Growth, Sporulation, δ-Endotoxins Synthesis, and Toxicity During Culture of Bacillus thuringiensis H14

Growth, sporulation, synthesis of δ-endotoxins, and toxicity against the larvae of Aedes aegypti and Culex pipiens were studied during fermentation of Bacillus thuringiensis H14 in a 20-L fermentor. Measurements of optical density and dielectric permittivity for biomass determination suggest a highly promising technique for on-line evaluation of sporulation. The synthesis of 65-, 25- and 130-kDa proteins started at 16, 18, and 23 h, respectively. These proteins were enriched in different ways until the end of culture (48 h). Toxicity in the course of sporulation was significantly different for the larvae of both mosquito species. Maximal activity against Ae. aegypti was obtained at the end of culture, whereas for Cx. pipiens, the sample at 38 h was the most active.     

Redirection of metabolism during nutrient feeding in fed-batch cultures of Bacillus thuringiensis

During sporulation, Bacillus thuringiensis produces insecticidal crystal inclusions (Cry proteins) encoded by cry genes. In fed-batch cultures (FBCs), spores and Cry protein yields are usually low, so we therefore studied the pattern of metabolic changes occurring in batch cultures and FBCs of a B. thuringiensis strain having a cry1Aa promoter-lacZ fusion, and their effect on sporulation and cry1A gene expression. In FBCs, there was a redirection of bacterial metabolism and a reduction in the specific growth rate during feeding, even when the nutrient concentration was higher than at the beginning of batch culture. These physiological changes suggest that the transition state is set up during feeding and this set-up seems to have a negative effect on both sporulation and cry1Aa expression. When the filtrate of a culture in the transition state was added to a batch culture early in the first exponential growth phase, it delayed sporulation and cry1Aa expression, thus suggesting that a soluble cellular factor that blocked sporulation might be excreted during the transition state. Citrate production usually started during the transition state but, when a medium rich in free amino acids was fed, citrate was produced from the first growth phase and sporulation was nearly blocked.     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Dielectric measurement to monitor the growth and the physiological states of biological cells

Measurement of capacitance, also referred to as dielectric permittivity, is a new method of estimating the concentration of cells, monitoring the growth and detecting the physiological changes during the cultivation of organisms in various bioprocess. Several types of biological cells were studied, namely; Saccharomyces cerevisiae, Escherichia coli, Perilla frutescens (plant cells) and AFP-27 hybridoma cells. Generally, a linear correlation between cell capacitance (C) and other biomass measurement technique such as optical density (OD) and dry weight (DW) was obtained using the different types of cell suspension. Therefore, this method could be used to monitor the growth of the organism during the active growth. It could be conveniently used to make a rapid estimate of the cell concentration such as in plant cell suspension culture. The capacitance sensor could also be designed to be installed and autoclaved in-situ in a bioreactor and used for on-line monitoring of cell growth. On the other hand, distinct deviations in the capacitance value were observed in relation with the growth stage of the organism. This was observed in all the organisms studied but the type of deviation depends on the physiology of the organism. This variation in cell capacitance showed the possibility of using this method as a means to indicate changes in the physiological state of cells during cultivation. This capability would be very useful in designing control strategies that would depend on the physiological states in the bioprocess. Present address: Miles Inc., Berkeley, CA 94701 U.S.A.The authors sincerely appreciated the generosity of Dr. K. Mishima and Dr. A. Mimura of Kobe Steel Co., Japan. The useful discussions with M. Nakajima and technical assistance of J. Zhong and R. Pambayun were also acknowledged. The work in hybridoma cell culture was done through the collaboration with C. Perusich-Kussow and Prof. W. S. Hu, University of Minnesota, USA.     

Multifrequency permittivity measurements enable on‐line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells

Lab and pilot scale batch cultivations of a CHO K1/dhfr^? host cell line were conducted to evaluate on‐line multifrequency permittivity measurements as a process monitoring tool. The β‐dispersion parameters such as the characteristic frequency (f_C) and the permittivity increment (Δε_max) were calculated on‐line from the permittivity spectra. The dual‐frequency permittivity signal correlated well with the off‐line measured biovolume and the viable cell density. A significant drop in permittivity was monitored at the transition from exponential growth to a phase with reduced growth rate. Although not reflected in off‐line biovolume measurements, this decrease coincided with a drop in OUR and was probably caused by the depletion of glutamine and a metabolic shift occurring at the same time. Sudden changes in cell density, cell size, viability, capacitance per membrane area (C_M), and effects caused by medium conductivity (σ_m) could be excluded as reasons for the decrease in permittivity. After analysis of the process data, a drop in f_C as a result of a fall in intracellular conductivity (σ_i) was identified as responsible for the observed changes in the dual‐frequency permittivity signal. It is hypothesized that the β‐dispersion parameter f_C is indicative of changes in nutrient availability that have an impact on intracellular conductivity σ_i. On‐line permittivity measurements consequently not only reflect the biovolume but also the physiological state of mammalian cell cultures. These findings should pave the way for a better understanding of the intracellular state of cells and render permittivity measurements an important tool in process development and control. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Analysis of nucleotide pools during protease production with Bacillus licheniformis

