Critical problems with extraction of ATP for bioluminescence assay of plankton biomass

Recovery of ATP by boiling tris extraction was 90–95 percent greater in 1 liter grab samples than in concentrated net samples. ATP losses were attributed to insulating effects promoted by accumulation of detritus on filters. A series of extractions over a concentration range of whole or size-segregated plankters and cultured algae was made to determine volume of water to be filtered for optimum extraction efficiency. Accuracy of ATP assays was optimized by: (i) using large diameter (i.e. 47 mm) acetate filters; (2) limiting sample volume filtered to 50 ml when particulate organic carbon (POC) exceeded 0.4 mg l^–1; and (3) performing extractions in boiling tris maintained initially on a laboratory hot plate at 400°C as opposed to hot water bath at 100°C.Additional problems were encountered in using published cellular carbon: ATP ratios for conversion of ATP data to biomass as carbon. Ratios of POC: ATP in cultures of sheathed blue-green algae reached 550 : i, while non-sheathed forms yielded ratios near values previously reported for plankton communities. Difficulties in applying a uniform conversion factor may be expected in plankton communities containing significant volumes of sheathed blue-greens.     

ATP as a biomass indicator in eight North Island Lakes, New Zealand

SUMMARY. 1. The relationship between microbial ATP and biomass- carbon in the near surface waters (0.5–5 m) of eight New Zealand lakes was studied to determine the constancy of the carbon:ATP ratio under natural growth conditions. Concentrations of microbial carbon were estimated indirectly from cell volume determinations.
2. The carbon:ATP ratio remained reasonably constant (interquartile range 248–291, n = 50), except during periods of nitrogen/phosphorus deficiency when carbon: ATP ratios increased to values greater than 400.
3. During periods of nitrogen/phosphorus sufficiency, corresponding estimates of microbial ATP and biomass-carbon were strongly correlated ( r =0.97, n =47) and related by the equation carbon = (287±20) ATP-(22±41) where carbon and ATP are expressed in mg m⁻³. From this relationship an average carbon:ATP ratio of 267 (SE=5) was calculated.
4. This ratio was not significantly affected by the relative proportions of bacterial and algal biomass in the surface water samples. However, because of the marked deviation of the carbon:ATP ratio during periods of nitrogen/phosphorus deficiency, the routine use of ATP as a biomass indicator is discouraged.     

ATP als indikator für die biomasse mariner sedimente

Zusammenfassung Aus Sedimentproben wurde ATP mit Trispuffer-Lösung bei 100° extrahiert. Bei Anwendung der Luciferin-Luciferase-Methode zur Bestimmung von ATP lag die untere Grenze der Erfaßbarkeit bei 5x10^-8 g ATP/ml Sediment. Durch Multiplikation mit dem Faktor 50 wurden die ATP-Gehalte in Biomasse-Kohlenstoff umgerechnet. In Abhängigkeit vom Sedimenttyp konnten 0,13–1,6% des organischen Gesamtkohlenstoffgehaltes der Sedimente der lebenden Substanz zugeordnet werden.

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ATP as an indecator of biomass in marine sediments

Summary ATP was extracted from sediments with Tris buffer at 100° C. Using the luciferine-luciferase assay for the determination of ATP the lowest detectable concentration in the sediments was 5x10^-8 g ATP/ml Sediment. By multiplication with the factor 50 ATP levels were converted into biomass-carbon. Comparison with total organic carbon content of the sediments led to the conclusion that, depending on the type of sediment, the organic carbon content of living matter amounts to 0.13–1.6 per cent of the total organic carbon.

     

ATP pools and transients in the blue-green alga,Anabaena cylindrica

Anabaena cylindrica grown in steady state continuous culture has an extractable ATP pool, measured on the basis of the luciferin-luciferase assay of 165±35 nmoles ATP mg chla ^-1. This pool is maintained by a dynamic balance between the rate of ATP synthesis and the rate of ATP utilization. Phosphorylating mechanisms which can maintain the pool in the short term are total photophosphorylation, cyclic photophosphorylation and oxidative phosphorylation. The alga can maintain its ATP pool by switching rapidly from one of these phosphorylating mechanisms to another depending on the environmental conditions. At each switch-over there is a transient drop in the ATP pool for a few seconds. On switching to conditions where only substrate level phosphorylation operates, the ATP pool falls immediately, but takes several hours to recover. The apparent rates of ATP synthesis by total photophosphorylation and by cyclic photophosphorylation are both much higher (210±30 and 250±13 moles ATP mg chla ^-1 h^-1 respectively) than the apparent rate of ATP synthesis by oxidative phosphorylation (22±3 moles ATP mg chla ^-1 h^-1). In long term experiments the ATP pool is maintained when total photophosphorylation is operating. It cannot be maintained in the long term by cyclic photophosphorylation alone in the absence of photosystem II activity or endogenous carbon compounds, or by oxidative phosphorylation in the absence of endogenous carbon compounds. Measurements of ATP, ADP and AMP show that the total pool of adenylates is similar in the light and in the dark in the short term. There is only limited production of ATP under dark anaerobic conditions when glycolysis and substrate phosphorylation can operate which suggests that these processes are of limited significance in providing ATP in Anabaena cylindrica.Abbreviations ADP adenosine 5-diphosphate - AMP adenosine 5-monophosphate - ATP adenosine 5-triphosphate - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCMU 3-(3,4-dichlorophenyl)1,1-dimethyl urea - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - PEP phosphoenolpyruvate     

