Calorific Content of Certain Bacteria and Fungi

Calorific contents of dried cells of several representative species of bacteria (gram-negative rods and gram-positive rods and cocci), two species of yeasts, and a filamentous fungus were determined by bomb calorimetry. The grand mean was 5,383 cal per g of ash-free dry weight. This value was then used to determine quantity of energy assimilated (E(a)) during growth. Subsequently, E(a) was employed in the equation: Y(kcal) = Y/(E(a) + E(d)), where Y(kcal) is the yield of cells per kilocalorie of energy taken from a culture medium, Y is the yield per mole of substrate utilized, E(a) is Y times caloric content of the cells, and E(d) is the energy expended by oxidative dissimilation. An estimate of E(d) was obtained for a number of experiments by multiplying the moles of oxygen consumed per mole of substrate utilized during growth by the average quantity of energy utilized to reduce a mole of oxygen with electrons from organic compounds (106 kcal). From previous studies in our laboratories, a value for Y(kcal) of 0.118 g/kcal was predicted. The mean value for data from five studies of aerobic growth of prototrophic heterotrophs was found to be 0.111.     

Regulation of growth and photosynthesis by Oscillatoria agardhii grown with a light/dark cycle

Abstract The cyanobacterium Oscillatoria agardhii was grown in turbidostat cultures with the light energy supply in either the continuous mode or in the pulsed mode (8/16 h light/dark (L/D) cycle). The light irradiance value used was sufficient to allow the maximal growth rate to be attained, when supplied continuously. Adaptation of O. agardhii to the L/D cycle was characterized by an increase in pigment content and photosynthetic performance, accompanied by a decrease in growth rate. This mode of adaptation resembled the adaptation of O. agardhii to continuous low light intensities. It is suggested that in this case the L/D cycle provokes this adaptation in order to allow the cells to accumulate carbohydrate rapidly during the light period. This was attributed to the storage of polyglucose, which served as a carbon and energy source for growth in the dark. The utilization of polyglucose in the dark was able to sustain the synthesis of all other cell components at the same rate as when cells were growing in the light. The growth yield in the dark, whilst metabolizing internally stored polyglucose, was 0.52 g cell C/g polyglucose C, or 0.62 g cell dry weight/g polyglucose. Although in the pulsed mode there is a 66% loss in light irradiance per 24 h when compared with a continuous light regime, the growth rate of the cyanobacteria grown in the pulsed mode was only 35% lower than the growth rate of a culture grown in continuous light. This can be explained by a high growth yield in the dark and by increased CO₂ fixation rates in the light of cells grown in the pulsed mode.     

Analysis of the productivity of a continuous algal culture system

We describe a first-principles analysis of a system for the continuous culture of the green alga Scenedesmus obliquus under light-limiting conditions. According to this analysis, the productivity of the algal culture is given by the relation Y = E(m)I(0)AK(1 - e(-alphacl)) - GRcV, where Y = yield (g cells/h), E(m) = 0.20 (the maximum attainable photosynthetic conversion on an energy basis), A = illuminated area (m(2)), K = 0.156[(g cells/h/W), the energy equivalent of the algae], I(0) = light intensity (W/m(2)), alpha = extinction coefficient (L/cm/g),c = cell concentration (g/L), I = light path (cm), R = respiration rate (g carbon/g cells/h), V = culture volume (L), and G = ratio of g cells to g carbon (2.04). This formula is completely determined and has no free adjustable parameters. Using parameter values determined independently, the model accurately predicted the relationship of productivity to cell density in the culture system.     

Characterization of energy conversion based on metabolic flux analysis in mixotrophic liverwort cells, Marchantia polymorpha

