Day-night changes of citric-acid levels in crassulacean acid metabolism: phenomenon and ecophysiological significance

Abstract A reappraisal is offered of old and new observations of substantial day-night changes of citric-acid levels in crassulacean acid metabolism (CAM). In contrast to malic acid, the biochemical consequences and the ecophysiological significance of nocturnal accumulation of citric acid in CAM have not received due attention. Considerations show that citric-acid accumulation does not provide a means for nocturnal storage of CO₂. It may, however, serve carbon retention by internal recycling and sustain the water budget affording a vacuolar osmoticum. Since citric-acid accumulation energetically is considerably more favourable than malic-acid accumulation, this may have important ecophysiological implications. The questions raised by these reflections can and need to be tackled experimentally.     

Addressing the critical challenge for rhamnolipid production: Discontinued synthesis in extended stationary phase

Rhamnolipids are commonly produced using N-limited Pseudomonas aeruginosa fermentations, with active production in the stationary phase. The production, however, stops after certain period. This causes lower productivity and yield because of downtime between batches and the substrate consumed for cell growth. This discontinued production is the significant problem addressed in this study. Rhamnolipid synthesis involves complex regulatory mechanisms, including quorum-sensing systems and alternative sigma factors. Current knowledge on these mechanisms, however, cannot adequately explain the discontinued rhamnolipid production at extended stationary phase. Four hypotheses to causes of this discontinued production were examined here with carefully planned fermentation designs using different initial N-source concentrations and different ways of adding oil and nutrients. Results rejected three of the hypothetical causes: accumulation of rhamnolipids or other inhibitory metabolites, presence of high amounts of oil phase, and exhaustion of non-N nutrients, and supported the hypothesis that the stopped production was caused solely by the extended N-starvation experienced by the cells. The discontinued production was found to be fully reactivated by partial broth replacements with fresh media containing N-source. The finding is important to production economics and promotes new designs to maximize rhamnolipid productivity and yield using extended stationary-phase production instead of short repeated batches.     

Effect of different carbon sources on the regulation of carbohydrate metabolism inSaccharomyces cerevisiae

The carbohydrate metabolism ofSaccharomyces cerevisiae is strongly influenced by the concentration and the nature of the carbon source. As long as glucose is present in the growth medium, the cells possess a predominantly glycolytic pathway of degradation and low levels of α-glucosidase and of those enzymes of the citric-acid cycle, the respiratory chain, and the glyoxylate cycle, which are localized in the mitochondria. After the depletion of glucose the level of these enzymes rises considerably. As long as the carbon source can be demonstrated in the medium, maltose-grown cells have a greater oxidative activity and a higher level of these enzymes than glucose-grown cells, unlike glucose-grown cells they easily adapt to ethanol and acetate. Catabolite repression is suggested as an important factor in the regulation of synthesis of enzymes of the citric-acid cycle, the glyoxylate cycle and the respiratory chain. There is an obvious correlation between the regulation of α-glucosidase and of the enzymes of oxidative carbohydrate metabolism.     

Effects of glucose limitation on biomass and spiramycin production by Streptomyces ambofaciens

Spiramycin production by Streptomyces ambofaciens Sp181110 with glucose as the carbon source was studied under a controlled nutritional environment. In a batch culture, the glucose excess after ammonium depletion led to pyruvate and α-ketoglutarate accumulation. 85 mg/l of spiramycin were produced in less than 70 h during the stationary and maintenance phase on these acids after glucose exhaustion. Fed-batch strategy was designed to study spiramycin production without by-product formation and glucose accumulation. In these conditions, up to 150 mg/l were produced in less than 80 h during the stationary phase on glucose. The antibiotic titre was found independent of the glucose feeding under carbon limitation and the importance of putative intracellular reserves formed after nutrient exhaustion was suggested. Besides, spiramycin production was not inhibited by the limiting flux of glucose.     

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.     

