Quantitative ecotoxicological impacts of sewage treatment plant effluents on plankton productivity and assimilative capacity of rivers.

Sewage treatment plants are sources of inorganic and organic matter as well as contaminants for the receiving watercourses. We analyzed the ecological consequences of such effluents by following a holistic and synecological ecotoxicological approach based on quantifying extracellular enzyme activities (EEA), primary production and bacterial cell, and biomass production rates. Samples were obtained at three locations at the Rivers Holtemme and Elbe, Germany and Lower Jordan River, Israel and West Bank, as well as from their adjacent sewage treatment plants. Blending river samples with sewage treatment plant effluents mainly resulted in a stimulation of EEAs, which was diminished in blends with 0.2-µm filtered sewage treatment plant effluents. Stimulation for primary production and bacterial cell and biomass production of River Holtemme and Elbe samples was observed, and inhibition of these rates for Lower Jordan River samples probably linked to generally high turbidity. The quantified bacterial biomass versus cell production rates showed almost unbalanced (≫ 1) growth. Very high biomass to cell production ratios were found for sewage and sewage-containing samples, which provides a semi-quantitative indicator function for high quantities of microbial easy utilizable dissolved organic matter as nutrition source. The presented approach enables the simultaneous quantification of inhibitory and stimulating toxic responses as well as supplying ecosystem-based data for policy decision-making, and for direct incorporation in models to derive management and remediation strategies.

Deciphering the mechanisms against oxidative stress in developing and mature akinetes of the cyanobacterium Aphanizomenon ovalisporum.

Cells of filamentous cyanobacteria of the orders Nostocales and Stigonematales can differentiate into dormant forms called akinetes. Akinetes play a key role in the survival, abundance and distribution of the species, contributing an inoculum for their perennial blooms. In the cyanobacterium Aphanizomenon ovalisporum, potassium deficiency triggers the formation of akinetes. Here we present experimental evidence for the production of reactive oxygen species (ROS) during akinete development in response to potassium deficiency. The function of ROS as a primer signal for akinete differentiation was negated. Nevertheless, akinetes acquired protective mechanisms against oxidative damage during their differentiation and maintained them as they matured, giving akinetes advantages enabling survival in harsh conditions.

Long-term changes in cyanobacteria populations in lake kinneret (sea of galilee), Israel: an eco-physiological outlook.

The long-term record of cyanobacteria abundance in Lake Kinneret (Sea of Galilee), Israel, demonstrates changes in cyanobacteria abundance and composition in the last five decades. New invasive species of the order Nostocales (Aphanizomenon ovalisporum and Cylindrospermopsis raciborskii) became part of the annual phytoplankton assemblage during summer-autumn. Concomitantly, bloom events of Microcystis sp. (Chroococcales) during winter-spring intensified. These changes in cyanobacteria pattern may be partly attributed to the management policy in Lake Kinneret's vicinity and watershed aimed to reduce effluent discharge to the lake and partly to climate changes in the region; i.e., increased water column temperature, less wind and reduced precipitation. The gradual decrease in the concentration of total and dissolved phosphorus and total and dissolved nitrogen and an increase in alkalinity, pH and salinity, combined with the physiological features of cyanobacteria, probably contributed to the success of cyanobacteria. The data presented here indicate that the trend of the continuous decline of nutrients may not be sufficient to reduce and to control the abundance and proliferation of toxic and non-toxic cyanobacteria.

Potassium deficiency triggers the development of dormant cells (akinetes) in Aphanizomenon ovalisporum (Nostocales, Cyanoprokaryota)(1).

Akinetes are spore-like nonmotile cells that differentiate from vegetative cells of filamentous cyanobacteria from the order Nostocales. They play a key role in the survival and distribution of these species and contribute to their perennial blooms. Various environmental factors were reported to trigger the differentiation of akinetes including light intensity and quality, temperature, and nutrient deficiency. Here, we report that deprivation of potassium ion (K(+) ) triggers akinete development in the cyanobacterium Aphanizomenon ovalisporum. Akinetes formation is initiated 3 d-7 d after an induction by K(+) depletion, followed by 2-3 weeks of a maturation process. Akinete formation occurs within a restricted matrix of environmental conditions such as temperature, light intensity or photon flux. Phosphate is essential for akinete maturation and P-limitation restricts the number of mature akinetes. DNA replication is essential for akinete maturation and akinete development is limited in the presence of Nalidixic acid. While our results unequivocally demonstrated the effect of K(+) deficiency on akinete formation in laboratory cultures of A. ovalisporum, this trigger did not cause Cylindrospermopsis raciborskii to produce akinetes. Anabaena crassa however, produced akinetes upon potassium deficiency, but the highest akinete concentration was achieved at conditions that supported vegetative growth. It is speculated that an unknown internal signal is associated with the cellular response to K(+) deficiency to induce the differentiation of a certain vegetative cell in a trichome into an akinete. A universal stress protein that functions as mediator in K(+) deficiency signal transduction cascade, may communicate between the lack of K(+) and akinete induction.

