Improving culture performance and antibody production in CHO cell culture processes by reducing the Warburg effect

Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in the cell culture development to improve growth, productivity, and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl coenzyme A (acetyl‐CoA). The PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA), resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed‐batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by more than 3 days, and the final antibody titer increased by more than twofold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by approximately 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes, whereas fluxes in the tricarboxylic acid (TCA) cycle were not affected, suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis, only few proteins were identified as being differentially expressed, indicating that DCA acts on a posttranslational level. Antibody quality in terms of aggregation, charge variant, and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell‐specific productivity or antibody quality.     

Dependence on glucose limitation of the pCO2 influences on CHO cell growth, metabolism and IgG production

The culture levels of glucose and CO(2) have been reported to independently have important influences on mammalian cell processes. In this work the combined effects of glucose limitation and CO(2) partial pressure (pCO(2)) on monoclonal antibody (IgG) producing Chinese Hamster Ovary cells were investigated in a perfusion reactor operated with controlled cell specific medium feed rate, pH and osmolality. Under high glucose conditions (14.3 +/- 0.8 mM), the apparent growth rate decreased (from 0.021 to 0.009 h(-1)) as the pCO(2) increased to approximately 220 mmHg, while the cell specific IgG productivity was almost unchanged. The lactate yield from glucose was not affected by pCO(2) up to approximately 220 mmHg and glucose was mainly converted to lactate. A feed medium modification from high (33 mM) to low (6 mM) glucose resulted in <0.1 mM glucose in the culture. As a result of apparently shifting metabolism towards the conversion of pyruvate to CO(2), both the ratio of lactate to glucose and the alanine production rate were lowered (1.51-1.14 and 17.7-0.56 nmol/10(6) cells h, respectively). Interestingly, when the pCO(2) was increased to approximately 140 mmHg, limiting glucose resulted in 1.7-fold higher growth rates, compared to high glucose conditions. However, at approximately 220 mmHg pCO(2) this beneficial effect of glucose limitation on these CHO cells was lost as the growth rate dropped dramatically to 0.008 h(-1) and the IgG productivity was lowered by 15% (P < 0.01) relative to the high glucose condition. The IgG galactosylation increased under glucose- limited compared to high-glucose conditions.     

Enhancement of erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate addition

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.     

Modeling kinetics of a large‐scale fed‐batch CHO cell culture by Markov chain Monte Carlo method

Markov chain Monte Carlo (MCMC) method was applied to model kinetics of a fed‐batch Chinese hamster ovary cell culture process in 5,000‐L bioreactors. The kinetic model consists of six differential equations, which describe dynamics of viable cell density and concentrations of glucose, glutamine, ammonia, lactate, and the antibody fusion protein B1 (B1). The kinetic model has 18 parameters, six of which were calculated from the cell culture data, whereas the other 12 were estimated from a training data set that comprised of seven cell culture runs using a MCMC method. The model was confirmed in two validation data sets that represented a perturbation of the cell culture condition. The agreement between the predicted and measured values of both validation data sets may indicate high reliability of the model estimates. The kinetic model uniquely incorporated the ammonia removal and the exponential function of B1 protein concentration. The model indicated that ammonia and lactate play critical roles in cell growth and that low concentrations of glucose (0.17 mM) and glutamine (0.09 mM) in the cell culture medium may help reduce ammonia and lactate production. The model demonstrated that 83% of the glucose consumed was used for cell maintenance during the late phase of the cell cultures, whereas the maintenance coefficient for glutamine was negligible. Finally, the kinetic model suggests that it is critical for B1 production to sustain a high number of viable cells. The MCMC methodology may be a useful tool for modeling kinetics of a fed‐batch mammalian cell culture process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

In-situ removal of ammonium and lactate through electrical means for hybridoma cultures

Ammonium and lactate are two known toxic products detrimental to mammalian cell growth and productivity. An electrokinetic technique, utilizing an electrophoretic mechanism, was developed to remove these cellular wastes in-situ from suspension hybridoma (ATCC CRL-1606) cultures to enhance cell growth and productivity. This technique applies continuously a dc electric field to selectively remove the electrically charged wastes. The experiments were shown to be successful in the removal of externally added 10 rnM ammonium and 45 mM lactate while maintaining the chemostatic condition of culture medium in a cell-free condition under an electric current density of 50 A/m(2). Toxic levels of ammonium were added, ranging from 7.5 to 12.5 mM, at the start of the hybridoma culture, and the applied dc electric fields were able to completely remove these added materials. This in turn released the inhibition and restored the cell growth. Finally, this electrokinetic technique was applied to the batch and glutamine fed-batch hybridoma cultures. At an applied electric current density of 50 A/m(2), this was able to completely remove cell-produced ammonium and increased the cell growth and antibody titer by 30% to 50%, respectively, compared to the control experiment in the absence of the electric field. Lastly, the applied electric current density of 50 A/m(2) did not affect cellular functionalities such as glucose and glutamine consumption and antibody productivity.(c) 1995 John Wiley & Sons, Inc.     

