Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma

Mutagenesis of Phaffia rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 g (g dry biomass)^-1, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h^-1. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthin content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 g g^-1 and 1926 g g^-1, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optimum pH for growth of P. rhodozyma was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3.     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Degradation of polyaromatic hydrocarbons by Burkholderia cepacia 2A-12

A new strain of bacterium degrading polyaromatic hydrocarbons (PAHs), Burkholderia cepacia 2A-12, was isolated from oil-contaminated soil. Of three PAHs, the isolated strain could utilize naphthalene (Nap) and phenanthrene (Phe) as a sole carbon source but not pyrene (Pyr). However, the strain could degrade Pyr when a cosubstrate such as yeast extract (YE) was supplemented. The PAH degradation rate of the strain was enhanced by the addition of other organic materials such as YE, peptone, glucose, and sucrose. YE was a particularly effective additive in stimulating cell growth as well as PAH degradation. When 1 g YE l^–1, an optimum concentration, was supplemented into the basal salt medium (BSM) with 215 mg Phe l^–1, the specific growth rate (0.30 h^–1) and Phe-degrading rate (29.6 mol l^–1 h^–1) were enhanced approximately ten and three times more than those obtained in the BSM with 215 mg Phe l^–1, respectively. Both cell growth and PAH degradation rates were increased with increasing Phe and Pyr concentrations, and B. cepacia 2A-12 had a tolerance against Phe and Pyr toxicity at the high concentration of 730–760 mg l^–1. Through kinetic analysis, the maximum specific growth rate ( _max) and PAH degrading rate ( _max) for Phe were obtained as 0.39 h^–1 and 300 mol l^–1 h^–1, respectively. Also, _max and _max for Pyr were 0.27 h^–1 and 52 mol l^–1 h^–1, respectively. B. cepacia 2A-12 could simultaneously degrade crude oil as well as PAHs, indicating that this bacterium is very useful for the removal of oils and PAHs contaminants.     

Energy requirements for growth and maintenance of Scenedesmus protuberans Fritsch in light-limited continuous cultures

Scenedesmus protuberans Fritsch was grown in light-limited continuous cultures with a light-dark cycle, at temperatures of 20° and 28° C. At 20° irradiances of 12 and 38 W m^–2 were used, at 28° 38 W m^–2.The relationships between growth rate and light uptake rate were of diphasic linear character. With the lower growth rates the relationships were defined with the parameters _e , i.e. the specific maintenance rate constant, and c, the true efficiency of light energy conversion into biomass. The _e -value was dependent on temperature, the c on irradiance.In cultures, incubated in prolonged darkness, decrease rates of biomass were comparable to the derived _e -values.Both diphasic linear relationships between growth rate and light uptake rate and the same order of magnitude of _e -values could be derived from literature data on other green algae.     

Formal kinetics of poly-β-hydroxybutyric acid (PHB) production in Alcaligenes eutrophus H 16 and Mycoplana rubra R 14 with respect to the dissolved oxygen tension in ammonium-limited batch cultures

Summary Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly--hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate proportional to the specific growth rates of total biomass. After complete consumption of ammonium, the increase of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass (cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutrophus, strain H 16, high concentrations of dissolved oxygen strongly influence growth as well as PHB synthesis.Abbrevations ^cO₂,L concentration of oxygen in the liquid phase (dissolved oxygen tension: d.o.t) - ^cH₂,L concentration of hydrogen in the liquid phase - ^cCO₂,L concentration of carbon dioxide in the liquid phase - S limiting substrate, concentration of - X total biomass, concentration of; total cell dry weight - P product; PHB, concentration of - R rest biomass: X-P, concentration of - ^rX dX/dt growth rate - ^rP dP/dt rate of PHB synthesis - ^rR dR/dt rate of rest biomass production - ^r0 ^dcO₂,L/dt rate of oxygen consumption - X dX/dt·1/X=r_X·1/X specific growth rate - P dP/dt·1/P=r_P·1/P specific rate of product formation - R dR/dt·1/R=r_R·1/R specific rate of rest biomass formation - r₀/R specific respiration rate     

Kinetics of inhibition in propionic acid fermentation

The inhibitory effect of propionic acid P and biomass concentration X is studied in batch and continuous fermentations with cell recycle.In batch fermentations, the specific growth rate decreases and cancels out at a critical propionic acid concentration Pc ₁; the formerly decreasing specific production rate becomes constant after Pc ₁ and cancels out when a second critical propionic acid concentration Pc ₂ is reached.In continuous fermentation with cell recycle, a similar inhibition is observed with biomass. The specific rates decrease and become constant at a critical biomass concentration Xc. They cancel out at different high biomass concentrations.In both cases, the specific production rate can be related to the specific growth rate by the Luedeking and Piret expression: =+, [1], where the constants and are determined by the fermentation parameters.List of Symbols t h time - X kg/m³ biomass concentration - P kg/m³ propionic acid concentration - A kg/m³ acetic acid concentration - S kg/m³ lactose concentration - dX/dt kg/(m³h) instantaneous rate of cell growth - dP/dt kg/(m³h) instantaneous rate of propionic acid production - h^–1 specific growth rate - h^–1 specific propionic acid production rate - D h^–1 dilution rate     

