Effects of anaerobiosis on ethylene production, respiration and flowering in iris bulbs

Ethylene production of iris bulbs (Iris hollandica cv. Ideal) was very low. When stored at 30°C, production was 12–20 pmol C₂H₄ (kg fresh weight)^?1 h^?1. Higher temperatures (35°C, 40°C) enhanced the ethylene production; a treatment with 40°C for ca 7 days caused a 3 times higher ethylene production than at 30°. During anaerobic storage (in 100% N₂) ethylene production was equal to that of control bulbs. When after a 7 day period of anaerobiosis the N₂ was replaced by air, a burstlike ethylene production was observed. Twenty-four h after the replacement, ethylene production was equal to control values again. The effects of this production of ethylene on mitochondrial respiration and flowering were investigated. When mitochondria were isolated immediately after the anaerobic treatment (before the enhanced ethylene production) alternative pathway capacity was not detectable, a situation also occurring in control bulbs. When mitochondria were isolated 24 h after the end of the anaerobiosis (after the ethylene burst) uninhibited respiration did not change significantly, but a capacity of the alternative pathway was observed. The increase in alternative pathway capacity after anaerobiosis was partly inhibited by 2,5-norbornadiene (NBD), an ethylene antagonist. Fermentation occurred during anaerobiosis: ethanol concentrations increased during the treatment and decreased when air was supplied. When bulbs were exposed to ethanol vapour the alternative pathway was induced but only when very high ethanol levels in the bulbs were reached. The amount of ethanol accumulated in the bulbs during a 7 day anaerobic treatment was far too low to explain the observed induction of alternative pathway capacity. Flowering percentages were enhanced after a 24 h treatment with ethylene and after a 7 day anaerobic treatment. NBD significantly inhibited the effect of exogenous ethylene and of anaerobiosis on flowering. Ethanol was not able to induce flowering. The burst-like production of ethylene after anaerobiosis probably is responsible for the effects on respiration and flowering.     

Continuous production of ethanol in high concentration using immobilized growing yeast cells

Summary Application of an immobilized growing yeast cell system to continuous production of ethanol in high concentration (10%) was investigated using Saccharomyces cerevisiae IFO 2363. When a medium containing 25% glucose was fed, the growth of yeast cells in gel was inhibited. The inhibitory effect was found to be reduced by a stepwise increase in concentration of glucose in the feed medium. The stepwise operation resulted in constant growth of cells in the gel even in the medium containing 25% glucose. By this stepwise feeding system, continuous production of ethanol of 114 mg/ml was maintained at a retention time of 2.6 h for over 2 months and a conversion rate of glucose to ethanol of over 95% of theoretical, was achieved.     

Ethylene biosynthesis and strigol-induced germination of Striga asiatica

Germination of witchweed [ Striga asiatica (L.) Kuntze], an important parasite on cereal crops, is stimulated by several natural and synthetic compounds. In the present study the role of ethylene in germination of Striga asiatica in response to strigol was examined. Unconditioned seeds and those conditioned for 3 days produced negligible amounts of ethylene in response to strigol. However, extending the conditioning period to 5 and 8 days increased ethylene evolution by more than 10-fold. Ethylene production preceded radicle protrusion and was detectable within 3h after treatment. No germination was observed in the first 6 h of exposure to strigol. Germination and ethylene production increased with strigol concentration. Strigol-induced germination was considerably reduced by the ethylene action inhibitors. 2. 5-norbornadiene, silver thiosulphate and CO₂. The ethylene precursor 1-aminocyclopropane-1-carboxyac acid (ACC) at 5 to 200 μ M elicited neither germination nor ethylene production. However, a combination of strigol and ACC resulted in a high germination rate and copious ethylene production. Both germination and ethylene production were reduced by CoCl₂ and cyclobeximide, inhibitors of the ethylene-forming enzyme and of protein synthesis, respectively. The results are consistent with a model in which conditioning and strigol are required to remove a restriction on the ethylene biosynthetic pathway and in which the ethylene-forming enzyme is rate limiting.     