ATP levels in algal cells as influenced by environmental conditions

The cellular content of adenosine triphosphate (ATP) relativeto cell size and cellular organic carbon has been investigatedin 30 different algal cultures representing 7 phyla. Duringexponential growth in batch culture, cellular contents of ATPremained at fairly uniform levels in all these unicellular algaeand averaged 0.35% of the cellular organic carbon content. Duringextreme nitrogen or phosphorus deficiency the cellular levelsof ATP decreased to 20–50% of that found in exponentially-growingcells, but these percentages may be low due to detrital carbonin the senescent cultures. The steady state levels of ATP in cells were similar in lightor in dark, although ATP concentrations fluctuated for a fewminutes upon any sudden change in light conditions. When thelight was turned on there was a rapid increase in ATP levels,followed by a slow decrease; when the light was turned off,there was a rapid fall in cellular ATP levels, which then rosewithin a few minutes to achieve the steady state concentration.The cellular concentrations of ATP in these algae and in othermicrobial groups are discussed relative to studies where ATPdeterminations are used to estimate microbial biomass. (Received June 2, 1970; )     

Use of the luciferin-luciferase assay of ATP for measuring the bacterial growth: Application to Escherichia coli

The relative constancy of ATP content per unit of dry biomass in different microorganisms enables to use the specific, sensitive and quick luciferin-luciferase method for ATP assay for indirect quantitation of microbial biomass by measuring the ATP content in biomass samples.The aim of this work was to investigate the feasibility of using the ATP concentration for the monitoring of the growth of Escherichia coli K 12 W 3350 in different cultivation conditions. Statistically significant high correlation coefficients (r > 0.9) between the ATP content (ATP/ml) and the other growth characteristics examined showed that the ATP content of the culture is as suitable characteristic as the optical density of the culture, total cell number per ml or total cell volume per ml for the monitoring of the growth of E.coliin batch culture in mineral medium with glucose at different growth temperatures (27–42 °C) as well as in fed-batch culture.     

Initial ATP Formation, NADP Reduction, CO(2) Fixation, and Chloroplast Flattening Upon Illuminating Pea Leaves

Chloroplasts in living cells of detached and sectioned leaves of Pisum sativum had a thickness of 2.68 ± 0.04 μ in the dark as determined from photographs made using a phase contrast microscope. Upon illumination with 4000 lux for 10 min, the chloroplasts flattened to 2.15 ± 0.04 μ. There was a short lag period of about 11 sec at 1000 lux and 2 sec at 4000 lux before appreciable light-induced flattening occurred. Both ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in detached pea leaves increased upon illumination and then fell during the initial 60 sec. The maximum ATP level was attained in 16 sec at 1000 lux and 10 sec at 4000 lux, while NADPH required about twice as long to reach a maximum. A sustained rate of carbon dioxide fixation occurred after a lag period coinciding in time with the drop in the NADPH level. ATP appeared to be involved not only with carbon dioxide fixation, but also with some reaction beginning sooner, perhaps the light-induced chloroplast flattening. Considering the initial photophosphorylation and the sustained CO₂ fixation rates, the ATP formation rate in vivo apparently increased after the leaves had been in the light for a few min.     

