Glycollate formation during the photoassimilation of acetate by Chlorella

Summary When ³H-¹⁴C-acetate was supplied to Chlorella pyrenoidosa in the light, glycollic acid became rapidly labelled with tritium and ¹⁴C. The ratio of glycollate was 10, whilst the ratio was 4 in the acetate added. Both ³H and ¹⁴C from acetate were present in glycollate before they were present in Calvin cycle intermediates, so that glycollate was not formed as a C₂-fragment from the Calvin cycle.     

CO2-Fixierung durch Knallgasbakterien

Zusammenfassung Die Gewinnung, Aufbereitung und papierchromatographische Analyse ¹⁴C-markierter Extrakte aus PHBS-speichernden Zellen von Hydrogenomonas H 16 wird beschrieben und diskutiert. Einige chromatographische Trennsysteme wurden verglichen, insbesondere mit dem von Metzner (1960) vorgeschlagenen System. R_f-Werte wichtiger Intermediärverbindungen und Positionskonstanten von Phosphatestern wurden ermittelt.Für die papierchromatographische Trennung von Zuckern, Aminosäuren und Hydroxamsäuren sowie für organische Säuren und Phosphatester wurden zweckmäßige Systeme angegeben, Sprühmittel verglichen und Farbreaktionen beschrieben.Chromatogramme von Extrakten aus Hydrogenomonas H 16 und Chlorella pyrenoidosa, die ¹⁴CO₂ im Kurzzeitversuch unter vergleichbaren Bedingungen eingebaut hatten, zeigten voneinander verschiedene Markierungsmuster. Unter den Phosphatestern sind bei Hydrogenomonas (nur 63% des fixierten ¹⁴C) vorwiegend Hexosemonophosphate, Sedoheptulose-7-phosphat, Phosphoglycerinsäure und AMP markiert, bei Chlorella (95% des fixierten ¹⁴C) hauptsächlich Phosphoglycerinsäure, Triosephosphat und Phosphoenolbrenztraubensäure. Bei Hydrogenomonas waren einige organische Säuren und Aminosäuren (Äpfelsäure, Citronensäure, Bernsteinsäure, Fumarsäure, Glutaminsäure, Alanin u.a.) relativ stark markiert, die bei Chlorella kaum oder nicht radioaktiv waren.

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CO₂-fixation by Knallgas bacteriaII. Chromatographical identification of early labeled fixation products

Summary The production, preparation, and analysis by paperchromatography of ¹⁴C-labeled extracts from cells of Hydrogenomonas H 16 storing poly--hydroxybutyric acid (PHBS) is described and discussed. Some chromatographical solvent systems were compared, especially with one proposed by Metzner (1960). R_f-values of important intermediates and phosphate ester position constants were determined.Separation by paperchromatography of sugars, amino acids, and hydroxamic acids was tested in various systems. Sprays for these compounds, as well as for organic acids and phosphate esters, were compared and their colour reactions described.Chromatograms of extracts from Hydrogenomonas H 16 and Chlorella pyrenoidosa after incorporation of ¹⁴CO₂ in short time experiments under comparable conditions showed labelling patterns different from each other. In the case of Hydrogenomonas, 63% of the activity fixed was found in the phosphate esters, mainly in hexose monophosphates (incl. sedoheptulose-7-phosphate), phosphoglyceric acid, and AMP. The phosphate esters of Chlorella contained 95% of the activity fixed, the bulk of which appeared in phosphoglyceric acid, triose phosphate and phosphoenolpyruvic acid. Much less was found in the hexose monophosphates.Some organic and amino acids such as malic, citric, succinic, fumaric glutamic acid and alanine of the Hydrogenomonas extracts were rather heavily labelled, while this was not the case with Chlorella.

     

Glycolic Acid Labeling During Photosynthesis with CO(2) and Tritiated Water

Chlorella pyrenoidosa were allowed to photosynthesize for short periods of time in the presence of ¹⁴CO₂ and HTO. Analysis of tritium and ¹⁴C labeling of photosynthetic intermediate compounds showed that the T/¹⁴C ratio of glycolic acid was comparable to that of intermediate compounds of the photosynthetic carbon reduction cycle when photosynthesis was performed in nearly 100% oxygen and only slightly higher under steady-state conditions. It is concluded that formation of labeled glycolic acid as a consequence of its proposed hydrogen transport role in photosynthesis is quantitatively of limited importance compared to the net synthesis of glycolic acid from CO₂.     

