COMPARATIVE ULTRASTRUCTURE OF THREE SPECIES OF CHLOROSARCINA (CHLOROSARCINACEAE,CHLOROPHYTA)1,2

Ultrastructural studies revealed that Chlorosarcina stigmatica Deason differs from C. brevispinosa and C. longispinosa Chantanachat and Bold because the vegetative cells possess a pyrenoid, and the zoospores have chlorophycean characteristics. Zoospores of the latter species exhibit pleurastrophycean characteristics including counterclockwise absolute orientation of the flagellar apparatus components. Nuclear division in C. longispinosa, like that of Friedmannia israelensis Chantanachat and Bold, is metacentric. It is suggested that a new genus, Desmotetra, be erected for Chlorosarcina stigmatica, and that it, along with Chlorosarcinopsis Herndon, be placed in the Chlorococcales, Chlorophyceae. Chlorosarcina brevispinosa and C. longispinosa belong in the Pleurastrales, Pleurastrophyceae.     

CARBON PARTITIONING WITHIN PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE) COLONIES IN THE ROSS SEA, ANTARCTICA

The haptophyte Phaeocystis antarctica Karsten is a dominant species within the seasonal bloom in the Ross Sea. One of the unique characteristics of this form is that carbon is partitioned between the cells and the colonial matrix, a relationship that is poorly documented for this region. We combined particulate organic carbon measurements and microscopic analysis of P. antarctica -dominated samples to assess the contribution of single cells, colony-associated cells, and mucilage to the carbon concentrations of waters with P. antarctica. Two cruises to the Ross Sea were completed, one in austral spring 1994 and one in summer 1995–1996. In 1994 the bloom was dominated by colonial P. antarctica that contributed up to 96% of the total autotrophic carbon, whereas in 1995–1996 a mixture of P. antarctica and diatoms occurred. P. antarctica colony volume ( V ) was related to colonial cell number ( N _C) by the relationship V = 417 × N _C^1.67. Total colony carbon (C_COL) was calculated as the sum of cell carbon (C_CC) and mucus-related carbon (C_M). We found the contribution of mucus carbon to be 213 ng C mm ^{− 3} of colony volume. For P. antarctica -dominated assemblages sampled at the peak of the bloom, C_M represented a minor fraction (14 ± 4%) of colony carbon, and during early summer conditions C_M was at most 33% of C_COL. This organism plays a cardinal role in the carbon cycle of many regions. These results constrain the partitioning of carbon between cellular material and the colony matrix, information that is necessary to accurately describe the biogeochemical cycles influenced by this species.     

DEVELOPMENT OF PROTOPLASTS OF ULVA FASCIATA (CHLOROPHYTA) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate protoplasts of the green alga Ulva fasciata Delile and subsequently induce them to form a microthallus suspension for algal seed stock. The protoplasts were covered with secreted mucilage following 6 h of culture when viewed with SEM. The mucilage fused to form thick layers during day 1 of culture. Microfibrillar cell walls were deposited into the thick layers of mucilage on the 5th day of culture. An average of about 10% of the freshly isolated protoplasts began to divide at 6–14 days. These protoplasts subsequently developed varied morphologies, depending on the time of collection during the year. Protoplasts isolated from U. fasciata collected in March to June developed frond thalli or microthalli when they were cultured in low or high densities (cells/area), respectively. The microthallus suspension was cultured for more than two years at 10–40 μ mol·m^{− 2} ·s^{− 1} . Frond thalli formed when the suspension was cultivated at 100–160 μ mol·m^{− 2} ·s^{− 1} . Therefore, microthallus suspension can serve as a seed stock of U. fasciata .     

COMPARATIVE ULTRASTRUCTURE OF THREE SPECIES OF CHLOROSARCINA (CHLOROSARCINACEAE,CHLOROPHYTA) 1,2

Ultrastructural studies revealed that Chlorosarcina stigmatica Deason differs from C. brevispinosa and C. longispinosa Chantanachat and Bold because the vegetative cell possess a pyrenoid, and the zoospores have chlorophycean characteristics. Zoospores of the latter species exhibit pleurastrophycean characteristics including counterclockwise absolute orientation of the flagellar apparatus components. Nuclear division in C. longispinosa, like that of Friedmannia israelensis Chantanachat and Bold, is metacentric. It is suggested that a new genus, Desmotetra, be erected for Chlorosarcina stigmatica, and that it, along with Chlorosarcinopsis Herndon, be placed in the Chlorococcales, Chlorophyceae. Chlorosarcina brevispinosa and C. longispinosa belong in the Pleurastrales, Pleurastrophyceae.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

On the abundance of marine naked amoebae on the surface of five species of macroalgae