Epilithic periphyton and detritus studies in a subalpine stream

The accumulation of epilithic periphyton in Ward Creek, a permanent stream within the Lake Tahoe basin, California, was measured weekly at three stations from July through September, 1972. Subsamples were analyzed for total carbon and adenosine triposphate content. The mean total carbon content at three stations over the period of investigation was 0.508 ± 0.263 mg carbon cm^–2. Live biomass, as estimated from ATP measurements, averaged 0.121 ± 0.115 mg carbon cm 2. It was estimated that approximately 76% of the organic carbon accumulating on rock substrates was present as detritus. Scanning electron microscopy of rock substrates suggested that much of this detrital accumulation may consist of diatom stalk materials.This work was supported by a grant from the National Science Foundation/RANN GI-22. C. R. Goldman, Principal Investigator.     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Energy-rich phosphates and transintestinal transport in rat intestine incubated in vitro at different temperatures

In the present work, the transported fluid and the tissue content of ATP, ADP and AMP has been evaluated in the jejunum rat intestine which was everted and incubated in vitro both at 28°C and 38°C for 1 h. The energy-rich phosphates have been measured in the tissue at the beginning and at the end of the experiment as well as in vivo. These determinations have been made in the total intestine and in the scraped mucosa. ATP and ADP content are higher in vivo and lower but constant at 28°C in vitro; on the contrary, at 38°C in vitro, the initial and final content of these adenilic nucleotides are both lower than at 28°C. Under all these conditions the AMP content does not vary appreciably.Wet weight to dry weight ratios have been reported for mucosal and submucosal tissues in unincubated and incubated intestines.In some experiments, fluid transport (measured as an actual serosal volume increase) was determined every 20 min during a 1-h incubation. At 28 °C, fluid transport is constant throughout the time of the experiment, but at 38 °C, there is a progressive decrease of the transported fluid.Fluid transport and ATP content of the intestine seem to be directly related. The transport activity which is lower at 38 °C than at 28°C, seems to be due to a low availability of energy-rich phosphates.     

Carbon and nitrogen content of Noctiluca scintillans in the Seto Inland Sea, Japan

The carbon and nitrogen content of Noctiluca scintillans cellsfrom the Seto Inland Sea, Japan was investigated in order toestimate its biomass in natural samples. The carbon contentof N.scintillans ranged from 123 to 627 ng C cell^–1 witha mean value of 353 ng C cell^–1, or 1.12 to 2.67 fg Cµm^–3 with a mean value of 1.98 fg C µm^–3.The nitrogen content ranged from 36.0 to 232 ng N cell^–1with a mean value of 131 ng N cell^–1, or 0.499 to 0.910fg N µm^–3 with a mean value of 0.694 fg N µm^–3.Total cell carbon and nitrogen increased but the carbon andnitrogen per cell volume decreased with increasing cell volume.The C/N ratio of the cells ranged from 2.3 to 4.4, which wasrelatively low compared with the Redfield ratio. The carbonand nitrogen content was extremely low (91.2 ng C cell^–1,41.8 ng N cell^–1) for starved cells, whereas it was extremelyhigh (528 ng C cell^–1, 205 ng N cell^–1) for cellswhich had ingested the large diatom, Coscinodiscus wailesii.Our results suggest that the carbon and nitrogen content ofN.scintillans varies depending on its physiological conditionand the type of food that it has recently consumed.     

Particle Counter Determination of Bacterial Biomass in Seawater

The applicability of the Elzone particle counter to the determination of marine bacterial biomass was investigated. The biomass of bacterial pure cultures and a mixed natural population were followed by using the particle counter, a CHN analyzer, and an ATP analyzer. The particle counter showed the precise size distribution of number and volume of submicron-size particles in seawater. For the pure cultured bacterial strains, the conversion factor from volume to carbon is 0.209 mg of C per mm³, and for natural bacterial cells of >0.6 μm in diameter, it is 0.184 mg of C per mm³. It is recommended that 0.2 be used as the conversion factor for both pure cultured marine bacterial cells and natural bacteria from coastal and near-shore marine environments.     

Volume decrease of glycerinated polymorphonuclear leukocytes induced by ATP and Ca : 1. Resemblances to actomyosin contraction

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.     

Seasonal variations of tintinnids (Ciliophora: Oligotrichida) in Narragansett Bay, Rhode Island, U.S.A.

Tintinnids exhibit a bimodal peak of abundance in NarragansettBay, with a minimum in late spring and a lesser peak in latesummer-early autumn, depending on location. Thirty-three speciesin eight genera were identified, with the fauna dominated bythe genus Tintinnopsis. Tintinnid abundance was not obviouslycorrelated with salinity, temperature, or nanoplankton chlorophyllconcentration. Patterns of change in seasonal abundance canserve as a framework for posing additional questions of therole tintinnids play in marine food webs.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

A comparative ultrastructural study on the nanoscale extrusomes of tintinnids (Alveolata,Ciliophora, Spirotricha) and their phylogenetic significance

