Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

Growth of Photorhabdus luminescens in batch and glucose fed-batch culture

Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration and oxygen uptake rate were recorded. The addition of 12.4 g l⁻¹ glucose prolonged the growth, and the yield almost doubled, from 6.85 g l⁻¹ to 12.45 g l⁻¹ dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift of P. luminescens to phase II variants was not detected. Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae

An efficient method of growing the protozoon Tetrahymena to high cell densities in a 2-1 bioreactor is described. The first phase of the fermentation is a batch phase with minimum generation times (1.4 h). During the next phase medium is exchanged continuously using a perfusion module based on microporous hollow fibres while cell are retained. Compared to standard batch fermentations of this organism 30- to 40-fold higher cell concentrations and dry weights were achieved routinely. A maximum cell concentration of 2.2 × 10⁷ cells/ml and a dry weight of 54 g/l have been obtained. As estimated from isocitrate dehydrogenase activity in the culture medium, no cell damage occurred even at high agitation rates. In addition, the cells remained viable for several weeks. Temporal limitation of the process was due to a decrease in the perfusion rate caused by blocking of the membranes. By X-ray microprobe analysis calcium phosphate depositions were detected in the pores of the clogged hollow-fibre membranes. However, even a T. pyriformis strain possessing mucocysts, dense core secretory organelles that may lead to early membrane clogging, was cultivated successfully for 3 weeks. Additionally, the consumption of nutrient protein and carbohydrates during fermentation was investigated and the effect of different perfusion rates and of glucose was studied in order to increase the efficieny of the system.Correspondence to: A. Tiedtke     

Detachment ofPseudomonas fluorescens from biofilms on glass surfaces in response to nutrient stress

The effects of glucose and nitrogen depletion on the colonization of glass Petri plates byPseudomonas fluorescens were studied in batch culture. Colonization of the surfaces was initiated before colonization of the bulk phase, and biofilm formation was observed. This resulted in an apparent lag in the batch growth curve for the cell suspension. The lag phase was an artifact caused by the partitioning of cells between the bulk and solid phase of the culture and was not due to a reduction in the growth rate of unattached cells. The specific growth rate of the unattached cells (0.331 hour^–1) was almost twice that determined for the total population (0.171 hour^–1). Consequently the growth rate of biofilm-forming bacteria cannot be determined in batch culture unless the growth of both attached and unattached cells is monitored, and batch growth curves may contain artifacts due to the formation and dispersion of biofilms. The depletion of either glucose or nitrogen led to the active detachment of cells from the biofilm. An increase in the hydrophobicity of unattached cells was noted on depletion of carbon. This increase was the result of emigration of cells from the surface into the bulk phase.Paper contribution number 128, Centre de Rechcrches Alimentaires de St Hyacinthe.     

Freeze Preservation of Cultured Plant Cells. III. The Pregrowth Phase

There is an inverse relationship between cell size and capacity to survive the freeze-preservation protocol. Pregrowth of cell suspensions in media rendered more negative in water potential by addition of mannitol enhances the survival capacity of Acer pseudoplatanus and Capsicum annuum cells but this effect can only partially be explained in terms of the associated reduction in mean cell size. Studies with cell suspensions of Daucus carota indicate the importance for successful freeze-preservation of the stage in the growth cycle of suspensions propagated in batch culture; highest survival was recorded for cells taken at lag phase or early exponential phase. Regrowth of recovered cells depends upon the establishment of an appropriate inoculum density of cells which have retained the capacity to divide. The dividing cells only achieve a growth rate equal to that of untreated cells after a number of cell generations. A proportion of the recovered cells giving an initial positive fluorescein diacetate reaction lose this capacity rapidly (within 24 h), others lose the capacity more slowly and others, in which the positive reaction persists, are incapable of division. These observations indicate that different levels of injury are inflicted by the freeze-preservation protocol and that only in a proportion of the cells is the injury reparable or compatible with growth by cell division.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Three-day-cultured cells of Vinca rosea L. (in the cell division phase) and 5-day-cultured cells (in the cell expansion phase) prelabelled with d -[U-¹⁴C] glucose were incubated in a medium containing unlabelled glucose. After various periods of chase, extra-cellular polysaccharides (ECP) and cell walls were isolated, and cell walls were fractionated into pectic substances, hemicellulose, and cellulose fractions. After acid hydrolysis, the radioactive constituents in the pectic substances and hemicellulose fractions were analyzed. Active turnover was observed in arabinose and galactose in the hemicellulose fraction of cell walls, while the constituents of the pectic substances, and xylose and glucose in the hemicellulose fraction did not undergo active turnover. The proportion of radioactivities of arabinose and galactose in total radioactivity of ECP increased markedly after chasing. These results indicate that arabinogalactan was synthesized, deposited in the cell wall, degraded rapidly, and made soluble in the medium as a part of ECP.     

