Utility of culture redox potential for identifying metabolic state changes in amino acid fermentation

We investigated the relationship of dissolved oxygen and culture redox potential (CRP) on amino acid production. Corynebacterium glutamicum ATCC 14296 was used for all experiments. The fermentation can be divided into a growth phase and a production phase. Our results indicate that in order to get higher amino acid production, a lower oxygen supply during the exponential phase is favored. A higher oxygen supply rate appears to be necessary during the production phase. Culture redox potential (CRP) was used to monitor the fermentation. CRP readings were observed to drop to a characteristic minimum value as the metabolic state changed from a growth to production phase. This was evidenced by the commencement of amino acid production and a simultaneous uptake of lactate. Upon lactate exhaustion, the CRP increased abruptly. At the same time, maximal amino acid yields were observed. By the use of minimum CRP as an indication of metabolic phase changes, the agitation rate was changed to increase oxygen supply during the production phase. This significantly increased amino acid production. These results show that culture redox potential measurements can be used to monitor and optimize amino acid production by process manipulation.     

Proliferation of human hematopoietic bone marrow cells in simulated microgravity

Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.     

Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential

On-line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb-synthesizing E. coli strain and the iso-genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8-fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen-limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb-expressing cells responds to rapidly changing oxygen tension, which is common in large-scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.(c) John Wiley & Sons, Inc.     

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O(2)/H(2)O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555-563, 1997.     

The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth-limiting glucose concentration in media without pH control was increased from 1.0 g carbon L(-1) to 2.2 g carbon L(-1), the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30-200 muM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH-dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (Y(ATP)) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y'(ATP)) was 9.4 and compared well with the true molar growth yield coefficient (Y(Max) (ATP)), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy-limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy-limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y'(ATP) of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.     

Characterization of nerolidol biotransformation based on indirect on-line estimation of biomass concentration and physiological state in batch cultures of Aspergillus niger

Biotransformation of the sesquiterpenoid trans-nerolidol by Aspergillus niger has previously been investigated as a method for the formation of 12-hydroxy-trans-nerolidol, a precursor in the synthesis of the industrially interesting flavor alpha-sinensal. We characterized biotransformations of cis-nerolidol, trans-nerolidol, and a commercially available cis/trans-nerolidol mixture in repeated batch cultures of A. niger grown in computer-controlled bioreactors. On-line quantification of titrant addition in pH control allowed characterization of (1) maximal specific growth rate in exponential growth phases, (2) exponential induction of acid formation in postexponential phases, (3) inhibition of organic acid formation after nerolidol addition, and (4) exponential recovery from this inhibition. Addition of a (+/-)-cis/trans-nerolidol mixture during exponential or postexponential phase to cultures grown in minimal medium at high dissolved oxygen tension (above 50% air saturation), to cultures at low dissolved oxygen tension (5% air saturation), or to cultures grown in rich medium demonstrated that the physiological state before nerolidol addition had a major influence on biotransformation. The maximal molar yield of 12-hydroxy-trans-nerolidol (9%) was obtained by addition of a (+/-)-cis/trans-nerolidol mixture to the culture in the postexponential phase at high dissolved oxygen tension in minimal medium. Similar yields were obtained in rich medium, where the rate of biotransformation was doubled.     

Batch, fed-batch, and microcarrier cultures with CHO cell lines in a pressure-cycle driven miniaturized bioreactor

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage-dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed-batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 10(7) /mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed-batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale-up.     

Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation

Human embryonic stem cells (hESCs) represent an important resource for novel cell-based regenerative medical therapies. hESCs are known to differentiate into mature cells of defined lineages through the formation of embryoid bodies (EBs) which are amenable to suspension culture for several weeks. However, EBs derived from hESCs in standard static cultures are typically non-homogeneous, leading to inefficient cellular development. Here, we systematically compare the formation, growth, and differentiation capabilities of hESC-derived EBs in stirred and static suspension cultures. A 15-fold expansion in total number of EB-derived cells cultured for 21 days in a stirred flask was observed, compared to a fourfold expansion in static (non-stirred) cultures. Additionally, stirred vessel mediated cultures have a more homogeneous EB morphology and size. Importantly, the EBs cultivated in spinner flasks retained comparable ability to produce hematopoietic progenitor cells as those grown in static culture. These results demonstrate the decoupling between EB cultivation method and EB-derived cells' ability to form hematopoietic progenitors, and will allow for improved production of scalable quantities of hematopoietic cells or other differentiated cell lineages from hESCs in a controlled environment.     