Ethylene Production by the Kudzu Strains of Pseudomonas syringae pv. phaseolicola Causing Halo Blight in Pueraria lobata (Willd) Ohwi

Significant amounts of ethylene was produced by Pseudomonassolanacearum (all strains), P. syringae pv. phaseolicola (Kudzustrains isolated from Pueraria lobata) and Erwinia rhapontici(2 strains out of 22) out of 24 species, 3 subspecies and 38pathovars of plant pathogenic bacteria tested in yeast extract-peptonebroth. The bean strains of P. syringae pv. phaseolicola causinghalo blight in kindney bean plants did not produce ethylene.The Kudzu strains produced ethylene at a rate of 7 to 100?10^–9nl cell^–1 h^–1, which was 500 to 1,000 times higherthan that of P. solanacearum and several times higher than thatof Penicillium digitatum, the most potent ethylene producerknown among microorganisms. The presence of living cells was essential for ethylene productionby the Kudzu strains. The bacterium effectively produced ethylenefrom amino acids such as glutamate, aspartate and their amides.Although glucose and succinate were also good substrates forethylene biosynthesis, the rate of ethylene production was significantlysmaller than that with glutamate. Methionine, which is knownas the precursor of ethylene in plants, had no effect on ethyleneproduction by the bacterium. 1-Aminocyclopropane-1-carboxylicacid (ACC) also had no effect on ethylene production, and therewas not enough ACC in the bacterial cells to account for thehigh rate of ethylene production. Ethylene production from glutamatewas inhibited by n-propylgallate and EDTA, but not by aminoethoxyvinylglycine.These results indicate that ACC is not involved as an intermediatein the process of ethylene biosynthesis by the bacterium, suggestingthe presence of a pathway different from that of plant tissues. (Received September 4, 1984; Accepted October 27, 1984)     

Ethylene production by metabolic engineering of the yeast Saccharomyces cerevisiae

The non-ethylene producing yeast, Saccharomyces cerevisiae, was transformed into an ethylene producer by introducing the ethylene forming enzyme from the plant pathogenic bacterium Pseudomonas syringae. Cultivation of the metabolically engineered strain was performed in well-controlled bioreactors as aerobic batch cultures with an on-line monitoring of ethylene production. The highest productivity was obtained during the respiro-fermentative growth on glucose but there was also a significant rate of formation during the subsequent phase of ethanol respiration. Furthermore, investigations were performed whether substitution of the original nitrogen source, NH(4)(+), for glutamate could improve productivity and yield of ethylene even more. The rationale being that one of the substrates for the enzyme is 2-oxoglutarate and this compound can be formed from glutamate in a single reaction. Indeed, there was a substantial improvement in the rate of production and the final yield of ethylene was almost three times higher when NH(4)(+) was replaced by glutamate.     

Novel Pathway for Alcoholic Fermentation of δ-Gluconolactone in the Yeast Saccharomyces bulderi

Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast. In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in delta-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2). (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism.     

Wound-induced Ethylene Formation in Albedo Tissue of Citrus Fruit

Excised albedo tissue of citrus fruit (Citrus unshiu and Citrus hassaku) produced ethylene at an increasing rate in response to wounding and aging. The application of 1-aminocyclopropane-1-carboxylic acid (ACC) enhanced ethylene production in both the fresh and aged tissues, but this increase was greater in the aged tissue than in the fresh tissue. ACC content was very low in fresh tissue but increased greatly in aging tissue, paralleling the rise in ethylene production. Aminoethoxyvinylglycine (AVG) strongly inhibited ethylene production in the aged tissue. In the presence of ACC, however, ethylene production was not inhibited by AVG. These results suggest that ACC is an intermediate in the pathway of ethylene biosynthesis in the albedo tissue and that both steps of ACC formation and ACC conversion to ethylene are enhanced by wounding and aging. Inhibitors of protein synthesis, cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide, strongly inhibited ethylene production in the albedo tissue, implying that protein synthesis is required to maintain the continuous evolution of ethylene. The stimulation of ethylene production by ACC was reduced by the addition of l-methionine, whereas d-methionine had very little inhibitory effect. Ethylene production in the albedo tissue was also inhibited by the addition of n-propyl gallate and 3,5-dibromo-4-hydroxybenzoic acid.     

Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation.

