Rapid physiological assays for nutrient demand by the plankton. I. Nitrogen

Three assays for nitrogen demand were compared on samples ofnatural plankton and on green and blue-green algal species inculture. The most reliable guide was selective luxury uptakeof nitrogen by the plankton after enrichment with a 10-to-1(wt/wt) mixture of inorganic N and P. Ammonium transport capacity,measured either directly (cultures) or by V_max for uptake ofthe NH₄⁺ analogue methylammonium (lakewaters), was generallyhigh in lakewaters with low dissolved inorganic N-to-P ratiosand in N-deficient cultures of green algae. By contrast, transportcapacity was much reduced in both natural and cultured populationsof heterocystous blue-green algae growing under conditions oflow combined inorganic nitrogen. Ammonium enhancement assays(heterotrophic CO₂, fixation after NH₄⁺ enrichment) were conductedat monthly intervals on eutrophic Lake Rotorua. There was astrong, negative correlation between this indicator of N deficiencyand dissolved inorganic N-to-P ratios below a threshold of 6:1.Ammonium enhancement was not, however, a reliable indicatorof combined inorganic N-demand by populations of heterocystousblue-green algae. All three assays provided strong evidenceof a persistent shortage of nitrogen relative to phosphorusfor algal production in Lake Rotorua.     

Phosphorus bioavailability of fluvial sediments determined by algal assays

Eroding bank soils and riverine suspended sediments from the Flathead River-Lake ecosystem, Montana, USA, were cultured with the alga Selenastrum capricornutum Printz in PAAP medium with the sediments as the sole source of phosphorus. Extraction of phosphorus by NaOH and nitrilotriacetic acid (NTA) solutions were compared to results from algal bioassays. The fine sediment particles transported into Flathead Lake during spring runoff had the highest availability (i.e. 6% of total phosphorus). Bank soils which contained the greatest percentage of fine clays exhibited similar (i.e. 4% of total phosphorus) availability. Bank soils containing predominantly organic phosphorus had the lowest availability. Spearman's rank correlation indicated significance at the 5% test level between algal assay estimates of available phosphorus and both chemical extraction techniques.     

Rapid antibody biosensor assays for environmental analysis

Traditionally, biosensor development has focused on molecules with a defined metabolic role that can be exploited by enzyme-based systems. Antibodies have the ability to move beyond this range of analytes, and are particularly useful in detecting small, hapten molecules. Electrochemically based biosensor developments have been less fruitful in this regard, as enzyme labelling is required, and such assays require the separation from bound and unbound species. These separations and the removal of background signals result in the increased complexity of the assay format, making it unsuitable for rapid sensor analysis. We have developed an electrochemical sensor based on antibodies that does not require the separation of bound and unbound molecules in a competition immunoassay format. This removes the need for several washing and separation steps as is normally employed in this type of assay. This allows single-step immunoassays to be performed using this system, and also allows for the real-time monitoring of antibody-antigen interactions. We have shown that such assays are possible in both batch and flow-injection formats and we are currently developing an assay for the pesticide atrazine. Tentative results show that analysis with this system is possible in the p.p.m. to p.p.b. range.     

Optical plankton analyser: a flow cytometer for plankton analysis, II: Specifications

An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton analysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 microns in width and over 1,000 microns in length can be analysed. Sample flow rates of up to 55 microliters/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 microns. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 10(6). A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.     

Response to nutrient enrichment by the plankton of Antarctic coastal lakes and the inshore Ross Sea

Summary Water samples from coastal lakes and ponds on Ross Island (77°S) and from the adjacent Ross Sea were analyzed to determine nutrient supply relative to the population size of planktonic algae and bacteria. An enrichment test was applied to each water to measure N and P accumulation by the seston as a guide to nutrient demand and deficiency. In all waters, including the sea, the dissolved inorganic N-to-P ratios were low, but inorganic nitrogen was generally present at concentrations that satisfied or exceeded the current growth requirements of the algae. None of the samples responded to added P, and only 3 of the 13 plankton assemblages tested responded positively to nitrogen enrichment. These waters are therefore characterized by an abundance of resources relative to demand. This apparently r-selecting coastal environment contrasts markedly in many ecosystem properties with the inland meromictic lakes of Antarctica.     

Rapid screening assays for soluble and particulate bacterial dehydrogenases

Two easy and quick assays are described for the semiquantitative determination of soluble and particulate dehydrogenases in bacteria. Soluble NAD- and NADP-linked dehydrogenases are determined by a spot test on paper. The presence of a dehydrogenase is recognized by the fluorescence of the reduced cofactor on the paper. Particle-linked dehydrogenases are detected by the bleaching of 2,6-dichlorophenol-indophenol. The advantages of these assays are that they are reliable, inexpensive to perform and require a minimum of time, labour and enzyme material. This may be useful as a comparative biochemical tool for bacterial taxonomy.     

Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles

We have used a model food chain composed of a natural bacterial assemblage, a pennate diatom and a bacterivorous microflagellate to investigate the factors controlling the relative importance of bacteria and protozoa as sources for regenerated nitrogen in plankton communities. In bacterized diatom cultures in which diatom growth was nitrogen-limited, the carbon:nitrogen (C:N) ratio of the bacterial substrate greatly affected which population was responsible for the uptake of nitrogen. When nitrogen was added as NH ₄ ⁺ and the cultures were supplemented with glucose, the bacteria competed successfully with the algae for NH ₄ ⁺ and prevented the growth of algae by rapidly assimilating all NH ₄ ⁺ in the cultures. Bacterivorous protozoa inoculated into these cultures grazed the bacterial population and remineralized NH ₄ ⁺ , thus relieving the nitrogen limitation of algal growth and allowing an increase in algal biomass. In contrast, bacteria in cultures supplemented with the amino acid glycine (C:N = 2) were major remineralizers of nitrogen, and the influence of protozoan grazing was minimal. We conclude that the relative importance of bacteria and protozoa as nutrient regenerators in the detrital food loop is dependent largely on the overall carbon:nutrient ratio of the bacterial substrate. The role of bacterivorous protozoa as remineralizers of a growth-limiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high.     

Rapid hybridization-based assays for identification by DNA probes of male-sterile and male-fertile cytoplasms of the sugar beet Beta vulgaris L.

Summary Methods are described whereby hybridization of mitochondrial (mt) DNA with different DNA probes can definitely distinguish male-fertile and and male-sterile (cms) cytoplasms of sugar beet Beta vulgaris L. We have developed two types of miniassays. (1) Comparative methods requiring the isolation and restriction of total cellular DNA, hybridization with cloned mtDNA fragments from either fertile or male-sterile cytoplasms, and comparison of the hybridization patterns to the fertile-and sterile-specific patterns of mtDNA of sugar beet for the given mtDNA probe. For these analyses, we routinely used 1 g of plant material to determine the type of cytoplasm. (2) Noncomparative (plus-minus) methods requiring neither the isolation of pure DNA nor restriction, electrophoresis, or Southern blotting. Instead, alkaline-SDS plant extracts from as little as 50 mg of plant material were dot-blotted and hybridized with fertile-specific (mitochondrial minicircular DNA) and/or cms-specific probes (consisting of a 2.3-kb mtDNA sequence exclusively occurring in the cms cytoplasm). The assays are simple to perform, give definitive results, are nonde-structive to the plants, and may be used in mass screening of sugar beet populations for hybrid production or in in vitro culture processes.     

A tripartite microbial reporter gene system for real-time assays of soil nutrient status

Plant-derived carbon is the substrate which drives the rate of microbial assimilation and turnover of nutrients, in particular N and P, within the rhizosphere. To develop a better understanding of rhizosphere dynamics, a tripartite reporter gene system has been developed. We used three lux-marked Pseudomonas fluorescens strains to report on soil (1) assimilable carbon, (2) N-status, and (3) P-status. In vivo studies using soil water, spiked with C, N and P to simulate rhizosphere conditions, showed that the tripartite reporter system can provide real-time assessment of carbon and nutrient status. Good quantitative agreement for bioluminescence output between reference material and soil water samples was found for the C and P reporters. With regard to soil nitrate, the minimum bioavailable concentration was found to be greater than that analytically detectable in soil water. This is the first time that bioavailable soil C, N and P have been quantified using a tripartite reporter gene system.     

Rapid and sensitive assays for phagosomal acidification in Paramecium and Tetrahymena

Biochemical and cytochemical procedures were developed to measure the rate of phagosomal acidification for phagosomal pH ranging from 5 to 2.5. These assays were based on the pH-dependent inactivation with time of horseradish peroxidase (HRP) activity, a result attributable to the dissociation of this enzyme to a colorless protein and ferriprotoporphyrin in acidic solutions. When preincubated in buffers of varying pH, the rate of HRP inactivation followed a sigmoid curve, with the highest rate of inactivation between 4.3 and 3.5 when using citrate-phosphate buffer and between pH 3.4 and 2.8 when using the universal ABC buffer. This inactivation was temperature but not concentration dependent. When Paramecium caudatum, members of the P. aurelia complex or Tetrahymena thermophila was pulsed briefly with HRP and small fluorescent beads, the loss of HRP activity, measured biochemically in cell homogenates and/or cytochemically in phagosomes, was rapid and followed the kinetics of a first-order rate reaction. Both assays gave similar values for the rate constant for acidification and similar rates of inhibition when P. caudatum was exposed to a proton ionophore, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. These assays can readily be adapted to other phagocytic cells as long as a rapid procedure is available for removing all unphagocytosed HRP and latex beads. These procedures are sensitive and rapid thus allowing many samples to be quickly prepared and analyzed.     