Summary During development of a fed-batch procedure for protease production with Bacillus licheniformis the nucleotide pools of the culture were assayed. Transitions between different growth phases or different nutrient limitations could easily be discerned by alterations in the nucleotide pool. As already described for B. subtilis, induction of sporulation was marked by a drop in the guanosine triphosphate (GTP) pool of the cells, which always preceded protease production. This was also valid for closed-loop controlled fed-batch processes in which the concentration of ammonium, which repressed protease biosynthesis, was kept constant at low levels. A marked decrease in the GTP content of the cells, e.g. after addition of mycophenolic acid during the exponential phase, increased protease formation during the stationary phase. During protease production the energy charge was lower (0.6–0.8) than during the exponential and early stationary phases (0.8–0.95) although very low energy charges (<0.5) did not support protease formation. Offprint requests to: G. Bierbaum     

The influence of seedling age and development on the infection of lettuce by Bremia lactucae

Laboratory methods are described for the culture and observation of Bremia lactucae on lettuce. Mycelial growth of the fungus within the cotyledons of the cultivar Cheshunt Early Giant progressed to a minimum density at which sporulation occurred, and then continued to a maximum density coinciding with peak sporulation. The timing and intensity of sporulation was influenced by the concentration of the inoculum. Seedlings often became systemically infected and this, together with intensity of sporulation, was a good indicator of fungal growth and hence host susceptibility. The severity of disease development on four susceptible lettuce cultivars (Cheshunt Early Giant, Paris White, Trocadero Improved and Crackling Ice) decreased with age and development of the seedlings at inoculation. The reasons for this are discussed.     

Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of <Emphasis Type="Italic">Thevetia peruviana</Emphasis>

Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige–Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17 ± 0.2 g g⁻¹ inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyl-jasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l⁻¹ of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l⁻¹ medium. The current results are the first report of an in vitro peruvoside production system.     

Quantitative modeling of viable cell density,cell size,intracellular conductivity,and membrane capacitance in batch and fed‐batch CHO processes using dielectric spectroscopy

Dielectric spectroscopy was used to analyze typical batch and fed‐batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole–Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The β‐dispersion was analyzed using the Cole–Cole distribution parameters Δε (magnitude of the permittivity drop), f_c (critical frequency), and α (Cole–Cole parameter). Furthermore, the dielectric parameters static internal conductivity (σ_i) and membrane capacitance per area (C_m) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Kinetics of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">israelensis</Emphasis> growth on high glucose concentrations

The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S ₀) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically well-defined culture phases, independent of S ₀ values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation and cell lysis. Important process parameters were also determined. The maximum specific growth rates (μ _X,m) were not affected with S ₀ up to 75 g/l (1.0–1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by a reduction in μ _X,m values. These higher S ₀ values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (X _m = 15.2–15.9 g/l) were achieved with S ₀ over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose to form other metabolites. In this case, with S ₀ from 30.8 to 152 g/l, cell yield (Y _X/S ) decreased from 0.58 to 0.41 g/g. On the other hand, with S ₀ = 10 g/l growth was limited by substrate, and Y _X/S has shown its maximum value (0.83 g/g).     

Simultaneous evaluation of on-line microcalorimetry and fluorometry during batch culture of Pseudomonas putida-ATCC 11172 and Saccharomyces cerevisiae ATCC 18824

Application of on-line sensors (flow calorimeter, fluorescence probe, dissolved oxygen and CO₂ probes) was assessed to monitor microbial biomass and physiological state of cells during a biological process. Two systems were studied; diauxic growth of Pseudomonas putida ATCC 11172 on glycerol and phenol, and the aerobic growth of Saccharomyces cerevisiae ATCC 18824 on glucose. The results showed that the cells produced a heat output which could be quantitatively related to the various phases of the growth cycle. The initial stage of enzymatic induction and substrate mobilization during the diauxic growth of P. putida was easily detected, and a clear oscillation phenomenon was observed during enzymatic rearrangement in shifting from phenol to glycerol metabolism. Glucose oxidation in ethanol and then in acetate was also clearly delineated from the growth of S cerevisiae. NADH (fluorescence probe) measurements gave a strong correlation with various biomass indicators such as optical density, dry weight, ATP content and cellular protein. The fluorescence signal appeared to be very sensitive to the quenching effect of the culture medium and of the cells themselves. The fluorescence emitted from the NADH molecules in a culture medium can be reduced from 30–70% depending on the chemical composition and the optical density.     