The languages spoken in the water body (or the biological role of cyanobacterial toxins).

Although intensification of toxic cyanobacterial blooms over the last decade is a matter of growing concern due to bloom impact on water quality, the biological role of most of the toxins produced is not known. In this critical review we focus primarily on the biological role of two toxins, microcystins and cylindrospermopsin, in inter- and intra-species communication and in nutrient acquisition. We examine the experimental evidence supporting some of the dogmas in the field and raise several open questions to be dealt with in future research. We do not discuss the health and environmental implications of toxin presence in the water body.

A CARD-FISH protocol for the identification and enumeration of cyanobacterial akinetes in lake sediments.

Akinetes are the dormant cells of Nostocales (cyanobacteria) that enable the organisms to survive harsh environmental conditions while resting in bottom sediments. The germination of akinetes assists the dispersal and persistence of the species. The assessment of the akinete pool in lake sediments is essential to predict the bloom formation of the Nostocales population. We present here the implementation of an improved catalysed reporter deposition (CARD)-fluorescence in situ hybridization (FISH) protocol to assist the identification and quantification of akinetes in sediment samples. Several 16S rRNA gene oligonucleotide probes were evaluated for labelling akinetes of various species of Anabaena, Aphanizomenon and Cylindrospermopsis. Akinetes of all the taxa studied were successfully labelled and could be easily detected by their bright fluorescence signal. The probes' specificity was tested with 32 strains of different taxa. All six Cylindrospermopsis raciborskii strains were labelled with a specific probe for its 16S rRNA gene. A more general probe labelled 73% of the Anabaena and Aphanizomenon strains. The counting data of field samples obtained with CARD-FISH and the regular light microscopy approach did not differ significantly, confirming the suitability of both methods. The CARD-FISH approach was found to be less time-consuming because of better visibility of akinetes.

Invasion of Nostocales (cyanobacteria) to Subtropical and Temperate Freshwater Lakes - Physiological, Regional, and Global Driving Forces.

Similar to the increased number of studies on invasive plants and animals in terrestrial and aquatic ecosystems, many reports were recently published on the invasion of Nostocales (cyanobacteria) to freshwater environments worldwide. Invasion and proliferation of Nostocales in new habitats have the potential to significantly alter the structure of the native community and to modify ecosystem functioning. But most importantly, they influence the water quality due to a variety of toxic compounds that some species produce. Therefore a special attention was given to the invasion and persistence of toxic cyanobacteria in many aquatic ecosystems. Here we summarize the currently published records on the invasion of two Nostocales genera, Cylindrospermopsis and Aphanizomenon, to lakes and water reservoirs in subtropical and temperate zones. These invading species possess traits thought to be common to many invasive organisms: high growth rate, high resource utilization efficiency and overall superior competitive abilities over native species when local conditions vary. Assuming that dispersion routes of cyanobacteria have not been changed much in recent decades, their recent establishment and proliferation in new habitats indicate changes in the environment under which they can exploit their physiological advantage over the native phytoplankton population. In many cases, global warming was identified as the major driving force for the invasion of Nostocales. Due to this uncontrollable trend, invasive Nostocales species are expected to maintain their presence in new habitats and further expand to new environments. In other cases, regional changes in nutrient loads and in biotic conditions were attributed to the invasion events.

Casting a net: fibres produced by Microcystis sp. in field and laboratory populations.

The reasons for the apparent dominance of the toxic cyanobacterium Microcystis sp., reflected by its massive blooms in many fresh water bodies, are poorly understood. We show that in addition to a large array of secondary metabolites, some of which are toxic to eukaryotes, Microcystis sp. secretes large amounts of fibrous exopolysaccharides that form extremely long fibres several millimetres in length. This phenomenon was detected in field and laboratory cultures of various Microcystis strains. In addition, we have identified and characterized three of the proteins associated with the fibres and the genes encoding them in Microcystis sp. PCC 7806 but were unable to completely delete them from its genome. Phylogenetic analysis of the most abundant one, designated IPF-469, showed its presence only in cyanobacteria. Its closest relatives were detected in Synechocystis sp. PCC 6803 and in Cyanothece sp. strains; in the latter the genomic organization of the IPF-469 was highly conserved. IPF-469 and the other two proteins identified here, a haloperoxidase and a haemolysin-type calcium-binding protein, may be part of the fibres secretion pathway. The biological role of the fibres in Microcystis sp. is discussed.

Multiannual variations in phytoplankton populations: what distinguished the blooms of Aphanizomenon ovalisporum in Lake Kinneret in 2010 from 2009?