Isothermic variation of the specific growth rate of Saccharomyces cerevisiae in batch culture

The specific growth rate () of a respiration-deficient mutant of Saccharomyces cerevisiae growing under defined experimental conditions in batch culture (mineral medium plus glucose and vitamins at 25°C) varied from experiment to experiment over a wide range (0.10–0.24 h^-1) and showed a normal distribution. Neither the age of the culture, the history of the inoculum, nor experimental error accounted wholy for the variability of . The variation was positively correlated with the specific rate of glucose transfer and negatively with the specific rate of production of non-fermentative CO₂. The yield decreased with implying higher maintenance requirements in batch culture (4.7 mmoles g^-1 h^-1) than in continuous culture (0.8 mmoles g^-1 h^-1). It was concluded that the strain is capable of establishing any one of several steady states of growth under the same experimental conditions, each steady state displaying some buildin inertia with respect to change. The variations of the specific rates of glucose transfer and non-fermentative CO₂ production, and of the yield appeared to be consequences rather than causes of the variation of . The ultimate causes of the variation of remained unidentified.Part of a doctoral thesis submitted by J. Martinez-Peinado to the University of Navarra Spain     

Modelling of alcohol fermentation in a tubular reactor with high biomass recycle

Fermentation in tubular recycle reactors with high biomass concentrations is a way to boost productivity in alcohol production. A computer model has been developed to investigate the potential as well as to establish the limits of this process from a chemical engineering point of view. The model takes into account the kinetics of the reaction, the nonideality of flow and the segregation in the bioreactor. In accordance with literature, it is shown that tubular reactors with biomass recycle can improve productivity of alcohol fermentation substantially.With the help of the computer based reactor model it was also possible to estimate the detrimental effects of cell damage due to pumping. These effects are shown to play a major role, if the biomass separation is performed by filtration units which need high flow rates, e.g. tangential flow filters.List of Symbols Bo d Bodenstein number - c kg/m³ concentration of any component - CPFR continuous plug flow reactor - CSTR continuous stirred tank reactor - d _h m hydraulic diameter - D _eff m²/s dispersion coefficient - f residence time distribution function - K _s kg/m³ monod constant for biomass production - K _s kg/m³ monod constant for alcohol production - p kg/m³ product concentration - P _i kg/m³ lower inhibition limit concentration for biomass production - p _i kg/m³ lower inhibition limit concentration for alcohol production - p _m kg/m³ maximum inhibition limit concentration for biomass production - p _m kg/m³ maximum inhibition limit concentration for alcohol production - q _p h^–1 specific production rate - q _p,max h^–1 maximum specific production rate for alcohol production - q _s h^–1 specific substrate consumption rate - Q _L m _gas ³ /m³h specific gas rate - r _p , r _s , r _x kg/(m³ · h) reaction rate for ethanol production substrate consumption and cell growth, respectively - S _F kg/m³ substrate concentration in feed stream - s kg/m³ substrate concentration - t h time - x kg/m³ biomass concentration - x _max kg/m³ maximum biomass concentration for biomass production - Y _p/s yield coefficient - h^–1 specific growth rate - _max h^–1 maximum specific growth rate - dimensionless time (t/) - h mean residence time - _s glucose conversion     

Estimation of specific growth rate from agitation speed in DO-stat culture

Summary A simple and effective method to estimate the specific growth rate estimation has been developed based on the observation of time changes in the agitation speed in dissolved oxygen(DO)-stat cultures of Brevibacterium ketoglutamicum. The estimation was compared with that using carbon dioxide evolution rate (CER). Estimated values of specific growth rates by both methods agreed well with the data directly calculated from cell concentration change although the use of agitation speed gave a slightly better result than CER.Nomenclature CER Carbon dioxide evolution rate (mmol/sec) - OUR Oxygen uptake rate (mmol/sec) - OTR Oxygen transfer rate (mmol/sec) - RPM Agitation speed (rev./min) - C* Saturated dissolved oxygen concentration (mmol/L) - Dissolved oxygen concentration (mmol/L) - k Time index - k _L a' Mass transfer coefficient (sec^-1) - Y _X/O2 Cellular yield based on oxygen consumed (g-cell/mmol O₂) - Specific growth rate (hr^-1) - Constant - t Fermentation time - t Sampling time for RPM and CER measurements     

