Measurements of Diel Rates of Bacterial Secondary Production in Aquatic Environments

Measurements of bacterial secondary production were carried out during 13 diel studies at one coastal marine station and in five lakes differing with respect to nutrient concentration and primary production. Bacterial secondary production was measured in situ every 3 to 5 h by [³H]thymidine incorporation into DNA. In some of the diel studies, these results were compared with results obtained from dark ¹⁴CO₂ uptake and frequency of dividing cells. Only minor diel changes were observed. The rate of [³H]thymidine incorporation into DNA and the frequency of dividing cells varied from 23 to 194% of the diel mean. The dark CO₂ uptake rate varied from 12 to 259% of the diel mean. An analysis of variance demonstrated that no specific time periods during 24 h showed significantly different production rates, supporting the idea that bacterial activities in natural assemblages are controlled by a variety of events. The best correction (r² = 0.74) was obtained between the [³H]thymidine incorporation and frequency of dividing cells procedures from the lake water samples. The actual production rates calculated by [³H]thymidine incorporation into DNA were appreciably lower than those obtained by the frequency of dividing cells and the dark CO₂ uptake techniques. Diel rates of bacterial production are discussed in relation to sampling frequency, statistical errors, and choice of method.     

Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.Gram’s method is the most important and fundamental orthodox method for bacterial identification. It classifies bacteria into two groups, gram-negative and gram-positive. The mechanism of Gram staining is based on the fundamental structural and chemical attributes of bacterial cell walls. The cell walls of gram-positive bacteria have a high percentage of peptidoglycan, while those of gram-negative bacteria have only a thin peptidoglycan layer (1–3, 6). In Gram’s method, an insoluble dye-iodine complex is formed inside bacterial cells and is extracted by alcohol from gram-negative but not gram-positive bacteria (6, 12, 16). There are taxonomically gram-variable species, but some cells of gram-negative or gram-positive species may show gram-variable characteristics due to environmental stress, such as unsuitable nutrients, temperature, pH, or electrolytes (3).Functional differences between gram-positive and gram-negative cell walls have been studied with special emphasis on nutrient uptake from the ambient environment. Gram-negative bacteria have a periplasmic space between the lipopolysaccharide layer and the plasma membrane. In this space, binding proteins initially attach to nutrients and take them to a membrane carrier. Gram-positive bacteria lack the periplasmic space and are believed to have no binding proteins (9). Therefore, nutrient uptake from the environment is easier for gram-negative bacteria than for gram-positive bacteria. Because of this difference, the population density of gram-negative bacteria in more oligotrophic environments could be higher than that of gram-positive bacteria (20).Gram staining is commonly used only to reflect cell wall structure. If Gram staining characterizes not only simple taxonomical dichotomy but also multiple biological functions, it may also be used to correlate bacterial cell wall structure with related physiological responses to the environment. In particular, Gram staining could supply ecological information on natural bacterial populations that are difficult to culture by the present technology.Membrane filter methods are widely used for microscopy in aquatic microbiology because of the low population densities of bacteria in many aquatic environments (4, 11, 16). However, these methods sometimes have problems associated with microscopic observations, causing unclear images of bacterial cells on Nuclepore filters when used with the conventional mounting medium (immersion oil; refractive index [nd] = 1.514). Hence, a suitable mounting agent must be applied to obtain precise image analyses of Gram-stained bacteria on Nuclepore filters.In this study, we have established a distinct method to characterize photometric Gram stain images; it involves the Gram stain index (GSI) for specifying natural bacterial populations in various aquatic environments. For this purpose, we have standardized the GSI of typical gram-negative and gram-positive bacteria by using Escherichia coli and Bacillus subtilis, respectively, and compared these GSI values to those of natural bacterial populations of several freshwater environments. The natural waters we investigated were Hyoutaro-ike pond, Matsumi-ike bog, and Lake Kasumigaura, which are oligotrophic, mesotrophic, and eutrophic water bodies, respectively, as previously determined (8, 10, 13, 18, 22, 23).     

Ecology of Vibrio mimicus in Aquatic Environments

[This corrects the article on p. 2077 in vol. 55.].     