Production of xylitol in cell recycle fermentations of Candida tropicalis

Xylitol was produced a in two-substrate, batch fermentation with cell recycling of Candida tropicalis ATCC 13803. A series of cell-recycle experiments showed that the feeding of xylose, glucose and yeast extract in the xylitol production phase was most effective in enhancing xylitol productivity. The optimized cell recycle fermentation resulted in 0.82 g xylitol/g xylose yield, 4.94 g xylitol l^–1 h^–1 productivity, and final xylitol concentration of 189 g l^–1. These results were 1.3 times higher in volumetric xylitol productivity and 2.2 times higher in final product concentration compared with the corresponding values of the optimized two-substrate batch culture.     

Production of hyaluronic Acid by a streptococcal strain in batch culture

A simple method for the production and preparation of hyaluronic acid (HA) is described. Three media were tested, which all supported relatively good yields of cells, but only in one of them were appreciable amounts of HA formed. In this medium, HA was produced during the logarithmic growth phase and showed a molecular weight of 7.3 × 10⁵. HA prepared from 24-hr cultures of the same organism had a molecular weight of only 2.6 × 10⁵. The kinetics of HA production in batch culture were studied, as well as of cell production and glucose consumption.     

Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to cell division activity in suspension cultures

Regulatory mechanisms of betacyanin biosynthesis in suspension cultures of Phytolacca americana and anthocyanin in Vitis sp. were investigated in relation to cell division activity.Betacyanin biosynthesis in Phytolacca cells clearly shows a positive correlation with cell division, as the peak of betacyanin accumulation was observed at the log phase of batch cultures. Incorporation of radioactivity from labelled tyrosine into betacyanin also showed a peak at early log phase. Aphidicolin, an inhibitor of DNA synthesis, and propyzamide, an antimicrotubule drug, reduced betacyanin accumulation and inhibited the incorporation of radioactivity from labelled tyrosine into betacyanin at concentrations which were inhibitory to cell division. Both inhibitors reduced the incorporation of radioactivity from labelled tyrosine to 3,4-dihydroxyphenylalanine (DOPA), but the incorporation of labelled DOPA into betacyanin was not affected. These results suggest that the conversion of tyrosine to DOPA is coupled with cell division activity.In contrast, the anthocyanin accumulation in Vitis cells showed a negative correlation with cell division. Accumulation occurred at the stationary phase in batch cultures when cell division ceased. Aphidicolin or reduced phosphate concentration induced a substantial increase in anthocyanin accumulation as well as the inhibition of cell division. Chalcone synthase (CHS) activity increased at the time of anthocyanin accumulation. Northern blotting analysis indicated that changes in CHS mRNA levels corresponded to similar changes in enzymatic activity. The pool size of endogenous phenylalanine was low during active cell division, but increased before anthocyanin began to accumulate and concomitantly with increasing levels of CHS mRNA. Exogenous supply of phenylalanine at the time of low endogenous levels induced the elevation of CHS mRNA and anthocyanin accumulation. These results indicate that the elevation of endogenous phenylalanine levels, when cell division ceases, may cause the increase in CHS mRNA levels, resulting in increased CHS activity and subsequently in anthocyanin accumulation in Vitis suspension cultures.Abbreviations CHS chalcone synthase - CHFI chalcone flavanone isomerase - DOPA 3,4-dihydroxyphenylalanine - PAL phenylalanine ammonia lyase     

The effect of lead on the cell cycle in the root meristem ofAllium cepa L.