Responses of wild plants to nitrate availability

Summary Two annual species of Bromus, an invader (B. hordeaceus, ex B. mollis) and a non-invader (B. intermedius), were grown for 28 days in growth chambers, at 5 and 100 M NO ₃ ^- in flowing nutrient solution. No differences between the two species were observed at either NO ₃ ^- level, in terms of relative growth rate (RGR) or its components, dry matter partitioning, specific NO ₃ ^- absorption rate, nitrogen concentration, and other characteristics of NO ₃ ^- uptake and photosynthesis. The effects of decreasing NO ₃ ^- concentration in the solution were mainly to decrease the NO ₃ ^- concentration in the plants through decreased absorption rate, and to decrease the leaf area ratio through increased specific leaf mass and decreased leaf mass ratio. Organic nitrogen concentration varied little between the two treatments, which may be the reason why photosynthetic rates were not altered. Consequently, RGR was only slightly decreased in the 5-M treatment compared to the 100-M treatment. This is in contrast with other species, where growth is reduced at much higher NO ₃ ^- concentrations. These discrepancies may be related to differences in RGR, since a log-linear relationship was found between RGR and the NO ₃ ^- concentration at which growth is first reduced. In addition, a strong linear relationship was found between the RGR of these species and their maximum absorption rate for nitrate, suggesting that the growth of species with low maximum RGR may be partly regulated by nutrient uptake.     

Unstable steady state operations of substrate inhibited cultures by dissolved oxygen control

Microorganism kinetic growth characterized by substrate inhibition was investigated by means of a continuous stirred tank reactor equipped with a feedback controller of the medium feeding flow rate. The aerobic growth of Pseudomonas sp. OX1 with phenol as carbon/energy source was adopted as a case study to test a new control strategy using dissolved oxygen concentration as a state variable. The controller was successful in steadily operating bioconversion under intrinsically unstable conditions. A simple model of the controlled system was proposed to set the feedback controller.The specific growth rate of Pseudomonas sp. OX1 was successfully described by means of the Haldane model. The regression of the experimental data yielded μ_M = 0.26 h⁻¹, K_Ph = 5 × 10⁻³ g/L and K_I = 0.2 g/L. The biomass-to-substrate fractional yield as a function of the specific growth rate did not change moving from substrate-inhibited to substrate-deficient state. The data was modelled according to the Pirt model: m = 1.7 × 10⁻² g/(g h), . The specific growth rates calculated for batch and continuous growth were compared.     

Mathematical modeling of production of microbial lipids

As a part of the investigations on the microbial lipid production using the yeast Rhodotorula gracilis, CFR-1, kinetics of the biomass synthesis has been studied using shake flask experiments. Using a medium containing a carbon to nitrogen ratio of 701, the rates of biomass production were followed at different initial substrate concentrations in the range of 20–100 kg/m³. A logistic model was found to be reasonably adequate to describe the kinetics of the growth of biomass; the maximum specific growth rate of 0.105 h^–1 was applicable for substrate concentrations less than 60 kg/m³, which gave reasonable agreement between predicted and actual biomass concentration values.List of Symbols S ₀, X ₀ kg/m³ Initial concentrations of sugar, non lipid biomass respectively - X, X(t) kg/m³ Concentrations of non lipid biomass at any time t - dX/dt kg/(m³ · h) Rate of biomass growth - h^–1 Specific growth rate - _max h^–1 Maximum specific growth rate - K _s mol/dm³ Monods constant - X _max kg/m³ Maximum biomass reached in a run     

Long term shear effects on a hybridoma cell line by dynamic perfusion devices

The long term shear effects on a hybridoma cell line were studied by the simulation of a hollow fiber perfusion system. Various mechanical/environmental stress conditions were applied and steady state concentrations of live, dead and lysed cells were measured or calculated in a continuous culture. From mathematical modeling, it is shown that inclusion of a lysed cell index (LCI) renders a better fit to the material balance equation at steady state. The specific cell death rate increased with increasing shear force as expected only when the LCI was included. Without the inclusion of the LCI, the calculated specific cell growth rates are about 25–60% of the value when included. The results reported may lend some insight to design improvements since most perfusion devices add shear stresses to the cells in the reactor.List of Symbols b ml/hr continuous culture flow rate - D hr^–1 dilution rate (b/V) - m g glucose/10⁹ cells/hr specific maintenance coefficient - S ₀ g/l feed substrate concentration - S g/l reactor substrate concentration - t hr time - V ml reactor volume - X ⁺ cells/ml live cell concentration - X ^– cells/ml dead cell concentration - X ⁰ cells/ml lysed cell concentration - Y _x/s 10⁹ cells/g glucose cell/substrate yield coefficient - hr^–1 specific growth rate - hr^–1 specific death rate - hr^–1 specific lysis rate - hr^–1 specific lysis rate for simultaneous death and lysis     

Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy     

Metabolic burden as reflected by maintenance coefficient of recombinant Escherichia coli overexpressing target gene

Metabolic burden as a consequence of overexpression of target gene in a recombinant strain of E. coli 1727 has been analyzed with respect to maintenance energy coefficient (m). The values of m for the host, uninduced recombinant and IPTG induced recombinant were determined to be 0.12, 0.17 and 0.32 g.g^-1.h^-1 respectively. Transient plasmid instability and nearly 33% fall in maximum specific growth rate were observed under conditions of enhanced requirements for maintenance energy.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

On-line parameter and state estimation of continuous cultivation by extended Kalman filter

Summary The on-line estimation of biomass concentration and of three variable parameters of the non-linear model of continuous cultivation by an extended Kalman filter is demonstrated. Yeast growth in aerobic conditions on an ethanol substrate is represented by an unstructured non-linear stochastic t-variant dynamic model. The filter algorithm uses easily accessible data concerning the input substrate concentration, its concentration in the fermentor and dilution rate, and estimates the biomass concentration, maximum specific growth rate, saturation constant and substrate yield coefficient. The microorganismCandida utilis, strain Vratimov, was cultivated on the ethanol substrate. The filter results obtained with the real data from one cultivation experiment are presented. The practical possibility of using this method for on-line estimation of biomass concentration, which is difficult to measure, is discussed.Nomenclature D dilution rate (h^-1) - DO₂ dissolved oxygen concentration (%) - E identity matrix - F Jacobi matrix of the deterministic part of the system equations g - g continuousn-vector non-linear real function - h m-vector non-linear real function - K Kalman filter gain matrix - K _S saturation constant (kgm^-3) - K_S expectation of the saturation constant estimate - M Jacobi matrix of the deterministic part of the measurement equations h - P(t₀) co-variance matrix of the initial values of the state - P(t_k/t_k) c-variance matrix of the error in (t _k|t _k) - P(t_k+1/t_k) co-variance matrix of the error in (t _k+1|t _k - Q co-variance matrix of the state noise - R co-variance matrix of the output noise - S substrate concentration (kgm^-3) - S _i input substrate concentration - t time - t _k discrete time instant with indexk=0, 1, 2,... - u(t) input vector - v(t_k) measurement (output) noise sequence - w(t) n-vector white Gaussian random process - x(t₀) initial state of the system - (t₀) expectation of the initial state values - x(t) n-dimensional state vector - x(t_k) state vector at the time instantt _k - (t_k|t_k) expectation of the state estimate at timet _k when measurements are known to the timet _k - (t_k+1|t_k) expectation of the state prediction - X biomass concentration (kgm^-3) - expectation of the biomass concentration estimate - y(t_k) m-dimensional output vector at the time instantt _k - Y _XIS substrate yield coefficient - _X|S expectation of the substrate yield coefficient estimate - specific growth rate (h^-1) - _M maximum specific growth rate (h^-1) - expectation of the maximum specific growth rate estimate - state transition matrix     

Effect of substrate concentration on ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.Nomenclature S glucose concentration (g/l) - X biomass concentration (g/l) - P ethanol concentration (g/l) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - YINX/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Factors influencing the stable carbon and oxygen isotopic composition ofPorites lutea coral skeletons from Phuket,South Thailand

We determined the ¹⁸O and ¹³C composition of the same fixed growth increment in severalPorites lutea coral skeletons from Phuket, South Thailand. Skeletal growth rate and ¹⁸O are inversely related. We explain this in terms of McConnaughey's kinetic isotopic disequilibria model. Annual trends in ¹⁸O cannot be solely explained by observed variations in seawater temperature or salinity and may also reflect seasonal variations in calcification rate. Coral tissue chlorophylla content and ¹³C of the underlying 1 mm of skeleton are positively related, suggesting that algal modification of the dissolved inorganic carbonate pool is the main control on skeletal ¹³C. However, in corals that bleached during a period of exceptionally high seawater temperatures in the summer of 1991, ¹³C of the outer 1 mm of skeleton and skeletal growth rate (over 9 months up to and including the bleaching event) are inversely related. Seasonal variations in °¹³C may reflect variations in calcification rate, zooxanthellae photosynthesis or in seawater ¹³C composition. Bleached corals had reduced calcification over the 9-month period up to and including the bleaching event and over the event they deposited carbonate enriched in¹³C and¹⁸O compared with unaffected corals. However, calcification during the event was limited and insufficient material was deposited to influence significantly the isotopic signature of the larger seasonal profile samples. In profile, overall decreases in ¹⁸O and ¹³C were observed, supporting evidence that positive temperature anomalies caused the bleaching event and reflecting the loss of zooxanthellae photosynthesis.