The rate of ethanol production per milligram of cell protein begins to decline in the early stage of batch fermentation before high concentrations of ethanol have accumulated. In yeast extract-peptone medium (20% glucose), this initial decline appears to be related to growth and to result in part from a nutrient deficiency. The addition of yeast extract, peptone, and ashed preparations of these restored the ability of glucose-reconstituted medium (in which cells had been previously grown) to support vigorous growth. Magnesium was identified as the active component. Supplementing fermentations with 0.5 mM magnesium prolonged exponential growth, resulting in increased yeast cell mass. The addition of magnesium also reduced the decline in fermentative activity (micromoles of CO2 evolved per hour per milligram of protein) during the completion of batch fermentations. These two effects reduced the time required for the conversion of 20% glucose into ethanol by 1/3 with no measurable loss in ethanol yield (98% of theoretical maximum yield). It is possible that some of the reported beneficial effects of complex nutrients (soy flour and yeast extract) for ethanol production also result from the correction of a simple inorganic ion deficiency, such as magnesium.     

Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation

The rate of ethanol production per milligram of cell protein begins to decline in the early stage of batch fermentation before high concentrations of ethanol have accumulated. In yeast extract-peptone medium (20% glucose), this initial decline appears to be related to growth and to result in part from a nutrient deficiency. The addition of yeast extract, peptone, and ashed preparations of these restored the ability of glucose-reconstituted medium (in which cells had been previously grown) to support vigorous growth. Magnesium was identified as the active component. Supplementing fermentations with 0.5 mM magnesium prolonged exponential growth, resulting in increased yeast cell mass. The addition of magnesium also reduced the decline in fermentative activity (micromoles of CO2 evolved per hour per milligram of protein) during the completion of batch fermentations. These two effects reduced the time required for the conversion of 20% glucose into ethanol by 1/3 with no measurable loss in ethanol yield (98% of theoretical maximum yield). It is possible that some of the reported beneficial effects of complex nutrients (soy flour and yeast extract) for ethanol production also result from the correction of a simple inorganic ion deficiency, such as magnesium.     

Induction by Electric Currents of Ethylene Biosynthesis in Cucumber (Cucumis sativus L.) Fruit

The effects of an electric current on ethylene biosynthesis were investigated in cucumber (Cucumis sativus L.) fruit that were producing almost no ethylene. Direct currents at 0.5 to 3.0 milliamperes induced much ethylene synthesis, with a rapid continuous increase in the rate, which reached a peak within 5 to 6 hours and then decreased. The rate of production was greater with a stronger current. Ethylene production was not observed after the use of a sine-wave alternating current (60 hertz) at 3 milliamperes, the magnitude at which a direct current had the greatest effect. The activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ethylene forming enzyme (EFE) increased before the rise in ethylene production. ACC synthase and EFE were activated sixfold and fourfold, respectively, by 2 hours. The concentration of ACC increased linearly up to 6 hours and then decreased. Ethylene induction by an electric current was suppressed almost completely by the infiltration of the cucumbers with 5 millimolar aminooxyacetic acid, an inhibitor of ACC synthase, and was also suppressed 70% by 5 millimolar salicylic acid, an inhibitor of EFE. The results indicate that the ethylene induced by the direct current was synthesized via the ACC-ethylene pathway as a result of electrical stress, a new kind of stress to be identified.     

Evolution of ethylene by Saccharomyces cerevisiae as influenced by the carbon source for growth and the presence of air.

Effects of the carbon source and oxygen on ethylene production by the yeast Saccharomyces cerevisiae have been studied. The amounts of ethylene evolved by the yeast culture were less than those detected in the blank (an equal volume of uninoculated medium), suggesting a net absorption of ethylene by the yeast cells. Addition of glucose to the lactate-grown yeast culture induced ethylene production. This glucose-induced stimulation of ethylene production was inhibited to a great extent by cycloheximide. Results suggested that the yeast cells in the presence of glucose synthesized an ethylene precursor and passed it into the medium. The conversion of this precursor to ethylene might be stimulated by oxygen. The fact that ethylene was produced by the yeast growing anaerobically and also by respiration-deficient mutants isolated from the wild-type yeast suggested that mitochondrial ATP synthesis was not an absolute requirement for ethylene biogenesis.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

Effect of ethanol on glucose transport, key glycolytic enzymes, and proton extrusion in Saccharomyces cerevisiae

The effect of ethanol on the activities of the key enzymes of the glycolytic pathway and on two membrane functions related with fermentation, the glucose uptake system, and proton extrusion rate are examined. The results indicate that ethanol, up to 2M, does not cause any change of the glucose uptake velocity nor any substantial change in the key glycolytic enzyme activities while the fermentation rate is reduced by about 50%. In a cell extract 3M ethanol as well as incubation of yeast cells with 4M ethanol caused a considerable decrease of pyruvate kinase and hexokinase activities. Phosphofructokinase remained unchanged even at higher ethanol concentrations. Transmembrane proton flow was found to be the most sensitive of the functions tested toward ethanol, and it could represent the first target of ethanol action on fermentation.     

