Development of Chemically Defined Media Supporting High-Cell-Density Growth of Lactococci, Enterococci, and Streptococci

Lactococcus lactis IL1403 was used as an experimental strain to develop a chemically defined medium for study of the physiology and metabolic pathways of lactococci. An experimental leave-one-out technique was employed to determine the necessity of each of the 57 chemical components used in medium development. A statistical experimental design approach including three fractional factorial designs and a central composite design was used to optimize the fermentation process with 21 variables composed of 19 nutritional factors grouped from the 57 components and two environmental factors (initial pH and temperature). For L. lactis IL1403, the maximum biomass concentrations obtained with the two optimal chemically defined media developed in this study (ZMB1 and ZMB2) were generally 3.5- to 4-fold higher than the maximum biomass concentrations obtained with the previously described best synthetic media (SA) and 50% to 68% higher than the maximum biomass concentrations obtained with M17, a complex medium commonly used for lactococci. The new chemically defined media support high-cell-density growth of numerous strains of L. lactis, Enterococcus faecalis, and Streptococcus thermophilus.     

Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hygroscopicus

Six non-amino acid nitrogen compounds were examined as nitrogen source for growth of Streptomyces hygroscopicus and biosynthesis of rapamycin. Of the nitrogen sources studied, ammonium sulfate was the best with respect to formation of rapamycin, and supported cell growth comparable to the organic nitrogen sources used in the control chemically defined medium, ie, aspartate, arginine plus histidine. In the new chemically defined medium, which is buffered with 200 mM 2-(N-morpholino)ethanesulfonic acid to prevent decline of pH during fermentation, an ammonium sulfate concentration of 40 mM was optimal for biosynthesis of rapamycin. Rapamycin production increased by more than 30% on both volumetric and specific bases as compared to the previous medium containing the three amino acids as nitrogen source. Received 08 November 1996/ Accepted in revised form 07 April 1997     

拟干酪乳杆菌发酵L-乳酸合成培养基的优化

为开发一种适合于乳酸发酵过程代谢流分析(MFA)的合成培养基,以拟干酪乳杆菌Lactobacillus paracasei为对象,考察主要营养成分对其生长和乳酸合成的影响.在合成培养基的优化过程中,先确定了影响菌体生长和乳酸合成的主要营养物质及其浓度范围,针对葡萄糖、混合氛基酸、核苷类物质、维生素等生长因子、混合磷源、CaCO3六大类营养成分,使用正交试验法先后设计了六因素五水平和四因素三水平正交试验以及氨基酸梯度试验和氨基酸缺失试验,得到了最佳培养基组成(1L):葡萄糖80g、醋酸钠2g、吐温-80 1 mL、柠檬酸氢二铵1g、金属离子0.72g、混合氨基酸3.925g、核苷类物质0.15g、维生素等生长因子0.075g、混合磷源0g、CaC03 35g.以半合成培养基为对照,考察优化后的合成培养基对菌体生长和乳酸合成的影响.结果表明,菌体在合成培养基中的乳酸产量、产率均比半合成培养基中高.这些结果为L.paracasei的代谢流定量研究莫定了基础.     

Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells

Advantages of using internally developed chemically‐defined (CD) media for cell culture‐based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in‐house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large‐scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large‐scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb‐expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1163–1171, 2015     

Increased xylitol production rate during long-term cell recycle fermentation of Candida tropicalis

Long-term cell recycle fermentations of Candida tropicalis were performed over 14 rounds of fermentation. The average xylitol concentrations, fermentation times, volumetric productivities and product yields for 14 rounds were 105 g l^–1, 333 h, 4.4 g l^–1 h^–1 and 78%, respectively, in complex medium; and 110 g l^–1, 284 h, 5.4 g l^–1 h^–1 and 81%, respectively, in a chemically defined medium. These productivities were 1.7 and 2.4 times those with batch fermentation in the complex and chemically defined media, respectively. The xylitol yield from xylose with cell recycle fermentation using the chemically defined medium was 81% (w/w), which was 7% greater than the xylitol yield with batch fermentation (74%); both modes of fermentation gave the same yield using the complex medium. These results suggest that the chemically defined medium is more suitable for production of xylitol than complex medium.     

Growth of neural cell cultures in a chemically defined,serum-free culture medium

The composition of a serum-free, completely chemically defined culture medium which supports active growth of dissociated neural-cells in culture is described. This serum-free medium can also be used to grow many types of human cell lines without modification. It is the first report which describes the development of a wholly chemically defined, synthetic culture medium for growth of neural cells.     

