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Protocol for laboratory testing of crude-oil bioremediation products in freshwater conditions.

In 1993, the Environmental Protection Agency, National Risk Management Research Laboratory (EPA, NRMRL), with the National Environmental Technology Application Center (NETAC), developed a protocol for evaluation of bioremediation products in marine environments [18]. The marine protocol was adapted for application in freshwater environments by using a chemically defined medium and an oil-degrading consortium as a positive control. Four products were tested using the modified protocol: two with nutrients and an oleophilic component; one with nutrients, sorbent, and organisms; and one microbial stimulant. A separate experiment evaluated the use of HEPES and MOPSO buffers as replacements for phosphate buffer. The oleophilic nutrient products yielded oil degradation similar to the positive control, with an average alkane removal of 97.1+/-2.3% and an aromatic hydrocarbon removal of 64.8+/-1.2%. The positive control, which received inoculum plus nutrients, demonstrated alkane degradation of 98.9+/-0.1% and aromatic degradation of 52.9+/-0.1%. The sorbent-based product with inoculum failed to demonstrate oil degradation, while the microbial stimulant showed less oil degradation than the positive control. Replacement of phosphate buffer with other buffers had no significant effect on one product's performance. Differences in product performance were easily distinguishable using the protocol, and performance targets for alkane and aromatic hydrocarbon degradation are suggested.

Alkanes↗

A medium for the detection of yeasts using a conductimetric method.

A new medium is described for the detection of yeasts using a Malthus microbial growth analyser which measures conductivity. This medium is superior to previous media devised for use in the Malthus, and allows the detection of a wide range of yeasts including those important in food spoilage. The new medium was inferior to existing media used to detect yeasts in the Bactometer 32 instrument which measures impedance.

Colony Count, Microbial↗

Bio-degradation of olive oil husks in composting aerated piles.

In this study, the composting performance of two olive oil husk piles was compared using two different aeration processes: aeration by mechanical turning or by forced air-injection. The results showed that after the maturation phase both piles had a similar chemical composition and the same level of organic matter degradation (around 55%). However, the time necessary to reach the thermophilic phase was shorter for the turned pile (2 months in comparison to 3 months for the air-injected pile) and the humification degree achieved was slightly higher (94% versus 83%). Both piles showed the presence of an active microbial community, with an increase by 2-3 orders of magnitude in the number of cultured microbial colonies during the composting process. No significant difference in quantitative or qualitative terms was found in the microbial populations of both piles, nor was a clear succession between a mesophilic and a thermophilic population observed. In terms of industrial application, the mechanical turning process seems to be better since it is a lower energy and time consuming process.

Agriculture↗

Detection of water-borne E. coli O157 using the integrating waveguide biosensor.

Escherichia coli O157:H7, the most common serotype of enterohemorrhagic E. coli (EHEC), is responsible for numerous food-borne and water-borne infections worldwide. An integrating waveguide biosensor is described for the detection of water-borne E. coli O157, based on a fluorescent sandwich immunoassay performed inside a glass capillary waveguide. The genomic DNA of captured E. coli O157 cells was extracted and quantitative real-time PCR subsequently performed to assess biosensor-capture efficiency. In vitro microbial growth in capillary waveguide is also documented. The biosensor allows for quantitative detection of as few as 10 cells per capillary (0.075 ml volume) and can be used in conjunction with cell amplification, PCR and microarray technologies to positively identify a pathogen.

Biosensing Techniques↗

Combined ozone and ultraviolet inactivation of Escherichia coli.

The kinetics of Escherichia coli inactivation using ozone and ultraviolet (UV) radiation, separately and simultaneously, was evaluated at 25 degrees C in buffered (pH 6.0, 7.0 and 8.0), demand-free media. While ozone was found to be a stronger disinfectant than UV radiation, using both simultaneously was more effective than using them individually. Inactivation kinetics was pseudo first-order for the three treatment processes, while the disinfection rate was a linear function of the disinfectant dose. The synergism observed in microbial inactivation when the disinfectant processes were combined was illustrated by estimates of kinetic model parameters. This synergy was attributed to the generation of hydroxyl radicals via ozone photolysis. Subsequently, dosage calculations, as based on disinfectant level and exposure time, indicated that the simultaneous use of UV and ozone could substantially reduce their individual doses.

Colony Count, Microbial↗

Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine.

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.

Bacteria↗

[Dynamics of microbial processes in the stratal waters of the Romashkinskoe oil field].

Dynamics of the microbial processes developing in parallel with the exploitation of the Romashkinskoe oil field (Tatarstan) was studied in two areas differing in the degree of stratal water freshening. Flooding the strata in conjunction with purposeful measures on stratal microflora activation was shown to increase the microbial population density and activate both methanogenesis and sulfate-reduction; the latter process was limited by the low sulfate concentration. Development of anaerobic processes correlated with changes in acetate concentration in the stratal water. High mineralization (over 200 g/l) inhibited the stratal water microflora even if other conditions were favorable. Isotopic analysis of the carbonate carbon showed that the bicarbonate concentration increased in the stratal water due to microbial degradation of oil hydrocarbons and further participation of the biogenic carbon dioxide in dissolution of the carbonate cement of the oil-bearing strata. In strongly desalinated stratal water, the proportion of the newly formed bicarbonate was as high as 80%.

