Kenneth Budd

Biotransformation of Hg(II) by cyanobacteria.

ABSTRACT The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl2 in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl2. Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that meta-cinnabar (β-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria: Limnothrix planctonica (Lemm.), Synechococcus leopoldiensis (Racib.) Komarek, and Phormidium limnetica (Lemm.). From the outset of mercury exposure, there was rapid synthesis of β-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra- and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into β-HgS. In addition, increasing the temperature enhanced the amount of β-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into β-HgS. Furthermore, the efficiency of conversion into β-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.

Phosphate uptake by synechococcus leopoliensis cyanophyceae enhancement by calcium ion

The influence of metallic, cations (added at 10 μM‐1 mM) on the uptake of orthophosphate from 0.2–10 μM solution by Synechococcus leopoliensis (Racib.) Komarek was investigated. All cations tested except Mg2+ and Zn2+ stimulated phosphate uptake. The most pronounced stimulation of phosphate uptake was caused by Ca2+·Ca2+ markedly decreased the half‐saturation concentration for orthophosphate uptake, apparently by acting upon the metabolic processes of phosphate transport into the cell. Phosphate did not influence Ca2+ fluxes across the cell‐surface.

Mercury Analysis of Acid- and Alkaline-Reduced Biological Samples: Identification of meta-Cinnabar as the Major Biotransformed Compound in Algae

ABSTRACT The biotransformation of HgII in pH-controlled and aerated algal cultures was investigated. Previous researchers have observed losses in Hg detection in vitro with the addition of cysteine under acid reduction conditions in the presence of SnCl2. They proposed that this was the effect of Hg-thiol complexing. The present study found that cysteine-Hg, protein and nonprotein thiol chelates, and nucleoside chelates of Hg were all fully detectable under acid reduction conditions without previous digestion. Furthermore, organic (R-Hg) mercury compounds could not be detected under either the acid or alkaline reduction conditions, and only β-HgS was detected under alkaline and not under acid SnCl2 reduction conditions. The blue-green alga Limnothrix planctonica biotransformed the bulk of HgII applied as HgCl2 into a form with the analytical properties of β-HgS. Similar results were obtained for the eukaryotic alga Selenastrum minutum. No evidence for the synthesis of organomercurials such as CH3Hg+ was obtained from analysis of either airstream or biomass samples under the aerobic conditions of the study. An analytical procedure that involved both acid and alkaline reduction was developed. It provides the first selective method for the determination of β-HgS in biological samples. Under aerobic conditions, HgII is biotransformed mainly into β-HgS (meta-cinnabar), and this occurs in both prokaryotic and eukaryotic algae. This has important implications with respect to identification of mercury species and cycling in aquatic habitats.

Polyol metabolism and osmotic adjustment in the mycelial ascomyceteNeocosmospora vasinfecta (E. F. Smith)

The polyols ofN. vasinfecta were predominantly mannitol and arabitol, with minor quantities of glycerol andm-erythritol, when this fungus was grown at minimal osmotic stress (180 mOsm). Growth at increasing osmotic stress (180 to 2640 mOsm) was associated with a progressive decrease in the molecular weight of the most abundant polyol. When the fungus was subjected to osmotic upshock (5 h, 180 to 1040 mOsm), a slight decrease in mannitol was accompanied by a large increase in arabitol, with smaller increases in erythritol and glycerol. This pattern was seen with either KCl or glucose as the osmoticum. This polyol response was inhibited by cycloheximide, suggesting that translational processes were required for its induction. Downshock in growth medium or in isotonic CaCl2 resulted in identical reductions in intramycelial total polyol, and similar polyol patterns by 1 h. There were different fates for the polyol carbon which was lost. Downshock in growth medium resulted in excretion of hexoses and mannitol, while downshock in CaCl2 induced loss to the medium as polyols, in a manner consistent with a simple excretion model.

RESPONSES OF CERTAIN FRESHWATER PLANKTONIC ALGAE TO FLUORIDE1

The effects of dissolved fluoride supplied as NaF at up to 150 p.p.m. F− (7.9 mM) on growth, photosynthesis, dark respiration, enolase activity and fluoride uptake were determined for six phytoplankters: Synechococcus leopoliensis (Racib.) Komarek (Cyanophyta), Oscillatoria limnetica Lemmermann (Cyanophyta), Ankistrodesmus braunii Brun (Chlorophyta), Scenedesmus quadricauda (Turp.) Bréb. (Chlorophyta), Cyclotella meneghiniana Kützing (Bacillariophyta) and Stephanodiscus minutus Grun. ex Cleve et Moll (Bacillariophyta). Growth (determined by absorbance at 660 nm or by cell‐numbers) was unaffected by fluoride at up to 50 p.p.m. (2.6 mM) in all algae except S. leopoliensis, in which growth ceased transiently followed by resumption of growth at reduced rate. These effects showed a threshold at ca. 25 p.p.m. (1.3 mM) F− and increased with increasing F− concentration above this threshold. Photosynthetic O2 evolution in the chlorophytes was unaffected by F− at up to 50 p.p.m., whereas in S. leopoliensis F− above ca. 25 p.p.m. caused a concentration‐dependent inhibition of photosynthesis which was most pronounced at saturating irradiance. Dark O2 uptake was unaffected at up to 50 p.p.m. in chlorophytes but was stimulated in S. leopoliensis. Enolase in clarified cell‐extracts of all six algae was inhibited by F−, with Ki values ranging from 27 to 319 μM. Fluorine (measured by proton‐induced gamma‐ray emission) could not be detected in chlorophytes exposed during growth to up to 50 p.p. m. F−, but was detected in S. leopoliensis, O. limnetica and C. meneghiniana. Fluorine associated with cells of these algae increased as the external F− concentration increased.