Improved method for extraction of hydroperoxide lyase from Chorella

Summary Hydroperoxide lyase converts fatty acid hydroperoxides to oxo-fatty acids which, after further oxidation, are suitable for the synthesis of higher polyamides. An improved method utilizing an acetone powder step for isolation of this enzyme from the unicellular alga Chlorella was developed. Using this procedure a five fold increase in hydroperoxide lyase activity from Chlorella pyrenoidosa was obtained compared to previously used extraction methods. Other Chlorella species were assayed, and it was found that C. fusca also contained significant hydroperoxide lyase activity.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

Taxonomic implications of Prototheca and Chlorella cell wall polysaccharide characterization

Summary Four polysaccharide fractions were obtained by acid and alkaline degradation of purified cell walls of Prototheca spp. and Chlorella spp. These fractions were further hydrolyzed and the component sugars identified. Five Prototheca strains and two Chlorella protothecoides strains have essentially similar polysaccharide compositions, which significantly differ from those of C. vulgaris and C. pyrenoidosa. This emphasizes the close affinity of C. protothecoides to Prototheca spp. not shared by other Chlorella spp.     

Transients in the Photosynthetic Carbon Reduction Cycle Produced by Iodoacetic Acid and Ammonium Chloride

Changes in ¹⁴C content of metabolites in photosynthesising Chlorellahave been investigated following the addition of iodoaceticacid or ammonium chloride. The ¹⁴C content of phosphoglyericacid increases in the former and decreases in the latter case.Both reagents divert ¹⁴C from sucrose and polysaccharide intocompounds of and associated with the tricarboxylic acid cycle.The principal site of iodoacetic acid inhibition is triose phosphatedehydrogenase whereas ammonium chloride stimulates the conversionof phosphoglyceric acid to phosphoenolpyruvate and pyruciv acid.The latter suggests a direct effect of the ammonium ion on phosphoglyceromutase,enolase, or pyruvic kinase in vivo. Changes in the concentrationof alanine, aspartic acid, glutamic acid, serine, and glutamineupon addition of the ammonium ion are not well correlated withtheir ¹⁴C labelling patterns.     

Metabolic adaptations to environmental changes in Caenorhabditis elegans

Metabolic adaptations to environmental changes were studied in Caenorhabditis elegans. To assess adjustments in enzyme function, maximum activities of key enzymes of main metabolic pathways were determined. After a 12 h incubation at varying temperatures (10, 20°C) and oxygen supplies (normoxia or anoxia), the activities of the following enzymes were determined at two measuring temperatures in tissue extracts: lactate dehydrogenase (LDH; anaerobic glycolysis), 3-hydroxyacyl-CoA-dehydrogenase (HCDH; fatty acid oxidation), isocitrate dehydrogenases (NAD-IDH, NADP-IDH; tricarboxylic acid cycle) and isocitrate lyase (ICL; glyoxylate cycle). Incubation at 20°C induced a strong increase in maximum LDH activity. Anoxic incubation caused maximum HCDH and NADP-IDH activities and, at 10°C incubation, LDH activity to increase. Maximum NAD-IDH and ICL activities were not influenced by any type of incubation. In order to study the time course of metabolic adaptations to varying oxygen supplies, relative quantities of free and protein-bound NADH were determined in living C. elegans using time-resolved fluorescence spectroscopy. During several hours of anoxia, free and protein-bound NADH showed different time courses. One main result was that just at the moment when the protein-bound NADH had reached a constant level, and the free NADH started to increase rapidly, the worms fell into a rigor state. The data on enzyme activity and NADH fluorescence can be interpreted on the basis of a two-stage model of anaerobiosis.     

Effects of temperature on development, survival, longevity, and fecundity of the Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) predator, Axinoscymnus cardilobus (Coleoptera: Coccinellidae)

Axinoscymnus cardilobus (Homoptera: Aleyrodidae) is an important predator of Bemisia tabaci (Coleoptera: Coccinellidae) that occurs in high population density of B. tabaci. Temperature among other factors is observed to play an important role in the development of arthropods. The effect of temperature on the development of A. cardilobus was studied at seven constant temperature regimes (14, 17, 20, 23, 26, 29, 32 °C). The results indicated that the duration of egg, larval and pupal stages were significantly influenced by increased temperature. The rate of development gradually increased with increase in temperature from 14 °C to 26 °C, but declined from 26 °C to 32 °C. The survival rates of different insect stages were stable at temperatures between 20 °C and 26 °C, but at extreme temperatures of 32 °C and 14 °C, a sharp decrease was evident. Ovipositional period of the female decreased when temperatures were increased from 17 °C to 32 °C. The highest fecundity of the female (225.7 eggs per female) was recorded at 23 °C. Life tables of A. cardilobus were constructed based on the experimental results at temperatures of 14–32 °C. The reproductive rate (R₀), the innate capacity for increase (r_m) and the finite rate of increase (λ) reached the maximum values at 23 °C, of 70.7, 0.059 and 1.062, respectively. The mean generation time (T) decreased with increased temperature from 17 °C to 32 °C, the highest and least values recorded at 17 °C and 32 °C were 112.7 and 38.7, respectively. These results offer valuable insight on the importation and establishment of A. cardilobus into new environments with diverse temperature regimes.     

