Measurement of Growth at Very Low Rates ((mu) >= 0), an Approach To Study the Energy Requirement for the Survival of Alcaligenes eutrophus JMP 134

Alcaligenes eutrophus JMP 134 was grown in a recycling-mode fermenter with 100% biomass retention on 2,4-dichlorophenoxyacetic acid (2,4-D), phenol, and fructose. The growth pattern obtained given a constant supply of substrates exhibited three phases of linear growth on all three substrates. The transition from phase 1 to phase 2, considered to correspond to the onset of stringent (growth) control as indicated by a significant increase in guanosine 5(prm1)-bisphosphate 3(prm1)-bisphosphate (ppGpp), took place at 0.016 h(sup-1) with 2,4-D and at about 0.02 h(sup-1) with phenol and fructose. In the final phase, phase 4, which was achieved after the growth rate on the respective substrates fell below 0.003 to 0.001 h(sup-1), a constant level of biomass was obtained irrespective of further feeding of substrate at the same rate. The yield coefficients decreased by 70 to 80% from phase 1 to phase 3 and were 0 in phase 4. The stationary substrate concentrations s(infmin) in phase 4, calculated from the kinetic constants of the strain, were 1.23, 0.34, and 0.23 (mu)M for 2,4-D, phenol, and fructose, respectively. These figures characterize the minimum stationary substrate concentrations required in a dynamic system to keep A. eutrophus alive. This is caused by a substrate flux which enables growth at a rate >=0 due to the provision of energy to an extent at least satisfying maintenance requirements. According to the constant feed rates of the substrates and the final and stable biomass concentrations, this maintenance energy amounts to 14.4, 4.0, and 2.4 (mu)mol of ATP (middot) mg of dry mass(sup-1) h(sup-1) for 2,4-D, phenol, and fructose, respectively, after correction for the fraction of living cells. The increased energy expenditure in the case of 2,4-D is discussed with respect to uncoupling.     

Measurement of Growth at Very Low Rates ((mu) >= 0), an Approach To Study the Energy Requirement for the Survival of Alcaligenes eutrophus JMP 134

Alcaligenes eutrophus JMP 134 was grown in a recycling-mode fermenter with 100% biomass retention on 2,4-dichlorophenoxyacetic acid (2,4-D), phenol, and fructose. The growth pattern obtained given a constant supply of substrates exhibited three phases of linear growth on all three substrates. The transition from phase 1 to phase 2, considered to correspond to the onset of stringent (growth) control as indicated by a significant increase in guanosine 5(prm1)-bisphosphate 3(prm1)-bisphosphate (ppGpp), took place at 0.016 h(sup-1) with 2,4-D and at about 0.02 h(sup-1) with phenol and fructose. In the final phase, phase 4, which was achieved after the growth rate on the respective substrates fell below 0.003 to 0.001 h(sup-1), a constant level of biomass was obtained irrespective of further feeding of substrate at the same rate. The yield coefficients decreased by 70 to 80% from phase 1 to phase 3 and were 0 in phase 4. The stationary substrate concentrations s(infmin) in phase 4, calculated from the kinetic constants of the strain, were 1.23, 0.34, and 0.23 (mu)M for 2,4-D, phenol, and fructose, respectively. These figures characterize the minimum stationary substrate concentrations required in a dynamic system to keep A. eutrophus alive. This is caused by a substrate flux which enables growth at a rate >=0 due to the provision of energy to an extent at least satisfying maintenance requirements. According to the constant feed rates of the substrates and the final and stable biomass concentrations, this maintenance energy amounts to 14.4, 4.0, and 2.4 (mu)mol of ATP (middot) mg of dry mass(sup-1) h(sup-1) for 2,4-D, phenol, and fructose, respectively, after correction for the fraction of living cells. The increased energy expenditure in the case of 2,4-D is discussed with respect to uncoupling.     

Growth Rates of a Pseudomonad on 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol

The growth of a pseudomonad on 2,4-D (2,4-dichlorophenoxyacetic acid) and 2,4-DCP (2,4-dichlorophenol) was studied in batch and continuous culture. The optimum growth rate using 2,4-D was 0.14/h at 25 C in a pH range from 6.2 to 6.9. Highest specific growth rate using 2,4-DCP was 0.12/h at 25 C in a pH range from 7.1 to 7.8. Growth was strongly inhibited by 2,4-DCP above a concentration of 25 mg/liter whereas no appreciable inhibition was observed with 2,4-D at concentrations up to 2,000 mg per liter. Growth on 2,4-DCP was described by Monod kinetics at subinhibitory concentrations but the inhibition by 2,4-DCP exhibited an unusual linear response to substrate concentration, and did not fit a model based on noncompetitive inhibition. The lag phase of batch cultures was found to depend on both 2,4-DCP concentration and prior adaptation of the inoculum. A study such as this on the kinetics of growth on related substrates may be useful as a method of finding the rate-limiting step in a metabolic sequence.     