In order to characterize the contributions of respiratory and photosynthetic actions to energy conversions, the mixotrophic cells of Marchantia polymorpha were cultivated in the medium containing 10kg/m(3) glucose as an organic carbon source. The cultures were conducted with the supply of ordinary air (0.03% CO(2)) at constant incident light intensities of 50 and 180W/m(2). From the results of metabolic analysis, it was found that the cell yield based on ATP synthesis was estimated to be 6.3x10(-3)kg-dry cells/mol-ATP in these cultures. Under the examined conditions, energy conversion efficiency through respiration was larger than that through photosynthesis, and efficiency of overall energy conversion to ATP was maximized when the sum of energies from glucose and light captured by the cells was approximately 7.2x10(5)J/(hkg-dry cells). Taking into account the efficiency of overall energy conversion, a batch culture of M. polymorpha in a bioreactor was carried out by regulating incident light intensity ranging from 9 to 58W/m(2). In the culture with light regulation, the cell yield of 6.2x10(-9)kg-dry cells/J was achieved on the basis of energy provided to the system throughout the culture, and this value was 2.3 and 9.3 times as large as those obtained in the cultures under constant incident light intensities of 50 and 180W/m(2), respectively.     

Kinetic analysis of the growth of Spirulina sp. in batch culture

Batch cultivation of Spirulina sp. was carried out under limited light at 30°C in the pH range of 9.2 to 9.7. The specific growth rate D was calculated from the tangent of the growth curve and the cell concentration at that time, and the amount of light energy absorbed per unit time per unit cell weight (E_x), namely, the specific absorption rate of light energy, was also calculated from the total amount of radiant flux of transmitted light at the surface of the culture vessel and cell concentration of the culture solution. A plot against E_x of D in the linear growth phase in batch culture and at various phases in continuous culture gave, for E_x of less than 1.0 kcal/g·h, points scattered near a straight line with slope m 0.037 g/kcal and an intercept on the ordinate, −b, of −0.0046 h⁻¹, and, for higher E_x values, points scattered near a curve of gradually decreasing slope which tended to approach a constant value.A Lineweaver-Burk plot of the reciprocal of D plus b against that of E_x yielded an equation for the growth rate which represented well the growth curve in batch culture. This equation also expressed the linear increase of D with increase of E_x at high cell concentration in the culture solution. The relation between cell growth rate and cell fluidity is discussed by use of a vector equation obtained by applying this relation to a culture solution contained in a given closed surface.     

Predicting Production in Light-Limited Continuous Cultures of Algae

Equations relating productivity, growth rate, cell concentration, and light absorption lead to the prediction that, when incident light is below saturating intensity, maximal productivity will occur at half the maximal growth rate. The freshwater alga Chlorella pyrenoidosa TX71105 and the marine alga Dunaliella tertiolecta were grown in a small continuous culture apparatus with turbidostatic control. With both cultures, the cell concentration showed a linear decrease with dilution rate. Productivity was maximal at about one-half the maximal dilution rate. Average mass per cell increased near the maximal dilution rate, causing some asymmetry in the productivity versus dilution rate curve. The chlorophyll content per unit mass decreased in this region, but the chlorophyll content per cell remained constant. Best production rate in a light-limited algal culture was obtained when the growth rate at very low cell concentration was determined in the apparatus and the dilution rate was set at one-half that value.     

Response of Rhodopseudomonas capsulata to illumination and growth rate in a light-limited continuous culture.

Rhodopseudomonas capsulata was grown under anaerobic, photosynthetic conditions in a continuous culture device. Under light limitation, at a constant dilution rate, it was shown that cell composition, including photopigment (bacteriochlorophyll and carotenoids) and ribonucleic acid content, was not affected by incident light intensity; however, steady state culture density varied directly and linearly with light intensity. On the other hand, photopigment and ribonucleic acid levels were affected by growth rate regardless of light intensity. Additional experiments indicated a high apparent Ks for growth of R. capsulata with respect to light. These results were interpreted to mean that near the maximum growth rate (D = 0.45 h-1) some internal metabolic process became the limiting factor for growth, rather than the imposed energy limitation. A mathematical expression for the relation between steady-state culture density and dilution rate was derived and was found to adequately describe the data. A strong correlation was found between continuous cultures limited either by light or by a chemical energy source.     

Galactose increases microvillus development in mouse jejunal enterocytes.

