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.     

Short-term thermal acclimation induces adaptive changes in the inner mitochondrial membranes of fish skeletal muscle

In the oxidative muscles (musculi laterales superficiales) of crucian carp Carassius carassius acclimated for 6 weeks to either 5 or 25° C, the volume density and the surface density of fibres per tissue did not differ significantly between the control and experimental groups. The correlation ratio (μ²) for these values was below 50, 39·3 and 43·9 respectively. After acclimation to 5° C, the surface density of outer mitochondrial membrane per fibre increased significantly from 0·93 to 1·23m² cm⁻³ in the summer population but dropped from 0·94 to 0·67 m² cm⁻³ in the winter population. The surface density of outer mitochondrial membrane per mitochondrion increased from 3·24 to 4·52 m² cm⁻³ in summer fish. After acclimation to 25° C, the surface density of inner mitochondrial membranes per muscle fibre decreased from 4·04 to 1·79 m² cm⁻³ in summer fish and from 3·86 to 1·07 m² cm⁻³ in winter fish. The surface density of inner mitochondrial membranes per mitochondrion increased from 14·17 to 15·60 m²cm⁻³ in summer fish but dropped from 13·91 to 10·67 m² cm⁻³ in winter fish. Correlation matrices demonstrate a negative correlation of the surface density of outer mitochondrial membrane per mitochondrion with the volume density of mitochondria per fibre and temperature, suggesting cold-induced proliferation of small mitochondria. It was concluded that short-term cold acclimation increased surface area of the inner mitochondrial membranes in summer fish.     

Effectiveness of a steam-vacuum sanitizer for reducing Escherichia coli O157:H7 inoculated to beef carcass surface tissue

A steam-vacuum sanitizer reduced aerobic plate counts associated with bovine faecal contamination from 5.5 log₁₀ cfu cm⁻² to 3.0 ± 0.21 log₁₀ cfu cm⁻² on beef carcass short plates. The same beef carcass short plates inoculated wiht 7.6 ± 0.09 log₁₀ cfu cm⁻² Escherichia coli O157: H7 in faeces, yielded an average residual level of E. coli O157: H7 of 2.1 ± 0.21 log₁₀ cfu cm⁻² after steam-vacuum treatments. This study demonstrates the effectiveness of a steam-vacuum sanitizer for removing E. coli O157: H7 from beef carcasses.     

Soil oxygen flux measured polarographically in an Alaskan tussock tundra

Soil oxygen flux was measured polarographically in undisturbed tussock tundra and in an old vehicle track at Eagle Creek, Alaska. Because some polarograms did not show effective voltage plateaus, the polarographic method was tested for use in the tundra by experimental manipulation of soil water content and microbial oxygen demand. The response of soil oxygen flux was as predicted for artificially waterlogged systems, and the response to increased microbial demand indicated relatively high oxygen availability. Soil oxygen flux ranged from 25.9 ng O₂ cm⁻² min⁻¹ in Sphagnum mats to 3.9 ng O₂ cm⁻² min⁻¹ in unvegetated ruts of the vehicle track. Most sites in unvegetated ruts did not have measurable soil oxygen flux. In contrast, oxygen flux beneath Eriophorum vaginatum tussocks growing in the vehicle track averaged 10.7 ng O₂ cm⁻² min⁻¹.     

Electroporation of abalone sperm enhances sperm-DNA association

The ability of sperm from the black-footed abalone Haliotis iris to take up foreign DNA in solution has been demonstrated. The efficiency of DNA uptake is related to the conditions of electroporation, including field strength (625 V cm⁻¹, 1000 V cm⁻¹), pulse length (18.6 ms, 27.4ms) and number of pulses (1, 2), and DNA concentration (20, 100 μg ml⁻¹). Sperm motility decreased with increased field strength and pulse number. At a field strength of 625 V cm⁻¹, neither the pulse length nor pulse number enhanced DNA uptake. A 40% enhancement in DNA uptake was observed when the sperm were shocked at 1000 V cm⁻¹ with two long pulses (27.4 ms each). Linear regression analysis revealed that pulse number ( p = 0.013) and field strength ( P =0.039) were the most important factors in sperm–DNA interaction. Higher DNA concentration enhanced sperm DNA uptake irrespective of field strength, pulse length and pulse number. The optimal electroporation conditions for DNA uptake were 1000 V cm', with two pulses of 27.4 ms each, and a DNA concentration of 100 μg ml⁻¹.     

Numerical abundance of myxomycetes (myxogastrids) in soils in the West of England

Abstract A most-probable-number technique was used to quantify the abundance of myxomycetes (myxogastrids) in soil samples taken from 23 'Sites of Special Scientific Interest' and one other site in the West of England. Associated organisms and soil conditions were also recorded. Sixteen of the 24 sites yielded myxomycete plasmodium-forming units (PFUs) in numbers averaging 230 cm⁻³ fresh soil. Where detectable in samples of grassland soil, the numbers ranged from 20–2750 cm⁻³, in woodland soil from 20–800 cm⁻³, and in sand dunes from 80–400 cm⁻³. Repeated sampling revealed changing numbers at single sites. The abundance of PFUs was correlated positively with numbers of soil amoebae, ciliates and nematodes, and with levels of magnesium and soil density, and negatively with organic matter content and levels of NH₄-nitrogen and phosphate. Each PFU probably corresponded to one or several uninucleate cells in the soil rather than to a plasmodium.     

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.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

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.     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

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.     

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.     

Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L.: subcellular localization

Abstract. Studies on the subcellular location of ethylene binding activity from developing cotyledons of Phaseolus vulgaris L. are described. Binding activity has been shown to be predominantly membrane bound. When separated by rate-zonal centrifugation more than 70% of this activity was of low sedimentation rate. The slowly sedimenting band of activity was further fractionated into three bands by isopycnic density gradient centrifugation. The three bands occur at sucrose densities of 1.125 g cm⁻³, 1.155 g cm⁻³ and 1.175 g cm⁻³, corresponding to the distribution of putative marker enzymes for the cell endomembrane system and to protein body membranes. Further circumstantial evidence was obtained by electron microscopy and sucrose step gradient centrifugation.     

Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

Abstract Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 μmol DMSP 1⁻¹) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 μmol DMSP 1⁻¹ h⁻¹, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 × 10⁷ cells cm⁻³ sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 × 10⁵ cells cm⁻³ in the diatom mat (23% cleavers, 77% demethylators), and 9 × 10⁴ cells cm⁻³ (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.     

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 .     

Interfering mechanism of sodium bicarbonate on spore germination of Bacillus stearothermophilus

Spore germination of Bacillus stearothermophilus was progressively inhibited as the concentrations of sodium bicarbonate (NaHCO₃) in the germination media increased from 0% to 1·0% (w/v). The inhibitory effect of NaHCO₃ was attributed to the release of HCO⁻₃ and its alkaline properties, each of which played a different role. At low concentrations (< 0·3%), the inhibitory effect of NaHCO₃ was mainly due to bicarbonate. As NaHCO₃ increased from 0·3% to higher concentrations, the effect of HCO⁻₃ reached a plateau while the alkalinating effect became the more dominant inhibitory factor. Fourier transform infrared (FTIR) analysis reveals that sodium bicarbonate reacted with the carboxyl group (1570 cm⁻¹) of some acidic amino-acid residues of protein in the spore, leading to a less orientated structure. A shift of two units towards the longer frequency for carboxyl groups indicates that a stronger interaction was formed between the carboxyl group and the Na⁺ ion. The largest ratio of peak height between the absorbance of carboxylate (1570 cm⁻¹) and of amide II (1546 cm⁻¹) of spores after pretreatment with 0·3% sodium bicarbonate reflects the biggest structural alterations of keratin-like proteins in the spore. The role of NaHCO₃ in enhancing the sporicidal effect of glutaraldehyde is discussed.     

Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland

SUMMARY 1. Viral and bacterial abundances were studied in relation to environmental attributes over an annual period, for both planktonic and attached (sediment, aquatic macrophyte and submerged wood) habitats, in a riverine wetland.
2. Annual mean abundance of planktonic viruses ranged from 2.3 × 10⁵−3.8 × 10⁵ particles mL⁻¹ and varied according to sampling site. Significant seasonal patterns in viral abundance were evident and appeared to be linked to variations in bacterial abundance, dissolved organic carbon and inorganic nutrients.
3. Annual mean abundance of viruses associated with surfaces ranged from 1.3 × 10⁶ particles cm⁻² on aquatic macrophytes to 1.1 × 10⁷ particles cm⁻² on wood and also showed seasonal patterns. The difference in viral dynamics among the different sites emphasizes the importance of considering habitat diversity within wetlands when examining microbial communities.     

Physiological stress effects of continuous- and pulsed-DC electroshock on juvenile bull trout

Juvenile bull trout Salvelinus confluentus exposed to continuous- or pulsed-DC electroshock exhibited rapid elevations in plasma cortisol and glucose, but plasma chloride did not change. In a 1-h experiment using 240 V at 1·4 A of 60-Hz pulsed DC (voltage gradient 0·81 V cm⁻¹), which proved lethal, plasma cortisol and glucose rose significantly within 15 min of a 10-s electroshock. Plasma cortisol reached a peak level of 156 ± 18 ng ml⁻¹ at 45 min and then decreased, whereas plasma glucose reached its highest level of 179 ± 7·5mg dl⁻¹ at 1 h. In a 24-h experiment using lower dosages, plasma cortisol increased from 6·1-16 ng ml⁻¹ to peak levels of 155–161 ng ml⁻¹ in 1 h in response to a 10-s electroshock of continuous (130 V, 0·5 A, 1·45 V cm⁻¹) or pulsed (120 V, 0·5 A, 60 Hz, 0·55 V cm⁻¹) DC. Although plasma concentrations declined thereafter, levels remained above control values at 24 h. Plasma glucose was elevated from 60–65 to 120–134 mg dl⁻¹ after 1h by both electroshock treatments and remained near or above those levels for the 24-h duration. Plasma cortisol and glucose levels were much higher in electroshocked bull trout at 1 h compared with those in fish 1 h after receiving a 30-s handling stressor (cortisol, 90 ± 12 ng ml⁻¹; glucose, 82 ± 6·1 mg dl⁻¹). The results indicate that both continuous and pulsed DC were more stressful to juvenile bull trout than handling and that recovery, at least for pulsed DC, may take longer than 24 h.     

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.     

Fish production in Oued Sebaou, a seasonal river in North Algeria

Fish were sampled by electrofishing at three contiguous sites 26 km from the source of Oued Sebaou, a seasonal river in North Algeria. River discharge ranges from 300 m³s⁻¹ in the peak of the rainy season to 0.1 m³s⁻¹ at the end of the dry season. Extensive gravel extraction from the river bed takes place during the dry season.
Two species were recorded at each site: Barbus callensis , which is heavily fished locally, and Anguilla anguilla . The three sample maximum likelihood Zippin method was used to estimate mean density and standing crops, which were: barbel, 452 fish ha⁻¹, 5.33 kg ha⁻¹; eels, 149 fish ha⁻¹, 3.04 kg ha⁻¹. Estimated average biomass and production were: barbel, 2.31 kg ha⁻¹, 4.27 kg ha⁻¹ year⁻¹; eels, 1.51 kg ha⁻¹, 2.60 kg ha⁻¹ year⁻¹.