Slime of Myxococcus virescens

The slime excreted by two strains of Myxococcus virescens during growth in liquid casitone medium was studied. Strain S1H, unable to grow in dispersion, excreted slime during growth later than strain D11, which grows in dispersion. Slime was precipitated from the cell-free culture solution with ethanol and the crude precipitate fractionately dissolved using first pH 5,4 and then pH 9.0 for the remainder of the precipitate. Comparatively more material from strain S1H than from strain D11 belonged to the pH 9.0 fraction. The fractions thus obtained were dialyzed and then lyophilized. The composition of the slime preparations varied with the density of the harvested cultures. The slime fraction dissolved at low pH contained 12–18 % (w/w) Folin reactive material, 2–4% lipid and 5–30% anthrone positive material (glucose equivalents). The fraction soluble at pH 9.0 was richer in Folin positive material. About 25% of the proteolytic activity in the culture solution was recovered in the slime preparations. No DNA was detected in the slime, unless the cultures were harvested daring the phase of decline. The high polymers of the slime were separated from material of low molecular weight and coprecipitated media constituents by gel filtration on Sepharose 2B. The relative amount of the high polymers increased during growth, although they seemed to be degraded in the culture during the phase of decline. The polymer had a molecular weight of about 20 million. In most preparations: it was Folin positive.     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION

The toxic dinoflagellate Alexandrium fundyense Balech was grown under temperature- and nutrient-limited conditions, and changes in labeling intensity on intact cells were determined for two probe types: an oligonucleotide probe targeting rRNA and a monoclonal antibody (MAb) targeting a cell surface protein. In nutrient-replete batch culture, labeling with the rRNA probe was up to 400% brighter during exponential phase than during stationary phase, whereas MAb labeling did not change significantly with growth stage at the optimal growth temperature. In cultures grown at suboptimal, low temperatures, there was a significant difference between labeling intensity in stationary versus exponential phase for both probe types, with exponential cells labeling brighter with the rRNA probe and slightly weaker with the MAb. The decrease in rRNA probe labeling with increasing culture age was likely due to lower abundance of the target nucleic acid, as extracted RNA varied in a similar manner. With the MAb and the rRNA probes, slower growing cultures at low, nonoptimal temperature labeled 35% and 50% brighter than cells growing faster at warmer temperatures. Some differences in labeling intensity per cell disappeared when the data were normalized to surface area or volume, which indicated that the number of target antigens or rRNA molecules was relatively constant per unit area or volume, respectively. Slow growth accompanying phosphorus and nitrogen limitation resulted in up to a 400% decrease in labeling intensity with the rRNA probe compared to nutrient-replete levels, whereas the MAb labeling intensity increased by a maximum of 60%. With both probes, labeling was more intense under phosphorus limitation than under nitrogen limitation, and for all conditions tested, labeling intensity was from 600% to 3600% brighter with the MAb than with the rRNA probe. Thus, it is clear that significant levels of variability in labeling intensity can be expected with both probe types because of the influence of environmental conditions and growth stage on cellular biochemistry, cell size,rRNA levels, and the number or accessibility of cell surface proteins. Of the two probes tested, the rRNA probe was the most variable, suggesting that in automated, whole-cell assays, it can be used only in a semiquantitative manner. For manual counts, the human eye will likely accommodate the labeling differences. The MAb probe was less variable, and thus should be amenable to both manual and automated counts.     

Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.     

Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 microg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 microg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r(2) = 0.83 to 0.98) and highly significant (P < 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

Effect of Culture Conditions on Ergosterol as an Indicator of Biomass in the Aquatic Hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 μg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 μg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r² = 0.83 to 0.98) and highly significant (P 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

The crystal violet nuclei staining technique leads to anomalous results in monitoring mammalian cell cultures

Nuclear counts determined by crystal violet staining from samples of stationary or microcarrier cultures of hybridomas, CHO or Vero cells were consistently and significantly higher than cell concentrations determined by the trypan blue or Coulter counter methods. This difference was attributed to the presence of a significant proportion of binucleated cells, which are assumed to be 35% of the cell population in the stationary phase of Vero cultures. The proportion of such cells during exponential growth was variable. However, continuous sub-culture of these cells induced a degree of synchrony during growth which resulted in a cyclic variation of the difference between the cell and nuclei counting techniques. This data indicates that care should be taken in interpreting cell culture profiles based solely on crystal violet nuclei staining counts.     

