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1.
ABSTRACT

In this study, we optimised the conditions for the production of micropropagules of Trichoderma harzianum EGE-K38 in static liquid culture in Modified Czapec Medium (MCM) containing 8?g/L glucose in an integrated tray bioreactor system designed by our research group. Incubation temperature, air flow rate, inoculum spore concentration, inoculation size, medium volume and the use of spores or agar plugs containing mycelia as inoculum were individually studied as one factor at a time. The maximum micropropagule count was 5.2?±?0.2?×?109?cfu/mL and dry cell weight was 17?±?2?g/L. For the subsequent drying processes, the maximum drying yield percentage ((viable micropropagule counts after drying/viable cells before drying)*100) after drying of micropropagules was 23.30% (cfu/cfu). Results obtained from our integrated tray bioreactor system showed that static liquid culture fermentation offers potential for industrial scale fungal BCAs production.  相似文献   

2.
The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two-stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 x 10(10) CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 x 10(9) CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h(-1) were used. (c) 1993 John Wiley & Sons, Inc.  相似文献   

3.
A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.  相似文献   

4.
Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.  相似文献   

5.
Production of probiotic cabbage juice by lactic acid bacteria   总被引:3,自引:0,他引:3  
Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice by lactic acid bacteria (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoculated with a 24-h-old lactic culture and incubated at 30 degrees C. Changes in pH, acidity, sugar content, and viable cell counts during fermentation under controlled conditions were monitored. L. casei, L. delbrueckii, and L. plantarum grew well on cabbage juice and reached nearly 10x10(8) CFU/mL after 48 h of fermentation at 30 degrees C. L. casei, however, produced a smaller amount of titratable acidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 degrees C, the viable cell counts of L. plantarum and L. delbrueckii were still 4.1x10(7) and 4.5x10(5) mL(-1), respectively. L. casei did not survive the low pH and high acidity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 degrees C. Fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.  相似文献   

6.
The paper describes a recombinant Schneider 2 (rS2) cell culture and protein expression in a bioreactor. S2 cells were transfected with a plasmid containing a fusion protein (human μ opioid receptor, hMOR, and green fluorescent protein, EGFP) under the control of inducible metallothionein promoter. A bioprocess in a bioreactor with 5% dissolved oxygen, 27°C and 120 rpm enabled the cell culture to attain 5.3×107 viable cells/mL at 96 h. The induction decreased the cell multiplication (2.5×107 viable cells/mL at 72 h). Glutamine and glucose and low levels of lactate were consumed. A fast recombinant protein synthesis took place and, at 6 h of induction, 2×104 receptors/cell could be detected by a functional binding assay. Fluorescence measurements showed a progressive increase of recombinant protein expression with a maximal value of 1.26×105 fluo counts/s at 24 h of induction. The data shown in this paper indicate a practical and scaleable cell culture bioprocess procedure for the preparation of recombinant proteins expressed in S2 cells.  相似文献   

7.
Cost-effective production of biopharmaceuticals on a large scale can be carried out by perfusion cultures of mammalian cells. One problem with this mode of operation for submerged free-cell cultures is the requirement for an efficient cell separation device located in the effluent stream. The present work investigates the potential for the development of a novel dielectrophoresis-based cell separator, capable of providing selective retention of viable cells in cell culture media, which are highly conductive. Predictions of the dielectrophoretic (DEP) response in culture media were first obtained through a series of DEP-levitation experiments. Subsequently, a prototype microelectrode "filter" was microfabricated and tested with C174 myeloma cell suspensions of density 1 x 10(6) cells/mL. The optimum frequency range for selective retention of viable cells was found in the range 5-15 MHz. A maximum separation efficiency of 98% was achieved at 10 MHz, with an applied peak-to-peak voltage of 30 V (maximum field strength of 10(5) V/m) and a flow rate of 30 mL/h which corresponds to a superficial velocity of 5.23 cm/h through the DEP-filter channels. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 239-250, 1997.  相似文献   

