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.     

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 × 10² to 2.3 × 10⁴ cell equivalents liter⁻¹, whereas GR-corrected abundances ranged from 4.7 × 10³ to 1.6 × 10⁶ cell equivalents liter⁻¹. GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.     

Evaluation of a Commercial Enzyme Linked Immunosorbent Assay (ELISA) for the Determination of the Neurotoxin BMAA in Surface Waters

The neurotoxin β-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA.     

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.     

Ecology of Vibrio mimicus in Aquatic Environments

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

Rate of Bacterial Mortality in Aquatic Environments

A method is proposed which provides a minimum estimate of the rate of bacterial mortality in growing natural populations of planktonic bacteria. This estimate is given by the rate of decrease of radioactivity from the DNA of a [³H]thymidine-labeled natural assemblage of bacteria after all added thymidine has been exhausted from the medium. Results obtained from river water, estuarine water, and seawater show overall bacterial mortality rates in the range 0.010 to 0.030 h⁻¹, in good agreement with the range of growth rates measured in the same environments. Use of selective filtration through Nuclepore filters (pore size, 2 μm) allowed us to determine the contribution of microzooplankton grazing to overall bacterial mortality. Grazing rates estimated by this method ranged from 0 to 0.02 h⁻¹.     

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     

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.     

Introduction to the Symposium: Ontogenetic Strategies of Invertebrates in Aquatic Environments

This symposium presents different ecological and physiologicalstrategies used by invertebrates to successfully adapt to aquaticenvironments. Adaptation has been studied mainly in adult animals,but the papers comprising the symposium emphasize ontogeneticstrategies, starting from the principle that natural selectionacts on all stages of development. Adaptive strategies may thusdiffer strikingly between developmental stages of the same organism.Invertebrates offer a wide array of ecophysiological modelsfor study, and these are exemplified by the contributions tothe symposium, which are briefly summarized. Future researchin the field will 1, expand the number of models for comparativepurposes; 2, examine the strategies, not only of larvae andjuveniles, but also of embryos, eggs and reproductive cells;and 3, investigate the genetic basis of ontogenetic strategies.     

极端微生物：一种新型的酶资源

极端微生物具有自身独特的特点和代谢产物 ,在食品工业、化工、药用工业和环境生物技术领域都有潜在的应用。一些酶已经得到纯化 ,其基因在宿主中已成功克隆。主要介绍和讨论极端微生物的类型、基因组及极端酶类的生产、分离与应用。     

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.     

A Novel Neurotoxin from Venom of the Spider,Brachypelma albopilosum

Spiders have evolved highly selective toxins for insects. There are many insecticidal neurotoxins in spider venoms. Although a large amount of work has been done to focus on neurotoxicity of spider components, little information, which is related with effects of spider toxins on tumor cell proliferation and cytotoxicity, is available for Brachypelma albopilosum venom. In this work, a novel spider neurotoxin (brachyin) was identified and characterized from venoms of the spider, Brachypelma albopilosum. Brachyin is composed of 41 amino acid residues with the sequence of CLGENVPCDKDRPNCCSRYECLEPTGYGWWYASYYCYKKRS. There are six cysteines in this sequence, which form three disulfided bridges. The serine residue at the C-terminus is amidated. Brachyin showed strong lethal effects on American cockroaches (Periplaneta americana) and Tenebrio molitor (common mealbeetle). This neurotoxin also showed significant analgesic effects in mice models including abdominal writhing induced by acetic acid and formalin-induced paw licking tests. It was interesting that brachyin exerted marked inhibition on tumor cell proliferation.     

Cyanobacterial Blooms and the Occurrence of the neurotoxin beta-N-methylamino-L-alanine (BMAA) in South Florida Aquatic Food Webs

Recent studies demonstrate that most cyanobacteria produce the neurotoxin beta-N-methylamino-L-alanine (BMAA) and that it can biomagnify in at least one terrestrial food chain. BMAA has been implicated as a significant environmental risk in the development of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). We examined several blooms of cyanobacteria in South Florida, and the BMAA content of resident animals, including species used as human food. A wide range of BMAA concentrations were found, ranging from below assay detection limits to approximately 7000 μg/g, a concentration associated with a potential long-term human health hazard.     

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

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。