Physiological difference during ethanol fermentation between calcium alginate-immobilized Candida tropicalis and Saccharomyces cerevisiae

Calcium alginate-immobilized Candida tropicalis and Saccharomyces cerevisiae are compared for glucose fermentation. Immobilized C. tropicalis cells showed a slight morphological alteration during ethanol production at 40 degrees C, but their fermentation capacity was reduced by 25%. Under immobilization conditions, the two species demonstrated two different mathematical patterns when the relationship between growth rate, respiration rate, and ethanol tolerance was assessed. The interspecific difference in behavior of immobilized yeast cells is mainly due to their natural metabolic preference. The production of CO(2) by calcium alginate-immobilized C. tropicalis, as well as the lower supply of oxygen to the cells, are the major factors that reduce ethanol production.     

Ethylene production by Botrytis cinerea in vitro and in tomatoes

A laser-based ethylene detector was used for on-line monitoring of ethylene released by the phytopathogenic fungus Botrytis cinerea in vitro and in tomato fruit. Ethylene data were combined with the results of a cytological analysis of germination of B. cinerea conidia and hyphal growth. We found that aminoethoxyvinylglycine and aminooxyacetic acid, which are competitive inhibitors of the 1-aminocyclopropane-1-carboxylic acid pathway, did not inhibit the ethylene emission by B. cinerea and that the fungus most likely produces ethylene via the 2-keto-4-methylthiobutyric acid pathway. B. cinerea is able to produce ethylene in vitro, and the emission of ethylene follows the pattern that is associated with hyphal growth rather than the germination of conidia. Ethylene production in vitro depended on the L-methionine concentration added to the plating medium. Higher values and higher emission rates were observed when the concentration of conidia was increased. Compared with the ethylene released by the fungus, the infection-related ethylene produced by two tomato cultivars (cultivars Money Maker and Daniela) followed a similar pattern, but the levels of emission were 100-fold higher. The time evolution of enhanced ethylene production by the infected tomatoes and the cytological observations indicate that ethylene emission by the tomato-fungus system is not triggered by the ethylene produced by B. cinerea, although it is strongly synchronized with the growth rate of the fungus inside the tomato.     

Construction of a xylose-metabolizing yeast by genome integration of xylose isomerase gene and investigation of the effect of xylitol on fermentation

A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.     

Nitrogen fertility and leaf age effect on ethylene production of cotton in a controlled environment

The effect of nitrogen (N) fertility and its subsequent impact on ethylene production varies with plant species. Additionally, ethylene production reportedly increases or decreases with leaf age for several species. We examined leaf age and N fertility effects on ethylene production of cotton (Gossypium hirsutum L.) during the early vegetative stages of development (14 to 42 days after emergence) in a controlled environment. Ethylene production was determined by sampling leaf discs from the topmost fully expanded, middle, and bottom leaves of the canopy at 14, 21, 28, 35, and 42 days after emergence. Ethylene was collected from leaf discs in sealed test tubes and quantified by gas chromatography. Early in development, a N deficiency was associated with elevated levels of ethylene, suggesting stress ethylene production was occurring in response to a N-deficiency stress. As plant development progressed, however, increased ethylene production was associated with higher levels of applied N. Additionally, higher ethylene production was linearly associated with higher chlorophyll levels in all three leaves sampled. Ethylene production within plants receiving any given rate of N initially increased and then decreased with leaf age. The dynamics of this relationship suggest that as the N status of the plant changes during plant development, the relative rate of ethylene production, with regard to leaf age, is significantly influenced.     

Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l⁻¹. At a glucose concentration of 100 g l⁻¹, SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD⁺/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.