Amine neuromediators,their precursors,and oxidation products in the culture of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The growth dynamics of the synthesis of monoamine neuromediators serotonin, norepinephrine, and dopamine in Escherichia coli K-12 was investigated for the first time using high performance liquid chromatography with electrodetection. Maximum (micromolar) concentrations of these compounds were detected in E. coli cells during the early growth phases; their intracellular content decreases after the transition to late growth phases. E. coli biomass contains (i) the substances DOPA and 5-hydroxytryptamine that serve in animal cells as neuromediator precursors and (ii) the products of their oxidative deamination. Presumably, the biosynthesis and degradation of monoamine neuromediators in bacterial cells involves enzyme systems analogous to those typical of animals. The culture fluid of E. coli contains micromolar concentrations of DOPA and nanomolar of serotonin, dopamine, and norepinephrine during the late growth phase. These concentrations are sufficient for animal/human receptors to bind them. This article deals with the potential biotechnological applications of the data obtained.     

Enhanced spore production ofBacillus thuringiensis by fed-batch culture

Summary Fed-batch culture was carried out to increase cell mass followed by batch culture for spore production ofbacillus thuringiensis. High cell mass obtained by increasing the feeding glucose concentration in constant fed-batch culture which supported fast cell growth resulted in good sporulation during subsequent batch culture, and the maximum cell mass of 72.6 g/L and spore concentration of 1.25×10¹⁰ spores/mL could be obtained.     

Changes of Exponential Growth Rates in Relation to Differentiation of Geotrichum candidum in Submerged Culture

Growth and differentiation of the imperfect fungus Geotrichum candidum were followed in submerged cultures containing a simple synthetic glucose salt medium. Uptake of glucose, ammonium and oxygen from the medium were measured during the entire growth perod. In 0.1% glucose the fungus grows with one exponential growth phase until all the glucose has been consumed. The arthrospores are formed in the stationary phase. In 0.5% glucose the growth curve has two exponential growth phases, one with a doubling time of 1.8 h and a second one with a doubling time of 4.9 h. The second exponential growth phase, which starts when less than 15% of the glucose and less than 30% of the ammonium have been consumed, is shown to be the sporulation phase. During this growth phase the oxygen saturation in the culture remained constant at about 50%.     

Verlust der Kompetenz für genetische Transformation in Bacillus subtilis-Zellen während des Überganges in die stationäre Wachstumsphase

Zusammenfassung Im Gegensatz zu den Hinweisen, daß kompetente Bacillus subtilis-Zellen solche sind, die sich in frühen Sporulationsphasen befinden, stehen die genannten Befunde, wonach Zellen sporulieren, ohne dabei kompetent zu werden. Wenn die Sporulation durch den Übergang einer Kultur in die stationäre Wachstumsphase ausgelöst wurde, nahm sofort die Zahl der kompetenten Zellen ab. Bei einem Wechsel der Stickstoffquelle von Ammoniumsulfat zu Histidin, der bei einem Drittel der Population zur Ausbildung thermoresistenter Sporen führte, verminderte sich sofort die Anzahl transformierbarer Zellen von 4,5·10⁵ ml^-1 auf 2,0·10¹ ml^-1, ohne später wieder den zuvor gemessenen Wert zu erreichen.Der genannte Widerspruch läßt sich aufheben durch die Annahme, daß Zellen, die während des exponentiellen Wachstums der Kultur in den stationären Zustand übergehen und sporulieren, sich von denen unterscheiden, deren Sporulation durch Substratlimitation induziert wurde. Nur die ersteren sind transformierbar. Wahrscheinlich ist bei den letztgenannten die Synthese oder Funktion der zur Kompetenzausbildung und Erhaltung notwendigen Substanzen gehemmt.

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Loss of competence in the Bacillus subtilis transformation system during transition to the stationary growth phase

Summary Former experiments of several authors indicated that competence in the Bacillus subtilis transformation system is linked to an early phase of sporulation. The results presented here show that B. subtilis cells may sporulate without becoming competent. If mass sporulation was induced by transition of the culture to the stationary growth phase the number of competent cells decreased abruptly. Substitution of ammonium sulphate by histidine as the sole source of nitrogen induced a third of the population to form thermoresistant spores. Simultaneously, the number of cells which could be transformed was reduced from 4.5×10⁵ ml^-1 to 2.0×10¹ ml^-1. After prolonged incubation no total recovery of competence could be observed.The differing results concerning the connection of sporulation and transformability may be explained by the assumption that there exists a difference between single cells which sporulate after entering the stationary state during the exponential growth of the culture and such cells the sporulation of which is induced by limitation of substrate. Only the former ones may be subject to transformation while in the latter cells synthesis or function of substances necessary for establishing and maintaining competence may be inhibited.

