Distribution and activity of microorganisms in subsurface sediments of a pristine study site in Oklahoma

Distribution and activity of microorganisms in surface soil and subsurface sediments were studied in depth profiles of six different microbial biomass and activity indicators (total direct counts, number of cells capable of electron transport system activity, viable cell plate counts, most Probable numbers of protozoa, and 4-hydroxybenzoate-degrading microorganisms, and ATP content). The profiles showed the same general trends on two different dates (January and June 1985). Seasonal variations were noted, but they were not extreme. Biomass and activity values declined sharply with depth in the unsaturated zone, reaching minima in a clay confining layer in the interface zone between 3 and 4 m. Contiguous 10-cm samples from the interface zone showed significant textural and microbiological variability. Higher and more stable biomass and activity values were detected in the saturated zone, the highest being a very permeable gravelly loamy sand layer at approximately 7.5 m. In this layer, viable counts were nearly equal to total counts and they approached the viable counts in surface soil. Surface-type protozoa and cyanobacteria also were detected in this layer, suggesting that it was connected hydrologically to a nearby river. Lowest values were detected in an underlying bedrock clay layer at 8 m, which, despite its impermeability and low viable counts, did contain measurable total counts, 4-hydroxybenzoate-degrading microorganisms, and ATP. Correlations were noted between sediment texture and microbial activity (i.e., sandy texture=high activity, clayey texture=low activity), but other hydrogeological and geochemical factors probably also influenced microbial distribution and activity in the profile.     

Distribution of iron- and manganese-oxidizing bacteria in the bottom sediments of Lake Baikal

The specific features of the distribution, abundance, and taxonomic diversity of bacteria oxidizing iron and manganese in the bottom sediments sampled from different areas of Lake Baikal are studied. These bacteria are widespread in Baikal bottom sediments. Cultivated iron- and manganese-oxidizing microorganisms are ascribed to six genera: Metallogenium, Leptothrix, Siderocapsa, Naumaniella, Bacillus, and Pseudomonas. The surface layers of the ground and the border between oxidized and reduced sediments serve as ecological niches for iron- and manganese-oxidizing microorganisms. Redox conditions of the environment and the availability of dissolved forms of iron and manganese, as well as of organic matter, which are controlled by the conditions of sedimentation in the lake, are the main factors affecting the abundance and distribution of iron bacteria.     

西双版纳次生林土壤微生物生态分布及其生化特性的研究

西双版纳次生林土壤微生物生态分布及其生化特性的研究张萍（中国科学院昆明生态研究所,６５０２２３）ＥｃｏｌｏｇｉｃａｌＤｉｓｔｒｉｂｕｔｉｏｎａｎｄＢｉｏｃｈｅｍｉｃａｌＰｒｏｐｅｒｔｉｅｓｏｆＳｏｉｌＭｉｃｒｏｏｒｇａｎｉｓｍｓｉｎＳｅｃｏｎｄａｒｙ...     

Potential Health Hazards from Microbial Aerosols in Densely Populated Urban Regions

Aerosolized bacteria were recovered up to 930 m downwind of three sewage treatment plants in Jefferson County, Ky. This distance includes homes in the proximity of several hundred such plants in that county. Bacterial counts were elevated on foliage near activated sludge tanks; although these counts decreased rapidly, at 48 h after exposure they were significantly higher than the counts on unexposed leaves. The 50% lethal dose of aerosolized Klebsiella pneumoniae was comparable to the 50% lethal dose of a virulent clinical isolate, and enteric bacteria were recovered from the respiratory organs of mice after forced inhalation adjacent to an aerated sludge tank. The coliform density in the effluents of the plants tested was inversely related to the airborne bacterial load at those plants. This relationship was attributed to the correlation between effluent quality and extent of aeration of activated sludge. Wind direction and distance influenced the airborne counts, but the extreme variation in counts indicates that it is not possible to predict emission rates accurately in an open ecosystem. Airborne enteric bacteria also were isolated near a decorative fountain used by humans for wading. The discovery of these sources of aerosolized microorganisms from polluted waters in densely populated areas suggests that a potential health hazard may be created by the increased probability of inhaling and ingesting microorganisms of fecal origin.     