Production and bioassay of bialaphos biosynthesized by Streptomyces hygroscopicus NRRL B-16256

Production and bioassays of the easily biodegradable herbicide bialaphos was investigated using the producer Streptomyces hygroscopicus NRRL B-16256. In glucose containing (1.5%) medium (pH 6.0) with complex N-source(peptone 0.5%) the herbicide production and growth attained peaks on around day 6. Production underwent catabolic repression in presence of glucose without complex N-source. The product was identified by HPTLC against authentic sample (R_f 0.49). Bioassays were conducted using Bacillus magaterium and green leaf.     

Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae.

Metabolic changes have been investigated during continuous growth of yeast cells inoculated in glucose-containing medium until the cells entered the stationary phase in response to glucose exhaustion. Well in advance of glucose exhaustion, a transition phase was observed, characterized by a decrease in the growth rate and a progressive reduction of protein and RNA accumulation. Two-dimensional gel analysis of the proteins synthesized during this stage showed that the pattern of proteins remained similar to that of log-phase cells. When the cells entered the stationary phase, protein accumulation was 10% of that in log-phase cells, and incorporation of labeled RNA precursor was undetectable. Analysis of protein synthesis gave evidence that the synthesis of 95% of the proteins present in log-phase cells was arrested in stationary-phase cells. Among the 20 proteins whose synthesis continues throughout the stationary phase were identified actin, aldehyde dehydrogenase, enolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and five heat shock proteins. In addition, the synthesis of six new proteins was observed. The occurrence of these new proteins in stationary-phase cells is presumed to result from the release of carbon catabolite repression due to glucose exhaustion.     

Interrelation between cyanophycin synthesis, L-arginine catabolism and photosynthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

Ultrastructural and immunocytochemical investigations gave evidence that cyanophycin (multi-L-arginyl-poly-L-aspartate) granules accumulate in the cyanobacterium Synechocystis sp. strain PCC 6803 under nutrient deficient growth conditions, especially under phosphate limitation. Besides nutrient deficiency, growth of Synechocystis PCC 6803 on L-arginine or L-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of L-arginine or L-asparagine with nitrate only caused minor cyanophycin accumulation. Growth of Synechocystis PCC 6803 on L-arginine as sole N-source caused substantial morphological and physiological changes, such as severe thylakoid membrane degradation with partial loss of pigments and photosynthetic activity leading to a phenotype almost like that seen under nutrient deficiency. In contrast to the wild type, the PsbO-free Synechocystis PCC 6803 mutant could grow on L-arginine as sole N-source with only minor morphological and physiological changes. Due to its fairly balanced growth, the mutant accumulated only few cyanophycin granules. L-arginine degrading activity (measured as ornithine and ammonium formation) was high in the PsbO-free mutant but not in the wild type when cells were grown on L-arginine as sole N-source. In both cells types the L-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on L-arginine in combination with nitrate, and as expected very low when cells were grown on nitrate as sole N-source. Thus, net cyanophycin accumulation in Synechocystis PCC 6803 is regulated by the relative concentration of L-arginine to the total nitrogen pool, and the intracellular L-arginine concentration is greatly influenced by the activity of the L-arginine degrading enzyme system which in part is regulated by the activity status of photosystem II. These results suggest a complex interrelation between cyanophycin synthesis, L-arginine catabolism, and in addition photosynthesis in Synechocystis PCC 6803.     

光合细菌(Rhodopseudomonas capsulata)固氮活性与钼的吸收

利用紫色非硫细菌能在厌气光照下和好气黑暗下交替生长的特点和同位素~(99)Mo示踪,来探讨Rhodopseudomonas capsulata中Mo的积累与固氮酶合成的关系。 用硫酸铵和谷氨酸盐作为氮源,把Rps. capsulata置于厌气光照下生长。由于硫酸铵阻遏固氮酶,所以菌体内既无固氮酶活性也无~(99)Mo积累。而谷氨酸盐解遏固氮酶的合成,菌体则显示固氮活性并有~(99)Mo积累。 黑暗好气生长的Rps. capsulata菌体既无固氮活性,也没有~(99)Mo的积累。将这样的菌体转移到含~(99)Mo(无谷氨酸)的培养液进行光照,固氮酶活性迅速出现,同时有~(99)Mo的积累。在Rps. capsulata中钼的吸收与固氮酶的合成及活性是紧密偶联的。     