The reasons for large multiannual fluctuations in phytoplankton biomass and composition in freshwater lakes are complex and involve many biotic and abiotic parameters. Here we studied the 2009 and 2010 summer-autumn blooms of the toxic, cylindrospermopsin producer, Aphanizomenon ovalisporum (hereafter Aphanizomenon) in Lake Kinneret (Sea of Galilee), Israel. During the summer the total dissolved phosphate concentration in the lake is very low, close to the detection level, limiting the development of phytoplankton. Earlier we showed that Aphanizomenon blooms are associated with a large rise in alkaline phosphatase (Apase) activity in the water body and that cylindrospermopsin produced by Aphanizomenon induces the PHO regulon, including secretion of Apase, in other alga thereby improving its own phosphate supply. Aphanizomenon transcripts of PHO and AOA (involved in cylindrospermopsin biosynthesis) genes in Lake Kinneret appeared much earlier in 2010 than in 2009 suggesting that the phytoplankton became phosphate-limited already at the beginning of its summer bloom in 2010 but much later in 2009. Water inflow and lake water temperatures were significantly higher in 2010 but the incoming nutrients were consumed by the much larger phytoplankton biomass early in 2010 before the beginning of the Aphanizomenon bloom. An analysis of abiotic and biological parameters provides an explanation for the very different development of Aphanizomenon populations during 2009 and 2010.

Enslavement in the water body by toxic Aphanizomenon ovalisporum, inducing alkaline phosphatase in phytoplanktons.

The hepatotoxin cylindrospermopsin (CYN) produced by certain cyanobacteria, including Aphanizomenon ovalisporum (hereafter Aphanizomenon) [1], seriously affects lake water quality [2], but its biological role is not known. Strong correlation between Aphanizomenon abundance in Lake Kinneret, Israel, and alkaline phosphatase (APase) activity suggests that inorganic phosphate (Pi) limitation induces the PHO regulon and APase secretion [3]. Staining lake samples with DAPI [4] revealed a high level of polyphosphate bodies (PPB) in Aphanizomenon. Application of enzyme-labeled fluorescence (ELF-APase) [5] showed APase in various organisms, but not in Aphanizomenon. ELF-APase signals and extracellular APase activity in Aphanizomenon were detected only after exploiting PPB under prolonged Pi deprivation in cultures or toward the end of its autumn bloom. Pi deprivation of Aphanizomenon induces CYN production, high-affinity Pi uptake, and an internal, not external, APase. Addition of Aphanizomenon spent media or CYN to various phytoplanktons, including Chlamydomonas reinhardtii, induced genes typically upregulated under Pi limitation and a rise in extracellular APase activity, despite ample surrounding Pi. Coculturing Aphanizomenon with Chlamydomonas or with Debarya sp. showed positive ELF-APase signals, but not in Aphanizomenon. CYN producers promote Pi supply by inducing APase secretion by other phytoplanktons, possibly explaining their increased abundance despite reduced Pi supply from watersheds.

Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment.

The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments.

Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples.

The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the betaAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations betaAMOf/betaAMOr, betaAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on betaAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

Sensitive measurement of NH4+ 15N/14N (delta 15NH4+) at natural abundance levels in fresh and saltwaters.

We report a new method for determining the 15N/14N of NH4+ at natural abundance level in both freshwater and seawater. NH4+ is first quantitatively oxidized to NO2- by hypobromite (BrO-) at pH approximately 12. After the addition of sodium arsenite to consume excess BrO-, yield is verified by colorimetric NO2- determination. NO2- is further reduced to N2O using a 1:1 sodium azide and acetic acid buffer solution using previously established procedures. The product N2O is then analyzed for isotopic composition using a continuous flow purge and cryogenic trap system coupled to an isotope ratio mass spectrometer. Reliable delta 15N values (standard deviation is 0.3 per thousand or better) are obtained over an NH4+ concentration range of 0.5-10 microM using 20 mL volumes of either freshwater or seawater samples. Higher concentration samples are readily diluted to lower concentration. Preexisting NO2- is removed by treatment with sulfanilic acid. There is no interference from any of the nitrogen-containing compounds tested except short-chain aliphatic amino acids (i.e., glycine) which typically are present at very low environmental concentrations. As compared to published methods, our approach is more robust, readily applicable at low concentrations and small sample volumes, and requires less time for preparation and analysis.

Statistical analysis of the fluctuating counts of fecal bacteria in the water of Lake Kinneret.

Counts of E. coli, Enteroccoci and fecal coliforms in four sites around Lake Kinneret (The Sea of Galilee), collected every 2-4 weeks for about 5 years during 1995-2002 showed irregular fluctuations punctuated by aperiodic outbursts of variable magnitude. Because of the haphazard nature of fecal contamination and large intervals between successive counts, these patterns were described by probabilistic models, based on the truncated Laplace or Extreme Value distribution. Their applicability was tested by comparing the predicted frequencies of counts exceeding different levels calculated from the first half of each record with those actually observed in its second half. Despite the records imperfections and minor violations of the underlying models' assumptions, there was a reasonable agreement between the estimated and actual frequencies. This demonstrated that it is possible to translate the irregular fluctuation pattern into a set of probabilities of future high counts. In principle, such probabilities can be used to quantify the water's fecal contamination pattern and as a tool to assess the efficacy of preventive measures to reduce it.

A Synechococcus PglnA::luxAB fusion for estimation of nitrogen bioavailability to freshwater cyanobacteria.

In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 micro M for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.