Some special problems in the determination of viable counts of groundwater microorganisms

Factors affecting viable cell counts in groundwater or sediments were studied with samples from the Segeberg Forest test area in northern Germany. There was very little variation in results with the season (April, August, November) or depth of sampling; generally there were 10³–10⁴ aerobic cells per ml or g sediment. Long incubation times resulted in higher cell counts; groundwater samples required 4–5 weeks, and sediment extracts had to be cultured for 7 weeks. Total cell counts in sediment were 10²–10⁴ cell/g higher than viable cell counts of aerobes. This was explained partly by the additional presence of anaerobes and partly by the observation that some morphotypes may not have grown under our conditions. Viable cell counts were not influenced by cell extraction from the sediment with either Na-pyrophosphate or groundwater extracts. However, iron-precipitating or manganese-oxidizing bacteria were better extracted with sterile groundwater. The microflora of wells was more numerous than that of the free aquifer; consequently it was better to pump off all well water before aquifer water was sampled. The diameter of the well was also important; thinner tubes had higher cell counts than those with wider diameter. For sampling, wells should be at least 1 year old, since young wells contain higher numbers of microorganisms due to underground disturbances from the drilling. Turbid water samples could be clarified by filtration, but this reduced the viable counts by 1–2 orders of magnitude. Two different media inoculated with a sample dilution resulted in the same cell counts, but their microbial diversity was different. Storage of groundwater samples before processing resulted in up to 17-fold increases in cell counts and loss of diversity in the first 24 hours. Cell numbers decreased slowly during longer storage.     

Influence of coal source and treatment upon indigenous microbial communities

Summary The leaching of six Eastern coals was investigated using experimental coal columns subjected to simulated leaching events. Measurements of CO₂ assimilation and specific enrichment cultures indicated that the microbial communities of all leachates were dominated by iron- and sulfur-oxidizing chemoautotrophic bacteria. Comparison of CO₂ assimilation rates in leachates and core samples of leached coal indicated that most chemoautotrophs remained within coal columns during leaching. Mean numbers of chemoautotrophic bacteria in leachate samples were correlated with concentrations of dissolved iron and sulfate. Leachates from unwashed, run-of-mine coals contained more chemoautotrophs and more iron and sulfate than did leachates from washed, final product coals. After several leachings, the ratio of sulfur oxidizers to iron oxidizers tended to increase. These data suggest that the chemoautotrophic community of final product coals may be pyritelimited. Aerobic heterotrophs constituted a minor component of the microbial community in leachates from the six coals and their abundance and metabolic activity were apparently not influenced by the beneficiation history of the coal. Changes in rates of acetate metabolism may have been related to microbial succession within the heterotrophic community of coal columns. In all leachates, rates of tritiated methylthymidine assimilation were correlated with rates of acetate incorporation but not with CO₂ assimilation, even though autotrophs dominated the microflora. Thus, thymidine assimilation rates appear to reflect activities or growth of mainly heterotrophic microorganisms in leachate.     

Evolution des populations bactériennes dans les sédiments des marais maritimes de l'Ile Grande pollués par I'Amoco Cadiz Evolution of Bacterial Populations in Salt Marsh Sediments of Ile Grande Polluted by Amoco Cadiz

Microbial investigations on heterotrophic bacteria (MPN counts, adenylates charge, growth rate and enzymatic activities) and hydrocarbon utilizing microorganisms (MPN counts) were carried out during three years in the salt marshes of Ile Grande. The aim was to follow their recovery after oil pollution. Oil degradation is related to bacterial activity (depending on site, depth in the soil, redox level and hydrocarbon concentrations). The results for the three years show a significant enhancement of the microflora during the first year (corresponding to removal of n. alkanes) followed by a decrease in bacterial activity which persisted for over two years. The activity of the heterotrophic bacterial population is deeply disturbed compared with unoiled sites, and these shifts remain detectable three years after oiling. This study does not modify our previous estimation regarding the recovery of the salt marshes.     