8.
The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.  相似文献   

9.
Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. (c) 1993 John Wiley & Sons, Inc.  相似文献   

10.
A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed. This process combined an integrated membrane-bioreactor-electrodialysis system. An energetic characterization of A. succiniciproducens during anaerobic cultured in a cell recycle bioreactor was done first. The very low value of Y(ATP) obtained suggests that an ATP dependent mechanism of succinate export is present in A. succiniciproducens. Under the best culture conditions, biomass concentration and succinate volumetric productivity reach values of 42 g/L and 14.8 g/L.h. These values are respectively 28 and 20 times higher compared to batch cultures done in our laboratory. To limit end-products inhibition on growth, a mono-polar electrodialysis pilot was secondly coupled to the cell recycle bioreactor. This system allowed to continuously remove succinate and acetate from the permeate and recycle an organic acids depleted solution in the reactor. The integrated membrane-bioreactor-electrodialysis process produced a five times concentrated succinate solution (83 g/L) compared to the cell recycle reactor system, at a high average succinate yield of 1.35 mol/mol and a slightly lower volumetric productivity of 10.4 g/L.h. The process combined maximal production yield to high productivity and titer and could be economically viable for the development of a biological route for succinic acid production.  相似文献   

11.
For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.  相似文献   

12.
Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions.  相似文献   

13.
TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.  相似文献   

14.
Kluyveromyces marxianus UCD (FST) 55-82 cells were immobilized in Na alginate beads and used in a packed-bed bioreactor system for the continuous production of ethanol from the extract of Jerusalem artichoke tubers. Volumetric ethanol productivities of 104 and 80 g ethanol/ L/h were obtained at 80 and 92% sugar utilization, respectively. The maximum volumetric ethanol productivity of the immobilized cell bioreactor system was found to be 15 times higher than that of an ordinary-stirred-tank (CST) bioreactor using cells of K. marxianus. The immobilized cell bioreactor system was operated continuously at a constant dilution rate of 0.66 h(-1) for 12 days resulting in only an 8% loss of the original immobilized cell activity, which corresponds to an estimated half-life of ca. 72 days. The maximum specific ethanol productivity and maximum specific sugar uptake rate of the immobilized cells were found to be 0.55 g ethanol/g/biomass/h and 1.21 g sugars/g biomass/h, respectively.  相似文献   

15.
A fed-batch cell culture process was developed that has general applicability to all evaluated Sp2/0 (n = 8) and NS0 (n = 1) antibody-producing cell lines. The two key elements of this generic process were a protein-free concentrated feed medium, and a robust, metabolically responsive feeding strategy based on the off-line measurement of glucose. The fed-batch process was shown to perform equivalently at the 15 L development scale and 750 L manufacturing scale. Compared to batch cultures, the fed-batch process yielded a 4. 3 fold increase in the average integral of viable cell concentration and a 1.7 fold increase in average specific antibody production rate, equivalent to a 7.6 fold increase in average final antibody concentration. The highest producing cell line reached a peak viable cell concentration of 1.0 x 10(7) cell mL(-1) and a final antibody concentration of 750 mg L(-1) in a 10 day process. For all lines evaluated, reducing bioreactor pH set point from 7.2 to 7.0 resulted in an additional 2.4 fold increase in average final antibody concentration. The optimized fed-batch process consistently yielded a volumetric productivity exceeding 50 mg L(-1) day(-1). This generic, high-yielding fed-batch process significantly decreased development time, and increased manufacturing efficiency, thereby facilitating the clinical evaluation of numerous recombinant antibodies.  相似文献   