Frequency of Dividing Cells, a New Approach to the Determination of Bacterial Growth Rates in Aquatic Environments

Frequency of dividing cells is suggested to be an indirect measure of the mean growth rate of an aquatic bacterial community. Seasonal changes in frequency of dividing cells were found which covariated with the bacterial uptake of ¹⁴C-labeled phytoplankton exudates. Batch and continuous culture growth experiments, using brackish water bacteria in pure and mixed enrichment cultures, were performed to establish a relationship between frequency of dividing cells and growth rate. An improved technique for bacterial direct counts, using fluorescent staining and epifluorescence microscopy, is presented. Based on a 6-month survey in a coastal area of the Baltic Sea, the bacterial production in the photic zone is estimated. Compared to the total primary production in the area, the bacterial population during this period utilized approximately 25% of the amount of carbon originally fixed by the primary producers.     

Denitrification in Aquatic Environments: A Cross-system Analysis

A meta-analysis was conducted on 136 data sets of denitrification rates (DR) recorded both during the period of highest water temperature and monthly in five types of aquatic ecosystems: oceans, coastal environments, estuaries, lakes and rivers. There was a gradual increase of DR from the ocean to rivers and lakes at both scales, with the rivers showing the highest DR variability. Denitrification peaked during summertime and showed highest seasonal variability in lakes and rivers. High concentrations of nitrate and interstitially-dissolved organic carbon as well as low oxygen concentration in the overlying water enhanced DR both during summer and at a seasonal scale whereas total phosphorus did at the seasonal scale only. There was a positive linear relationship between overlying nitrate and DR over the range of 1–970 μmol NO₃ (r ² = 0.86, P = 0.001). DR in lakes and rivers might reach values doubling those in the more denitrifying terrestrial ecosystems (e.g. agrosystems). Discrepancies in DR and its controlling factors between site-specific studies and this meta-analysis may arise from environmental variability at two, often confounded, scales of observation: the habitat and the ecosystem level. Future studies on denitrification in aquatic environments should address the topic of spatial heterogeneity more thoroughly.     

Detection of the Antiviral Drug Oseltamivir in Aquatic Environments

Oseltamivir (Tamiflu®) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2–58 ng L⁻¹ was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.     

Optimal Recombination Rate in Fluctuating Environments

The optimal recombination rate which maximizes the long-term geometric average of the population fitness is studied for a two-locus haploid model, assuming that the fitnesses of genotypes AB, Ab, aB and ab are 1 + s(t), 1 - s(t), 1 - s(t), and 1 + s(t), respectively, where s(t) follows various stationary stochastic processes with the average zero. With positive recombination, the polymorphism is stably maintained at both loci. After an initial transient phase, the dynamics are reduced to one dimension, and are analyzed for weak selection limit, strong selection limit, and selection with two state Markovian jump. Results are: (1) If the environmental fluctuation has a predominant periodic component, ropt is approximately inversely proportional to the period irrespective of selection intensity. (2) If the fluctuation is a superposition of many periodic components, the one with the longest period is the most effective in determining ropt because the genetic dynamics cannot track very quick fluctuations (low pass filter effect). (3) If the power spectrum density is decreasing with the frequency, as in pink, or 1/f noises, ropt is small when selection is weak, and increases with the selection intensity. Numerical calculation of the genetic dynamics of a recombination modifier supports all these predictions for the evolutionarily stable recombination rate.     

Simplified Method for Dissolved DNA Determination in Aquatic Environments

A method was developed for the determination of dissolved DNA in aquatic environments. The method is based upon the concentration of dissolved DNA by ethanol precipitation of 0.2-μm-pore-size filtered water. The DNA in concentrated extracts was quantified by the fluorescence of Hoechst 33258-DNA complexes. Fluorescence not attributable to DNA was corrected for by DNase I digestion of the extracts and averaged 25% of the total fluorescence for all samples. The effectiveness of the procedure for concentrating dissolved DNA was demonstrated by the efficient (>90%) recovery of internal standards. Concentrations of dissolved DNA from a variety of marine and freshwater environments ranged from 0.2 to 44 μg/liter, with the highest values being obtained for estuarine and river environments. The method is simple, specific for DNA, and more sensitive than previously described methods for the determination of extracellular DNA.     

