Comparison of Light and Electron Microscopic Determinations of the Number of Bacteria and Algae in Lake Water

Determinations of the number of microorganisms in lake water samples with the bright-field light microscope were performed using conventional counting chambers. Determinations with the fluorescence microscope were carried out after staining the organisms with acridine orange and filtering them onto Nuclepore filters. For transmission electron microscopy, a water sample was concentrated by centrifugation. The pellet was solidifed in agar, fixed, dehydrated, embedded in Epon, and cut into thin sections. The number and area of organism profiles per unit area of the sections were determined. The number of organisms per unit volume of the pellet was then calculated using stereological formulae. The corresponding number in the lake water was obtained from the ratio of volume of solidified pellet/volume of water sample. Control experiments with pure cultures of bacteria and algae showed good agreement between light and electron microscopic counts. This was also true for most lake water samples, but the electron microscopic preparations from some samples contained small vibrio-like bodies and ill-defined structures that made a precise comparison more difficult. Bacteria and small blue-green and green algae could not always be differentiated with the light microscope, but this was easily done by electron microscopy. Our results show that transmission electron microscopy can be used for checking light microscopic counts of microorganisms in lake water.     

Microbiological characterization of the accreted ice of subglacial Lake Vostok, Antarctica]

The accreted ice of subglacial Lake Vostok extends upward from the lake water level (a depth of 3750 m) to the bottom surface of the overlying Antarctic ice sheet. All of the accreted ice samples, taken from depths between 3541 and 3611 m, were found to contain pro- and eukaryotic microorganisms, whose number and diversity varied in different ice horizons and correlated, to a certain degree, with the occurrence of organic and inorganic impurities in a given horizon. Some biological objects found in the accreted lake ice, including bacteria, microalgae, and the pollen of higher plants, were morphologically similar to those found earlier in the glacier ice bulk. The others were not. It is suggested that the microorganisms found in the lake ice may come from different locations--the bottom layer of the glacier ice, the bedrock underlying the glacier, and the lake water.     

Microbiological Characterization of the Accreted Ice of Subglacial Lake Vostok,Antarctica

The accreted ice of subglacial Lake Vostok extends upward from the lake water level (a depth of 3750 m) to the bottom surface of the overlying Antarctic ice sheet. All of the accreted ice samples, taken from depths between 3541 and 3611 m, were found to contain pro- and eukaryotic microorganisms, whose number and diversity varied in different ice horizons and correlated, to a certain degree, with the occurrence of organic and inorganic impurities in a given horizon. Some biological objects found in the accreted lake ice, including bacteria, microalgae, and the pollen of higher plants, were morphologically similar to those found earlier in the glacier ice bulk. The others were not. It is suggested that the microorganisms found in the lake ice may come from different locations—the bottom layer of the glacier ice, the bedrock underlying the glacier, and the lake water.     

Survival and detection of bacteria in an aquatic environment

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistence of Enteroviruses in Lake Water

Two enteroviruses were inactivated more rapidly in a lake than in sterile lake water; then their coat proteins were degraded and, perhaps, used by microorganisms.     

Survival and detection of bacteria in an aquatic environment.

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inorganic nutrients on the acclimation period preceding mineralization of organic chemicals in lake water

The addition of phosphate, nitrate, or sulfate (each at 10 mM) decreased the acclimation period for the mineralization of low concentrations of p-nitrophenol (PNP) in lake water. Added phosphate shortened the acclimation period for biodegradation of 2 ng to 2 micrograms of PNP per ml in various lake water samples and of 2,4-dichlorophenoxyacetate at 100 ng/ml. Added P enhanced the rate of growth of PNP-mineralizing microorganisms in waters containing 200 ng or 2 micrograms of PNP per ml. We suggest that the effect of P on the acclimation period results from an increase in the growth rate of the initially small population of microorganisms able to mineralize the synthetic chemicals.     

Effect of inorganic nutrients on the acclimation period preceding mineralization of organic chemicals in lake water.

The addition of phosphate, nitrate, or sulfate (each at 10 mM) decreased the acclimation period for the mineralization of low concentrations of p-nitrophenol (PNP) in lake water. Added phosphate shortened the acclimation period for biodegradation of 2 ng to 2 micrograms of PNP per ml in various lake water samples and of 2,4-dichlorophenoxyacetate at 100 ng/ml. Added P enhanced the rate of growth of PNP-mineralizing microorganisms in waters containing 200 ng or 2 micrograms of PNP per ml. We suggest that the effect of P on the acclimation period results from an increase in the growth rate of the initially small population of microorganisms able to mineralize the synthetic chemicals.     