1. Mice fed low carbohydrate and galactose-containing diets have been used to determine both positional and temporal aspects of microvillus development during enterocyte migration from intestinal crypts towards the tips of jejunal villi. 2. The positional dependence of microvillus growth was found to be similar in mice fed low carbohydrate (3.0 kcal/g), galactose-containing lipid substituted (2.9 kcal/g) and galactose-containing agar substituted (5.1 kcal/g) diets. The daily calorific intake by mice fed these diets was about 10.4 kcal/mouse. The maximal microvillus length reached by enterocytes fed galactose was nearly twice that measured in mice fed the low carbohydrate diet. 3. Enterocyte migration rate in mice fed the low carbohydrate and the high calorie galactose-containing diet was twice that measured in mice fed the low calorie galactose-containing diet. These changes were not associated with any noticeable alteration in the size of intestinal crypts. 4. Changes in maximal microvillus length (M) can be predicted from the equation M = 0.0016 CD + 0.073 CD/R, where CD and R refer to crypt depth and enterocyte migration rate respectively, Smith M. W. and Brown D. (1989). Dual control over microvillus elongation during enterocyte development. Comp. Biochem. Physiol. 93A, 623-628. Substituting measured values for CD and R in this equation revealed a specific capacity of galactose to potentiate microvillus development when presented in the form of a high calorie diet. 5. The possibility that galactose, which is poorly metabolized in mice, can increase microvillus expression by interfering specifically with some aspect of carbohydrate metabolism is discussed.     

Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection

Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of Escherichia coli MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (D = 0.08 h(-1)) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar T(90) (time until 90% of the population is inactivated) values (T(90, chemostat) = 2.66 h; T(90, batch) = 2.62 h), whereas T(90) for cells growing at a mu of 0.9 h(-1) was 0.2 h. We present evidence that the stress sensitivity of E. coli is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that E. coli rpoS mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (mu = 1.0 h(-1)) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (mu = 0.3 h(-1)), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained.     

The ‘true’ growth efficiency of phytoplankton as influenced by light attenuation and insolation: implications of the photosynthesis-irradiance relationship

The true growth efficiency (c) relates the light energy absorbed by phytoplankton to the production of biomass corrected for constant energy requirement of maintenance. Continuous culture studies have shown that, at constant incident irradiance, the value of c for both prokaryotic and eukaryotic species is constant. Culture data for the relevant conditions of incident light may be used for directly estimating the growth rate from daily insolation of optically deep, fully mixed lakes, when the light absorption by the phytoplankton can be established. In order to examine the influence of vertical light attenuation and daily insolation on c, computations were made on a basis of a photosynthesis-irradiance curve of light-limited Oscillatoria limnetica. For steady state growth, the true growth efficiency is linearly related to the areal quantum efficiency of photosynthesis ( _a ). The computations showed that _a remains constant at fluctuating vertical light attenuation, no matter whether the concentration of tripton or phytoplankton changes. The effect of insolation is great: _a is 0.108 mol O₂/E at very low light, but only 0.014 mol O₂/E at 400 W m^–2 subsurface downward irradiance. The results imply that a c-value obtained from cultures for summer averaged insolation must be corrected: between cloudy and clear days the value may vary by a factor of 2. The true growth efficiency for cultures will decrease by about 10% when the same daily irradiation is dosed sinusoidally instead of constantly.     

Performance assessment of two patent strains ofZymomonas mobilis in batch and continuous fermentations