Role of the membrane potential in the transport of zinc byNeocosmospora vasinfecta

Abstract The distribution of tetraphenyl phosphonium between mycelium and external medium was used to estimate the membrane potential ( E m ) in Neocosmospora . A minimum E m of −68 mV (interior negative) was calculated for aerobic mycelium in a buffer at pH 6.5 lacking metallic cations. The membrane was depolarized by carbonylcyanide- m -chlorophenylhydrazone, anaerobiosis, and KCl, whereas it was hyperpolarized by gramidicin, trifluoperazine (TFP), and prior treatment with acetate at low pH. TFP caused K + to be lost from the mycelium and stimulated zinc uptake. Acetate pretreatment also enhanced zinc uptake. These and previous results (K. Budd, 1988 , Exp. Mycol. 12 , 195–202) indicate that zinc transport in Neocosmospora is correlated with the membrane potential.

A high-affinity system for the transport of zinc in Neocosmospora vasinfecta

Abstract Zinc-sufficient mycelium of Neocosmospora vasinfecta ATCC 11686 incubated in a buffer lacking metallic cations displayed complex velocity/concentration kinetics for the transport of zinc across the mycelial surface. Two transport “systems” were identified from Hofstee plots, one with a high affinity for zinc ( K 0.5 approx 1.1 μ M ) and one with a low affinity for zinc ( K 0.5 approx 770 μ M ). Zinc transport via the high-affinity system was relatively insensitive to the presence of other metallic cations in the uptake medium but was strongly inhibited by anaerobiosis and by carbonylcyanide- m -chlorophenylhydrazone at uncoupling concentrations. Diethylstilbestrol and dicyclohexylcarbodiimide only weakly inhibited zinc transport. The transport of zinc was also influenced by mycelial zinc and magnesium status, and to a lesser extent by mycelial P content.

Elemental analysis of algae with PIXE and PIGME

Abstract With an external proton beam, layers of algae a few μm thick produced by the simple procedure of filtering fixed amounts of a colony onto millipore filters have been examined. The biological aspects of the investigation dealt with the influence of F− on the growth of the algae; therefore proton inelastic scattering was used in addition to PIXE for the elemental analysis. Since the samples were thin (∼8 μm), the broad resonance structure in the 19 F( p , p ′γ) 19 F yield curve at Ep=2.03 Me V was used to increase the sensitivity. A sensitivity of 10 ppm was reached for these thin specimens, quite adequate to show that the algae take up fluorine from the environment. With beams of a few nanoamperes, the counting rate for all elements was found to decrease linearly with flux, reaching 70% of the initial value after about 1000 s. The concentration of fluorine in the algae was found to be proportional, up to 150 ppm, to the F− ion concentration in the nutrient. For one species of algae, S. leopoliensis, the presence of fluoride ion in the nutrient caused a marked change in the growth pattern. Furthermore, this change was found to be correlated with the concentration in the algae of one essential element, potassium, which the algae seemed to lose and then recover.

THE MECHANISMS OF FLUORIDE TOXICITY AND FLUORIDE RESISTANCE IN SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYCEAE)1

Fluoride was supplied as dissolved NaF at concentrations ranging from 0.26 to 7.9 mM (5–150 ppm) to three freshwater microalgae: Synechococcus leopoliensis (Racib.) Komarek (Cyanophyta), Oscillatoria limnetica Lemmermann (Cyanophyta) and Chlorella pyrenoidosa Chick (Chlorophyta). Growth of C. pyrenoidosa was unaffected by fluoride, and uptake of fluoride by this organism was not detectable. Growth of the cyanophytes was temporarily inhibited by NaF. The duration of this growth lag increased markedly as the pH was lowered at constant external fluoride concentration. In S. leopoliensis, fluoride uptake and inhibition of photosynthesis by NaF increased in the same way as did the growth lag in response to pH. Growth‐inhibitory NaF treatments decreased the ATP level in cells of S. leopoliensis by 75% and also abolished phosphate uptake. Cells of S. leopoliensis in which fluoride‐resistance was induced by prior growth in non‐growth‐inhibitory levels of NaF accumulated much less fluoride than did normal (“sensitive”) cells, and also did not respond to fluride by reduction of the ATP pool. It is suggested (1) that fluoride enters sensitive cells of S. leopoliensis principally as undissociated HF; (2) that its major inhibitory effect in these cells is the reduction in cellular ATP; (3)that fluoride‐resistant cells accumulate less fluoride by developing incresed permeability to the fluoride anion.

Osmotic adjustment in the mycelial ascomyceteNeocosmospora vasinfecta (E. F. Smith)

Abstract Osmotic adjustment in the ascomyceteNeocosmospora vasinfecta was investigated by determining intramycelial water, mycelial solutes, and total mycelial osmolality. Major organic and inorganic solutes as well as proline and glycine betaine were determined under conditions of osmotic stress and shock, imposed by 0.5M KCl. Comparison to glucose as a nonelectrolytic osmoticum was also made. Results quantitatively implicated the polyhydric alcohols as the osmotic adjusters. Changes in amino acids were due to growth and were not osmoregulatory in nature. The osmoticum was not utilized for osmotic adjustment. The growth ofN. vasinfecta in the presence of KCl indicated that this organism is moderately sensitive to osmotic stress.