Microbial metabolism of N-nitrosodiethanolamine in lake water and sewage.

The carcinogenic nitrosamine, N-nitrosodiethanolamine (NDElA), was degraded in samples of sewage and two lake waters, and microorganisms were responsible for the transformation. However, the rate of NDElA disappearance was slow. In the samples of lake water, the rate and extent of NDElA metabolism varied with the time of year, and no disappearance occurred in samples taken in winter. The products formed from NDEIA were persistent in lake water. In sewage, no seasonal effect on the microbial conversion was evident, and the products of metabolism were slowly mineralized. NDElA is apparently converted to the same organic products in samples of all three environments. Although the products were not identified, the data suggest that they were modified dimers of NDElA.     

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.     

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments.

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

Chemical composition of labile fractions in DOM

Dissolved organic matter in natural water was classified into a labile and a refractory fraction according to the decomposition properties for microorganisms. In decomposition experiments of dissolved organic matter from an eutrophic small lake in Japan, dissolved amino acids and dissolved carbohydrates fractions could be confirmed to be the labile fractions of dissolved organic matter.Tokyo Metropolian University, Department of Chemistry, Faculty of Science     

The toxic effects of rubber contaminants on microbial food webs and zooplankton of two lakes of different trophic status

Rubber is commonly used in recreational equipment and devicesforsampling in lakes, but there have been few studies of theeffectsof rubber on planktonic organisms. We investigated the toxiceffects of rubber on the microbial food webs of a mesotrophiclakeand a eutrophic lake. Lake water was collected by pumping via,(i) a polyvinylchloride hose and, (ii) a rubber hose. Samplesoflake water collected by each method were incubated insitu in4.25 l enclosures for four days. The lake water was sampledbeforeand after incubation to determine the concentrations ofinorganicnutrients, chlorophyll a, microorganisms (bacteria,picophytoplankton, flagellates, ciliates) and zooplankton.In the mesotrophic lake, momentary exposure (ten seconds) oflakewater to the rubber hose significantly lowered theconcentrationsof chlorophyll a, bacteria, picophytoplankton and somespecies of zooplankton (Boeckella hamata, Bosmina,androtifers), relative to those in water exposed to the plastichose;flagellates, ciliates and Ceriodaphnia dubia were notsignificantly affected. In the eutrophic lake, the effects oftherubber hose on components of the microbial food web were muchlesssevere, and were consistent with the lake's high levels ofdissolved organic carbon (DOC), which is known to chelatetoxicmetals in water.     

Structure of planktonic microbial food web in a brackish stratified Siberian lake

The distribution of primary components of the microbial community (autotrophic pico- and nanoplankton, phototrophic bacteria, heterotrophic bacteria, microscopic fungi, heterotrophic flagellates, ciliates and heliozoa) in the water column of Lake Shira, a steppe brackish-water, stratified lake in Khakasia, Siberia (Russia), were assessed in midsummer. Bacterioplankton was the main component of the planktonic microbial community, accounting for 65.3 to 75.7% of the total microbial biomass. The maximum concentration of heterotrophic bacteria were recorded in the monimolimnion of the lake. Autotrophic microorganisms contributed more significantly to the total microbial biomass in the pelagic zone (20.2–26.5%) than in the littoral zone of the lake (8.7–14.9%). First of all, it is caused by development of phototrophic sulphur bacteria at the oxic-anoxic boundary. The concentrations of most aerobic phototrophic and heterotrophic microorganisms were maximal in the upper mixolimnion. Heterotrophic flagellates dominated the protozoan populations. Ciliates were minor component of the planktonic microbial community of the lake. Heterotrophic flagellates were the most diverse group of planktonic eucaryotes in the lake, which represented by 36 species. Facultative and obligate anaerobic flagellates were revealed in the monimolimnion. There were four species of Heliozoa and only three of ciliates in the lake.     