Substrate inhibition under stationary growth conditions – nutristat experiments with Ralstonia eutropha JMP 134 during growth on phenol and 2,4-dichlorophenoxyacetate

Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP 134 was continuously grown on phenol and 2,4-dichlorophenoxyacetate at elevated levels of stationary substrate concentration by using the nutristat principle in order to study the physiological impact exerted by these toxic substrates. Growth at stationary concentrations of both the substrates resulted in the reduction of growth efficiency and growth rate. The growth yield data revealed a pronounced dependence on the substrate concentration, and the growth yield increasingly diminished with rising substrate concentration. Inhibition was more pronounced with 2,4-dichlorophenoxyacetate, which reduced the growth yield coefficient by 50% at a substrate concentration of 0.1–0.25 mM. The same effect was obtained with phenol at about 5 mM. The growth rate profile had two distinct phases: after an initially strong reduction, the rate levelled-off at higher substrate concentrations. Standardizing the inhibition profiles, by taking into account the maximum effect after extrapolating the data to zero growth yield, revealed an almost identical pattern with both substrates, indicating some common mechanism. The growth yield data show that an increased amount of energy is required for both growth and maintenance. Homeostatic work was increased by a factor of 8 at 75% inhibition; growth collapsed once this amount of energy was no longer available. The effects are discussed with respect to the properties of these substrates functioning as potential uncouplers of energy conservation. Received: 5 June 1997 / Received revision: 7 July 1997 / Accepted: 12 July 1997     

In vitro regeneration in Primula ssp. via organogenesis

Culturing pedicle segments of primroses on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2, 4-D) and thidiazuron (TDZ) resulted in callus induction rates of about 80%. The highest shoot regeneration rate (1.8 shoots per explant; mean of ten genotypes) was achieved with the combination of 2.0 mg/l 2, 4-D and 2.0 mg/l TDZ. Culture on a medium containing a high concentration of nitrate (for example, B5 medium) negatively affected the survival of regenerated shoots of one genotype, Gelb IV 48, probably due to an increase in the pH value of the medium. Consequently, the highest efficiency was obtained using a basal medium containing half-strength Murashige and Skoog macroelements. A protocol to regenerate shoots of Primula vulgaris and P. elatior is described.     

Adsorption and bio-degradation of phenol by chitosan-immobilized Pseudomonas putida (NICM 2174)

Biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different conditions. Pseudomonas putida (NICM 2174) cells immobilized in chitosan were used to degrade phenol. Adsorption of phenol by the chitosan immobilized matrix played an important role in reducing the toxicity of phenol. In the present work, results of the batch equilibrium adsorption of phenol on chitosan from its aqueous solution at different particle sizes (0.177 mm, 0.384 mm, 1.651 mm) and initial concentration of phenol (20, 40, 60, 80, 100, 120, 140, 160, 180, 200 mg/l) have been reported. The adsorption isotherms are described by Langmuir, Freundlich and Redlich-Peterson types of equations. These indicate favourable adsorption with chitosan. From the adsorption isotherms, the adsorption capacity, energy of adsorption, number of layers and the rate constants were evaluated. In batch kinetic studies the factors affecting the rate of biodegradation of phenol, were initial phenol concentration (0.100 g/l, 0.200 g/l, 0.300 g/l), temperature (30v°C, 34v°C, 38v°C) and pH (7.0, 8.0, 9.0). Biodegradation kinetic data indicated the applicability of Lagergren equation. The process followed first order rate kinetics. The biodegradation data generally fit the Lagergren equation and the intraparticle diffusion rate equation from which adsorption rate constants, diffusion rate constants and diffusion coefficients were determined. Intraparticle diffusion was found to be the rate-limiting step. Cell growth contributed significantly to phenol removal rates especially when the degradation medium was supplemented with a utilizable carbon source.     