Rapid and flexible biochemical assays for evaluating 4'-phosphopantetheinyl transferase activity

PPTases (phosphopantetheinyl transferases) are of great interest owing to their essential roles in activating fatty acid, polyketide and non-ribosomal peptide synthetase enzymes for both primary and secondary metabolism, as well as an increasing number of biotechnological applications. However, existing techniques for PPTase characterization and development are cumbersome and technically challenging. To address this, we have developed the indigoidine-synthesizing non-ribosomal peptide synthetase BpsA as a reporter for PPTase activity. Simple co-transformation allows rapid assessment of the ability of a PPTase candidate to activate BpsA in vivo. Kinetic parameters with respect to either CoA or BpsA as variable substrate can then be derived in vitro by continuously measuring the rate of indigoidine synthesis as the PPTase progressively converts BpsA from its apo into holo form. Subsequently, a competition assay, in which BpsA and purified carrier proteins compete for a limited pool of CoA, enables elucidation of kinetic parameters for a PPTase with those carrier proteins. We used this system to conduct a rapid characterization of three different PPTase enzymes: Sfp of Bacillus subtilis A.T.C.C.6633, PcpS of Pseudomonas aeruginosa PAO1, and the putative PPTase PP1183 of Ps. putida KT2440. We also demonstrate the utility of this system for discovery and characterization of PPTase inhibitors.     

Rapid CE microbial assays for consumer products that contain active bacteria

Recent advances in high efficiency separation methods of bacteria allow their rapid identification and quantitation in some cases. A specific capillary electrophoresis (CE) technique is used to identify and quantitate Lactobacillus acidophilus in both pill and syrup health products as well as Bifidobacterium infantis in a powdered formula supplement. Cell viability can be evaluated as well. In some cases, both the living and dead bacterial cells as well as the molecular excipients can be evaluated in a single run.     

Production and decomposition of new DOC by marine plankton communities: carbohydrates, refractory components and nutrient limitation

The accumulation and biodegradation of dissolved organic carbon (DOC) and dissolved and particulate combined neutral sugars (DCNS, PCNS) were followed during a period of 22 days in experimental marine phytoplankton incubations. Five different growth regimes were established in 11 m³ coastal mesocosms to test whether an alternate Redfield ratio with either N or P depletion and ±diatom dominance would induce accumulation of refractory DOC (RDOC) and DCNS. The highest accumulation of DOC, DCNS and PCNS was found in the diatom dominated mesocosms. Sixteen percent of the newly accumulated DOC in the mesocosms with diatoms dominating could be explained by DCNS, while only 6% was explained in the mesocosms with few diatoms. PCNS composition was similar in all mesocosms and with dominance of glucose and mannose, while DCNS were more evenly distributed with the following mole percentages fucose 15, rhamnose 14, arabinose 6, galactose 27, glucose 20 and mannose 18%. The DCNS composition did not reflect the PCNS composition at any time during the experiment. Accumulated DCNS were quickly degraded and only 1% of the new RDOC was explained by DCNS. RDOC accumulated after day # 17 in the two mesocosms driven towards the most severe P limitation both with and without silicate. This shows that RDOC can be produced directly by the phytoplankton or indirectly in food web processes during the later stages of a bloom where the phytoplankton is P limited.     

Rapid and complete degradation of thymidine by human peripheral blood platelets: implications for genotoxicity assays

Small-scale washed cell preparations obtained by Percoll density-gradient fractionation of whole blood were used to study the metabolic fate of [3H]thymidine supplied to isolated human blood mononuclear cells and platelets incubated for up to 24 h in vitro. Two cell fractions were monitored: low molecular weight compounds which were soluble in Triton X-100 and TCA were investigated by thin-layer chromatography, and high molecular weight components, distinguished by their Triton and TCA insolubility, were examined by agarose-gel electrophoresis. Under the conditions used, greater than 99% of added [3H]thymidine was very rapidly degraded. Catabolites were recovered in the Triton-soluble (cytoplasmic) fraction and the extracellular medium. A negligible proportion of added label was associated with Triton- and TCA-insoluble cell fractions. These results confirm and clarify previous data and have important implications for genotoxicity tests which employ in vitro leukocyte cultures.