Growth In Continuous Culture Research Articles

The Effect of Growth Conditions on the Respiratory System of a Succinoglucan-producing Strain of Agrobacterium radiobacter

SUMMARY: The respiratory system of Agrobacterium radiobacter NCIB 11883, a producer of succinoglucan exopolysaccharide under ammonia-limited conditions, was studied following growth in continuous culture (D = 0·045 h-1)under glucose, oxygen and ammonia limitation. The respiratory chain contained b- and c-type cytochromes, plus two terminal oxidases (aa 3 and co) under all growth conditions, and exhibited a low transhydrogenase activity. An inactive apoenzyme form of the quinoprotein glucose dehydrogenase was also present which could be activated by adding pyrroloquinoline quinone (PQQ) to cell suspensions. The activity of the terminal region of the respiratory chain increased approximately fourfold following growth under oxygen limitation, and this was accompanied by a significant increase in the concentration of cytochrome oxidaseco. Whole cells exhibited →H+/O quotients of 5·5-6·3 for the oxidation of endogenous substrate depending on the nature of the growth-limiting nutrient. It is concluded that the respiratory chain energy conservation system of this organism is not significantly modified during ammonia-limited growth to offset the increased energy demands for exopolysaccharide synthesis.

Investigation of the effect of growth environment on the stability of low-copy-number plasmids in Escherichia coli.

The stability of a low-copy-number plasmid, pHSG415, in Escherichia coli, was investigated in batch and continuous culture. The plasmid was unstable in batch culture, but was significantly stabilized by growth in continuous culture with phosphate, nitrogen or potassium limitation. However, the plasmid was very unstable when grown in continuous culture with sulphate limitation. These results contrast with those obtained with multicopy plasmids such as pBR322, which is particularly unstable in carbon- or phosphate-limited continuous culture. The effect of growth rate on the stability of E. coli(pHSG415) grown in continuous culture with glucose limitation was also investigated. The plasmid was significantly more stable in cells grown at higher growth rates. The segregational instability (R) of the plasmid and the difference in growth rate between plasmid-free and plasmid-bearing cells (dmu) were calculated for each condition using the method of Cooper et al. (accompanying paper: Journal of General Microbiology 133, 1871-1880). It was found that the primary cause of the loss of pHSG415 from the cell population was the segregational instability of the plasmid.

Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellobiose dehydrogenase remain to be elucidated.

Evaluation of Inorganic Phosphate on Growth and Lactose Metabolism of Lactic Streptococci in Batch and Continuous Culture

Addition of 1.9% inorganic phosphates (K2HPO4, 1.33% + KH2PO4, 0.57% wt/vol) in place of 1.9% disodium (β-glycerophosphate (GP) in M17 medium (M17P) resulted in increased buffering capacity. Even at equimolar concentration (88 mM), Na2HPO4 (M17P1) or K2HPO4 (M17P2) showed higher buffering than GP(M17). Cultures consistently showed lower cell density in M17P2 than in other buffered media after 7 h of growth at 32°C, suggesting that buffering media with Na2HPO4 is better than buffering with K2HPO4 for cultivation of lactic streptococci. The effect of buffering media with Na2HPO4 on culture growth and appearance of lactose-negative (Lac−) variants was also tested under continuous culture growth conditions in a chemostat. Growth of Streptococcus lactis C2 continuously at pH 6.8 for 168 h in M17P1, and M17 broths at 32°C failed to yield any lac− strains. Results showed that successful propagation of lactic streptococci in continuous culture growth can be achieved without enriching for cells with undesirable metabolic characteristics.

5] Large-scale production and recovery of human leukocyte interferon from peripheral blood leukocytes

Publisher Summary This chapter discusses the large-scale production and recovery of human leukocyte interferon (IFN-α) from peripheral blood leukocytes. Advances in the assessment of the biological and clinical efficacy of IFN-α have depended on the establishment of satisfactory protocols for their induction and purification from peripheral blood leukocytes or lymphoblastoid cell lines capable of continuous growth in culture. More recently, IFN-α has been produced in prokaryotic organisms through recombinant DNA technology. Together, these approaches have assured an ample supply of IFN-α for characterization. It is now evident that IFN-α derived from leukocytes consists of multiple subtypes, each with subtle differences in structure and biologic activity, whereas that derived following gene cloning consists of a single subtype with a restricted expression of biologic activity. More recently, a two-step chromatographic process has been used for the purification of IFN-α. The chapter describes the processes used for IFN-α production in facility and some of the characteristics of the isolated interferon.