Symbiotic Chlorella enhances the thermal tolerance in Paramecium bursaria

Symbiotic Chlorella enhanced the tolerance to high temperature in Paramecium bursaria. We found that 50% of Chlorella-free P. bursaria died within 85 s of exposure to 41°C in a standard saline solution, while the presence of Chlorella almost doubled the survival time of P. bursaria (160 s, P<0.001). The degree of tolerance in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor for photosynthesis) treated Chlorella-containing P. bursaria and Chlorella-containing organisms kept in the dark for 24 h was as low as in Chlorella-free organisms. The degree of tolerance to high temperature in Chlorella-free P. bursaria in solutions containing maltose, glucose, fructose or O₂, was as high as that of normal Chlorella-containing organisms. The degree of thermal tolerance in Chlorella-containing P. bursaria was not affected in the presence of these carbohydrates or oxygen.     

Food selectivity of the herbivore <Emphasis Type="Italic">Daphnia magna</Emphasis> (Cladocera) and its impact on competition outcome between two freshwater green algae

Freshwater green algae, Chlorella, have heavy cell walls and their size usually exceeds the lower limits of limb size of herbivorous Daphnia (Cladocera). According to the optimal foraging theory, we speculated that Daphnia would graze more exposed and relatively large Clamydomonas rather than Chlorella, and this process would lead to small-sized Chlorella becoming a superior competitor in the presence of Daphnia. We used Daphnia magna, Clamydomonas sajao and Chlorella pyrenoidosa to test this hypothesis. Our grazing experiments showed that Daphnia preferred C. sajao to C. pyrenoidosa, regardless of the concentration and relative abundance of these two algae. The decrease in relative abundance of high-quality Clamydomonas in Clamydomonas–Chlorella assemblages did not diminish the grazing efficiency of Daphnia on this algal species, but increased selectivity of low-quality Chlorella. However, when the concentration of Clamydomonas was extremely high, the grazing of Daphnia on Clamydomonas decreased. In competition experiments, we observed that the presence of Clamydomonas restrained the growth potential of Chlorella; however, the introduction of herbivorous Daphnia into the competing environment weakened this influence and to some extent enhanced the growth ability of Chlorella. Moreover, we also observed that the intensity of herbivory, imposed by different densities of Daphnia, had an obvious influence on the competition outcome between Clamydomonas and Chlorella. At the highest intensity of herbivory (10 Daphnia), C. sajao was eliminated from the culture medium whereas C. pyrenoidosa could persist, but at low cell density.     

Mould growth on building materials under low water activities. Influence of humidity and temperature on fungal growth and secondary metabolism

The influence of relative humidity (RH) and temperature on growth and metabolism of eight microfungi on 21 different types of building material was investigated. The fungi were applied as a dry mixture to the materials, which were incubated at 5°C, 10°C, 20°C and 25°C at three humidity levels in the range 69–95% RH over 4–7 months. The lower limit for fungal growth on wood, wood composites and starch-containing materials was 78% RH at 20–25°C and increased to 90% RH at 5°C. An RH of 86% was necessary for growth on gypsum board. Ceramic materials supported growth at RH >90%, although 95% RH was needed to yield chemically detectable quantities of biomass. Almost exclusively only Penicillium, Aspergillus and Eurotium (contaminant) species grew on the materials. Production of secondary metabolites and mycotoxins decreased with humidity and the quantities of metabolites were insignificant compared with those produced at high RH (RH >95%), except in the case of Eurotium.     

The glycollate pathway during the photoassimilation of acetate by Chlorella

Summary When Chlorella pyrenoidosa photoassimilates ³H–¹⁴C-acetate glycollic acid rapidly becomes labelled with both tritium and ¹⁴C. The ³H/¹⁴C ratio was 10 in glycollate, (compared with 4 in the acetate added) and the only other intermediates showing similar ³H/¹⁴C ratios to glycollate were glycerate and serine. This suggests a glycollate pathway for the formation of serine was operating in Chlorella pyrenoidosa during the photoassimilation of acetate. When Chlorella pyrenoidosa assimilated ³H–¹⁴C-acetate in the dark glycollate was not labelled with either ¹⁴C or tritium. Although glycerate and serine both became labelled with ¹⁴C and tritium in the dark they did not show the high ³H/¹⁴C ratios recorded in the light. When cells were aerated with unlabelled 5% CO₂ during the photoassimilation of ³H–¹⁴C-acetate, the ³H/¹⁴C ratios of glycollate, glycerate and serine were slightly decreased. Similarly, under anaerobic conditions in the light the ³H/¹⁴C ratio was decreased compared with aerobic conditions.     

