronectin were detected in the medium by enzyme-linked immunosorbent assay (ELISA) for 48 d in vitro. Immunohistochemical analysis of sections through the cultures confirmed the presence of these proteins as well as cytokeratin at the cellular level; the extracellular matrix stained for both collagen type III and laminin. Long-term PC proliferation and function were enhanced by the presence of stromal cells as well as by a meshwork template whose geometry allows the interaction of PC with stroma and matrix on several different planes. To permit transplantation, co-cultures of hepatic PC and stromal cells were established on PGA felt constructs instead of nylon screens. After 24 d in vitro, these constructs were grafted into sites in the mesentery, omentum, and sub-cutaneous tissues of adult Long-Evans rats. The growth of hepatocytes after 30 din situ was evident by histological analysis; grafts of co-cultures regenerated a liver-like architecture consisting of sinusoids and putative biliary structures. In addition, PC at these extrahepatic graft sites were positive for albumin, transferrin, and fibrinogen synthesis by immunohistochemistry. Graft survival was enhanced when recipients were subjected to 40% hepatectomy. Hepatic PC:stromal cell cocultures may prove useful in the restoration of liver function either by direct transplantation using PGA or similar templates, or as extracorporeal devices, using nylon screens.     

Acid Pullulanase from Bacillus polymyxa MIR-23

Within the frame of a screening program aimed at the isolation of amylolytic sporeformers, one strain with high amylolytic activity designated MIR-23 was selected. The microbial characterization was carried out by morphological and biochemical tests and, by means of statistical treatment, was identified asBacillus polymyxa. The organism could grow in acidic conditions (pH 5.0) on a starch medium and produce α-amylase, pullulanase, and α-glucosidase. Batch cultures showed the highest enzyme activities in the stationary phase. Pullulanase activity exhibited an optimal temperature of 52–57°C at pH 4.5–5.5. These properties would allow its use in the saccharification processes in the starch industries.     

Assessment of various carbon sources and nutrient feeding strategies for Panax ginseng cell culture

Ginseng (root of Panax ginseng C. A. Meyer) cells were cultivated on medium supplemented with various carbohydrates including sucrose, glucose, and fructose, at initial concentrations ranging from 10 to 110 g/L. Sucrose was shown to be the superior carbon source to the monosaccharides for ginseng cell growth and the optimal concentration was between 30 and 50 g/L. An increase in the initial concentration within this range increased the maximum cell density and growth index significantly, whereas much higher concentrations inhibited cell growth. Feeding of sucrose and some other medium components during the growth (fed-batch mode) was more effective in enhancing the cell growth and biomass productivity, increasing the growth index by more than 60–70% and biomass productivity by more than 50%.     

Methane production from synthesis gas using a mixed culture ofR. rubrum M. barkeri,and M. formicicum

The components of synthesis gas, CO, H₂, and CO₂, may be converted into CH₄ biologically through either acetate or H₂/CO₂ as intermediates. Of these two routes, conversion through H₂/CO₂ is preferred. This paper presents results of mixed-culture studies employing the photosynthetic bacteriumR. rubrum for converting CO to CO₂ and H₂ by the water gas shift reaction and two methanogens,M. formicicum andM. barkeri, for converting CO₂ and H₂ into CH₄. Results are presented for triculture operation in two types of reactors, the packed bubble column and the trickle-bed reactor.     

Effects of glucose,glutamine, and malate on the metabolism of spodoptera frugiperda clone 9 (sf9) cells

Optimal design and operation of bioreactors for insect cell culture is facilitated by functional relations providing quantitative information on cellular metabolite consumption kinetics, as well as on the specific cell growth rates (μ_G). Initial specific consumption rates of glucose, malate, and oxygen, and associated changes in μ_G, were measured forSpodoptera frugiperda clone 9 (Sf9) cells grown in batch suspension culture in medium containing 7–35 mM glucose, 0–16 mM malate, and 4–16 mM glutamine. The initial specific glucose consumption rate (q _G ) could be described by a modified Michaelis-Menten equation treating malate as a “competitive” inhibitorK ₁ = 6.5 mM) and glutamine as a “noncompetitive” inhibitorK _I = 14 mM) ofq _G , with aK _m of 7.1 mM for glucose. All three carbon sources were found to increase μ_G in a saturable manner, and a modified Monod equation was employed to describe this relationship (μ_Gmax = 0.047 h^-1). The initial specific oxygen consumption rate (q_O2) in Sf9 cells could be related to μ_G by the maintenance energy model, and it was calculated that, under typical culture conditions, about 15–20% of the cellular energy demand comes from functions not related to growth. Fitted parameters in mathematical expression for μg: K₄, Monod constant for glucose (mM); K₅, modified Monod constant for malate (mM); K₆, Monod constant for glutamine (mM); m_o2, specific consumption rate of oxygen by the cells under zero-growth conditions (nmol/cell/h); q_F, initial specific fumarate production rate (nmol/cell/ h);q _G , initial specific glucose consumption rate (nmol/cell/h); q_Gmax, maximum initial specific glucose consumption rate (nmol/cell/h);q _M , initial specific malate consumption rate (nmol/cell/h); q_o2, initial specific oxygen consumption rate (nmol/cell/h); Y_o2, cell yield on oxygen (cells/nmol); μ, initial specific cell growth rate (h^-1); μ_g, initial specific cell growth rate (h^-1); μG_max, maximum initial specific cell growth rate (h^-1).     

Optimization of ammonium nitrate concentration in single-stage continuous cultures of Aspergillus niger with biomass retention

The effect of ammonium nitrate concentration in the citric acid biosynthesis by Aspergillus niger NC-12 in single-stage continuous cultures with biomass retention was investigated. Experiments were carried out in a BIOMER laboratory fermenter with 5 dm³ working volume. At the initial stage of each cultivation, the substrate in the bioreactor contained 1.5 g NH₄NO₃ dm⁻³. After 120 h onwards, the bioreactor was fed continuously at a constant dilution rate of 0.009 h⁻¹. NH₄NO₃ concentration in the feed was varied from one culture to another, ranging between 0.5 g dm⁻³ and 2.5 g dm⁻³. Promising results were obtained when NH₄NO₃ concentration of 1.5 g dm⁻³ was used. The observed concentration of citric acid (c _P) and yield of citric acid with respect to the introduced sucrose (Y _P/S) were 117.88 g dm⁻³ and 78.59 %, respectively. The efficiency coefficient of citric acid biosynthesis (K _ef) was very high, amounting to 83.38. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.