Fatty acid composition and microbial activity of benthic marine sediment from McMurdo Sound, Antarctica

Abstract Signature lipids from the phospholipid esterlinked fatty acids (PELFA) of cell membranes were used to describe benthic microbial communities of 4 Antarctic sediments. Metabolic activities of the communities were determined by incorporation of [³H]thymidine into bacterial DNA and sodium [¹⁴C]acetate into membrane lipids. Biomass measurements from extractable phospholipid fatty acids per g dry wt. ranged between 6 to 76 nmol, or when converted to number of bacteria, 3.7 × 10⁸ to 4.5 × 10⁹ cells per g dry wt. The West Sound site at New Harbor contained the lowest biomass, while Cape Evans on the East Sound contained the greatest. A marked difference was also noted between sites in their sediment microbial community structure. The East Sound sites at Cape Armitage and Cape Evans contained a greater abundance of diatom marker lipids, whilst both sides of the Sound contained approximately the same relative amounts of bacterial groups distinguished using PELFA. Activity of sediment microorganisms measured by radiolabel incorporation under ambient conditions followed the trends of the biomass measurements. The East Sound sites were more active by an average of 45–73% for [³H]thymidine and possibly also for sodium [¹⁴C]acetate.     

Environmental factors affecting the occurrence of mycobacteria in brook sediments

The occurrence of mycobacteria was studied in aerobic brook sediments from 39 drainage areas in Finland. The culturable counts of mycobacteria were related to climatic conditions, characteristics of the drainage area, chemical characteristics of the sediment and water, culturable counts of other heterotrophic bacteria, and microbial respiration rate in the sediment. The counts of mycobacteria varied from 1·1 × 10² to 1·5 × 10⁴ cfu g⁻¹ dry weight of sediment. They correlated positively with the proportion of the drainage area consisting of peatland, total content of C, content of Pb, microbial respiration rate in the sediment, and chemical oxygen demand of the water. The correlations of the mycobacterial counts with pH of sediment and alkalinity of water were negative. The results of the present sediment study and of the forest soil study published earlier strongly suggest that an increase in acidity increases the counts of mycobacteria and decreases the counts and activity of other heterotrophic bacteria. Mycobacterial counts were more than 100 times higher (per dry weight) in forest soils with pH 3·5–4·3 than in sediments with pH 4·5–6·3.     

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.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

Spatiotemporal distribution of microbial communities in a coastal, sandy aquifer system (Doñana, SW Spain)

The aquifer system of Doñana (SW Spain) represents the most important freshwater source in the Doñana Natural Area. Its spatiotemporal dynamics favours the hydrological connection between surface and subsurface ecosystems, and promotes matter fluxes among the different terrestrial and aquatic systems present here. This aquifer has been intensively studied from a hydrogeological point of view but little is known from an ecological perspective. In order to understand the ecological roles played by microbial communities in this system, we conducted a long-term seasonal study of bacterial abundance, cell biomass, bacterial biomass and functional activities over a 2-year period. Bacterial abundance ranged between 2.11 ± 1.79 × 10⁵ and 8.58 ± 6.99 × 10⁷ bacteria mL⁻¹ groundwater, average cell biomass was estimated to be 77.01 ± 31.56 fgC and bacterial biomass varied between 8.99 ± 4.10 × 10⁻² and 5.65 ± 0.70 µgC mL⁻¹. Iron-related bacteria showed the highest activities among the functional groups studied. Moreover, among the variables that usually control spatial distributions of microbial communities in aquifer systems, depth did not have a relevant effect on this aquifer, at least in the range of depths studied, but grain size, probably due to its direct effects on hydrogeological parameters, such as permeability or porosity, appeared to exert moderate control, principally in terms of bacterial abundance. Finally, significant seasonal differences in the means of these microbiological variables were also observed; temperature seems to be the main factor controlling the temporal distribution of microbial communities in this aquifer system.     

Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

Quantitative analysis and mapping of micro-organisms in anaerobic digester granules using a combination of transmission electron microscopy with immunotechnology

The precise localization of microbes within the matrix of digester granules has not previously been fully elucidated. To address this problem, a dual approach was applied for the first time, combining a method that identifies microbes with another that defines the structure in which the microbes are located. Bacterial and archaea morphotypes within granules from an upflow anaerobic sludge blanket digester purifying brewery waste water were quantified and localized in situ from TEM micrographs. Cell numbers were translated to biomass using image analysis techniques. Cells in granule homogenates were counted using a haemocytometer while methanogen and acidogen strains were enumerated using antibody probes. Granules showed stratification ; an outer cortex consisting primarily of cells, and an inner medulla virtually devoid of cells. Methanothrix- and Methanobrevibacter- like cells (26·2% and 37% of the population, or 46·3% and 27·1% of the biomass, respectively) concentrated in two bands within the granule whereas Syntrophobacter- like cells (9·6% of the population or 19·0% of the biomass) were evenly distributed throughout the cortex. Haemocytometer counts indicated 6·95×10⁶ cells ml⁻¹ which was lower than values obtained from TEM quantification because of cell losses during granule homogenization. Only 39% of the cells counted using the haemocytometer, or from TEMs, were identified using antibody probes, suggesting that combined methodologies provide more accurate cell quantification within granules or similar uniform attached microbial associations. Moreover, errors arising from specific techniques can be detected and quantified.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase-producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×10⁶ to 3·7×10⁸ cfu g⁻¹ and from 1·3×10³ to 1·6×10⁸ cfu g⁻¹, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml⁻¹. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas , Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter , coryneforms, Enterobacteriaceae, Moraxella , Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml⁻¹) was found in 12 strains, 11 from Aeromonas and one Pseudomonas . The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

The seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves

The epidemiology of clinical cases of campylobacter in temperate climates shows a striking seasonality. In the search for a seasonal environmental reservoir changes in the carriage rate and population size of campylobacters in bovine hosts with time have been measured. Most probable number (MPN) methodology was used to enumerate thermophilic campylobacters in samples taken from the small intestines of beef cattle at slaughter and the fresh faeces of four dairy herds and new-born calves. Statistical analyses revealed significant evidence for seasonal periodicity in the data from dairy herds ( P = 0·044). Not only was there a departure from constancy within a 12-month interval but these data revealed a true seasonality, that is, the same periodicity in numbers from one year to the next. Each herd had two peaks per year, in approximately spring and autumn. Peaks coincided in herds on neighbouring farms but those on farms in the north preceded those on farms in the south by 2 and 1 months, respectively ( P = 0·0057). Intestinal carriage by beef cattle at slaughter was 89·4% ( n = 360) with an average MPN campylobacters per gram fresh weight (MPN gfw⁻¹) of 6·1 × 10². Average MPN gfw⁻¹ in faeces from the dairy herds and calves were 69·9 ( S.D. 3) and 3·3 × 10⁴ ( S.D. 1·7 × 10²). There was no evidence of seasonal periodicity in the size of the campylobacter population in beef cattle at slaughter. Calves were campylobacter free at birth but became colonized within a few days.     

Sperm allocation in the three-spined stickleback

Male three-spined stickleback Gasterosteus aculeatus have a fixed amount of sperm during the breeding season because spermatogenesis is inhibited at this time. A method was developed to estimate ejaculate size in situ by removing the sperm from the male's nest. The reliability of the method was tested using known numbers of sperm. In their first mating, males ejaculated 11·64 × 10⁶ sperm (median), representing c. 5% of the male's sperm store (median 27·88 × 10⁷ sperm). The amount of sperm in the testes was significantly reduced in males that had mated several times (median 8·09 × 10⁷). Additionally, ejaculate size was smaller in these experienced males (median 8·79 × 10⁵). Heavier and larger fish invested absolutely and relatively more sperm in a mating than did lighter and smaller fish. Ejaculate size did not correlate with the mass of the egg clutch.     

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.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

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 .     

Clarification of small volume microbial suspensions in an ultrasonic standing wave

The removal of Saccharomyces cerevisiae and Escherichia coli from 2·5 ml suspensions in ultrasonic standing wave formed at 1 or 3 MHz has been characterized. The standing wave was set up by a plane transducer and reflector mounted in the vertical plane. Cells in the ultrasonic field first concentrated in vertical planes at half wavelength separations. The ultrasound was then pulsed to allow clumps of concentrated cells to sediment in a controlled way during the short 'off' intervals. Yeast removal from suspension at a concentration of 3 × 10⁹ ml⁻¹ (14% volume v/v) was 99·5% in a total time of 4·5 min. Almost total (99·5%) clarification of prokaryote ( E. coli ) suspension was achieved here for the first time in a standing wave field. The clarification of a 1·3 × 10¹¹ ml⁻¹ (16% v/v) E. coli suspension occurred over 11·5 min. The period decreased to 7 min in the presence of a polycationic flocculant, polyethyleneimine. The implications of the results for design of systems to further reduce clarification times are discussed. Removal efficiency for both S. cerevisiae and E. coli decreased with decrease in cell concentration. This concentration dependence is shown not to be simply a consequence of acoustic interaction between single cells. Flow cytometry of stained cells detected no loss of cell viability arising from the ultrasonic procedure.     

Microbial Spoilage of Fresh Refrigerated Beef Liver

The types and numbers of micro-organisms involved in the spoilage of refrigerated beef liver were studied together with pH, hydration and organoleptic changes of the material. Fresh liver harboured a mixed population ( c . 1 × 10⁵ organisms/g) of Gram positive cocci, chromogens and non-chromogens, sporeformers, presumptive coliforms and Gram negative rods. When samples were rejected organoleptically, after 7–10 days at 5°, the contamination attained levels of c . 7–8 × 10⁷ organisms/g. Spoilage was due to souring; the pH fell from 6·3 (fresh liver) to c . 5·9. Lactic acid bacteria were predominant and Gram negative bacteria did not exceed 1·0 × 10⁶ organisms/g. This type of spoilage is explained by the carbohydrate content of c . 5% in liver. The value of pH appears to be a reliable indicator of liver freshness, with a pH of 6·1 indicating incipient spoilage.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.