Pleiotropic Effects of Adaptation to a Single Carbon Source for Growth on Alternative Substrates

It is frequently assumed that populations of genetically modified microorganisms will perform their intended function and then disappear from the environment due to inherent fitness disadvantages resulting from their genetic alteration. However, modified organisms used in bioremediation can be expected to adapt evolutionarily to growth on the anthropogenic substrate that they are intended to degrade. If such adaptation results in improved competitiveness for alternative, naturally occurring substrates, then this will increase the likelihood that the modified organisms will persist in the environment. In this study, bacteria capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were used to test the effects of evolutionary adaptation to one substrate on fitness during growth on an alternative substrate. Twenty lineages of bacteria were allowed to evolve under abundant resource conditions on either 2,4-D or succinate as their sole carbon source. The competitiveness of each evolved line was then measured relative to that of its ancestor for growth on both substrates. Only three derived lines showed a clear drop in fitness on the alternative substrate after demonstrable adaptation to their selective substrate, while five derived lines showed significant simultaneous increases in fitness on both their selective and alternative substrates. These data demonstrate that adaptation to an anthropogenic substrate can pleiotropically increase competitiveness for an alternative natural substrate and therefore increase the likelihood that a genetically modified organism will persist in the environment.     

Organogenesis and Plantlet Formation from Organ- and Seedling-Derived Calli of Rice (Oryza sativa)

Callus was induced in different somatic organs of Oryza sativa L. Specific minimum 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations in the medium were necessary for the induction of callus from different organs while high levels of 2,4-D (6–10 mg/l) induced callus formation in each organ tested. The optimum 2,4-D concentration for callus induction and growth for root-derived calli was 2 mg/l and for leaf-derived 6 mg/l. Root and shoot organogenesis were induced in both root- and leaf-derived calli by sub-culturing to a medium lacking 2,4-D. Root organogenesis occurred at a higher frequency than shoot organogenesis. Shoot organogenesis rarely occurred in calli without differentiated roots. Increased age of callus cultures almost completely inhibited shoot development. The addition of the cytokinin 6-γ,γ-dimethylallyl-amino purine partially restored the potential for shoot organogenesis. Whole plants were easily recovered from the calli and grown to maturity with some plants exhibiting phenotypic abnormalities.     

Adaptation of Delftia acidovorans for degradation of 2,4-dichlorophenoxyacetate in a microfluidic porous medium

Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (Cucurbita pepo l.) and melon (Cucumis melo l.)

Leaf explants of squash (Cucurbita pepo L.) and melon (Cucumis melo L.) were pretreated initially with 113.1, 226.2 or 452.4 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 46.5, 93 or 186 µM kinetin or a combination of both at the above concentrations, for 6, 24 or 48 h. After pretreatment, explants were transferred to an agar-solidified medium that was not supplemented with growth regulators or to a species-specific standard induction medium. Control explants from each species were incubated directly on the species-specific standard induction medium. Initial pretreatment of squash explants with 186 µM kinetin and of melon explants with 226.2 µM 2,4-D for 48 h significantly promoted the formation of somatic embryos which developed further to the torpedo-shape stage and germinated. Under these conditions at least four plants can be regenerated per square centimeter of explant surface, thus achieving an increase over non-pretreated cultures of 143% and 130% for squash and melon, respectively.     

Mathematical modeling of phenol degradation system using fuzzy comprehensive evaluation

New mathematical model for phenol degradation is developed that uses fuzzy comprehensive evaluation. Biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different conditions. In the present work, results of batch study on P. putida (NICM 2174) and its degradation activity on phenolic compounds such as phenol, o-cresol, p-cresol, p-nitrophenol and resorcinol each of concentration 0.300 g/l are considered. In the present study, the effect of glucose, yeast extract, ammonium sulphate and NaCl each at 0.5, 1.5, 2.0, 3.0 and 4 g/l on degradation of aforementioned phenolic compounds have been investigated and a fuzzy control model has been developed to predict the extent of degradation. Main aspect of this study is to establish a fuzzy relation matrix R for objective evaluation of phenol degradation. A series of membership functions for the degradation are being evaluated after investigating the growth properties of bacteria at various levels of carbon and nitrogen sources. Important element is the factor vector A, which is deduced from a survey of panel of judges (n=25). A in conjunction with R generates a multifactorial equation which can be used to calculate the extent of phenol degradation. Biomass growth contributed significantly to phenol degradation rates especially when the degradation medium was supplemented with a utilizable carbon and nitrogen sources.     

