Raymond J. Ritchie

Universal chlorophyll equations for estimating chlorophylls a, b, c, and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents

A universal set of equations for determining chlorophyll (Chl) a, accessory Chl b, c, and d, and total Chl have been developed for 90 % acetone, 100 % methanol, and ethanol solvents suitable for estimating Chl in extracts from natural assemblages of algae. The presence of phaeophytin (Ph) a not only interferes with estimates of Chl a but also with Chl b and c determinations. The universal algorithms can hence be misleading if used on natural collections containing large amounts of Ph. The methanol algorithms are severely affected by the presence of Ph and so are not recommended. The algorithms were tested on representative mixtures of Chls prepared from extracts of algae with known Chl composition. The limits of detection (and inherent error, ±95 % confidence limit) for all the Chl equations were less than 0.03 g m−3. The algorithms are both accurate and precise for Chl a and d but less accurate for Chl b and c. With caution the algorithms can be used to calculate a Chl profile of natural assemblages of algae. The relative error of measurements of Chls increases hyperbolically in diluted extracts. For safety reasons, efficient extraction of Chls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of Chls in natural assemblages of aquatic plants.

A CRITICAL ASSESSMENT OF THE USE OF LIPOPHILIC CATIONS AS MEMBRANE POTENTIAL PROBES

A critical review has been made of the literature on the use of lipophilic cations, such as triphenylmethyl phosphonium (TPMP ÷ ) as membrane potential probes in prokaryotes, eukaryote organelles in vitro, and eukaryote cells. An ideal lipophilic cation should be capable of penetrating through a biological membrane and obey the Nernst equation between a membrane bound phase and its environment. Many different forms of the Nernst equation are presented, useful in the calculation equilibrium potentials oflipophilic cations across membranes. Lipophilic cations appear to behave as valid membrane potential probes in prokaryotes and eukaryote organelles in vitro and even in vivo although some technical difficulties may be involved. On the other hand invalid forms of the Nernst equation have often been used to calculate the equilibrium potential of lipophilic cations across the plasma membranes of eukaryotic cells. In particular, the problem of intracellular compartmentation of lipophilic cations has often not been appreciated. Lipophilic cations do not appear to behave as reliable plasma membrane potential probes in eukaryotic cells. Some other avenues are discussed which might be useful in the determination of the plasma membrane potentials of small eukaryotic cells, e.g. the use of lipophilic anions as membrane potential probes.

Modelling photosynthesis in shallow algal production ponds

Shallow ponds with rapidly photosynthesising cyanobacteria or eukaryotic algae are used for growing biotechnology feedstock and have been proposed for biofuel production but a credible model to predict the productivity of a column of phytoplankton in such ponds is lacking. Oxygen electrodes and Pulse Amplitude Modulation (PAM) fluorometer technology were used to measure gross photosynthesis (PG) vs. irradiance (E) curves (PGvs. E curves) in Chlorella (chlorophyta), Dunaliella salina (chlorophyta) and Phaeodactylum (bacillariophyta). PGvs. E curves were fitted to the waiting-in-line function [PG = (PGmax × E/Eopt) × exp(1 — E/Eopt)]. Attenuation of incident light with depth could then be used to model PGvs. E curves to describe PGvs. depth in pond cultures of uniformly distributed planktonic algae. Respiratory data (by O2-electrode) allowed net photosynthesis (PN) of algal ponds to be modelled with depth. Photoinhibition of photosynthesis at the pond surface reduced PN of the water column. Calculated optimum depths for the algal ponds were: Phaeodactylum, 63 mm; Dunaliella, 71 mm and Chlorella, 87 mm. Irradiance at this depth is ≈ 5 to 10 μmol m−2 s−1 photosynthetic photon flux density (PPFD). This knowledge can then be used to optimise the pond depth. The total net PN [μmol(O2) m−2 s−1] were: Chlorella, ≈ 12.6 ± 0.76; Dunaliella, ≈ 6.5 ± 0.41; Phaeodactylum ≈ 6.1 ± 0.35. Snell’s and Fresnel’s laws were used to correct irradiance for reflection and refraction and thus estimate the time course of PN over the course of a day taking into account respiration during the day and at night. The optimum PN of a pond adjusted to be of optimal depth (0.1–0.5 m) should be approximately constant because increasing the cell density will proportionally reduce the optimum depth of the pond and vice versa. Net photosynthesis for an optimised pond located at the tropic of Cancer would be [in t(C) ha−1 y−1]: Chlorella, ≈ 14.1 ± 0.66; Dunaliella, ≈ 5.48 ± 0.39; Phaeodactylum, ≈ 6.58 ± 0.42 but such calculations do not take weather, such as cloud cover, and temperature, into account.

The use of pulse amplitude modulation (PAM) fluorometry to measure photosynthesis in a CAM orchid, Dendrobium spp. (D. cv. Viravuth Pink).