Summary The performance ofZymomonas mobilis strains ATCC 31821 and ATCC 31823 was assessed in batch and continuous culture. In batch culture using a medium containing 250 g/l glucose, identical maximum specific growth rates of 0.16/h were found, though final biomass concentration and growth yield were significantly lower for ATCC 31 823 than for ATCC 31 821. Final ethanol concentrations in this medium were about 110 g/l vor both organisms. In continuous culture at increasing dilution rates using a medium containing 100 g/l glucose, no significant differences were seen between the two strains with respect to the fermentation parameters studied. For ATCC 31 821, maximum rates of glucose uptake (Q_s) and ethanol produktion (Q_p) of 8.7 g glu/g/h and 4.4 g eth/g/h, respectively, were found. Both strains showed a similar performance at a fixed dilution rate of 0.1/h, where maximum ethanol concentrations of about 68 g/l were reached at a feed glucose concentration of about 139 g/l. At this dilution rate the maximum values of Q_s and Q_p were about 5.8 g glu/g/h and 2.8 g eth/g/h, respectively. Test tube experiments showed that growth, measured as optical density, decreased with increasing concentrations of exogenous ethanol with complete inhibition of growth at ethanol concentrations >8% (v/v). As evidenced by the results presented here, we have been unable to practice the invention as described in U.S. Patent 4,403,034 (Rogers and Tribe 1983).Nomenclature D Dilution rate, 1/h - _max maximum specific growth rate, 1/h - S_R Initial substrate concentration, g glucose/1 - S Residual substrate concentration, g glucose/1 - S₀ Effluent substrate concentration, g glucose/1 - X Blomass concentration; g cells/l - OD₆₂₀ Optical density at 620 nm, dimensionless - [P] Product concentration, g ethanol/1 - Y_x/s Growth yield, g cells/g glucose used - Y_p/s Product yield, g ethanol/g glucose used - %, Yield Percentage yield, Y_p/sx100/Y _p ^s/max =Y_p/sx100/0.51 - Q_s Specific rate of glucose uptake, g glucose/g cells/h - Q_p Specific rate of ethanol formation, g ethanol/g cells/h - m_e Maintenance energy coefficient, g glucose/g cells/h - VP Volumetric productivity, g ethanol/l/h - t Fermentation time, h     

High cell density culture of Anabaena variabilis with controlled light intensity and nutrient supply

Controlling the light energy and major nutrients is important for high cell density culture of cyanobacterial cells. The growth phase of Anabaena variabilis can be divided into an exponential growth phase and a deceleration phase. In this study, the cell growth in the deceleration phase showed a linear growth pattern. Both the period of the exponential growth phase and the average cell growth rate in the deceleration phase increased by controlling the light intensity. To control the light intensity, the specific irradiation rate was maintained above 10 micromol/s/g dry cell by increasing the incident light intensity stepwise. The final cell density increased by controlling the nutrient supply. For the control of the nutrient supply, nitrate, phosphate, and sulfate were intermittently added based on the growth yield, along with the combined control of light intensity and nutrient concentration. Under these control conditions, both final cell concentration and cell productivity increased, to 8.2 g/l and 1.9 g/l/day, respectively.     

Lasers-an effective artificial source of radiation for the cultivation of anoxygenic photosynthetic bacteria

The laser diode (LD) is a unique light source that can efficiently produce all radiant energy within the narrow wavelength range used most effectively by a photosynthetic microorganism. We have investigated the use of a single type of LD for the cultivation of the well-studied anoxygenic photosynthetic bacterium, Rhodobacter capsulatus (Rb. capsulatus). An array of vertical-cavity surface-emitting lasers (VCSELs) was driven with a current of 25 mA, and delivered radiation at 860 nm with 0.4 nm linewidth. The emitted light was found to be a suitable source of radiant energy for the cultivation of Rb. capsulatus. The dependence of growth rate on incident irradiance was quantified. Despite the unusual nearly monochromatic light source used in these experiments, no significant changes in the pigment composition and in the distribution of bacteriochlorophyll between LHII and LHI-RC were detected in bacterial cells transferred from incandescent light to laser light. We were also able to show that to achieve a given growth rate in a light-limited culture, the VCSEL required only 30% of the electricity needed by an incandescent bulb, which is of great significance for the potential use of laser-devices in biotechnological applications and photobioreactor construction.     

Application of light-emitting diodes in bioreactors: flashing light effects and energy economy in algal culture (Chlorella pyrenoidosa)