Relationships among Bacterial Cell Size, Productivity, and Genetic Diversity in Aquatic Environments using Cell Sorting and Flow Cytometry

Abstract The study of relationships between cell size and productivity is of key importance in microbial ecology to understand which members of natural aquatic communities are responsible for the overall activity and/or productivity. Flow sorting of microorganisms from different environmental samples was used to analyze the activity of bacterial cells depending on their biovolume. Bacterial cells from five different natural samples taken along the Mediterranean coast including fresh- and seawaters were incubated with tritiated leucine, then stained with SYTO 13 and sorted by flow cytometry according to their average side-angle-scattered (SSC) light. In all samples, a bell-shaped relationship was found between cell biovolume and activity, whereas activity of a given cell-size class varied between samples. In contrast, an inverse relationship was found between biovolumes and abundances. These results suggest that medium-sized cells with highest growth rates are probably submitted to intense grazing. For one sample, bacteria within five different size classes were sorted and the genetic diversity of cells within each sorted size class and that of the whole community were analyzed by the denaturing gradient gel electrophoresis (DGGE) method. The genetic diversity, as determined at the community level was highly represented into the pool of small cells, whereas only few species were present into larger cell subpopulations. The results suggest that only a few genotypes may be dominant within the largest and most productive cells. Furthermore, cell size polymorphism as well as heterogeneous cellular activities were found within some species. Received: January 2000; Accepted: April 2000; Online Publication: 28 August 2000     

Detection of Mercury in Aquatic Environments Using EPRE Reporter Zebrafish

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase–green fluorescent protein (LUC–GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl₂ concentrations. GFP expression from the EPRE–LUC–GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl₂. We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl₂ concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.     

The Challenges of Living in Hypoxic and Hypercapnic Aquatic Environments

Organisms living in coastal waters, and especially estuaries,have long been known to have behavioral or physiological mechanismsthat enable them toexist in water containing low amounts ofoxygen. However, the respiratory consumption of oxygen thatgenerates hypoxia is also responsible for producing significantamounts of carbon dioxide. An elevation of carbon dioxide pressurein water will cause a significant acidosis in most aquatic organisms.Thus, the combination of low oxygen and elevated carbon dioxidethat occurs in estuaries represents a significant environmentalchallenge to organisms living in this habitat. Organisms maymaintain oxygen uptake in declining oxygen conditions by usinga respiratory pigment and/or by making adjustments in the convectiveflow of water and blood past respiratory surfaces (i.e., increasecardiac output and ventilation rate). Severe hypoxia may resultin an organism switching partially or completely to anaerobicbiochemical pathways to sustain metabolic rate. There is alsoevidence to suggest that organisms lower their metabolism duringhypoxic stress. Elevated water CO₂ (hypercapnia) produces anacidosis in the tissues of organisms that breathe it. This acidosismay be wholly or partially compensated (i.e., mechanisms returnpH to pre-exposure levels), or may be uncompensated. Some studieshave examined the effects on organisms of exposure simultaneouslyto hypoxia and hypercapnia. This article reviews some of thespecific adaptations and responses of organisms to low oxygen,to high carbon dioxide, and to the cooccurrence of low oxygenand high carbon dioxide     

Methods for Detection of Conjugative Plasmid Transfer in Aquatic Environments

Donor and recipient counter selection was evaluated by selecting bacteria that received plasmid RP4 by conjugation on filters and in lake water microcosms. Three counter selection systems were compared; (i) Use of antibiotic-resistant recipients, (ii) use of an auxotrophic donor, and (iii) use of a donor with chromosomal suicide genes. Transfer efficiencies of transconjugants per recipient obtained with the three different counter selection systems in filter-matings were not significantly different. Some nalidixic acid-resistant recipients became partly sensitive to nalidixic acid after receiving the plasmid. Use of an auxotrophic donor was a feasible and easy way to recover indigenous transconjugants. A strain with two copies of the suicide gene gef was successfully eliminated in filter-matings, but elimination of the donor in microcosms by induction of the suicide genes did not succeed. Thus, this counter selection system was not usable in microcosm experiments. Received: 3 March 1998 / Accepted: 15 May 1998     