Abstract Naked amoebae constituted a numerically significant component of the surface microbial community of five species of seaweeds. They were most abundant in the summer months on the brown macrophytes, Fucus and Laminaria , where numbers up to 23 cells cm⁻² of algal surface were recorded. This implies that littoral algal stands can support populations of up to 3.2 × 10⁶ amoebae m⁻². A total of 27 different species were recognized and, of these, six species were less than 10 μm in length, a size class overlooked in previous studies. Damaged tissue contained higher numbers of amoebae, up to 43 cells cm⁻², presumably due to higher bacterial densities and increased shelter at these sites. Some amoebae may have been utilizing algal carbon exudates directly, especially isolates of Trichosphaerium , which showed evidence of algal digestion. The numbers of amoebae found in this study suggest that these protozoa may play a significant, and previously overlooked, role in the cycling of estuarine carbon.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

Arginine utilization by Mycoplasma fermentans is not regulated by glucose metabolism: A 13C-NMR study

Abstract Electrofusion of protoplasts of two mutant strains of Hansenula polymorpha resulted in high fusion and hybrid yields when the calcium ions present in the conventional fusion medium replaced by zinc ions. The optimal fusion conditions were an alignment field of 0.4 kV cm⁻¹ strength and 2 MHz frequency for 30 s, followed by two consecutive pulses of 12 kV cm⁻¹ strength and 15 μs duration. With 0.05–0.1 mM zinc ions in the fusion medium an average clone number of 10⁴–10⁵ clones per 10⁸ input cells was reached. The presence of about 0.6 mM magnesium ions in the zinc fusion medium was essential.     

Inactivation of bacteria and yeasts on agar surfaces with high power Nd: YAG laser light

G.D. WARD, I.A. WATSON, D.E.S. STEWART-TULL, A.C. WARDLAW AND C.R. CHATWIN. 1996. Near infrared light from a high-powered, 1064 nm, Neodymium : Yttrium Aluminium Garnet (Nd : YAG) laser killed a variety of Gram-positive and Gramnegative bacteria and two yeasts, lawned on nutrient agar plates. A beam (crosssectional area, 1.65 cm²) of laser light was delivered in 10 J, 8 ms pulses at 10 Hz, in a series of exposure times. For each microbial species, a dose/response curve was obtained of area of inactivation vs energy density (J cm⁻²). The energy density that gave an inactivation area (IA) equal to 50% of the beam area was designated the IA₅₀-value and was plotted together with its 95% confidence limits. Average IA₅₀-values were all within a threefold range and varied from 1768 J cm⁻² for Serratia marcescens to 4489 J cm⁻² for vegetative cells of Bacillus stearothermophilus. There were no systematic differences in sensitivity attributable to cell shape, size, pigmentation or Gram reaction. At the lowest energy densities where inactivation was achieved for the majority of organisms (around 2000 J cm⁻²), no effect was observed on the nutrient agar surface, but as the energy density was increased, a depression in the agar surface was formed, followed by localized melting of the agar.     

Electrotransformation of whole cells of Brevibacterium sp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Abstract: A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 10⁸ transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm⁻¹), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml⁻¹ and cell density (10¹⁰ cells ml⁻¹). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.     

Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

Generation of a reproducible nutrient-depleted biofilm of Escherichia coli and Burkholderia cepacia

An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 μmol cm⁻² glucose for E. coli and up to 2·7 × 10⁻⁹ mol cm⁻² iron for B. cepacia . Escherichia coli growing exponentially had a growth rate of μ = 0·30 h⁻¹ in a biofilm and μ = 0·96 h⁻¹ in planktonic culture. The growth rate, μ, for exponentially growing B. cepacia in a biofilm was 1·12 h⁻¹ and in planktonic culture 0·78 h⁻¹. This method allows the limitation of the size of a biofilm population to a chosen value.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

GRAPPLING WITH CONFLICT AMONG INFORMATION SOURCES IN RECONSTRUCTION OF THE EARLY EVOLUTION OF LAND PLANTS

In an assay addressing the toxicity of copper to Dictyosphaerium pulchellum , a 48 hours' exposure of axenic cultures of the alga to metal concentrations ranging from 0 to 300 mg L⁻¹ caused a significant decrease in the growth rate and cell chlorophyll content. Extracellular mucilage remotion by controlled sonication before exposure to copper emphasized chlorophyll content reduction, but not cellular density diminution. Because mucilage acts as a cation interchanger, these results suggested that, during at least the first 48 hours, division rates might be affected by lower Cu⁺² concentrations than chlorophyll content. Ultrastructural observations showed that the normal organization of the chloroplasts was altered; they presented dilated lamellae, irregularly arranged with practically no stacking. Copper precipitates were mainly observed outside the plasmalemma or within the extracellular mucilage. Electron probe microanalysis confirmed these observations, showing only minor precipitates inside the cells.     