Effects of temperature on cadmium toxicity to the green alga Scenedesmus acutus. II. Light-limited growth in continuous culture.

Use b culture was pumped off via a volume-regulating vertical tube. The nutrient solution used was based on the $300 medium of Mur (1971). The C-source was supplied by an air stream with 5~ CO2. The energy source was fluorescent light provided by one or two Osram-L 22W/25C circular tubes. In all experiments presented in this paper, pH was maintained at 7.0 + 0.I and temperature at 25.0 _+ 0.5~ Because in this set-up self-shading of algal cells causes light to be the growth-limiting factor, the limiting energy flow is not, as usual with continuous cultures of heterotrophic organisms, contained in the infiuent stream, but is supplied via the cylindrical surface of the culture vessel. Therefore, the 'classical' chemostat theory (Roels , 1983) does not apply and hence a new mathematical model had to be derived. Assuming ideai mixing and a constant volume as well as cylindrical symmetry, the biomass balance for steady state growth is

Activation of c-myb expression by phytohemagglutinin stimulation in normal human T lymphocytes.

The expression of c-myb in normal human T lymphocytes directly derived from a normal subject and not adapted to continuous growth in culture was found to be markedly increased after phytohemagglutinin stimulation. In the same cells, the expression of c-myc mRNA is a much earlier event compared with the appearance of c-myb mRNA, which takes place soon after that of histone H3 mRNA. The increase in c-myb expression was not due to a particular T-lymphocyte subset, as shown by in situ hybridization assays.

Activation of c-myb expression by phytohemagglutinin stimulation in normal human T lymphocytes.

The expression of c-myb in normal human T lymphocytes directly derived from a normal subject and not adapted to continuous growth in culture was found to be markedly increased after phytohemagglutinin stimulation. In the same cells, the expression of c-myc mRNA is a much earlier event compared with the appearance of c-myb mRNA, which takes place soon after that of histone H3 mRNA. The increase in c-myb expression was not due to a particular T-lymphocyte subset, as shown by in situ hybridization assays.

Cybernetic modeling of microbial growth on multiple substrates

The internal regulatory processes, which underlie a variety of behavior in microbial growth on multiple substrates, are viewed as a manifestation of an invariant strategy to optimize some goal of the cells. A goal-seeking or cybernetic model is proposed here, with the optimization obased on a short-term perspective of response to the environment. The model parameters are determined from the growth data on single substrates. The model predicts the entire range of microbial growth behavior on multiple substrates from simultaneous utilization of all sugars to sequential utilization with pronounced diauxic lags. It is shown to predict the many variations of the diauxic phenomenon in different growth conditions. The transients in continuous culture growth on mixed substrates caused by varying the feed strategies are easily simulated by this model. The framework of this model can be applied to batch or continuous culture growth of many bacteria on different combinations of substrates.

Nitrogen-fixing Growth in Continuous Culture of a Strain of Rhizobium sp. Isolated from Stem Nodules on Sesbania rostrata

A strain of Rhizobium sp. (ORS571), isolated from stem nodules on Sesbania rostrata, was grown as a N2-fixing continuous culture for up to five weeks (> 80 culture replacements). Under optimum conditions of O2 supply [< 9 m dissolved O2, 100160 nmol O2 (mg dry wt)−1 min−1], N2 fixation rates were 300400 nmol N2 (mg dry wt)−1 h−1 and nitrogenase activity in culture samples reached 2000 nmol C2H4 (mg dry wt)−1 h−1, the highest values yet recorded for any Rhizobium species. Other properties of the continuous cultures are described and a comparison is made with previous reports of N2 fixation by cultures of Rhizobium spp. Significant features were the greater tolerance to O2 of ORS571 and the retention of fixed N in the cells of this strain.