Destruction of chlorinated derivatives of phenol: ortho-chlorophenol, para-chlorophenol, and 2,4-dichlorophenoxyacetic acid by bacteria communities in anaerobic sludge

The bacterial community of anaerobic sludge could degrade ortho-chlorophenol, para-chlorophenol, and 2,4-dichlorophenoxyacetic acid at concentrations as high as 100 mg/l. The time needed for the degradation of a given chlorinated phenol derivative increased 1.5- to 2-fold upon a twofold increase in its concentration (from 50 to 100 mg/l). The duration of the adaptation period depended on the compound studied and on its concentration. The degradation of 2,4-dichlorophenoxyacetic acid proceeded via 2,4-dichlorophenol and p-chlorophenol as intermediates; the degradation of o-chlorophenol occurred with the formation of phenol. The dynamics of p-chlorophenol degradation and chloride ion accumulation were studied.     

Determination of the Ks values during the growth of Alcaligenes eutrophus on phenol, 2,4-dichlorophenoxyacetic acid and fructose

The determination of the K_S values presented here is based on the estimation of the stationary substrate concentrations in continuous cultivation experiments. The separation of biomass from the suspension was performed by an ultrafiltration step which succeeded within one second. The decay of substrate concentration during sampling was calculated to amount to less than 6% of the stationary substrate concentration at relevant growth rates. The K_S values derived from these reduced substrate concentrations deviated by only 10% from the theoretical values at a biomass concentration of about 1 g/1. Thus relevant kinetic parameters can be calculated from the data obtained by this procedure. Values of 11, 59 and 14 μM were obtained with 2,4-dichlorophenoxyacetic acid (2,4-D), phenol and fructose, respectively. Similar K_S values were derived with 2,4-D and fructose by using a respirationbased determination for reasons of comparison. With phenol this value was only 7 μM which is as cribed to a physiological background.     

Isolation and characterization of a 2,4-dichlorophenoxyacetic acid-degrading soil bacterium

Summary A 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, Xanthobacter sp. CP, was isolated after enrichment in aerated soil columns. A limited number of chlorinated phenols and chlorinated phenoxyalkanoic acids with an even number of carbon atoms in the side chain served as substrates for growth, although whole cells exhibited oxygen uptake with a wide range of those compounds. The maximal growth rate with 2,4-D was 0.13·h^-1 at a growth yield of 0.1 g biomass/g 2,4-D. Chloride ions were released quantitatively from 2,4-D and related chlorinated aromatic compounds which served as growth substrates. No by-products of 2,4-D metabolism were detected in oxygen-sufficient cultures of Xanthobacter sp. CP and catechols were cleaved exclusively by catechol 1,2-dioxygenase.     

Effect of auxins on berberine synthesis in cell suspension culture of Coscinium fenestratum (Gaertn.) Colebr--a critically endangered medicinal liana of Western Ghats

Cell suspension culture of critically endangered Coscinium fenestratum was established from young leaf segments on WPM supplemented with auxins. Effect of 2,4-D, IAA, IBA and NAA was examined on cell growth and berberine production. Berberine was synthesized and released continuously into the liquid medium. Presence of 2,4-D stimulated cell growth, but was not inhibitory on berberine synthesis. On the contrary, NAA stimulated berberine biosynthesis, but was not favourable for cell growth. Among the auxins tested, highest yield of berberine (5.79 mg/30 ml; 4.14 times to that of control) was obtained with 4 mg/l of NAA, while the best cell growth (214.43 mg dry wt., 1.96 times to that of control) was observed in the presence of 2 mg/l of 2,4-D. IAA and IBA were not favourable for cell growth and berberine synthesis.     

Early cellular events during induction of carrot explants with 2,4-D

Summary The cellular events occurring in carrot hypocotyl explants during long-term and pulse treatment with 2,4-D were followed using different techniques (light and transmission electron microscopy, flow cytometry, PCNA staining). Different morphogenetic pathways were induced under the various experimental conditions. Nevertheless, in the explants the activated cells were the same (provascular cells) and they showed very similar structural and ultrastructural changes. The long-term treatment with 2,4-D induced rapid re-activation of the cell cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - PCNA proliferating cell nuclear antigen - ER endoplasmic reticulum - GA Golgi apparatus     

向日葵悬浮培养再生芽

从54份向日葵(Helianthus annuus L.)材料中筛选出三个再生能力较强的基因型,取其下胚轴诱导愈伤组织,再以愈伤组织制备悬浮细胞系。在 MS 添加 BA(0.5mg/l)和2,4-D(0.001mg/l)液体培养基上确定细胞生长量,悬浮培养细胞在去掉2,4-D 的 MS 培养基上能够诱导芽的再生。     

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.     

Uptake kinetics of 2,4-dichlorophenoxyacetate by Delftia acidovorans MC1 and derivative strains: complex characteristics in response to pH and growth substrate

The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20-100 microM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 microM 2,4-D at either pH, and amounted to 15-20 nmol/min x mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min x mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.