On-line monitoring of growth of Escherichia coli in batch cultures by bioluminescence

Bioluminescence was used to monitor growth of Escherichia coli in batch cultures on-line. Light emission of a strain engineered for constitutive bioluminescence was monitored with a simple set-up consisting of a photodiode, a photodetector amplifier and a recorder. Bioluminescence and colony forming units (CFU) of the cultures increased and decreased proportionally and were correlated during every growth phase at temperatures between 28 °C and 40 °C. Up to the late log (deceleration) phase, both light emission and CFU increased rapidly. Beyond the stationary phase these characteristics decreased very slowly at lower temperatures, while at higher ones they declined more rapidly. Towards the end of the cultivation, light emission of the cultures dropped to undetectable levels, even though CFU were recovered. This was particularly marked at lower temperatures where non-luminescent cultures retained very high CFU. This indicates that the actual metabolism of cells in a culture can be at a very low level or completely shut down, yet cells retain their capability to be culturable. The on-line technology described here has a number of potential uses in the laboratory and industry. Received: 30 September 1999 / Received revision: 29 November 1999 / Accepted: 3 December 1999     

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.     

Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.     

Regulation of phenylalanine ammonia-lyase activity in cell-suspension cultures of Petroselinum hortense. Apparent rates of enzyme synthesis and degradation.

The time courses for induced changes in the phenylalanine ammonia-lyase activity at five different stages during the growth cycle of cell-suspension cultures from parsley (Petroselinum hortense Hoffm.) were investigated. Large increases in the enzyme activity, induced either by irradiation or by dilution of a cell culture into fresh medium, were followed by an expotential decline to the initial low level. The maximum inducible level of specific enzyme activity varied within a range of about six-fold, depending on the mode of induction and the growth stage of the cell culture.     

Production of type 5 capsular polysaccharide by Staphylococcus aureus grown in a semi-synthetic medium

The concentration of the type 5 capsular polysaccharide (CP) antigen of Staphylococcus aureus can be measured directly in cultures or cell suspensions by a two-step inhibition enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies. CP was synthesized during growth on a variety of carbon substrates and its production was not affected by the nature of the carbon source. High levels of yeast extract inhibited CP formation. CP was synthesized in batch culture at the same rate during exponential growth as in the post-exponential phase. Post-exponential CP production contributed at least half the final amount of CP measured. This phenomenon was observed in different culture media, although the specific yield of polysaccharide varied from one medium to another. Post-exponential CP production was observed in the pH range 6-7, but not at pH 8. Post-exponential production was strictly dependent on oxygen availability and did not occur under anaerobic conditions.     

GROWTH CHARACTERISTICS OF PHYTOPLANKTON DETERMINED BY CELL CYCLE PROTEINS: PCNA IMMUNOSTAINING OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

By immunohistochemistry and immunofluorescence methods, we observed that the analog of proliferating cell nuclear antigen (PCNA) in Dunaliella tertiolecta Butcher (Chlorophyceae) was exclusively located in the nucleus. Among positively stained cells, PCNA abundance varied, being highest in S-phase cells, lower in others, and undetectable in early G1- or late M-phase cells. In exponentially growing and partially synchronized cultures, the percentage of PCNA-stained cells (% PCNA-stained cells) oscillated in the photocycle (12:12 h LD). It increased during the light period and reached a peak (75%) before the onset of the dark period when the culture was mainly (71%) in the S phase of the cell cycle. The DNA synthesis inhibitor, hydroxyurea, depressed PCNA abundance, whereas no effect was detected for the mitosis inhibitor colchicine. We conclude that PCNA in D. tertiolecta is associated with the S phase of the cell cycle where it is accumulated and functioning. PCNA was used to characterize the growth pattern of cultures grown in different media, temperatures, and growth stages. The time lag between the PCNA-stained phase and the M phase was very short in a continuous culture grown in reduced f/2 medium at 22°C and was considerably longer in the cultures grown in f/2 at 15°C. When an exponentially growing culture grew older, % PCNA-stained cells decreased. In a late stationary culture where there was no net growth, a small number of cells were still cycling through the PCNA-stained phase and cell division. In the continuous culture grown at 22°C, the duration of the PCNA-stained phase (T_s) was 13 h. Calculations with this T_s and % PCNA-stained cells yielded a growth rate of 0.77 d^?1, which was close to that obtained by cell counts (0.69 d^?1). Taken together, the results suggest that PCNA is a useful indicator of growth status and a promising cell cycle marker for estimation of species-specific growth rate.     

Plant cell culture monitoring using an in situ multiwavelength fluorescence probe

A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe.     

