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.     

Use of ATP bioluminescence to determine the bacterial sensitivity threshold to a bacteriocin

A new ATP bioluminescence‐based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic‐nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin–luciferase. Nisin‐induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quanti?cation of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a AN_tot:AN_ext ratio = 1. For an initial concentration of 1.4 × 10⁷ bacteria/mL, the nisin effectiveness threshold is 3.4 ± 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells. Copyright © 2003 John Wiley & Sons, Ltd.     

Caged ATP - an internal calibration method for ATP bioluminescence assays

ATP bioluminescence, based on the firefly luciferase system, is used for the rapid determination of hygienic practices in the food industry. This study has demonstrated the use of caged ATP as an internal ATP standard and quantified the effects of industrial cleansing solutions, alcoholic beverages and pH on firefly luciferase activity. The light signal was quenched 6-47% by five cleansing solutions at standard working concentrations. Ethanol at 1% (v/v) inhibited bioluminescence by 15% (w/v) whereas concentrations above 4% enhanced the light output. The light signal was quenched by 20-25% at pH values below pH 4 and above pH 10.     

Use of lipophilic anions for estimation of biomass and cell viability

A method is described to estimate the microbial biomass of a sample, to enumerate the cells, and to distinguish the portion of metabolically active cells in the population by measuring the binding of phenyldicarbaundecaborane (PCB(-)) to the cells. This method can also be used for the analysis of a complex population of microorganisms if the cells composing the sample are sensitive to different biocidal agents. In addition, the analysis of PCB(-) binding is useful for the enumeration of the phage-infected cells and phage particles.     

Salivary biomass assessed by bioluminescence ATP assay related to (bacterial and somatic) cell counts

The present work aimed (1) to evaluate ATP content in saliva by the bioluminescent luciferin-luciferase method, (2) to evaluate the relationships between ATP content, bacterial count and epithelial cell numbers in saliva, (3) to study the effect of two different antiseptics (peroxidase system producing hypothiocyanite and chlorhexidine) on the salivary biomass. In 45 young adults, the salivary ATP content ranged from 8 to 1515 nM. Salivary ATP content was significantly and directly correlated to bacterial count and epithelial cell numbers (Spearman-Rank correlation, P< or =0.001). Regression analysis allowed the inference of a mean epithelial cell and bacterial ATP content of 152.7 fg and 8.3 fg per cell, respectively. The salivary ATP content decreased significantly to 38. 8+/-12.3 per cent (mean+/-SEM, N=6) of its initial value after a 30-min incubation in the presence of a peroxidase system producing hypothiocyanite (OSCN(-)). Chlorhexidine (CHX) reduced salivary ATP content to 52.0+/-16.7 per cent. OSCN(-) did not affect the transformed logarithm of bacterial count but CHX reduced it from 7. 02+/-0.26 to 0.52+/-0.33. No effect of OSCN(-) was seen on the ratio of epithelial cell viability while CHX reduced it from 46.7+/-5.1 to 3.9+/-1.1 per cent. It is concluded that the combination of the evaluations of the ATP content and cell numbers in saliva can provide reliable data about the effects of oral antiseptics on salivary biomass.     

Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence.

Measurement of extracellular ATP in biological solutions is complicated by protein-binding and rapid enzymatic degradation. We hypothesized that the concentration of extracellular ATP could be determined luminometrically by limiting degradation and measuring the free and protein-bound fractions. ATP was added (a) at constant concentration to solutions containing varying albumin concentrations; (b) at varying concentrations to a physiological albumin solution (4 gm/dL); (c) at varying concentrations to plasma. After centrifugation, a fraction of each supernatant was heated. ATP in heated and unheated samples was measured luminometrically. Blood was drawn into saline or an ATP-stabilizing solution and endogenous plasma ATP measured. ATP-albumin binding was a linear function of albumin concentration (3.5% ATP bound at 100 micromol/L to 33.2% ATP bound at 1000 micromol/L) but independent of ATP concentration (29.3%, 10-1000 nmol/L ATP in 602 micromol/L albumin). Heating released the majority of bound ATP from albumin-containing solutions (94.8 +/- 1.7%) and plasma (97.6 +/- 5.1%). Total endogenous plasma ATP comprised 93 +/- 27 nmol/L (free) and 150 +/- 40 nmol/L (total fraction). Without stabilizing solution, degradation of free endogenous plasma ATP occurred. Within a physiological range (10-1000 nmol/L), ATP binds albumin independently of ATP concentration. Heating releases bound ATP, enabling accurate luminometric measurement of total extracellular ATP (free and bound) in biological samples.     

On-line estimation of the biomass activity during animal-cell cultivations

A software sensor was developed to determine the volumetric biomass activity of animal cell cultivations on-line. It was based on the on-line estimation of the ATP-production rate from the oxygen uptake and the lactic-acid production rate. The sensor was verified for a batch culture of Vero cells, and a batch and a continuous culture of hybridoma cells. For the hybridoma cells, the sensor showed a good correlation with the biomass concentration. However, this was not the case for the Vero cells. As soon as glutamine was exhausted, the biomass activity stabilized, whereas the amount of biomass almost doubled. Because the sensor developed responds to nutrient limitations much faster than becomes visible through cell density measurements, and because the volumetric biomass activity can be related to the volumetric consumption rates and production rates of important metabolites, it shows excellent possibilities for control purposes. (c) 1996 John Wiley & Sons, Inc.     

Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87^N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.     

Improved ATP methodology for biomass assays

ATP methodology needs to be further standardized and improved in order to avoid the pitfalls that have sometimes hampered its application to biomass assays. The following steps have been reconsidered as far as the bacteriological applications is concerned:

• destruction of free and somatic ATP: replacement of apyrase by mammalian ATPase, more readily accessible to specific inhibition;
• extraction of bacterial ATP: protection of luciferase by lipids against inhibitory effect of cationic detergents with production of a constant light response.

New methods are proposed for the calibration of luminometers and for the matching of sample holders in multichannel instruments. The limit of sensitivity of ATP assays is discussed in the light of currently available reagents and instruments.     

Using ATP bioluminescence technique for monitoring microbial activity in sludge

This study demonstrates, for the first time, that the presence of suspended solids in waste‐activated sludge interferes with adenosine triphosphate (ATP) bioluminescence tests. The sludge subject to acid/alkaline treatment represented the test sample. Without consideration of the effect of solid concentrations, one would erroneously estimate the density levels of heterotrophic bacteria in the sludge using ATP data. A light blockage model was proposed to evaluate the luminescence reading without the interference of suspended solids. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 469–474, 2001.     

An enzyme-linked immunosorbent assay for the estimation of fungal biomass during solid-state fermentation

An enzyme-linked immunosorbent assay for sensitive, specific and quantitative estimation of fungal biomass during solid-state fermentation is described. Using this method, differential growth rates and colonization of the substrate can be studied. The assay has potential application for the efficient monitoring of solid-state fermentation involving specific fungus, for which available methods are not adequate. Received: 18 November 1997 / Received last revision: 8 June 1998 / Accepted: 14 June 1998     

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.     

Use of a newly developed rapid microbial ATP bioluminescence assay to detect microbial contamination on poultry carcasses

A newly developed rapid microbial ATP bioluminescence test (R-mATP) was shown to be an adequate means to assay the microbial load of poultry carcasses. This assay utilizes differential extraction and filtration to separate somatic from microbial ATP in a very rapid timeframe. The assay requires approximately 5 min to complete; approximately 3.5 min to sample and 90 s analytical time. Correlation coefficient (r) between aerobic colony counts and R-mATP test results (n=329) was 0.82. Post-test probabilities to correctly classify carcasses with different levels of microbial contamination were as high as 98% for samples of ≥3.5 log aerobic CFU per ml. Given the rapidity of this assay, the R-mATP holds potential for monitoring the microbial load of carcasses at poultry-processing critical control points. Other potential applications of this new version of the microbial ATP bioluminescence test are discussed.     

Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase

Apoptosis is a distinct form of cell death, which requires energy. Here, we made real-time continuous measurements of the cytosolic ATP level throughout the apoptotic process in intact HeLa, PC12 and U937 cells transfected with the firefly luciferase gene. Apoptotic stimuli (staurosporine (STS), tumor necrosis factor alpha (TNFalpha), etoposide) induced significant elevation of the cytosolic ATP level. The cytosolic ATP level remained at a higher level than in the control for up to 6 h during which activation of caspase-3 and internucleosomal DNA fragmentation took place. When the STS-induced ATP response was abolished by glucose deprivation-induced inhibition of glycolysis, both caspase activation and DNA laddering were completely inhibited. Annexin V-binding induced by STS or TNFalpha was largely suppressed by glycolysis inhibition. Thus, it is suggested that the cells die with increased cytosolic ATP, and elevation of cytosolic ATP level is a requisite to the apoptotic cell death process.     

On-line estimation of the specific growth rate based on viable biomass measurements: experimental validation

The application of model based control techniques to biotechnological processes is often hampered due to the lack of reliable on-line sensors. This problem can be tackled by the application of software sensors, in which the available hardware measurements are combined with the model equations. The resulting estimates serve as additional measurements useful for process monitoring and control. In this paper, an observer based estimator for the specific growth rate based on on-line viable biomass measurements is studied. Several fed-batch experiments with baker's yeast in a stirred tank bioreactor illustrate the design, tuning, and implementation from a practical point of view. The main contributions of this paper are to illustrate (i) the implementation and validation of the presented algorithm in real-time, (ii) the use of an advanced on-line biomass measurement, and (iii) the design and tuning of the algorithm from a practical point of view. Real-time knowledge of the specific growth rate is important because it yields information on the viability of the cells and it can be used in real-time feedback control algorithms.     

Soft sensors for on-line biomass measurements

One of the difficulties encountered in control and optimisation of bioprocesses is the lack of reliable on-line sensors for their key state variables. This paper investigates the suitability of using on-line recurrent neural networks to predict biomass concentrations. Input variables of the proposed recurrent neural network are feed rate, liquid volume and dissolved oxygen. Experimental results revealed that the proposed neural network is able to predict biomass concentrations with an accuracy of ±11%.