Growth and lipid production of Umbelopsis isabellina on a solid substrate—Mechanistic modeling and validation

Microbial lipids are an interesting feedstock for biodiesel. Their production from agricultural waste streams by fungi cultivated in solid-state fermentation may be attractive, but the yield of this process is still quite low. In this article, a mechanistic model is presented that describes growth, lipid production and lipid turnover in a culture of Umbelopsis isabellina on κ-carrageenan plates containing the monomers glucose and alanine as C-source and N-source, respectively, and improves the understanding of the complex solid-state system. The model includes reaction kinetics and diffusion of glucose, alanine and oxygen. It is validated empirically and describes the different phases of the culture very well: exponential growth, linear growth because of oxygen limitation, accumulation of lipids and carbohydrates after local N-depletion and turnover of lipids after local C-depletion. Extending the model with an unidentified extracellular product improved the fit of the model to the data. The model shows that oxygen limitation is extremely important in solid-state cultures using monomers, and explains the difference in production rate with submerged cultures. However, the results also show that the specific lipid production rate in solid-state cultures is much lower than in submerged cultures, which results in a low lipid yield.     

The glucose metabolic frame of conditions induce the overproduction of a co-culture pigment: induction mechanism of the pigment and development of a specific eutrophic–oligotrophic transition cultivation system

Induction mechanism of a potential red pigment (RPc) was investigated in the present paper. A typical competition relationship exists between Penicillium sp. HSD07B and Candida tropicalis during co-culture, and C. tropicalis converts glucose into glycerol, organic acids and other substances, resulting in a stricter glucose limitation and the secretion of RPc. Moreover, a novel eutrophic–oligotrophic transition cultivation system (E-OTCS) was developed to produce red pigment during monoculture of Penicillium sp. HSD07B. However, the monoculture pigment (RPm) is different from RPc in components, and RP3 and RP4 only occur in RPm when glycerol is supplied. In addition, the additions of glycerol and organic acids to glucose exhaustion medium can significantly improve the pigment yield. These facts not only prove the feasibility of producing RPm using E-OTCS, but also reveal that, besides glucose exhaustion, the accumulation of metabolites of glucose including glycerol and organic acids is also an important factor influencing the production of RPc.     

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     

Characterization of the stringent and relaxed responses of Streptococcus equisimilis.

The 739-codon rel(Seq) gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3'-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel(Seq) gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel(Seq)-dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, approximately 0.7 min(-1)). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel(Seq) insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel(Seq) fulfils functions that reside separately in the paralogous reL4 and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.     

Changes in the yeast metabolism at very high-gravity wort fermentation

The rate of ethanol production increased with increasing wort gravity up to the initial wort concentration of 24%, reaching the maximum ethanol concentration of 6.2%, but its attenuation reached only 49%. The intracellular trehalose accumulation was proportional to the inital wort gravity, at 24 or 30% wort fermentation increased 3 or 4.5 times, respectively, compared to 12% wort fermentation. Trehalose accumulation began after exhaustion of glucose, ceased after uptake of approximately 65% reducing saccharides, despite of increasing ethanol or remaining saccharide concentration in the environment.     