Improved Method for Determination of Respiring Individual Microorganisms in Natural Waters

A method is reported that combines the microscopic determinations of specific, individual, respiring microorganisms by the detection of electron transport system activity and the total number of organisms of an estuarine population by epifluorescence microscopy. An active cellular electron transport system specifically reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, which is recognized as opaque intracellular deposits in microorganisms stained with acridine orange. In a comparison of previously described sample preparation techniques, a loss of >70% of the counts of INT-reducing microorganisms was shown to be due to the dissolution of INT-formazan deposits by immersion oil (used in microscopy). In addition, significantly fewer fluorescing microorganisms and INT-formazan deposits, both ≤0.2 μm in size, were found for sample preparations that included a Nuclepore filter. Visual clarity was enhanced, and significantly greater direct counts and counts of INT-reducing microorganisms were recognized by transferring microorganisms from a filter to a gelatin film on a cover glass, followed by coating the sample with additional gelatin to produce a transparent matrix. With this method, the number of INT-reducing microorganisms determined for a Chesapeake Bay water sample was 2-to 10-fold greater than the number of respiring organisms reported previously for marine or freshwater samples. INT-reducing microorganisms constituted 61% of the total direct counts determined for a Chesapeake Bay water sample. This is the highest percentage of metabolically active microorganisms of any aquatic population reported using a method which determines both total counts and specific activity.     

Bacterial Activity in a Reservoir Determined by Autoradiography and its Relationships to Phyto- and Zooplankton

In the drinking water reservoir Římov (Southern Bohemia) bacterioplankton was studied during 1983. Special attention was given to the relationships between parameters of bacterial abundance, total and individual activity. Bacterial counts and biomass was assessed and autoradiographic determinations of the proportion of active bacteria incorporating thymidine (Th) and mixture of amino acids (AA) and total uptake rate of AA were made over a year in the surface layer and during summer stratification from the thermocline and 15 m depth. Specific activity of metabolically active bacteria (SAMAB) and specific activity per unit of biomass (SAUB) were negatively correlated with counts of metabolizing cells and with bacterial biomass, respectively. Total and individual heterotrophic activity and counts of bacteria coincided with the changes of phytoplankton biomass, whereas bacteria incorporating Th were more tightly correlated with primary production. The most significant relation of metabolically active bacteria was found to cladoceran biomass. Thus, this part of heterotrophic bacterial activity seems to be stimulated by leakage of dissolved organic matter from phytoplankton being disrupted and incompletely digested by cladocerans rather than from healthy photosynthetizing cells.     

Diversity and distribution of the prokaryotic community in near-surface permafrost sediments in the Tianshan Mountains, China

The community structures and diversity of bacteria and archaea were investigated at 4 depths (1.5, 2.0, 2.5, and 3.0 m) in permafrost sediments in the Tianshan Mountains, using denaturing gradient gel electrophoresis of 16S rRNA gene amplified by polymerase chain reaction. Phylogenetic analysis of the dominant bands sequenced revealed the presence of rich diversity of bacteria, which could be related to the Proteobacteria, Actinobacteria, Acidobacteria, Gemmatimonadetes, Bacteroidetes, Firmicutes, and Chloroflexi. The Proteobacteria, consisting of the alpha, beta, gamma and epsilon subdivisions, were clearly the dominant group at all depths studied. Archaeal diversity was relatively low and archaeal 16S rRNA gene sequences were grouped into 3 phylogenetic clusters within the 2 kingdoms Euryarchaeota and Crenarchaeota. Within the Euryarchaeota, methanogen-related group II was most abundant at shallow depth (1.5 m), whereas halobacterium-related group I dominated at greater depths. The low-temperature Crenarchaeota group was detected only at 2.5 and 3.0 m. Specific-depth distribution of methanogen-related Euryarchaeota group II and denitrifying bacteria of the genus Pseudomonas dominated at 1.5 m depth, accompanied by a distinct peak in the ratio of NH4-N to NO3/NO2-N, implying the potential capacity of these organisms in near-surface permafrost to release the greenhouse gases N2O and CH4.     