Transient kinetics of yeast growth in batch and continous culture with an inhibitory carbon and energy source

Growth and acetate metabolism by Candida utilis (ATCC 9226) is reported for both acetate- and zinc-limited cultures in defined media. Acetate concentrations were varied from suboptimal to inhibitory levels in both types of media in differential shake flask culture and in batch and continuous cultures in stirred fermentors. Transient responses of steady-state cultures to small or large additions of concentrated sodium acetate, or to shifts in dilution rate or inlet acetate concentration are compared with one another and with simple mathematical models of growth and acetate metabolism. Exponential growth was observed during unrestricted growth (differential shake flask and batch cultures) with both types of media. Addition of acetate during unrestricted growth always caused lags and for larger pulses, lower specific growth rates were observed after exponential growth resumed. Inhibition by high acetate concentrations was much greater in acetate–limited than in zinc–limited cultures. During restricted growth (steady-state, continuous cultures), high acetate concentrations again consistently caused growth lags but stimulated, inhibited, or temporarily stopped acetage uptake. Qualitative agreement between the predictions of a simple mathematical model of acetate inhibition fitted to differential shake flask data and the observed transient data was surprisingly good.     

Regulation of catalase synthesis in Salmonella typhimurium.

The specific activity of catalase in Salmonella typhimurium and other enteric bacteria decreased during the logarithmic phase of growth and increased at the onset and during the stationary phase. The increase in catalase synthesis at the end of the exponential phase in S. typhimurium cells coincided with the lowest pH value reached by the culture. Maintenance of the pH at a constant neutral value did not alter the typical pattern of synthesis in contradiction of the results previously reported (McCarthy and Hinshelwood. 1959). A sudden decrease in the pH value of an S. typhimurium culture during exponential growth by addition of HC1 did not cause an alteration in the catalase synthesis pattern. Addition of hydrogen peroxide to S. typhimurium cultures within the range 1 muM TO 2MM during the exponential growth phase stimulated catalase synthesis. The extent of catalase synthesis depended on the concentration of hydrogen peroxide; the maximum stimulation was observed at 80 muM. Increased catalase synthesis was not detected for 10 to 15 min after hydrogen peroxide addition. Hydrogen peroxide was produced by S. typhimurium cultures during the exponential and stationary growth phases. However, no direct relationship between hydrogen peroxide accumulation and synthesis of catalase was observed.     

PROTEIN AND AMINO ACID COMPOSITION OF THE OBLIGATE HALOPHILE APHANOTHECE HALOPHYTICA (CYANOPHYTA)1

Total protein was determined for cells of Aphanothece halophytica Fremy harvested during early log, mid-log and linear growth phases in media containing 1, 2, and 3 M NaCl. Cells grown in medium containing 1 M NaCl showed a progressive increase in protein content up to a maximum of 76% of dry weight (linear phase). Total protein also increased in cells grown in 2 M NaCl. medium (56.5–72.0%). Cells grown in 3 M NaCl medium showed a progressive decrease in total protein (59.9–43%). Although amounts of protein varied, the percentages of the respective amino acids of hydrolyzed bulk protein were consistent to within 1% for linear phase cells grown in 1, 2, and 3 M NaCl cultures. Percentages of acidic amino acids were 2.3–2.6 times greater than those of the basic amino acids. The amino acid composition of phycocyanin was similar to that of bulk protein. Free amino acids varied with both age of the culture and the concentration of NaCl. The high quantity and quality of the protein observed suggest that A. halophytica might be a useful food organism.     

Growth and toxin production in batch cultures of a marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea

Nutritional and environmental conditions were characterized for a batch culture of the marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea for its growth (cells ml⁻¹), cellular toxin content (Qt in fmol cell⁻¹) and toxin composition (mol%). Under a nutrient replete condition, Qt increased with cell growth and peaked at the late stationary phase. Toxin content increased with the nitrate concentration in the culture while it reached a maximum at 5 μM phosphate. When nitrate was replaced with ammonia, Qt decreased by 4.5-fold. Salinity and light intensity were important factors affecting Qt. The latter increased two-fold over the range of salinity from 15 to 30‰, while decreased 38% as light intensity increased from 80 to 220 μE m⁻² s⁻¹. Toxin composition varied with growth phase and culture conditions. In nutrient replete cultures, toxin composition varied greatly in the early growth phase (first 3 days) and then C1/C2, C3/C4 and GTX1 remained relatively constant while GTX4 increased from 32 to 46% and GTX5 decreased from 28 to 15%. In general, the composition of GTXs was affected in a much greater extent than C toxins by changes in nutrient conditions, salinity and light intensity. This is especially true with GTX4 and GTX5. These data indicate that the cellular toxin content and toxin composition of A. tamarense HK9301 are not constant, but that they vary with growth phase and culture conditions. Use of toxin composition to identify a toxigenic marine dinoflagellate is not always valid. The data also reveal that high salinity and low light intensity, together with high nitrate and low phosphate concentrations, would favor toxin production by this species.     

In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene

The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture).     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.