Summary Allium cepa (L.) adventitious roots were treated with lead (2.5 mg of Pb²⁺ [from Pb(NO₃)₂] per dm³) for 30–72 h. The cell cycle was studied by pulse labeling with [³H]thymidine. Mitotic activity kinetics, occurrence of disturbed mitoses (c-mitoses), and level of DNA synthesis were examined. It was found that lead prolonged the cell cycle and that cells in two phases of the cycle, G₂ and S, differed in their sensitivity to lead. Cells in G₂ were more sensitive; lead lengthened their cycle by 216% and disturbed the course of cell division by causing c-mitoses. Cells in S phase were less sensitive. Their cell cycle was longer by 55%. They went through their G₂ phase without major disturbances, mitosis in these cells was normal. During treatment ofA. cepa with lead, its destructive effects on cells were exerted only during the first few hours (around 6 h) of incubation. That is when the inhibition of mitotic activity, numerous disturbances of cell division, a decline in the number of cells synthesizing DNA, and a lower level of DNA synthesis were observed. As the incubation continued, the above processes were found to return to normal. In the discussion, data are presented supporting the hypothesis that during the initial period of exposure ofA. cepa to lead, this metal enters both the root apoplast and symplast, exerting a destructive effect on cells, while later, lead penetrates only into the root apoplast, and in this way remains harmless to cells.     

Evaluation of alginate-immobilized Lactobacillus casei for lactate production

Summary Lactate production by immobilized Lactobacillus casei has been studied. The cells were immobilized in alginate and the effect of variations in different parameters on product formation and productivity was investigated. The performance of the reaction was evaluated in stirred batch as well as in packed-bed conditions. pH control was a problem in the packed-bed reactor. In stirred batch experiments, nearly total glucose utilization was observed with a lactate yield of 90–99% and a total productivity of 1.6 g·l^–1·h^–1. Under standard conditions only a low percentage (3–4%) of the total lactate formed was the abetd-isomer. When immobilized cells were reused, increased formation of abetd-lactate took place, especially when the cell conditions were sub-optimal. After revitalization by exposure to growth nutrients the balance was restored.On leave from Microbiology Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou-310021, China Offprint requests to: Bo Mattiasson     

Production of α-tocopherol by sequential heterotrophic–photoautotrophic cultivation of Euglena gracilis

Photoautotrophic cultivation of Euglena gracilis results in cells with high α-tocopherol content but the final cell concentration is usually very low due to the difficulty of supplying light efficiently to the photobioreactor. On the other hand, Euglena grows heterotrophically to high cell concentrations, using various organic carbon sources, but the α-tocopherol contents of heterotrophically grown cells are usually very low. Sequential heterotrophic/photoautotrophic cultivation, by which cells are grown heterotrophically to high cell concentrations and then transferred to photoautotrophic culture for accumulation of α-tocopherol was therefore investigated for efficient α-tocopherol production. In batch culture, using glucose as the organic carbon source, the cellular α-tocopherol content increased from 120 μg g⁻¹ at the end of heterotrophic phase to more than 400 μg g⁻¹ at the end of the photoautotrophic phase. By using ethanol as the organic carbon source during the heterotrophic phase, adding corn steep liquor as a nitrogen source and optimizing light supply during the photoautotrophic phase, the α-tocopherol content of the cells at the end of the photoautotrophic phase increased to 1700 μg g⁻¹. A system consisting of a mini-jar fermentor (for the heterotrophic phase) and an internally illuminated photobioreactor (for the photoautotrophic phase) was then constructed for continuous sequential heterotrophic/photoautotrophic cultivation. The cells were continuously cultivated heterotrophically in the mini-jar fermentor and the effluent was continuously passed through the photobioreactor for α-tocopherol accumulation. In this way, it was possible to produce 7 g l⁻¹ cells containing about 1100 μg α-tocopherol per g-cell continuously for more than 420 h. The continuous process resulted in α-tocopherol productivity of 100 μg l⁻¹ h⁻¹ which is about 9.5 and 4.6 times higher than those obtained in batch photoautotrophic culture and batch heterotrophic cultures, respectively.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 °C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 °C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 °C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 °C than at 22 °C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell⁻¹) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x^−0.858. Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29–46%), followed by SPX-D (21–28%), 13-desMeC (21–23%), and 13-desMeD (17–21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 × 10⁴ cells mL⁻¹. As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x^−0.8986. In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60–87%) and lower proportions of SPX-D (3–12%) and 13-desMeD (1.6–10%) as compared to batch culture.     