C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts

A new mathematical model, based on the observation of ¹³C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-¹³C]Glc or [1,2-¹³C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the ¹³C-enrichment of acetyl-CoA can be deduced from ¹³C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-¹³C]glucose in the absence of insulin, was investigated by ¹³C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate ↔ 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G_o) or aspartate (A_O) were approximately the same for all hearts and remained constant during perfusion: G₀ = (0.74 ±0.03) μmol/g; A₀ = (1.49±0.05) μmol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min⁻¹ and 0.72 min⁻¹, respectively; the flux of this cycle is about (1.07±0.02) μmol min^-1 g^-1. Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

Gas chromatography–electron-capture detection of urinary methylhippuric acid isomers as biomarkers of environmental exposure to xylene

Methylhippuric acid isomers (MHAs), urinary metabolites of xylenes, were determined, after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GC with ECD detection, on an SPB-35 capillary column (30 m, 0.32 mm I.D., 0.25 μm film thickness, β=320). S-benzyl-mercapturic acid was used for internal standardization. Chromatographic conditions were: oven temperature 162°C, for 14.2 min; ramp by 30°C/min to 190°C, for 3.5 min; ramp by 30°C/min to 250°C, for 4 min; helium flow rate: 1.7 ml/min; detector and injector temperature: 300°C. The sample (1 μl) was injected with a split injection technique (split ratio 5:1). MHA recovery was >95% in the 0.5–20 μmol/l range; the limit of detection was <0.25 μmol/l; day-to-day precision, at 2 μmol/l, was Cv<10%. Urinary MHAs were determined in subjects exposed to different low-level sources of xylenes: (a) tobacco smoking habit and (b) BTX urban air pollution (airborne xylene ranging from 0.1 to 3.7 μmol/m³). Study (a) showed a significant difference between urinary MHA median excretion values of nonsmokers and smokers (4.6 μmol/l vs. 8.1 μmol/l, p<0.001). Study (b) revealed a significant difference between indoor workers and outdoor workers (4.3 μmol/l vs. 6.9 μmol/l, p<0.001), and evidenced a relationship between MHAs (y, μmol/mmol creatinine) and airborne xylene (x, μmol/m³) (y=0.085+0.34x; r=0.82, p<0.001, n=56). Proposed biomarkers could represent reliable tools to study very low-level exposure to aromatic hydrocarbons such as those observed in the urban pollution due to vehicular traffic or in indoor air quality evaluation.     

Optimisation of physical factors on the production of active metabolites by Bacillus subtilis 355 against wood surface contaminant fungi

A Surface Response Model was used to study the effect of pH, temperature and agitation on growth, sporulation and production of antifungal metabolites by Bacillus subtilis CCMI 355.Strong agitation, temperature between 27 and 34 °C and pH 6 favoured cell growth. Alkaline pH, strong agitation and temperature between 28 and 34 °C favoured spore formation. No relationship was found between sporulation and the production of antifungal metabolites. According to the model, pH 8, 37 °C and the absence of agitation were the optimal conditions for the production of broad-spectrum antifungal metabolites against Botrytis cinerea, Penicillium expansum, Trichoderma sp, Trichoderma harzianum, Trichoderma koningii and Trichoderma virgatum.In situ assays using green wood impregnated with Bacillus subtilis CCMI 355 inoculated in Yeast Extract Glucose Broth medium in the conditions above, displayed an efficient protection against wood surface contaminant fungi.     

Comparisons Between the Kinetic Behaviour of the Muscle Carnitine Palmitoyltransferase I of a Higher and Lower Vertebrate

The V_max of rat muscle mitochondrial CPT I toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 were far lower than those recorded previously for this enzyme in rat liver at the same temperature (37°C). However, the V_max of 7.0 nmol · min⁻¹ · mg mitochondrial protein⁻¹ for linoleoyl CoA (18:2n-6), which was the greatest recorded for the five acyl CoAs examined in muscle, was similar to that in liver. These comparisons presumably reflect a difference in the essential fatty acid requirements of these two rat tissues. Although the V_max values for CPT I in the musculature of a lower vertebrate (larval lamprey) at 20°C were similar to those exhibited toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 by the CPT I of rat musculature at 37°C, the corresponding V_max toward 18:2n-6 (3.2 nmol · min⁻¹ · mg mitochondrial protein⁻¹) was lower. The latter relatively low activity may spare from oxidation this essential fatty acid, which is in low abundance in the diet of larval lampreys. Although the V_max values toward the four nonessential fatty acids in larval lamprey muscle were similar to those in rat muscle, the corresponding K_0.5 values were lower, thus indicating that the musculature of larval lampreys has a high capacity for energy generation through β-oxidation.