Abundance and activity of nitrate reducers in an arable soil are more affected by temporal variation and soil depth than by elevated atmospheric [CO2

Elevated atmospheric carbon dioxide concentrations ([CO(2) ]) might change the abundance and the function of soil microorganisms in the depth profile of agricultural soils by plant-mediated reactions. The seasonal pattern of abundance and activity of nitrate-reducing bacteria was studied in a Mini-FACE experiment planted with oilseed rape (Brassica napus). Three depths (0-10, 10-20 and 20-30 cm) were sampled. Analyses of the abundances of total (16S rRNA gene) and nitrate-reducing bacteria (narG, napA) revealed strong influences of sampling date and depth, but no [CO(2)] effects. Abundance and activity of nitrate reducers were higher in the top soil layer and decreased with depth but were not related to extractable amounts of nitrogen and carbon in soil. Dry periods reduced abundances of total and nitrate-reducing bacteria, whereas the potential activity of the nitrate reductase enzyme was not affected. Enzyme activity was only weakly correlated to the abundance of nitrate-reducing bacteria but was related to NH(4) (+) and NO(3) (-) concentrations. Our results suggest that in contrast to the observed pronounced seasonal changes, the elevation of atmospheric [CO(2) ] has only a marginal impact on nitrate reducers in the investigated arable ecosystem.     

Comparison of techniques to determine the abundance of predatory bacteria attacking chromatiaceae

Abstract This work quantifies the number of bacterial predators attacking the population of Chromatiaceae in the hypolimnion of Lake Estanya to assess the potential role of these microorganisms in controlling phototrophic bacterial populations. The abundance of predators was estimated from total counts of infected prey cells and by counting plaque-forming units. In spite of the large difference between both determinations, their variations with depth and time followed very similar patterns. During the summer, in the hypolimnion, and during the winter in the entire lake, up to 60% of the prey cells had potential predators attached. In comparison, plaque counts showed that viable predators represented less than 1% of the population of the prey. Our results demonstrated that predatory bacteria were far more abundant than indicated by the low viable counts obtained, suggesting that they play a more important role in controlling phototrophic bacterial populations than is currently assumed.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

Growth and survival of non-O157:H7 Shiga-toxin-producing Escherichia coli in cow manure

AIMS: The main objective of this study was to evaluate the behaviour of non-O157:H7 Shiga-toxin-producing Escherichia coli (STEC) strains in cow manure. METHODS AND RESULTS: A mixture of eight green-fluorescent-protein-labelled STEC strains was inoculated around 10(6)-10(7) CFU g(-1) into four manure heaps. Two heaps were regularly turned and the two others remained unturned. STEC counts and physical parameters (temperature, pH, moisture content and oxido-reduction potential) were monitored for 1000 manure samples. The highest mean pH values were obtained near the surface at the base of all manure heaps. At the surface, the moisture content decreased from 76.5% to 42% in turned heaps. Temperatures reached 65 degrees C near the main body of all manure heaps, and only 35 degrees C near the superficial parts located at the base of them. These two sites (the centre and the base) were associated with D values for the STEC counts of 0.48 and 2.39 days, respectively. We were able to detect STEC strains during 42 days in turned manure heaps and during at least 90 days in unturned ones. CONCLUSIONS: These results emphasize the long-term survival of non-O157:H7 STEC in cow manure. SIGNIFICANCE AND IMPACT OF THE STUDY: Good management practices (e.g. turning) should be respected in order to minimize the risk of environmental contamination by STEC.     