Mass collection of magnetotactic bacteria from the permanently stratified ferruginous Lake Pavin,France

Obtaining high biomass yields of specific microorganisms for culture-independent approaches is a challenge faced by scientists studying organism's recalcitrant to laboratory conditions and culture. This difficulty is highly decreased when studying magnetotactic bacteria (MTB) since their unique behaviour allows their enrichment and purification from other microorganisms present in aquatic environments. Here, we use Lake Pavin, a permanently stratified lake in the French Massif Central, as a natural laboratory to optimize collection and concentration of MTB that thrive in the water column and sediments. A method is presented to separate MTB from highly abundant abiotic magnetic particles in the sediment of this crater lake. For the water column, different sampling approaches are compared such as in situ collection using a Niskin bottle and online pumping. By monitoring several physicochemical parameters of the water column, we identify the ecological niche where MTB live. Then, by focusing our sampling at the peak of MTB abundance, we show that the online pumping system is the most efficient for fast recovering of large volumes of water at a high spatial resolution, which is necessary considering the sharp physicochemical gradients observed in the water column. Taking advantage of aerotactic and magnetic MTB properties, we present an efficient method for MTB concentration from large volumes of water. Our methodology represents a first step for further multidisciplinary investigations of the diversity, metagenomic and ecology of MTB populations in Lake Pavin and elsewhere, as well as chemical and isotopic analyses of their magnetosomes.     

Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts

Cryptosporidium parvum is an enteric protozoan parasite of medical and veterinary importance. Dissemination of environmentally resistant oocysts in surface water plays an important role in the epidemiology of cryptospridiosis. Although the polymerase chain reaction (PCR) is a well-established technique and is widely used for detecting microorganisms, it is not routinely applied for monitoring waterborne C. parvum. In order to facilitate the application of PCR to the detection of waterborne C. parvum oocysts, a comparison of published PCR protocols was undertaken and different sample-preparation methods tested. The sensitivity of a one-step PCR method, consisting of 40 temperature cycles, was 10 purified oocysts or fewer than 100 oocysts spiked in raw lake water. The detection limit of two primer pairs, one targeting the ribosomal small subunit and another specific for a C. parvum sequence of unknown function, was approximately ten-fold lower than achieved with a primer pair targeting an oocyst shell protein gene. Three cycles of freezing/thawing were sufficient to expose oocyst DNA and resulted in higher sensitivity than proteinase K digestion, sonication or electroporation. Inhibition of PCR by surface water from different local sources was entirely associated with the soluble fraction of lake water. Membrane filtration was evaluated in bench-scale experiments as a means of removing lake water inhibitors and improving the detection limit of PCR. Using gel and membrane filtration, the molecular size of inhibitory solutes from lake water was estimated to less than 27 kDa. Received: 14 November 1996 / Received revision: 18 March 1997 / Accepted: 27 March 1997     

Reproducing Authigenic Carbonate Precipitation in the Hypersaline Lake Acıgöl (Turkey) with Microbial Cultures

In the present study, laboratory precipitation experiments using similar water chemistry and two different bacterial cultures from Lake Ac?göl sediments, a hypersaline lake in Turkey, were performed to reproduce mineral assemblages similar to those found in the lake. Two different bacterial cultures induce various calcium/magnesium carbonates precipitation under all the experimental conditions (solid vs. liquid): Hydromagnesite, dypingite, huntite, monohydrocalcite, and aragonite. The geochemical program PHREEQC was used to calculate the mineral saturation indexes in the cultures and in lake water. Carbonate mineral assemblages identified in the experiments seem to be independent of the type of microorganisms but rather controlled by the chemical composition and physical conditions of the media. The relative amounts of monohydrocalcite, hydromagnesite, and dypingite are controlled by varying sulfate concentration from 0 to 56 mM. This demonstrates a kinetic effect that could similarly affect the mineral assemblage in the lake. Also the spherical morphology of hydromagnesite points to growth of these minerals under partial inhibition in the brine under high concentrations of ions and organic polymers produced by the microbial communities. As reproduced by the culture experiments, the authigenic carbonate mineral assemblage of Lake Ac?göl most likely results from interplay of ionic composition of the brine and microbial effects.     

Halobacterial Community Analysis of Mierlei Saline Lake in Transylvania (Romania)

In this study a combination of both molecular and biochemical methods have been used to characterize the bacterial microbiota in water and sediment of a saline lake in Transylvania. The physicochemical characterization of the water samples from different lake depths indicated a stratification of the lake that affects the distribution of resident microorganisms, confirmed also by the significant differences in terms of functional diversity of the microbial communities in different water layers. The superficial bacterial community shows a good oxidative capability, degrading a wide range of organic substrates, yet the bottom layer community exhibits a major level of specialization. The membrane fatty acid analysis of the sediment bacterial community shows the prevalent presence of Gram negative bacteria, confirmed by the culturing techniques. Among the 24 collected isolates, 16S rRNA gene sequencing analysis permitted to identify 10 different species, belonging to Bacillus, Halomonas, Idiomarina, Marinobacter, Pseudoalteromonas, Salinivibrio, Staphylococcus genera and prevalently classified as halophiles.