Survival of largemouth bass, bluegill and channel catfish embryos after electroshocking

Survival to hatching was determined after electroshocking embryos of largemouth bass Micropterus salmoides , bluegill Lepomis macrochirus and channel catfish Ictalurus punctatus . Embryos at different developmental stages were exposed for 20 s to homogeneous electric fields (4–16 V cm⁻¹) of direct current (DC) or 60 or 120 Hz pulsed direct current (PDC) in water of 100 μS cm⁻¹ ambient conductivity. For all species, DC reduced survival of embryos at developmental stages before, during, or soon after epiboly; but survival did not differ from controls during later developmental stages. Survival of largemouth bass and bluegill was not reduced by 60 or 120 Hz PDC except for bluegill exposed at 12 h post‐fertilization. Channel catfish embryo survival was <5% when exposed to 60 or 120 Hz PDC at 8 h post‐fertilization, survival improved for embryos exposed at 67 h to 60 Hz but not to 120 Hz, and all embryos survived exposure to PDC at 150 h post‐fertilization. Exposure durations as short as 5 s resulted in <10% survival of largemouth bass during sensitive stages. All bluegill embryos aged 22 h post‐fertilization hatched prematurely after exposure to 16 V cm⁻¹ DC, but survival was not affected. The use of PDC for electroshocking near largemouth bass and bluegill nests could reduce the negative effects on survival of these species; however, PDC can reduce survival of channel catfish embryos.     

GROWTH OF HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE) ON NITRATE AND AMMONIUM AT THREE PHOTON FLUX DENSITIES: EVIDENCE FOR N STRESS IN NITRATE-GROWING CELLS

The marine alga Heterosigma carterae Hulburt (Raphidophyta) was grown in N-limiting batch cultures using either nitrate or ammonium as the N source, at photon flux densities (PFDs) of 50, 200, and 350 μmol·m^-2·s^-1 in a 12:12 h LD cycle. Carbon content could be estimated from biovolume (μg C = 0.278 × nL; R = 0.98) but not reliably from pigment content. During exponential growth, ammonium-grown cells (in comparison with nitrate-grown cells at the same PFD) attained higher growth rates by at least 20%, contained more N, and had a lower C:N ratio, higher concentrations of intracellular free amino acids, and higher ratios of glutamine: glutamate (Gln: Glu) and asparagine: aspartate (Asn:Asp). Growth was nearly light-saturated on ammonium at 200 μmol·m^-2·s^-1 (cell-specific growth rate of 1.2 d^-1) but probably not saturated in nitrate-grown cells at 350 μmol·m^-2·s^-1. PFD did not affect Gln: Glu or Asn: Asp for a given N source. These results indicate that the nitrate-growing cells were more N-stressed than those using ammonium (which in contrast were relatively C-stressed) and that this organism would show an enhanced competitive advantage against other species when supplied with a transient supply of ammonium rather than nitrate .     

Antimicrobial effect and shelf-life extension by combined thermal and pulsed electric field treatment of milk

Aims:  The impact of a combined hurdle treatment of heat and pulsed electric fields (PEF) was studied on native microbiota used for the inoculation of low-fat ultra-high temperature (UHT) milk and whole raw milk. Microbiological shelf-life of the latter following hurdle treatment or thermal pasteurization was also investigated.
Methods and Results:  UHT milk was preheated to 30°C, 40°C or 50°C over a 60-s period, pulsed for 50  μ s or 60  μ s at a field strength of 40 kV cm⁻¹ or for 33  μ s at 50 kV cm⁻¹. Heat and PEF reduced the microbial count by a maximum of 6·4 log in UHT milk (50°C; 50 kV cm⁻¹, 33  μ s) compared to 6·0 log ( P  ≥ 0·05) obtained by thermal pasteurization (26 s, 72°C). When raw milk was treated with a combination of hurdles (50°C; 40 kV cm⁻¹, 60  μ s) a 6·0 log inactivation of microbiota was achieved and microbiological milk shelf-life was extended to 21 days under refrigeration (4°C) vs 14 days in thermally pasteurized milk. Native microbiota was decreased by 6·7 log following conventional pasteurization.
Conclusions:  The findings suggest that heat and PEF achieved similar inactivation of native microbiota in milk and longer stabilization of microbiological shelf-life than thermal pasteurization.
Significance and Impact of the Study:  A hurdle approach of heat and PEF could represent a valid milk processing alternative to conventional pasteurization. Hurdle treatment might also preserve native milk quality better due to less thermal exposure.     

Influence of red light on the activity of phosphofructokinase in Chlorella kessleri

In crude extracts of the unicellular green alga Chlorella kessleri Fott et Novákóva grown in red light the activity of the glycolytic enzyme phosphofructokinase (PFK, EC 2.7.1.11) is about 40% higher compared to white light conditions giving the same dry matter production. Application of cycloheximide and density labelling with D₂O indicate that this increase depends on the de novo synthesis of the enzyme: Twelve h of illumination at a fluence rate of 7 × 10¹⁸ quanta m⁻² s⁻¹ (11.6 μmol m⁻² s⁻¹) suffice to saturate the effect. In autotrophically grown algae maximal increase in enzyme saturate the effect. In autotrophically grown algae maximal increase in enzyme activity is reached in light of 680 nm, while in 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU)-poisoned, glucose-fed cells, light of wavelengths around 727 nm is most effective. Involvement of a phytochrome-like photoreceptor is discussed.