环境因素对细菌群体感应的影响

一种海洋费氏弧菌(Vibrio fischeri)的发光现象在20世纪60年代引起了科学家的兴趣,Nealson等在1970年首次报道了该菌的菌体密度与发光呈正相关,该发光现象受细菌本身的群体感应调节系统所控制[1]。尽管细菌是单细胞原核生物,但是越来越多的研究发现细菌在自然环境中常常表现出多细胞的群体行为。细菌利用自诱导物进行相互交流并调控其群体行为的现象被称为群体感应(Quorum sensing,QS)[2],这个概念最早由Fuqua等     

微生物在水环境污染物降解中的应用

每年有大量来自工业、农业、养殖业和城市污水处理厂的废水被排入到水环境中,因此,地球上的水环境面临大量来自生活废水、工农业废水、非法排放的废水及其它废水的污染物质(如抗生素、杀虫剂,除草剂、烃等)的严重挑战,特别是近年来随着集约化养殖的发展,废水污染问题日益突出,并且随着分析手段的进步,能够检测到被排入水环境中的化学污染物质也越来越多,这些化学污染物对水环境中的生物产生有害影响.但是,微生物在污染控制上具有许多重要的作用.因此,本文对微生物在水环境污染物降解中的应用进行了评论.结果表明微生物主要是应用在水产养殖水中,而在其它的水体系(如河、湖、海)的应用较少.     

微生物在水环境污染物降解中的应用（英文稿）

每年有大量来自工业、农业、养殖业和城市污水处理厂的废水被排入到水环境中, 因此, 地球上的水环境面临大量来自生活废水、工农业废水、非法排放的废水及其它废水的污染物质(如抗生素、杀虫剂、除草剂、烃等)的严重挑战, 特别是近年来随着集约化养殖的发展, 废水污染问题日益突出, 并且随着分析手段的进步, 能够检测到被排入水环境中的化学污染物质也越来越多, 这些化学污染物对水环境中的生物产生有害影响。但是, 微生物在污染控制上具有许多重要的作用。因此, 本文对微生物在水环境污染物降解中的应用进行了评论。结果表明微生物主要是应用在水产养殖水中, 而在其它的水体系(如河、湖、海)的应用较少。     

微生物在水环境污染物降解中的应用

每年有大量来自工业、农业、养殖业和城市污水处理厂的废水被排入到水环境中, 因此, 地球上的水环境面临大量来自生活废水、工农业废水、非法排放的废水及其它废水的污染物质(如抗生素、杀虫剂、除草剂、烃等)的严重挑战, 特别是近年来随着集约化养殖的发展, 废水污染问题日益突出, 并且随着分析手段的进步, 能够检测到被排入水环境中的化学污染物质也越来越多, 这些化学污染物对水环境中的生物产生有害影响。但是, 微生物在污染控制上具有许多重要的作用。因此, 本文对微生物在水环境污染物降解中的应用进行了评论。结果表明微生物主要是应用在水产养殖水中, 而在其它的水体系(如河、湖、海)的应用较少。     

Protein Method for Investigating Mercuric Reductase Gene Expression in Aquatic Environments

A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r² = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10² CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 ± 2)% over the range of population densities investigated (10² to 10⁸ CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.     

The American Alligator Detects Food Chemicals in Aquatic and Terrestrial Environments

American alligators (Alligator mississippiensis) were offered raw meat or animal carcasses under water or on land in experiments where only chemoreception could be used to discriminate between these and control materials. Alligators also were presented with aqueous extracts and airborne chemicals from meat. Juvenile alligators, tested in indoor tanks, opened more cheesecloth packets containing meat than control packets when these materials were placed under water and on platforms above the water surface. Adult alligators, tested in outdoor semi-natural enclosures, removed more cheesecloth-wrapped meat presented under water, and more of both meat and raccoon (Procyon lotor) carcasses placed under perforated baskets on land, than control materials. Juvenile alligators, tested in tanks partially filled with water, exhibited more lateral head movements and mouth-openings to an aqueous beef extract than to water alone. Juvenile alligators, tested in an olfactometer, exhibited more gular pumps to airborne chemicals from beef than to distilled water. These experiments indicate that alligators may use chemical cues to locate food both on land and under water, and that they detect both water- and airborne chemicals from meat.