In situ delivery of passive immunity by lactobacilli producing single-chain antibodies

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.     

Changes in the biofilm microflora of limestone caused by atmospheric pollutants

Historic limestone materials in urban environments are continually exposed to air pollutants, including sulfur compounds and hydrocarbons. We investigated the effects of air pollution on the biofilm microflora of historic limestone gravestones located at two locations Massachusetts, USA. Our data showed that the culturable populations of chemolithotrophic and heterotrophic bacteria, and fungi were suppressed in the polluted habitat comparing with the unpolluted location. The diversity of the microflora was also reduced in the surface biofilms on gravestones in the city contaminated by air pollution. However, both the sulfur-oxidizing and hydrocarbon-utilizing microflora were enriched in the biofilms exposed to air pollution. In a laboratory study, low concentrations of the polluting chemicals stimulated growth of these bacteria, and resulted in rapid acid production. Scanning electron microscopy demonstrated that the biofilms of both the sulfur-oxidizing bacteria and the hydrocarbon-degrading microflora penetrated into the limestone. The enrichment of sulfur- and hydrocarbon-utilizing bacteria in the biofilms may contribute to dissolution of the stone. However, further research is required to determine the effects of specific metabolites of these microorganisms on stone deterioration.     

Investigation of Microorganisms in Bentonite Deposits

Microbiological characteristics of bentonite deposits were investigated as a natural analogue of microbial behavior in the buffer material for geological disposal of radioactive waste. Distributions of microorganisms in bentonite were examined at four sites in two different bentonite deposits in Japan. The sites included pond bottom, wetland, and wet mine gallery environments where bentonite layers have been left undisturbed for 2 to 30 years. Excavation was performed without using drilling water and the center parts of the cores were used for microbial examination. Plate counts with R2A medium of aerobic and anaerobic bacteria at the drilling mouth ranged from 10⁵ to 10⁷/g DW (dry weight) and from 10³ to 10⁶/g DW, respectively. The CFDA-AM (Carboxyfluorescein diacetate acetoxymethyl ester) cell counts ranged from 10⁶ to 10⁹/g DW. Bacterial numbers in the bentonite layers declined with distance from the drilling mouth; both aerobes and anaerobes were less than 10²/g DW and CFDA-AM cell counts less than 10⁶/g DW for core samples taken from approximately 1 m depth, except at the pond bottom. These results suggest that microbial activity in natural bentonite is lower than in typical soils and aquatic sediments and does not spread easily.     

Synthesis and antimicrobial activity of novel 2-thiazolylimino-5-arylidene-4-thiazolidinones

New 2-thiazolylimino-5-arylidene-4-thiazolidinones (compounds 4a-j), unsubstituted or carrying hydroxy, methoxy, nitro and chloro groups on the benzene ring, were synthesized and assayed in vitro for their antimicrobial activity against Gram positive and Gram negative bacteria, yeasts and mould. The compounds were very potent towards all tested Gram positive microorganisms (MIC ranging from 0.03 to 6 microg/mL in most of the cases) and Gram negative Haemophilus influenzae (MIC 0.15-1.5 microg/mL), whereas no effectiveness was exhibited against Gram negative Escherichia coli and fungi up to the concentration of 100 microg/mL. The 5-arylidene derivatives showed an antibacterial efficacy considerably greater than that of the parent 2-(thiazol-2-ylimino)thiazolidin-4-one 3, suggesting that the substituted and unsubstituted 5-arylidene moiety plays an important role in enhancing the antimicrobial properties of this class of compounds. The remarkable inhibition of the growth of penicillin-resistant staphylococci makes these substances promising agents also for the treatment of infections caused by microorganisms resistant to currently available drugs.