Optimization of Escherichia coli growth by controlled addition of glucose

During aerobic growth of Escherichia coli (recombinant K-12 and strain B) on protein hydrolysate (L-broth) and a carbon source (glucose), acetic acid is produced via glucose metabolism until the late log phase. At this point, the culture pH starts to increase and the growth rate decreases. In cultures without further glucose supplementation, these changes are associated with the accumulation of ammonia, the utilization of acetic acid, the depletion of amino acids, and the complete depletion of glucose. We hypothesize that, after depletion of the glucose, the bacteria catabolize amino acids for energy and carbon and give off the nitrogen as ammonia. Also contributing to the overall increase in pH is the depletion of the acetic acid produced earlier as it is metabolized upon exhaustion of glucose. However, there is a lag time of about 1 hour after the initial pH increase before the sustained accumulation of ammonia begins. This lag indicates that an unidentified factor, in addition to the increase in ammonia, contributes to the increase in pH. Advantage was taken of the turnaround from acid production to base production as reflected in the culture pH to implement the addition of glucose. In growth experiments during which the pH was controlled in the basic direction by glucose addition, the observed decrease in growth rate was significantly postponed and the pH change in the basic direction was reversed as a result of acid production by the cells from the newly added glucose. Furthermore, coll densities of twice that obtained without glucose feeding were demonstrated. Based on the media cost per unit cell density, the data indicate a 31% cost savings.     

Effect of prolonged exercise on the level of triglycerides in the rat liver

This study examined the effect of prolonged exercise on the level of triglycerides (TG) in rat liver. The rats were divided into groups: 1-control, 2-treated with nicotinic acid, 3-fed with glucose during exercise, 4-fasted, 5-adrenalectomized, 6-adrenalectomized and fed with oil. In the control group, there was gradual accumulation of TG in the liver and their level was doubled at exhaustion as compared to the resting value. Nicotinic acid lowered the resting level of TG and prevented their accumulation during exercise. Administration of glucose during exercise partially prevented the increase in TG level in the liver. In rats fasted for 24 h before exercise, the net increase in liver TG level during exercise was similar to that in the controls. Adrenalectomy, like nicotinic acid, lowered TG level at rest and prevented its increase during exercise. Feeding the adrenalectomized rats with oil elevated the plasma free fatty acid level but did not result in accumulation of TG in the liver, either at rest or during exercise. It is concluded that prolonged exercise results in accumulation of TG in the liver and that the process depends on the supply of free fatty acids and glucose and requires the presence of glucocorticoids.     

Effect of carbon and nitrogen sources on neutral proteinase production byPseudomonas aeruginosa

A strain ofPseudomonas aeruginosa from soil produced large quantitaties of extracellular neutral proteinase and could utilize several organic substances as carbon and nitrogen sources for enzyme production. The growth media required the presence of a high amount of phosphate when glucose was the carbon source. The intermediates of citric-acid cycle acids supported the proteinase production more than any other carbon sources. However, complex nitrogenous substances supported enzyme production more efficiently. Higher concentration of casamino acids suppressed the proteinase synthesis.     

Variation and evolution of the citric-acid cycle: a genomic perspective.

The presence of genes encoding enzymes involved in the citric-acid cycle has been studied in 19 completely sequenced genomes. In the majority of species, the cycle appears to be incomplete or absent. Several distinct, incomplete cycles reflect adaptations to different environments. Their distribution over the phylogenetic tree hints at precursors in the evolution of the citric-acid cycle.     

Improvement of erythromycin production by Saccharopolyspora erythraea in molasses based medium through cultivation medium optimization

In the present work, erythromycin production was carried out in submerged culture using Saccharopolyspora erythraea. Different experiments were conducted to optimize the cultivation medium through the change of carbon and nitrogen sources to cheaper one in order to reduce the cost of medium and to utilize sugar cane molasses as one of major sugar industry by-products in Egypt. It was found that the addition of sugar cane molasses a sole carbon source at a concentration of 60 g/l accompanied by corn steep liquor (as organic N-source) in combination with ammonium sulphate (as inorganic N-source) gave the maximal erythromycin production. The antibiotic production in this medium reached about 600 mg/l which is about 33% higher than the value obtained in glucose based medium. On the other hand, the addition of n-propanol in concentration of 1% (v/v) increased the antibiotic production reaching about 720 mg/l after 144 h. Concluding, the new medium formulation based on cheap carbon source, sugar cane molasses, was a good alternative solution for the production of erythromycin economically.