On-line assessment of metabolic activities based on culture redox potential and dissolved oxygen profiles during aerobic fermentation.

We report here on the utility of on-line culture redox potential and dissolved oxygen measurements to identify metabolic changes in fermentation by Corynebacterium glutamicum under aerobic conditions. Metabolic changes were identified by observing discrepancies in the profile of culture redox potential and dissolved oxygen. On the basis of these measurements, we can identify the end of the lag phase, threonine exhaustion, and glucose exhaustion during fermentation.     

Stroma-dependent maintenance of cytokine responsive hematopoietic progenitor cells derived from long-term bone marrow culture

Hematopoietic cells maintained for long periods on primary cultures of bone marrow stromal cells formed cobblestone colonies (Dexter's long-term bone marrow culture, LTBC). These stably maintained hematopoietic cells (for 4 months) were transferred to a coculture on an established spleen stromal cell line (MSS62), and maintained under stromal cell layer, where they retained their invasive ability in the restricted space between the stromal cell layer and culture substratum (DFC culture). DFC contained lineage-negative (Lin-), c-Kit+, Sca-1- cells and spontaneously produced Mac-1+, Gr-1+ cells. DFC could not grow in the absence of MSS62 stromal cells, although, GM-CSF, IL-3, or IL-7 stimulated its growth. Production of granulocyte and monocytic cells was maintained by GM-CSF or IL-3 while it was decreased by IL-7. RT-PCR analysis showed that the IL-7 responsive cell population expressed early lymphoid markers (Ikaros, Pax-5, Oct-2, Rag-1, TdT, IL-7R and Imu), while lacking expression of receptors for G-CSF (G-CSFR) and for M-CSF (M-CSFR), or myeloperoxidase (MPO). These results suggested that DFC simultaneously contained lymphoid-committed progenitors and myeloid-committed progenitors, and that cytokines may expand their responding progenitor cells under the influence of signals provided by the stromal cells. Such a stromal cell-dependent culture system may be useful to analyze the switching mechanism from constitutive to inducible hematopoiesis in vitro.     

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.     

Ex vivo differentiation of umbilical cord blood progenitor cells in the presence of placental conditioned medium

Hematopoetic stem cells (HSC) are the progenitors for the lympho-hematopoietic system, with long lifespan and high proliferation potential. Transplantation of HSC from bone marrow or peripheral blood represents a standard therapy in severe hematological conditions. A possible alternative source of HSC is the umbilical cord blood, prepared by various separation procedures followed by expansion in cultures supplemented with hematopoietic growth factors. In order to check the effects of placental conditioned medium (PCM) from placental cells culture upon viability of HSC, we added plasma, PCM, dimetil sulfoxyde or hemin in HSC cultures. Flow cytometry or direct scoring of solid cultures using CD45+, CD34+, CD71+ and CD14+ fluorescent-labeled monoclonal antibodies evaluated the effects upon cell proliferation and colony forming ability of HSC cultures, versus controls. PCM produced the highest proliferation, followed by plasma, DMSO and hemin. PCM improved the survival time and maintained a higher proportion of immature cells. PCM stimulates the differentiation towards myeloid lineage progenitor cells (>90% being CD45+), increasing the percentage of CD14+, granulocites /monocytes precursors. It is highly suggestive that PCM contains growth factors or cytokines, which regulate the development of HSC. Characterization of these factors is in progress.     