Acetates↗

Rapid separation and counting of viable microbial cells in food by nonculture method with bioplorer, a focusing-free microscopic apparatus with a novel cell separation unit.

A nonculture method utilizing a novel apparatus, the bioplorer, was developed. The bioplorer is composed of an efficient cell separation unit, a focusing-free microscopic device, and an image analysis program. A meat or vegetable suspension is poured into the cell separation funnel, and insoluble matter in the sample suspension is trapped by prefilters. Microbial cells passing through the two prefilters are then trapped by the membrane filter (pore size, 0.4 microm). Trapped cells are double-stained with 4',6'-diamidino-2-phenylindole and propidium iodide, and the membrane filter is removed and set on the focusing-free microscope. A fluorescent image is then recorded. Total numbers of viable and dead cells on the membrane filter can thus be determined automatically. One assay can be performed within 10 min, which is much faster than the culture method. The results obtained with both the nonculture method and the culture method for meat and vegetable samples were highly correlated (r = 0.953 to 0.998). This method is feasible for the practical purpose of food safety control.

Bacteria↗

Optimization of direct cell counting in sediment.

This study reports a method for optimizing direct counts of bacteria in sediment, designed to reduce the masking by sediment particles. The protocol was designed to determine appropriate dilution factors by incorporating counting statistics and was used to measure depth-associated changes in microbial abundance in metal-impacted freshwater sediments. We demonstrated a direct method to determine appropriate sample dilution for accurate counting by adding a known amount of cells to the sediment. For accurate counting in our sediment samples, we determined that the average number of bacteria per microscope ocular field must be between 8.5 and 10. This is well below the 30 bacteria/field previously suggested for accurate counting. These results indicate that an optimal dilution rate must be determined before accurate direct counts in sediment can be achieved.

Bacteria↗

Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages.

In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (> 100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.

Bacteria↗

Long-term aerobic cometabolism of a chlorinated solvent mixture by vinyl chloride-, methane- and propane-utilizing biomasses.

The aerobic cometabolic biodegradation of a mixture of chlorinated aliphatic hydrocarbons (CAHs) including vinyl chloride (VC), cis- and trans-1,2-dichloroethylene (cis-DCE, trans-DCE), trichloroethylene (TCE), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA) was investigated at both 25 and 17 degrees C by means of bioaugmented and non-bioaugmented sediment-groundwater slurry microcosm tests. The goals of the study were (i) to study the long-term aerobic biodegradation of a CAH mixture including a high-chlorinated solvent (1,1,2,2-TeCA) generally considered non-biodegradable in aerobic conditions; (ii) to investigate the efficacy of bioaugmentation with two types of internal inocula obtained from the indigenous biomass of the studied site; (iii) to identify the CAH-degrading bacteria. VC, methane and propane were utilized as growth substrates. The non-bioaugmented microcosms were characterized, at 25 degrees C, by an average 18-day lag-time for the direct metabolism of VC (accompanied by the cometabolism of cis- and trans-DCE) and by long lag-times (36-264 days) for the onset of methane or propane utilization (associated with the cometabolism of the remaining CAHs). In the inoculated microcosms the lag-phases for the onset of growth substrate utilization and CAH cometabolism were significantly shorter (0-15 days at 25 degrees C). Biodegradation of the 6-CAH mixture was successfully continued for up to 410 days. The low-chlorinated solvents were characterized by higher depletion rates. The composition of the microbial consortium of a propane-utilizing microcosm was determined by 16s rDNA sequencing and phylotype analysis. To the best of our knowledge, this is the first study that documents the long-term aerobic biodegradation of 1,1,2,2-TeCA.

Aerobiosis↗

Diversity and distribution of pigmented heterotrophic bacteria in marine environments.

A systematic investigation of marine pigmented heterotrophic bacteria (PHB) based on the cultivation method and sequencing analysis of 16S rRNA genes was conducted in Chinese coastal and shelf waters and the Pacific Ocean. Both the abundance of PHB and the ratio of PHB to CFU decreased along trophic gradients from coastal to oceanic waters, with the highest values of 9.9 x 10(3) cell mL(-1) and 39.6%, respectively, in the Yangtze River Estuary. In contrast to the total heterotrophic bacteria (TB) and CFU, which were present in the whole water column, PHB were primarily confined to the euphotic zone, with the highest abundance of PHB and ratio of PHB to CFU occurring in surface water. In total, 247 pigmented isolates were obtained during this study, and the phylogenetic analysis showed a wide genetic diversity covering 25 genera of six phylogenetic classes: Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacilli, Flavobacteria and Sphingobacteria. PHB belonging to Alphaproteobacteria, Flavobacteria and Sphingobacteria were obtained mainly from the South China Sea and East China Sea; PHB from the Pacific Ocean water were predominantly affiliated with Gammaproteobacteria, and most isolates from the Yangtze River Estuary fell into the classes Actinobacteria and Bacilli. The isolates exhibited various colours (e.g. golden, yellow, red, pink and orange), with genus or species specificity. Furthermore, the pigment of PHB cells absorbed light mainly in the wavelength range between 450 and 550 nm. In conclusion, our work has revealed that PHB with broad genetic diversity are widely distributed in the marine environment, and may account for up to 39.6% of culturable bacteria, equivalent to 1.4% of the total microbial community. This value might even be underestimated because it is probable that not all pigmented bacteria were isolated. Their abundance and genetic distribution are heavily influenced by environmental properties, such as light and nutrition, suggesting that they have important roles in the marine ecosystem, especially in the absorption of visible light.