The capsules, putative extrusomes in tintinnid ciliates, are known since 1971. Based on their ultrastructure, shape, and size, five capsule types were distinguished and suggested to be of phylogenetic significance. However, detailed morphometric data and transmission electron micrographs are lacking to verify former conclusions. In the current study, comprehensive analyses of transmission electron microscopic data were performed, investigating 14 species from 13 genera and more than seven families collected in European coastal waters and in the Northeast Pacific. Our data suggest two main capsule types (large and ampulliform vs small and ellipsoidal/ovoidal) each including two subtypes characterised by their internal structures. Species groupings inferred from the capsule (sub-)types emerge also as closely related in gene trees. Additionally, the ampulliform type unites the Undellidae, Xystonellidae, and Tintinnid clade 2, while the shared possession of the small ellipsoidal type proposes a close relationship of Tintinnid clade 11 with the Rhabdonellidae and Cyttarocylididae. Thus, the capsules provide promising features to shed light on several unresolved evolutionary relationships among tintinnid genera and families; yet, information on capsules is still missing for many monophyletic groupings. Finally, we provide the first ultrastructural clues for the extrusive character of these organelles.     

Impact de l'échantillonnage sur la mésure des nucléotides adényliques (ATP,ADP, AMP) du microplancton

The influence of sampling and sample treatment upon adenylic nucleotide (ATP, ADP, AMP) content of microplankton is studied. Changes in light conditions during nigh-sampling and extracting do not induce significant variations, in the adenylic nucleotide content of microplankton or in energy charge values.The contribution of zooplankton (size up to 200 µm) to microplankton adenosine values can be neglected for inshore surface waters and traditional sample volumes (about one liter). This result can been explained by the low density of zooplankton in such a small sample volume and by differences in efficiency of the extracting method used.

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Impact de l'échantillonnage sur la mésure des nucléotides adényliques (ATP, ADP, AMP) du microplancton

     

Opposite effects of d-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells

d-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM d-glucose. Even in the absence of d-glucose, d-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of d-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of d-glucose and/or d-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). d-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM d-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, d-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of d-glucose and/or d-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either ⁸⁶Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.     

In vitro effects of bisphenol A on the quality parameters,oxidative stress,DNA integrity and adenosine triphosphate content in sterlet (Acipenser ruthenus) spermatozoa

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be responsible for adverse effect on reproductive ability of various species. In the present study the effect of BPA on the quality parameters, oxidative stress, the DNA integrity and intracellular ATP content of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were exposed to concentrations of BPA possibly occurring in nature (0.5, 1.75, 2.5, 5 and 10 μg/L) for 2 h. Results revealed that BPA significantly decreased spermatozoa motility and velocity of spermatozoa at concentration of BPA 2.5–10 μg/L. Significant positive correlation (r = 0.713, P < 0.05) was found between percent motile spermatozoa and ATP content. Oxidative stress was observed at concentrations 1.75–10 μg/L, as reflected by significantly higher levels of protein and lipid oxidation and superoxide dismutase activity. Intracellular ATP content of spermatozoa decreased with increasing concentrations of BPA. A dramatic increase in DNA fragmentation expressed as percent tail DNA (2.2% ± 0.46) and Olive tail moment (0.37 ± 0.09 arbitrary units) was recorded at concentrations of 1.75 μg/L and above. The present study confirms that concentrations of BPA that can be encountered in nature are capable to induce oxidative stress, leading to impaired sperm quality, DNA fragmentation and intracellular ATP content.     

福州市森林碳储量定量估算及其对土地利用变化的响应

基于RS与GIS技术,以遥感影像数据、土地利用数据、森林资源二类调查数据为主要数据源,采用逐步回归法建立森林蓄积量定量估测模型。根据"蓄积量-生物量-碳储量"推算方法,对福州市森林植被碳储量和碳密度进行估算。建立福州市土地利用转移矩阵,分析2000—2010年土地利用变化影响下的福州市森林碳储量变化特征。结果表明:(1)根据不同的森林类型,即常绿阔叶林、常绿针叶林、针阔混交林分别建立的多元线性回归模型修正决定系数分别为0.599、0.679、0.694,通过模型适用性检验和精度验证。(2)2000年、2010年福州市森林植被碳储量总量分别为12.499Tg、12.642Tg,植被碳密度分别为18.694、18.708 t/hm~2,森林植被碳储量增加了1.430×10~5t。(3)福州市闽清县、永泰县、闽侯县的森林植被碳密度常年保持较高水平,并呈现出增长趋势;罗源县、长乐市、连江县森林植被碳密度较低,并呈现下降趋势。(4)2000—2010年,灌木和耕地是主要土地利用类型转出者,森林和建设用地是主要土地利用类型转入者。森林主要由灌木和耕地转化,主要向建设用地、耕地进行转化。由于土地利用变化,10年间福州市总碳储量减少了1.711×10~4t,其中土壤碳储量减少2.230×10~3t,植被碳储量减少1.489×10~4t。