16.
The Continuous fermentation of Jerusalem artichoke juice to ethanol by free cells of Kluyveromyces marxianus UCD (FST) 55-82 has been studied in a continuous-stirred-tank bioreactor at 35 degrees C and pH 4.6. A maximum yield of 90% of the theoretical was obtained at a dilution rate of 0.05 h(-1). About 95% of the sugars were utilized at dilution rates lower than 0.15 h(-1). Volumetric ethanol productivity and volumetric biomass productivity reached maximum values of 7 g ETOH/L/h and 0.6 g dry wt/L/h, respectively, at a dilution rate of 0.2 h(-1). The maintenance energy coefficient for K. marxianus culture was found to be 0.46 g sugar/g biomass/h/ Oscillatory behavior was following a change in dilution rate from a previous steady state and from batch to continuous culture. Values of specific ethanol production rate and specific sugar uptake were found to increase almost linearly with the increase of the dilution rate. The maximum specific ethanol production rate and maximum specific sugar uptake rate were found to be 2.6 g ethanol/g/ cell/h and 7.9 sugars/g cell/h, respectively. Washout occurred at a dilution rate of 0.41 h(-1).  相似文献   

17.
VERO细胞生物反应器放大培养初探   总被引:1,自引:0,他引:1  
目的:研究用生物反应器放大进行Vero细胞微载体培养,实现生物反应器之间Veto细胞放大培养.方法:5L微载体生物反应器以10g/L微载体浓度培养Vero细胞,96h时经漂洗、消化、接种于30L微载体生物反应器,实现放大后的30L微载体生物反应器细胞怏速增殖,期间对不同时期的微载体细胞进行细胞计数、细胞代谢分析和形态观察.结果:5L生物反应器细胞经过96h灌注培养,平均细胞密度达到7.81×10~6cells/mL.5L微载体细胞放大到30L微载体生物反应器,平均细胞收获率为32.3%;放大到30L生物反应器后经过144h培养,细胞密度达到9.19×10~6cells/mL;放大后的细胞代谢途径依然以葡萄糖氧化代谢乳酸为主.结论:生物反应器由5L到30L进行Veto细胞放大培养是可行的.  相似文献   

18.
Lasiodiplodan, an exopolysaccharide of the (1→6)-β-D: -glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO(3), (NH(4))(2)SO(4), urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan's anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 ± 0.05 g L(-1)), maltose (2.08 ± 0.04 g L(-1)) and yeast extract (2.46 ± 0.06 g L(-1)) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 ± 0.006 vs. 0.22 ± 0.008 g g(-1)). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 ± 0.07 g L(-1)) with a specific yield of 0.25 ± 0.57 g g(-1) and a volumetric productivity of 0.06 ± 0.001 g L(-1) h(-1). A factorial 2(2) statistical design developed to evaluate the effect of glucose concentration (20-60 g L(-1)) and impeller speed (100-200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L(-1)) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60°C in the presence of CaCl(2). Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC(50) of 100 μg lasiodiplodan mL(-1).  相似文献   

19.
Defined protein and animal component-free NS0 fed-batch culture   总被引:1,自引:0,他引:1  
A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.  相似文献   

20.
Bioreactor headspace pressurization represents an excellent means of enhancing oxygen mass transfer to a culture. This method is particularly effective in situations where stirring or vigorous aeration is difficult. Because it in itself introduces no undesirable hydrodynamic force, the proposed method is also attractive for cells susceptible to agitation and sparging. Experiments were first conducted in an ideal fermentor by sparging air into a sulfite solution free from extraneous microbial effects. An increased oxygen mass transfer rate resulting from pressurization led to a superior cell growth rate and a higher maximum cell density in both of the microbial systems studied: a bacterial (Escherichia coli) culture up to 2.72 bar and a fragile algal (Ochromonas malhamensis) culture with pressure programming. Applying pressurization increased the maximum dry cell weight from 1.47 g/L to 1.77 g/L in the E. coli culture and increased the maximum viable cell density from 4 x 10(7) cells/mL to 10(8) cells/mL in the algal culture. An additional advantage is that formation of undesirable products under oxygen limitation, e.g., acetic acid in the E. coli culture, can be suppressed. A significant (over 250%) improvement in the oxygen transfer rate can be achieved with existing fermentors with little modification as they are already designed to withstand reasonable pressure from autoclaving. This method is simple, clean, inexpensive, and easily implemented, and it can be applied alongside other existing methods of oxygen mass transfer enhancement.  相似文献   

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