Growth of NS0 cells in protein-free, chemically defined medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

适合龟裂链霉菌~(13)C代谢通量分析合成培养基的优化及应用

为开发一种适合龟裂链霉菌13C代谢通量分析的合成培养基,以龟裂链霉菌模式菌株M4018为研究对象,比较其在各种有机氮源和无机氮源的生长和土霉素合成特性。首次筛选到以硝酸钾为主要氮源的合成培养基,通过响应面分析法进一步优化,将土霉素合成能力由75.2 mg/L提高到145.6 mg/L。并应用到100%的1-13C葡萄糖标记实验,首次从同位素标记代谢流分析上证实了龟裂链霉菌中不存在2-酮-3-脱氧-6-磷酸葡糖酸裂解途径(Entner-Doudoroff pathway,ED),为龟裂链霉菌13C代谢通量分析提供了重要基础。     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

Comparison of batch culture growth and fermentation of a poultry Veillonella isolate and selected Veillonella species grown in a defined medium

The objective of this study was to develop a defined medium for quantitating nutritional requirements and fermentation products of a poultry cecal isolate of Veillonella and to compare these parameters with representative Veillonella species. The poultry isolate is one of 29 organisms from a continuous-flow culture that has been shown to be effective against Salmonella colonization in broilers. When the Veillonella species were grown in anaerobic batch culture, propionate and acetate were the only volatile fatty acids detected. Lactate was needed to provide energy for the growth of the Veillonella in the defined medium. The poultry isolate had significantly (p< 0.05) higher Y(lactate)(g of dry cell weight per mole of lactate utilized) and dry cell weight than the other Veillonella species when grown on amino acid supplemented defined media. Cultures of the Veillonella species in the defined medium grown with supplemented amino acids aspartate, threonine, arginine, and serine indicated that these amino acids were metabolized to acetate and propionate. Amino acid analysis on media inoculated with either V. atypica or the poultry isolate also indicated that these organisms may have different amino acid preferences. For nearly all of the amino acid supplemented media combinations the poultry isolate utilized significantly (p< 0.05) more threonine and serine whereas V. atypica utilized significantly (p< 0.05) more aspartate. The defined medium supported growth of all of the Veillonella species tested and should enable further in-depth physiological studies to be conducted on the poultry Veillonella studies.     

Nutritional requirements ofLysobacter lactamgenus for the production of cephabacins

Summary To optimize the fermentation medium for the production of new cephem compounds, cephabacins, by an eubacteriaLysobacter lactamgenus IFO 14,288, the effects of medium components on cephabacin production were investigated. Supplementation of glucose as a sole carbon source in liquid media was the best for the antibiotic production as well as for the cell growth. Casamino acid was the best nitrogen source for antibiotic biosynthesis and cell growth among nitrogen sources tested, and this strain could utilize sulfate or thiosulfate as a sulfur source. No significant effects of growth factors (vitamins) on the antibiotic production and cell growth were observed, but ferrous, magnesium and nickel ions slightly enhanced the cephabacin production.     

Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus

A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media. Received: 4 October 1996 / Accepted: 25 October 1996     

Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus

The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.     

Protein Production in Chemically Defined Medium by Bacillus brevis No. 47

A simple chemically defined medium was devised for exoprotein production by Bacillus brevis No. 47. About 2 mg/ml of proteins was produced in the synthetic medium containing 4% glucose and 1% ammonium sulfate. An essential component of fermentation medium was Ca salt which is required by this organism for assimilating glucose.

Studies on the effects of various medium components on protein production revealed that the conditions appropriate for growth are also suitable for protein accumulation. Some compounds, especially inhibitors of cell wall synthesis and certain detergents, were found to enhance protein production.     

Leakage and delivery of liposome-encapsulated methotrexate-gamma-aspartate in a chemically defined medium

A chemically defined medium was developed to study liposome-mediated delivery of methotrexate-gamma-aspartate to cells under conditions where dilute suspensions of negatively charged liposomes to not leak extensively. The defined medium induced 14% leakage of methotrexate-gamma-aspartate from egg phosphatidylglycerol/cholesterol (67:33) liposomes diluted to 53 nM lipid. In contrast, commercially available serum replacements induced up to 91% leakage from the same liposomes. The growth inhibitory properties of non-loaded phosphatidylglycerol liposomes were greater in the chemically defined medium that they were in medium supplemented with 10% serum. Egg phosphatidylglycerol, dioleoylphosphatidylglycerol and dilaurylphosphatidylglycerol liposomes inhibited cell growth more than dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes. In 10% serum, phosphatidylglycerol liposomes with widely varying phase-transition temperatures were nearly equally effective to deliver drug to CV1-P and L929 cells, despite great differences in liposome stability. Liposome encapsulated methotrexate-gamma-aspartate was more potent when the cells were grown in the defined medium, and the increase in drug delivery was observed from phosphatidylglycerol liposomes of different phase-transition temperatures. The minimum fraction of negatively charged phospholipid required for optimal liposome-mediated drug delivery varied between cell types and among growth media. The growth inhibitory effects of liposome-encapsulated methotrexate-gamma-aspartate was also determined under conditions where the cells were exposed to drug for periods shorter than the entire growth assay. Reduction of the exposure time decreased the potency of both encapsulated and free drug in medium containing 10% serum, and decreased the potency of free drug in the defined medium. However, the potency of encapsulated drug in the defined medium was similar for all exposure lengths between 1 and 48 hours.     

Development and advantages of serum-free, chemically defined nutrient media for culturing of nerve tissue

This paper describes in a historical perspective the development of serum-free nutrient media suitable for long-term culturing of nerve tissue. Several disadvantages of the use of serum are discussed, coupled with an acknowledgement that it is not always advisable to replace a routinely used serum-supplemented medium by a chemically defined medium with the expectation of immediate success. Therefore a strategy is given on how to develop a chemically defined medium that is thoroughly tuned to the specific needs of the cell type to be cultured. It is argued that such a medium has several substantial advantages over the use of serum.     

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.