Stress-induced alignment of division plane in plant tissues grown in vitro

The architectural nature of cell patterns in plants, which are visible under the light microscope, offers strong intuitive evidence for a structural relationship between mechanical forces acting through the tissue and cell wall orientation at cytokinesis. However, a direct relationship between the two has never been convincingly established. We find that compressive forces at intensities between 500 and 800 mg mm−2 can induce a conspicuous spatial ordering of division activity when applied to sterile plant tissues during continued growth in culture; this results in cambium-like areas of cell wall co-planarity which are clearly distinguishable from the characteristically disorganized growth of unstressed regions of the explant, and implies the presence of an intracellular structural–mechanical sensor capable of directing the installation of the new cell partition. The experiment reported here, in which compressive forces were applied to plant tissue growing in vitro, provides an example of mechanical control of division plane.

SOME ASPECTS OF THE PHYSIOLOGY AND BIOCHEMISTRY OF MARINE FUNGI

Summary1. This review deals with certain aspects of the physiology and biochemistry of marine fungi. Though it is not easy to define what is meant by a marine fungus, the information presented here relates to those species that by consensus can be accepted as being truly marine. The material is in two parts, that relating to the higher fungi (Ascomycotina, Basidiomycotina and Deuteromycotina) and that relating to the lower fungi, particularly those zoosporic fungi that require sodium for growth.2. Higher marine fungi appear to have a similar carbon, nitrogen and vitamin nutrition to their terrestrial counterparts.3. Growth of higher marine fungi can be optimal in 100% sea water, but more frequently optimal growth is at a lower percentage. The percentage giving optimal growth may be determined by the rate of ion uptake required to generate the necessary turgor for growth. The tolerance of the vegetative phase of many terrestrial fungi to salinity appears little different from that of marine fungi. In general, members of the Basidiomycotina are particularly sensitive to salinity, while those of the Ascomycotina and Deuteromycotina are much more tolerant.4. The degree of tolerance to salinity may be dependent upon temperature and whether or not adaptation by the fungus has occurred.5. Maintenance of a suitable internal potassium concentration by a higher marine fungus is important for growth in a saline medium. Calcium appears to be necessary for the retention of potassium and organic solutes within the hyphae.6. It appears that higher marine fungi are able to maintain a ratio of potassium: sodium higher than that of sea water by the presence of a plasma membrane ATPase which may have a higher pH optimum than the equivalent enzyme from terrestrial counterparts.7. Higher fungi produce more glycerol as the salinity of the external medium is increased. Whether or not the compound is involved in osmoregulation has yet to be determined.8. Turgor is thought to be generated in higher marine fungi growing in sea water by organic solutes (predominantly glycerol and arabitol) and by ions, with the latter playing the major role. Though interpretation of the data depends on several assumptions, the high concentrations of sodium that seem likely to be present in hyphae or cells have implications for the activity of enzymes, if they have similar properties to those of higher plants. There is a need for information on the effects of high concentrations of ions on enzymes located in the cytoplasm of higher fungi.9. In spite of some experimental uncertainties, it seems that reproduction and spore germination of higher marine fungi are very much less affected by salinity than are the same processes of terrestrial counterparts.10. The zoosporic marine fungi require sodium for growth. Though the ion is required at high concentration for growth, sodium cannot be replaced by potassium. Evidence indicates that sodium is involved in the transport of solutes across the plasma membrane.11. The carbon and nitrogen requirements for the growth of the zoosporic marine fungi demand further investigation, particularly at the biochemical level. There is evidence that the respiration of these fungi possesses many interesting features.12. Vitamin requirements of the zoosporic marine fungi depend on the isolate under investigation. Vitamin B., does not now seem to be an obligatory requirement. The ability of phospholipids and sterols to stimulate growth requires further investigation.13. Further studies on marine fungi in the laboratory should focus particularly on growth in continuous culture. Physiological and biochemical studies would be helped by more precise guidance from those concerned with the ecology of these fungi.