Pulse amplitude modulation (PAM) fluorometer techniques provide unique information on photosynthetic activity of CAM (crassulacean acid metabolism) plants such as the orchid Dendrobium spp. (cv. Viravuth Pink). CAM plants close their stomata for at least part of the day, creating a sealed compartment in the stems and leaves that precludes measurement of the light reactions of photosynthesis by any gas exchange–based method. PAM machines calculate photosynthesis as the electron transport rate (ETR) through PSII (four electrons per O2 produced) as mol m−2 s−1. Photosynthesis‐versus‐irradiance (P‐vs.‐E) curves fitted the waiting‐in‐line function ( \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape

A simplified model for assessing critical parameters during associative 15N2 fixation between Azospirillum and wheat

\mathrm{ETR}\,=( \mathrm{ETR}\,_{\mathrm{max}\,}\times E/ E_{\mathrm{opt}\,}) \times e^{1-E/ E_{\mathrm{opt}\,}}

Breakdown of food waste by anaerobic fermentation and non-oxygen producing photosynthesis using a photosynthetic bacterium

\end{document} ), allowing half‐saturating and optimal irradiances (Eopt) to be estimated. Effective quantum yield (Ymax), ETR (ETRmax), and the nonphotochemical quenching parameter NPQmax all vary on a diurnal (circadian) cycle, but the parameter qNmax does not show a systematic variation over a diurnal period. Dendrobium Viravuth is a “sun plant,” with Eopt between 404 and 539 μmol m−2 s−1 PAR but photosynthetic capacity is very low in the late afternoon. Total CO2 fixed nocturnally as C4‐dicarboxylic acids by leaves was only ≈120 mg C m−2 d−1. Titratable acid of leaves was depleted by ∼1500 hours and shows a classical CAM diurnal cycle. Dendrobium Viravuth stores only enough CO2 as C4 acids to account for ∼10% of the daily gross photosynthesis estimated with the PAM machine (≈1.4 g C m−2 d−1). Dendrobium Viravuth is an orchid cultivar with very limited capacity to fix and store C4 acids at night. It is probably a CAM‐cycling orchid.

Photosynthetic electron transport in an anoxygenic photosynthetic bacterium Afifella (Rhodopseudomonas) marina measured using PAM fluorometry.

Uptake, assimilation and compartmentation of phosphate were studied in the opportunist green macroalgaUlva lactucaand the estuarine red algal epiphyteCatenella nipae. The Michaelis–Menten model was used to describe uptake rates of inorganic phosphate (Pi) at different concentrations. Maximum uptake rates (Vmax) of P-starved material exceededVmaxof P-enriched material; this difference was greater forC. nipae. Uptake and allocation of phosphorus (P) to internal pools was measured using trichloroacetic acid (TCA) extracts and32P. Both species demonstrated similar assimilation paths: when P-enriched, most32P accumulated as free phosphate. When unenriched,32P was rapidly assimilated into the TCA-insoluble pool.C. nipaeconsistently assimilated more32P into this pool thanU. lactuca, indicatingC. nipaehas a greater P-storage capacity. In both species,32P release data showed two internal compartments with very different biological half-lives. The rapidly exchanging compartment had a short half-life of ≈2 to 12 min, while the slowly exchanging compartment had a much longer half-life of 12 days in P-starvedC. nipaeor 4 days in P-starvedU. lactuca. In both species, the slowly exchanging compartment accounted for more than 90% of total tissue.U. lactucaandC. nipaeresponded differently to high external Pi.U. lactucarapidly took up Pi, transferring this Piinto tissue phosphate and TCA-soluble P in a few hours (≈90% of total P).C. nipaetook up Piat lower rates and stored much of this P in less mobile TCA-insoluble forms. Long-term storage of refractory forms of P makesC. nipaea useful bioindicator of the prevailing conditions of Piavailability over at least the previous 7 days, whereas the P-status ofU.lactucamay reflect conditions over no more than the previous few hours or days.C. nipaeis a more useful bioindicator for P status of estuarine and marine waters thanU. lactuca.

Comparison of a lipophilic cation and microelectrodes to measure membrane potentials of the giant-celled algae,Chara australis (Charophyta) andGriffithsia monilis (Rhodophyta)

Detailed studies in field experiments have shown repeatedly that the transfer of 15 N 2 fixed by diazotrophic bacteria to wheat tissue is minimal. Here, a simple and convenient laboratory co-culture model was designed to assess important features of the association between Azospirillum brasilense and wheat, such as the rate of nitrogen fixation (acetylene reduction), ammonia excretion from the bacterium and the transfer of newly fixed 15 N 2 from the associative diazotroph to the shoot tissue of wheat plants. After 70 h, in this model, insignificant amounts of newly fixed N 2 were transferred from an ammonia-excreting strain of A. brasilense to the shoot tissue of wheat. However, when malate was added to the co-culture the 15 N enrichment of the shoot tissue increased 48-fold, indicating that 20% of shoot N had been derived from N 2 fixation. Thus, the inability of the host plant to release carbon in the rhizosphere is a significant constraint in the development of associative N 2 -fixing systems. These specific results suggest that wheat plants with an increased release ofphotosynthate to the rhizosphere should be a priority for the future development of broad-acre agricultural systems that are more self-sufficient for nitrogen nutrition. The simplicity of the model for assessing the critical parameters of associative 15 N 2 fixation may allow large-scale surveys of plant-bacterial interactions to be conducted and a selection of improved associations for further study.