Carbon-nitrogen relations during batch growth ofNannochloropsis oculata (Eustigmatophyceae) under alternating light and dark

Nannochloropsis oculata (strain CCAP 849/1) was sampled at least every 12 h over a 26-d period of batch culture growth in a 12 h/12 h light/dark illumination cycle. Exponential cell-specific growth rate was 0.5 d^–1. Cell division occurred during the dark phase, while ammonium uptake, pigment synthesis and cell volume increase occurred mainly during the light. Stationary phase cells were on average larger that the largest exponentially growing cells. The lag phase prior to cell division was short with the C/N ratio returning to 6.25 (from 28) within 2 d of refeeding with ammonium. Significant Chl.a synthesis commenced after this period; net synthesis of Chl.a ceased on exhaustion of the N-source with a 40% fall in levels by the end of the stationary phase. Levels of carotenoids per cell also declined during N-deprivation although per ml of culture levels remained constant. Ammonium-refeeding of N-deprived cells resulted in a very rapid rise in glutamine (Gln) and very high ratios of glutamine/glutamate (Gln/Glu peaking at 35 within 1 h); peak Gln/Glu was lower in cells refed in the dark or after a shorter period of N-deprivation. The major intracellular amino acids during exponential phase were Glu, Gln, alanine and arginine, but on exhaustion of the N-source, levels of Gln fell rapidly (Gln/Glu falling to below 0.1 from 0.5–0.9 in the light and 0.3 in darkness during exponential growth). During N-deprivation tyrosine accumulated within the cells. Comparisons are drawn with the growth ofIsochrysis galbana, another alga used in aquaculture, under identical conditions.Author for correspondence     

Mitochondrial function plasticity in <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> during growth in batch culture

The alterations in mitochondrial bioenergetics during growth in a batch culture of Acanthamoeba castellanii were studied. The capacity of cytochrome pathway-dependent respiration measured in vitro decreased from the intermediary phase, when cell division slowed down. The pattern of the cytochrome pathway capacity changes was paralleled from the intermediary phase by alterations in the amount of total (and reducible) membranous ubiquinone. These changes were accompanied by a decrease in mitochondrial reactive oxygen species production in vitro (when no energy-dissipating system was active), and almost no change in superoxide dismutase activity and protein level, thus indicating an equivalent need for this enzyme in oxidative stress defence in A. castellanii culture. On the other hand, a decrease in the activity and protein level of alternative oxidase and uncoupling protein was observed in vitro, when cells shifted from the exponential growth phase to the stationary phase. It turned out that the contribution of both energy-dissipating systems in the prevention of mitochondrial reactive oxygen species generation in vivo could lead to its constant level throughout the growth cycle of A. castellanii batch culture. Hence, the observed functional plasticity insures survival of high quality cysts of A. castellanii cells.     

COMPARATIVE PHYSIOLOGICAL STUDY OF MARINE DIATOMS AND DINOFLAGELLATES IN RELATION TO IRRADIANCE AND CELL SIZE. I. GROWTH UNDER CONTINUOUS LIGHT1,2

Cell division rates and chlorophyll a and protein contents for ten diatom and dinoflagellate species were measured. Species were chosen to include a wide range of cell size in terms of both cell volume and cell protein: from 0.004 ng protein/cell for a small Chaetoceros sp. to 2.2 ng protein/cell for Prorocentrum micans Ehrenberg. Experiments were conducted in batch or semi-continuous cultures at 21 C under continuous illumination from 8–256 μEin ^.m^-2'^.s^-1. Light saturation of cell division occurred at 32–80 μEin m^-1 s^-1 for all species, with no observable difference between the two phylogenetic groups. When the light-saturated cell division rates were plotted against cell size as protein/cell, the diatoms and dinoflagellates fell on two separate lines with the diatoms having higher rates. Chl a /protein ratios (μg/μg) decreased with increasing irradiance. The diatoms had higher chl a per unit protein. The relationship between cell division rate and the chl a/protein ratio is discussed.     

Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015