Bacteria↗

Microorganisms in the accreted ice of Lake Vostok, Antarctica.

Analysis of a portion of Vostok ice core number 5G, which is thought to contain frozen water derived from Lake Vostok, Antarctica (a body of liquid water located beneath about 4 kilometers of glacial ice), revealed between 2 x 10(2) and 3 x 10(2) bacterial cells per milliliter and low concentrations of potential growth nutrients. Lipopolysaccharide (a Gram-negative bacterial cell biomarker) was also detected at concentrations consistent with the cell enumeration data, which suggests a predominance of Gram-negative bacteria. At least a portion of the microbial assemblage was viable, as determined by the respiration of carbon-14-labeled acetate and glucose substrates during incubations at 3 degrees C and 1 atmosphere. These accreted ice data suggest that Lake Vostok may contain viable microorganisms.

Acetates↗

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity.

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.

Animals↗

Residual effects of fluoride on a severe cariogenic challenge in rats.

The influence of temporary dietary restriction with and without fluoridated drinking water on the progression of caries was studied in 60 desalivated Sprague-Dawley rats. Dietary restriction alone had little effect on caries progression when the challenge was interrupted and then resumed, in contrast to the effects of dietary restriction plus fluoride. Animals subjected to gavage plus fluoride in the drinking water for 11 days developed significantly less severe lesions on their lingual surfaces than did any of the other groups. No significant differences were observed in the microbial flora of the animals at the end of the study. The results show that temporary dietary restriction alone has little permanent effect on caries progression and that fluoride administered even for a short time in the absence of cariogenic challenge has a statistically significant, but probably less than optimum, protective effect against a subsequent caries challenge.

Animals↗

Microbial contamination of "in use" bar soap in dental clinics.

Bar soap from 18 different dental clinics were investigated for microbial contamination, while it was "in-use". Of the 32 samples obtained from the bar soap, 100% yielded positive culture. A total of 8 different genera of organisms were isolated. Each bar soap was found to harbor 2-5 different genera of micro organisms. Heavily used soap had more micro organisms compared to less used soap. The microbial load of the "in-use" bar soap constituted a mixed flora of gram positive, gram negative, aerobes, anaerobes, and fungi. The results indicate that the bar soap under "in-use" condition is a reservoir of microorganisms and handwashing with such a soap may lead to spread of infection.

Aspergillus niger↗

Microbial growth and air pollution in carbonate rock weathering. Preliminary results of a in situ experimental study.

Preliminary results on limestone weathering caused by air pollution and microbial colonization are presented in this study. Outdoor exposure experimental assays were performed on Scaglia limestone samples. Samples were exposed in two areas in Perugia (Italy) that differ for degree of urban air pollution. At different times of exposure, ranging from 1 to 12 months, microbial contamination and textural modifications of sampled surfaces were evaluated by microbiological procedures, X-ray diffraction, and scanning electron microscopy. After one year of exposure a significant fungal colonization and the presence of weathering products (i.e. gypsum) were detected on sampled surfaces.

Air Pollutants↗

Periodontal pathogens on polytetrafluoroethylene membrane for guided tissue regeneration inhibit healing.

This study determined the microbial composition of the apical parts of the expanded polytetrafluoroethylene membrane surfaces facing the gingiva and the tooth in guided tissue regeneration. Microbial and clinical features of 2-to-3 wall periodontal bony defects treated with membranes with and without concomitant use of systemic Augmentin therapy were also determined. 18 patients with 18 study sites participated. 9 patients received systemic 500 mg Augmentin 1 h prior to surgery, and 500 mg TID for 8 days thereafter. 9 patients received no systemic antimicrobial therapy. Microbiological examination was performed 1 h prior to surgery, at the time of membrane removal at week 6, and at 6 months post-surgery. Microbial morphotypes, total viable counts, and the occurrence of selected microbial species were determined by phase-contrast microscopy, selective and non-selective culture, and DNA probes. Study sites were examined for probing pocket depths and attachment levels. At baseline, no microbial or clinical parameter showed statistical differences between groups. At 6 months, the Augmentin group demonstrated a significantly higher (P = 0.032; Student t-test) mean probing attachment gain (36.5% of potential gain to the cemento-enamel junction) than the 9 control patients (22.4% of potential gain). At the time of removal, membranes in the Augmentin group showed significantly fewer organisms than membranes in the control group (52.2 x 10(6) versus 488.6 x 10(6)). Sites free of pathogens on the membrane surface toward the tooth gained the most clinical attachment, even in the presence of various pathogens on the gingiva-facing membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