高渗条件下利用蔗糖提升2-酮基-L-古龙酸生产效率

旨在进一步提升维生素C前体2-酮基-L-古龙酸(2-KLG)的生产效率。在详细考察了2-KLG工业化生产过程中渗透压变化规律的基础上,研究了高渗对混合菌系细胞生长和2-KLG合成的影响,提出蔗糖促进伴生菌巨大芽胞杆菌Bacillus megaterium生长,进而促进普通生酮古龙酸菌Ketogulonigenium vulgare生长和产酸的策略。结果表明,2-KLG的积累和碱性物质的流加使渗透压上升了832mOsmol/kg;高渗抑制了巨大芽胞杆菌的生长(15.4%),从而抑制普通生酮古龙酸菌(31.7%)的生长,导致2-KLG产量和生产强度分别下降67.5%和69.3%(以1250mOsmol/kg为例);蔗糖的添加则显著促进巨大芽胞杆菌的生长,使高渗条件下(摇瓶,1250 mOsmol/kg)2-KLG产量(40.6g/L)提高87%;在3L发酵罐中,补加10mmol/L蔗糖使2-KLG发酵周期缩短10.8%,2-KLG生产强度提高10.4%。研究成果为在环境胁迫下提高混菌生产目标代谢产物的产量提供了潜在的策略。     

Reassessing culture media and critical metabolites that affect adenovirus production

Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell‐specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell‐specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 × 10⁶ cells/mL. In comparison, only 50% of reduction in the cell‐specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 × 10⁶ cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 × 10⁶ cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells

The aim of the present work was to study the effect of ammonia and lactate on growth, metabolism, and productivity of BHK cells producing a recombinant fusion protein. Results show that cell growth was reduced with the increase in ammonia or lactate: k(1/2) of 1.1 mM and 3.5 mM for stirred and stationary cultures, respectively, for ammonia and of 28 mM for both stationary and stirred cultures for lactate, were obtained. The cell-specific consumption rates of both glucose (q(Glc)) and glutamine (q(Gln)) increased, whereas that of oxygen (q(O2)) decreased, with the increase in ammonia or lactate concentrations. The cell-specific production rates of lactate (q(Lac)) increased with an increase in ammonia concentration; similarly for the cell-specific production rates of ammonia (q(Amm)), which also increased with an increase in lactate concentration; on the other hand, both q(Lac) and q(Amm) markedly decreased when lactate or ammonia concentrations were increased, respectively; lactate was consumed at lactate concentrations above 30 mM and ammonia was consumed at ammonia concentrations above 5 mM. In vivo (31)P NMR experiments showed that ammonia and lactate affect the intracellular pH, leading to intracellular acidification, and decrease the content in phosphomonoesters, whereas the cell energy state was maintained. The effect of lactate on cell growth and q(Gln) is partially due to osmolarity, on q(Glc) and q(Amm) is entirely due to osmolarity, but on q(Lac) is mainly due to lactate effect per se. An increase in ammonia from 0 to 20 mM induced a 50% reduction in specific productivity, whereas an increase in lactate from 0 to 60 mM induced a 40% decrease.     

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.     

The gel microdrop secretion assay: Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells

The long-term stability of high-level expression is the mostimportant factor to consider when choosing cell lines for the expression of recombinant proteins. Declining volumetricyields in large-scale fermentation can be caused by changes affecting the cell population as a whole such as loss in viability, depletion of nutrients or accumulation of metabolites affecting cell growth. Alternatively, geneticinstability may lead to the outgrowth of a less productive,metabolically favored sub-population. Currently a variety ofparameters are measured to monitor the condition of cells infermenters including glucose uptake, lactate accumulation andoxygen consumption; in addition, periodic viable cell countsallow the determination of the growth rate and viability of the population. All of these methods measure the condition ofthe cell population as a whole and changes must involve a significantly large proportion of the total culture in orderto be detectable. Here we report on a method that allows theevaluation of the productivity of individual cells. Using the gel microdrop secretion assay, we detected the appearance ofa sub-population of cells with lower productivity. Subsequentanalysis of the culture confirmed the existence of lower productivity cells with a lower vector copy number. Therefore,the single cell secretion assay proved to be a rapid method todetect and isolate a low productivity variant of the producer cell line.     

Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

Tuning metabolic efficiency for increased product yield in high titer fed-batch Chinese hamster ovary cell culture

High demand in manufactured biologics drives the continued need for increased productivity. In this study elevated lactate metabolization resulted in improved metabolic efficiency and cellular productivity for a readily intensified high titer fed-batch process. Scheduled base or lactate feeds during the stationary growth phase led to increased titers (+9% and +8% respectively) without impacting the overall growth performance. The higher lactate consumption induced by either feed strategy substituted for glutamate catabolism and consequently reduced ammonia build-up. Direct correlation between increased titers and reduced ammonia levels was shown. Product quality attributes were impacted by both feeding strategies but could be matched with the control process by shortening the cell culture duration while maintaining titer constant.     

Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.     

High-end pH-controlled delivery of glucose effectively suppresses lactate accumulation in CHO fed-batch cultures

A simple method for control of lactate accumulation in suspension cultures of Chinese hamster ovary (CHO) cells based on the culture's pH was developed. When glucose levels in culture reach a low level (generally below 1 mM) cells begin to take up lactic acid from the culture medium resulting in a rise in pH. A nutrient feeding method has been optimized which delivers a concentrated glucose solution triggered by rising pH. We have shown that this high-end pH-controlled delivery of glucose can dramatically reduce or eliminate the accumulation of lactate during the growth phase of a fed-batch CHO cell culture at both bench scale and large scale (2,500 L). This method has proven applicable to the majority of CHO cell lines producing monoclonal antibodies and other therapeutic proteins. Using this technology to enhance a 12-day fed-batch process that already incorporated very high initial cell densities and highly concentrated medium and feeds resulted in an approximate doubling of the final titers for eight cell lines. The increase in titer was due to additional cell growth and higher cell specific productivity.     

Hyperosmotic pressure on HEK 293 cells during the growth phase, but not the production phase, improves adenovirus production

Hyperosmotic stress has been widely explored as a means of improving specific antibody productivity in mammalian cell cultures. In contrast, a decrease in cell-specific productivity of adenovirus production has been reported in several studies in which virus production in HEK 293 cell cultures was conducted under hyperosmotic conditions. However, production of viral vectors and, in particular, adenoviral vectors is the result of two consecutive phases: the growth phase and the virus production phase. In this study, the singular and combined effects of osmolality on the phases of cell growth and virus production were evaluated in culture media with osmolalities ranging from 250 to 410 mOsm. A two-factor, five-level full factorial design was used to investigate the effect of osmotic stress on cell physiology, as determined through the characterization of cell growth, cell metabolism, cell viability, cell cycle, cell RNA and total protein content, and total virus yield/cell-specific virus productivity. Overall, the results show that the growth of cells under hyperosmotic conditions induced favorable physiological states for viral production, and the specific virus productivity was improved by more than 11-fold when the medium's osmolality was increased from 250 to 410 mOsm during the cell growth phase. Both hypo- and hyperosmotic stresses in the virus production phase reduced virus productivity by as much as a factor of six. Optimal virus productivity was achieved by growing cells in media with an osmolality of 370 mOsm or greater, followed by a virus production phase at an osmolality of 290 mOsm. Compared to standard culture and production conditions in isotonic media, the shift from high to low osmolality between the two phases resulted in a two- to three-fold increase in virus yields. This hyperosmotic pressure effect on virus productivity was reproduced in five different commercial serum-free media.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Considerations on the lactate consumption by CHO cells in the presence of galactose

A CHO cell line producing t-PA was cultured using glutamate and glucose or galactose to decrease the formation of metabolic end-products and therefore improving the process. In batch cultures using glutamate (6 mM) with glucose at two different levels (5 and 20 mM) or with glucose and galactose (5 and 20 mM, respectively) a remarkable difference in cell culture parameters was evidenced. For 20 mM glucose, a usual cell pattern was observed with lactate built-up in the medium. For 5 mM glucose, cell growth was arrested due to glucose depletion and only a limited use of the excreted lactate could be observed, not supporting cell growth sufficiently. However, when glucose 5 mM and galactose 20 mM were used together, cells consumed the glucose first and, interestingly, in a second phase they continued growing on galactose with the simultaneous consumption of the endogenous lactate. Under these conditions, cell growth was even improved with respect to growth on 20 mM glucose, used as a control. This metabolic behavior is further investigated by using metabolic flux analysis, suggesting that the lactate produced is not used in the oxidative metabolism through the TCA cycle. Metabolic fate of the lactate consumed is discussed.     

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in <Emphasis Type="Italic">Stigmatella</Emphasis> WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC₅₀ values were 9.94, 7.33, 11.34 and 11.66 μg ml⁻¹ respectively. The IC₅₀ value was above 700 μg ml⁻¹ on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.