     

Effect of recombinant Rv1009 protein on promoting the growth of Mycobacterium tuberculosis

Aims: To determine whether resuscitation-promoting factor (RPF) from Mycobacterium tuberculosis can promote mycobacterial growth and shorten culture time. Method and Results: We cloned, expressed and purified an RPF from M. tuberculosis, Rv1009 protein and subsequently studied the biological activity of the recombinant Rv1009 (rRv1009) in liquid and on solid media. Our results indicate that the molecular weight of rRv1009 protein expressed in Escherichia coli BL21 was approximately 39 kDa. At picomolar and micromolar concentrations, rRv1009 protein could increase the optical density of freeze-dried Mycobacterium bovis BCG three to fivefold in Middlebrook 7H9 medium, stimulate the growth of viable mycobacteria on solid medium, and shorten positive growth detection time of a small number of M. tuberculosis in BACTEC 960 medium. Conclusions: The rRv1009 could promote proliferation of mycobacteria. It may be useful for culture of mycobacteria presented in clinical samples. Significance and Impact of the Study: rRv1009 protein can be used as a growth-promoting reagent of mycobacteria in the medium to shorten the time of culture.     

Novel bioreactor internals for the cultivation of spore‐forming fungi in pellet form

This study introduced an automated long‐term fermentation process for fungals grown in pellet form. The goal was to reduce the overgrowth of bioreactor internals and sensors while better rheological properties in the fermentation broth, such as oxygen transfer and mixing time, can be achieved. Because this could not be accomplished with continuous culture and fed‐batch fermentation, repeated‐batch fermentation was implemented with the help of additional bioreactor internals (“sporulation supports”). This should capture some biomass during fermentation. After harvesting the suspended biomass, intermediate cleaning was performed using a cleaning device. The biomass retained on the sporulation support went through the sporulation phase. The spores were subsequently used as inocula for the next batch. The reason for this approach was that the retained pellets could otherwise cause problems (e.g., overgrowth on sensors) in subsequent batches because the fungus would then show undesirable hyphal growth. Various sporulation supports were tested for sufficient biomass fixation to start the next batch. A reproducible spore concentration within the range of the requirements could be achieved by adjusting the sporulation support (design and construction material), and an intermediate cleaning adapted to this.     

The color of mass culture: spectral characteristics of a shallow water column through shade‐limited algal growth dynamics1

It is envisioned that mass algal cultivation for commercial biofuels production will entail the use of large raceway pond systems, which typically have shade‐limited photosynthetic growth within depths of 20–30 cm. The attenuation of light and spectral qualities of red, green, and blue wavelengths in a 20‐cm water column as a function of Chl‐a concentration during exponential and linear phases of growth dynamics for the marine diatom Thalassiosira pseudonana was examined under laboratory conditions. While photosynthetically available radiation (PAR) was in excess throughout the water column during the phase of exponential growth, PAR became rate limiting differently for red, green, and blue wavelengths during the phase of linear growth. The transition from exponential to linear growth occurred at 1–2 mg Chl‐a · L?1, whereby a scalar ~5 μmol photons · m?2 · s?1 at 20‐cm depth was found to occur as would be anticipated having the compensation point for where rates of photosynthesis and respiration are equal. During the phase of linear growth, red wavelengths became increasingly dominant at depth as Chl‐a concentrations increased, being contrary to the optical conditions for those natural bodies of water that forced the evolution of phytoplankton photosynthesis. It is hypothesized this dramatic difference in water column optics between natural and synthetic environments could influence a variety of biological reactions, importantly non‐photochemical quenching capacities, which could negatively impact crop yield.     

Life‐cycle kinetic model for endospore‐forming bacteria,including germination and sporulation

We develop a mechanistic life‐cycle model for endospore‐forming bacteria (EFB) and test the model with experiments with a Bacillus mixed culture. The model integrates and quantifies how sporulation and germination are triggered by depletion or presence of a limiting substrate, while both substrates affect the rate of vegetative growth by a multiplicative model. Kinetic experiments show the accumulation of small spherical spores after the triggering substrate is depleted, substantially more rapid decay during sporulation than for normal decay of vegetative cells, and a higher specific substrate utilization rate for the germinating cells than that for growth of vegetative cells. Model simulations capture all of these experimental trends. According to model predictions, when a batch reactor is started, seeding with EFB spores instead of active EFB delays the onset of rapid chemical oxygen demand (COD) utilization and biomass growth, but the end points are the same. Simulated results with low aeration intensity show that germination can consume some substrate without dissolved oxygen (DO) depletion. Biotechnol. Bioeng. 2009; 104: 1012–1024. © 2009 Wiley Periodicals, Inc.