Light-emitting diodes (LEDs) were used as the sole light source in continuous culture of the green alga Chlorella pyrenoidosa. The LEDs applied show a peak emission at 659 nm with a half-power bandwidth of 30 nm. Selection of this wavelength range, which is optimal for excitation of chlorophylls a and b in their "red" absorption bands makes all photons emitted potentially suitable for photosynthesis. No need for additional supply of blue light was found. A standardized panel with 2 LEDs cm(-2) fully covered one side of the culture vessel. At standard voltage in continuous operation the light output of the diode panel appeared more than sufficient to reach maximal growth. Flash operation (5-mus pulse duration) enables potential use of higher operating voltages which may render up to three times more light output. Flat airlift fermentor-type continuous culture devices were used to estimate steady state growth rates of Chlorella pyrenoidosa as a function of the light flux (micromol photons x m(-2) x s(-1)) and the flashing frequency of the light-emitting diodes (which determines the duration of the dark "off" time between the 5-micros "on" pulses). At the fixed voltage and turbidostat setting applied a 20-kHz frequency, which equals dark periods of 45 mus, still permitted the maximum growth rate to become nearly reached. Lower frequencies fell short of sustaining the maximal growth rate. However, the light flux decrease resulting from lowering of the flash frequency appeared to reduce the observed growth rates less than in the case of a similar flux decrease with light originating from LEDs in continuous operation. Flash application also showed reduction of the quantum requirement for oxygen evolution at defined frequencies. The frequency domain of interest was between 2 and 14 kHz. LEDs may open interesting new perspectives for studies on optimization of mixing in mass algal culture via the possibility of separation of interests in the role of modulation on light energy conversion and saturation of nutrient supply. Use of flashing LEDs in indoor algal culture yielded a major gain in energy economy in comparison to luminescent light sources. (c) 1996 John Wiley & Sons, Inc.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones     

The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells

A novel perfusion culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the perfusion culture. Furthermore, the significance of the control strategy of perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel perfusion culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.     

Kinetics and metabolism of cellulose degradation at high substrate concentrations in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h(-1), biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose x liter(-1) the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose x liter(-1), respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose x liter(-1). Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119-130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD+, and q(NADH produced)/q(NADH used) ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y(max)X/S) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y(max)ATP) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327-335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells(-1) x h(-1) could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells(-1) x h(-1); (ii) while the NADH/NAD+ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.     

Oxygen mass transfer enhancement via fermentor headspace pressurization.

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

Using the expolinear growth equation for modelling crop growth in year-round cut chrysanthemum

The aim of this study was to predict crop growth of year-round cut chrysanthemum (Chrysanthemum morifolium Ramat.) based on an empirical model of potential crop growth rate as a function of daily incident photosynthetically active radiation (PAR, MJ m-2 d-1), using generalized estimated parameters of the expolinear growth equation. For development of the model, chrysanthemum crops were grown in four experiments at different plant densities (32, 48, 64 and 80 plants m-2), during different seasons (planting in January, May-June and September) and under different light regimes [natural light, shading to 66 and 43 % of natural light, and supplementary assimilation light (ASS, 40-48 micro mol m-2 s-1)]. The expolinear growth equation as a function of time (EXPOT) or as a function of incident PAR integral (EXPOPAR) effectively described periodically measured total dry mass of shoot (R2 > 0.98). However, growth parameter estimates for the fitted EXPOPAR were more suitable as they were not correlated to each other. Coefficients of EXPOPAR characterized the relative growth rate per incident PAR integral [rm,i (MJ m-2)-1] and light use efficiency (LUE, g MJ-1) at closed canopy. In all four experiments, no interaction effects between treatments on crop growth parameters were found. rm,i and LUE were not different between ASS and natural light treatments, but were increased significantly when light levels were reduced by shading in the summer experiments. There was no consistent effect of plant density on growth parameters. rm,i and LUE showed hyperbolic relationships to average daily incident PAR averaged over 10-d periods after planting (rm,i) or before final harvest (LUE). Based on those relationships, maximum relative growth rate (rm, g g-1 d-1) and maximum crop growth rate (cm, g m-2 d-1) were described successfully by rectangular hyperbolic relationships to daily incident PAR. In model validation, total dry mass of shoot (Wshoot, g m-2) simulated over time was in good agreement with measured ones in three independent experiments, using daily incident PAR and leaf area index as inputs. Based on these results, it is concluded that the expolinear growth equation is a useful tool for quantifying cut chrysanthemum growth parameters and comparing growth parameter values between different treatments, especially when light is the growth-limiting factor. Under controlled environmental conditions the regression model worked satisfactorily, hence the model may be applied as a simple tool for understanding crop growth behaviour under seasonal variation in daily light integral, and for planning cropping systems of year-round cut chrysanthemum. However, further research on leaf area development in cut chrysanthemum is required to advance chrysanthemum crop growth prediction.