Improved methods for investigating the external redox potential in hybridoma cell culture

Because of the interest in understanding and optimizing secretion of proteins from mammalian cells, reliable and more reproducible methods are needed to monitor the external redox potential of animal cells in suspension culture. An improved off-line method was established that greatly reduces the typically long response time of redox electrodes in cell culture media and improves the standardization of redox probes. In addition, the dependence of medium redox potential on dissolved oxygen concentrations and pH was investigated using cell-free medium. Off-line as well as on-line redox potential measurements were then applied to spinner or bioreactor cultures of murine hybridoma cells. Serum containing or protein-free medium were used. The time dependence of the experimentally determined external redox potential was found to be affected not only by oxygen, pH, and medium composition. but to a significant extent by the rate of generation of reductants by hybridoma cells. The observed specific rate of medium reduction by generation of reductants (mV h^–1 viable cell^–1) decreased during exponential growth while cell number increased from 2×10⁵ viable cells ml^–1 to 3.5×10⁶ viable cells ml^–1. This rate, however, was essentially constant at –7.3 mV h^–1±3.7 mV h^–1 per 10¹⁰ viable cells during growth under conditions of constant dissolved oxygen tension and constant pH. Using these observations, the quantity of reductants synthesized and secreted into the medium by viable hybridoma cells was estimated to be approximately 1.3 mole h^–1 per 10¹⁰ viable hybridoma cells. The time course of specific monoclonal antibody secretion rate did not correlate with changes in the external oxidation/reduction potential in either serum containing or protein-free medium.     

Basic Aspects of Electrode Potential Change in Submerged Fermentation

The platinum electrode potentials relative to the standard half cell depended on a pH value, dissolved oxygen concentration, equilibrium constant and oxidation reduction potentials of the liquid The overall potential change in submerged fermentation gave no independent information on these individual factors A thermostatic and pH-static apparatus excluded influences of temperatures and pH values on the electrode pontentials If the determination was completed for short time duration, potentials were governed by the dissolved oxygen tension. While the oxygen concentration was maintained at a same level, redox potential changes became a dominant. This measurement of redox potential, which gave the concentration of extremely low dissolved oxygen that could not be detected by the membrane-coated oxygen electrode, was practically useful for the control of aerobic fermentation     

Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

Expansion of mesenchymal stem cells under atmospheric carbon dioxide

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system – which relies upon an elevated CO₂ environment – typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO₂ levels negatively affecting cell growth, but more importantly, the need to sparge CO₂ into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO₂ environment in static cultures. MSC expanded under atmospheric CO₂ with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self‐renewal properties, and the ability to differentiate into multiple lineages after expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1298–1306, 2013     

Chimeric cytokine receptor can transduce expansion signals in interleukin 6 receptor alpha (IL-6Ralpha)-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin(-)), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin(-)Sca-1(+)c-kit(+) progenitors and increased either mixed colony-forming unit or cobblestone area-forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin(-)Sca-1(+)c-kit(+)CD34(-) further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Ralpha-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Residual radiation injury exhibited in long-term bone marrow cultures

Residual radiation injury was demonstrated in long-term primary cultures of mouse bone marrow. Control cultures underwent three phases of hematopoietic activity as distinguished by initial establishment, steady high (plateau) production of granulocytes, and gradual decline. Irradiation with 50, 300, or 550 rads, given at the end of the initial phase, did not prevent any culture flasks from entering the plateau phase. However, actual production levels and the time they were maintained varied inversely with the radiation dose so that the accumulated postradiation cell production corresponded to an exponential dose-response relationship at any time after treatment. The accumulated cell productions were found to be similar in all groups when expressed by the number of stem cell doublings necessary to produce them. The findings cannot be explained by reproductive cell death and are consistent with the notion of a limited division capacity in hematopoietic stem cells.     

The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture

Fusobacterium nucleatum ATCC 10953 was grown in continuous culture and the atmosphere changed stepwise from nitrogen (anaerobiosis) to a mixture of air: oxygen (40:60). No significant differences in biomass were observed and the baseline low level of superoxide dismutase increased only slightly; catalase and peroxidase activities were never detected but the level of NADH oxidase increased more than three-fold when oxygen was introduced into the system. In relation to acidic end-products, the relative proportion of acetate increased while that of butyrate decreased. Due mainly, it would seem, to NADH oxidase activity, the culture maintained a low redox potential (E(h)=-274 mV) even under an atmosphere of 40% oxygen in air and dissolved oxygen was not detected. This may, in part, explain the protective role of F. nucleatum for anaerobes in both biofilm and planktonic phases of aerated, mixed cultures of oral bacteria.