The Utilization of 5-Oxoproline, Ammonia and Glutamine by Rhizobium leguminosarum in Chemostat Culture

The growth in continuous culture of Rhizobium leguminosarum on the nitrogen sources glutamine, ammonia, or ammonia and 5-oxoproline (formed when aqueous glutamine solutions are autoclaved) is described. When growth was nitrogen-limited with autoclaved glutamine the specific rate of 5-oxoproline utilization was independent of dilution rate, whereas the specific rate of ammonia utilization increased with dilution rate. Although the culture was nitrogen-limited, excess nitrogen (as 5-oxoproline) was still present in the supernatant, especially at high dilution rates. Values obtained for Y max glucose and Y max oxgen were 72·2 and 32·5 g dry wt bacteria (mol substrate)−1, respectively, under ammonia/5-oxoproline limitation and 95·7 and 30·2 g dry wt bacteria (mol substrate)−1, respectively, under glutamine limitation.

Host and T-DNA determinants of cytokinin autonomy in tobacco cells transformed byAgrobacterium tumefaciens

The hormone autonomy of tobacco (Nicotiana tabacum L.) cells transformed byAgrobacterium tumefaciens containing mutations attmr (the "rooty" locus) of the pTiT37 plasmid has been examined. These cells require cytokinin, but not auxin for continuous growth in culture, indicating that the function of thetmr locus is to specify or induce cytokinin autonomy. Examination of tissues from plants regenerated from cells transformed by the mutant bacteria showed that the auxin independent phenotype is suppressed, but can be reinitiated in culture by exposure to an exogenous supply of auxin. In addition the developmental state of the cells from such regenerated plants can exert a profound influence on their cytokinin autonomy phenotype.

Continuous culture growth of photoautotrophic cell suspensions from Chenopodium rubrum

The construction and operation of a continuous culture system for the propagation of cell suspensions from Chenopodium rubrum under photoautotrophic conditions has been described. A dilution rate of 0.16/day gave an equilibrium culture density of 1,100,000 cells/ml and a mean doubling time of 150 hours. During continuous culture steady state conditions with respect to nutrient uptake, cell protein and chlorophyll content, starch accumulation, in vitro activities of enzymes related to different metabolic pathways could be established. Photosynthetic CO2 assimilation of steady state cells was about 100 mol CO2/mg chlorophyll x hour. Dark CO2 fixation was 3% of the light values.

The effect of amino acids and amino acid analogues on growth of an obligate methanotroph, Methylosinus trichosporium OB3b

AbstractThe sensitivity of the obligate methanotroph, Methylosinus trichosporium OB3b to a number of amino acid analogues and amino acids has been examined. Sulphaguanidine (5 μg ml−1) norleucine (100 μg ml−1) m‐fluorophenylalanine (50 μg ml−1), p‐fluorophenylalanine (50 μg ml−1) and S2‐aminoethyl‐C‐cysteine (1000 μg ml−1) inhibited growth. Proline, threonine, methionine and lysine (2–4 μg ml−1) also inhibited growth, but arginine and leucine at similar concentrations had no effect. Attempts were made to isolate amino acid analogue resistant mutants, which would be expected to overproduce amino acids. Two approaches were made, selection of spontaneous and induced mutants in plate culture, and selection of a spontaneous mutant during growth in continuous culture under selective conditions, but no mutants were obtained. Possible reasons for failure to obtain such mutants are discussed.

The Relationship of Indole-3-Acetic Acid Content and Growth of Crown-Gall Tumor Tissues of Tobacco in Culture

We have measured the content of the auxin, indole-3-acetic acid (IAA), in cloned, crown-gall teratoma line of Nicotiana tabacum L. cv. "Turkish" by a highly specific and sensitive radioimmunoassay. This tissue line, which does not require auxin for continuous growth in culture, exhibits two phases of growth. During the first phase, which lasts about two weeks after subculturing, growth is exponential on a fresh weight basis and the content of IAA is about 10-7-10-6 mol.kg-1, but variable. During the second phase, growth rate declines gradually and the IAA content of the tissue drops dramatically; however, this drop does not result from a net loss of auxin by the tissue. The rate of growth during the exponential phase was not correlated with the IAA content of the tissue, but was strongly correlated with the IAA content of the inoculum. We found that rapidly growing leaves of Turkish tobacco have roughly the same IAA content as cultured teratoma tissues. Moreover, both tissues exhibited a similar relationships between auxin content expressed per leaf or per tissue explant and growth. These findings do not support the hypothesis that the autonomous growth of plant tumors results from an abnormally high content of auxin in the tissue.

Response of freshly isolated strains of Streptococcus mutans and Streptococcus mitior to change in pH in the presence and absence of fluoride during growth in continuous culture.

A study was undertaken to compare the effects of pH and fluoride on the growth and metabolic properties of Streptococcus mutans 2452 and Streptococcus mitior 572, strains recently isolated from 8-year-old school children and grown in continuous culture with a glucose limitation. Each experiment had four consecutive stages of growth: (i) pH 7.0, (ii) no pH control, (iii) pH 7.0, and (iv) no pH control plus 50 mug of fluoride per ml in the medium. At a dilution rate (D) of 0.13 h (-1), cells of S. mitior possessed high glycolytic activity at pH 7.0 in the initial stage, but were washing out of the chemostat within 24 h after the pH control was shut off and the pH fell to 5.1. Once the culture was reestablished at pH 7.0, fluoride (50 mug/ml) was added to the medium and the pH control was again turned off. Whereas cell numbers fell from 24.0 x 10(8) to 0.9 x 10(8)/ml within 24 h, the culture remained relatively constant during the following 6 days despite the fall in pH to 5.4. The cells from this culture also maintained an intermediate glycolytic rate of 0.44 mumol mg(-1) min(-1). The cells in this latter stage developed phenotypic resistant to fluoride at concentrations up to 16 mM. Growth of S. mitior at D = 0.034 h(-1) resulted in a slower response to environmental change such that cells were able to grow to pH values as low as 5.2 in the absence of fluoride. In contrast to S. mitior, S. mutans 2452 under the same conditions at D = 0.13 h(-1) grew to higher cell numbers and higher yields and was able to maintain significant cell numbers to pH 4.8 once the pH control was shut off in the presence and absence of fluoride. S. mutans had 40% less glycolytic activity but was fourfold more resistant to fluoride at the start of the experiment, and cells were shown to adapt to growth at low pH and to fluoride at levels as high as 20 mM. This fluoride resistance by freshly isolated S. mutans 2452 was significantly higher than that of S. mutans DR0001 grown under identical conditions in the chemostat. S. mutans DR0001 is a strain which has been subcultured in vitro for several years. This study demonstrated that S. mutans 2452 was more aciduric than S. mitior 572 and, unlike the latter organism, could grow at pH values below 5.1. The addition of fluoride to the medium stabilized the S. mitior culture in the absence of pH control, indicating that whereas fluoride does suppress growth and glycolytic activity it also results in higher environmental pH values, which permit the survival of the less aciduric bacteria.

Intracytoplasmic membranes in oxygen-limited chemostat cultures of Methylosinus trichosporium OB3b: Biocatalytic implications of physiologically balanced growth

The continuous culture growth conditions for induction of intracytoplasmic membranes in Methylosinus trichosporium OB3b are described. During oxygen-limited, nitrate-excess chemostat culture, organisms have an extensive intracytoplasmic membrane system and particulate, cell-free methane mono-oxygenase (MMO). Under methane limitation fewer intracytoplasmic membranes are seen, while under all other conditions tested, membranes are absent and cell-free MMO is entirely soluble. These findings may be important in relation to the development of oxidative biotransformation processes using this bacterium.

Mathematical models for continuous culture growth dynamics of mixed populations subsisting on a heterogeneous resource base. II. Predation and trophic structure

Models are presented for the joint dynamics of predators and prey, maintained in continuous flow chemostat culture. The predators are visualized as subsisting on one or more prey organisms, which in turn are visualized as subsisting on one or more substrate resources supplied by the investigator. The dynamic equations are translated into an analogous Lotka-Volterra predation model, and the criteria for the existence and stability of various equilibria are indicated. Denoting the number of different predator organisms as N H , the number of different prey organisms by N I and the number of different substrates as N J , it is shown that the joint coexistence of all components requires 0 ⩽ N I − N H ⩽ N J . The model is extended to more complex situations by including additional trophic layers and by allowing trophic layer “leap-frogging.” The model may always be translated into an approximately quadratic differential equation of the Lotka-Volterra type. The α- and β-coefficients of these latter are really variables, and become quite complex for some of the multi-layered models.