Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms.

Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all <96% which is the previously proposed cutoff value for differentiating species within this genus. The in silico DDH values of the three type strains of the new species also showed a similarity<70% with the most closely related species indicating they belong to different taxa. The three groups of strains could be differentiated from each other and from other known Aeromonas species on the basis of several phenotypic characters. This polyphasic study revealed that the 3 groups of strains represent 3 novel Aeromonas species for which the names Aeromonas aquatica sp. nov. (type strain AE235T=CECT 8025T=LMG 26712T), Aeromonas finlandiensis sp. nov. (type strain 4287DT=CECT 8028T=LMG 26709T) and Aeromonas lacus sp. nov. (type strain AE122T=CECT 8024T=LMG 26710T) are proposed.

Removal of cyanobacteria, cyanotoxins, heterotrophic bacteria and endotoxins at an operating surface water treatment plant.

The removal of cyanobacteria, hepatotoxins produced by them (microcystins), phytoplankton, heterotrophic bacteria and endotoxins were monitored at a surface water treatment plant with coagulation, clarification, sand filtration, ozonation, slow sand filtration and chlorination as the treatment process. Coagulation-sand filtration reduced microcystins by 1.2-2.4, and endotoxins by 0.72-2.01 log10 units. Ozonation effectively removed the residual microcystins. The treatment process reduced phytoplankton biomass by 2.2-4.6 and heterotrophic bacteria by 2.0-5.0 log10 units. In treated water, the concentration of microcystins never exceeded the WHO guide value (1 microg/L), but picoplankton and monad cells were often detected in high numbers. The heterotrophic bacterial isolates from the treated waters belonged to genera Sphingomonas, Pseudomonas, Bacillus, Herbaspirillum and Bosea.

Evaluation of the purification capacity of nine portable, small-scale water purification devices.

A test was performed to evaluate the microbial and chemical purification capacity of nine portable, small-scale water purification filter devices with production capacity less than 100 L/h. The devices were tested for simultaneous removal capacity of bacteria (cultured Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae and Enterobacter cloacae), enteric protozoans (formalin-stored Cryptosporidium parvum oocysts), viral markers (F-RNA bacteriophages) and microcystins produced by toxic cyanobacterial cultures. In general, the devices tested were able to remove bacterial contaminants by 3.6-6.9 log10 units from raw water. Those devices based only on filtration through pores 0.2-0.4 microm or larger failed in viral and chemical purification. Only one device, based on reverse osmosis, was capable of removing F-RNA phages at concentrations under the detection limit and microcystins by 2.5 log10. The present study emphasised the need for evaluation tests of water purification devices from the public safety and HACCP (Hazard Analysis and Critical Control Point) points of view. Simultaneous testing for various pathogenic/indicator microbes and microcystins was shown to be a useful and practical way to obtain essential data on actual purification capacity of commercial small-scale drinking-water filters.

Effect of nitrogen and phosphorus on growth of toxic and nontoxic Microcystis strains and on intracellular microcystin concentrations.

The growth and intracellular microcystin concentration of two hepatotoxic and two nontoxic axenic Microcystis strains were measured in batch cultures with variable nitrogen (0.84-84 mg L(-1)) and phosphorus (0.05-5.5 mg L(-1)) concentrations. Growth was estimated by measuring dry weight, optical density, chlorophyll a, and cellular protein concentration. Microcystin concentrations in cells and in culture medium were measured by HPLC analysis. Both nontoxic strains needed less nutrients for their growth at low nutrient concentrations. With high nutrient concentrations the toxic strains grew better than the nontoxic strains. Growth and intracellular microcystin concentration did not correlate in the hepatotoxic strains. Multivariate regression analysis together with mathematical modeling revealed a significant interactive effect of nitrogen and phosphorus, which partly explains the controversial results obtained in previous studies. In this study we have shown that variation of nitrogen and phosphorus concentrations influence the growth and the microcystin production of Microcystis strains and that the strains differ in their response to nutrients. High levels of nitrogen and phosphorus in freshwaters may favor the growth of toxic Microcystis strains over nontoxic ones.

Occurrence of microcystins in raw water sources and treated drinking water of Finnish waterworks.

Problems caused by cyanobacteria are common around the world and also in raw water sources of drinking water treatment plants. Strains belonging to genera Microcystis, Anabaena and Planktothrix produce potent hepatotoxins, the microcystins. Laboratory and pilot scale studies have shown that microcystins dissolved in water may pass the conventional surface water treatment processes. In 1998 the World Health Organization proposed a guide value of 1 microgram/L for microcystin-LR (MC-LR) in drinking water. The purpose of this research was to study the occurrence of microcystins in raw water sources of surface waterworks and in bank filtration plants and to evaluate the removal of microcystins in operating waterworks. Four bank filtration plants and nine surface waterworks using different processes for water treatment were monitored. Phytoplankton was identified and quantified, and microcystins analysed with sensitive immunoassay. Microcystin occurrence in selected water samples was verified with HPLC and a protein phosphatase inhibition method. Microcystins were detected sporadically in raw water sources of most of the waterworks. In two raw water supplies toxins were detected for several months. The highest microcystin concentrations in incoming raw water were approximately 10 micrograms/L MC-LR equivalents. In treated drinking water microcystins were detected occasionally but the concentrations were always below the guide value proposed by WHO.

Characterization of cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene.

Planktonic, filamentous cyanobacterial strains from different genera, both toxic and nontoxic strains, were characterized by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene. Total protein pattern analysis revealed the mutual relationships at the genus level. Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene with reference strains proved to be a good method for the cyanobacterial taxonomy. The nonheterocystous strains outgrouped from the nitrogen-fixing ones. With both methods, Aphanizomenon clustered with Anabaena, and Nodularia with Nostoc. In the RFLP study of Anabaena, the neurotoxic strains were identical, but the hepatotoxic ones formed a heterogeneous group. Genetic distances found in the RFLP study were short, confirming that close genotypic relationships underlie considerable diversity among cyanobacterial genera.

Variation of microcystins, cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli.

Cyanobacterial hepatotoxins, microcystins, are specific inhibitors of serine/threonine protein phosphatases and potent tumor promoters. They have caused several poisonings of animals and also pose a health hazard for humans through the use of water for drinking and recreation. Different strains of the same cyanobacterial species may variously be nontoxic, be neurotoxic, or produce several microcystin variants. It is poorly understood how the amount of toxins varies in a single strain. This laboratory study shows the importance of external growth stimuli in regulating the levels and relative proportions of different microcystin variants in two strains of filamentous, nitrogen-fixing Anabaena spp. The concentration of the toxins in the cells increased with phosphorus. High temperatures (25 to 30 degrees C), together with the highest levels of light studied (test range, 2 to 100 mumol m-2 s-1), decreased their amount. Different structural variants of microcystins responded differently to growth stimuli. Variants of microcystin (MCYST)-LR correlated with temperatures below 25 degrees C, and those of MCYST-RR correlated with higher temperatures. Nitrogen added into the growth medium and increasing temperatures increased the proportion of microcystin variants demethylated in amino acid 3. All variants remained mostly intracellular. Time was the most important factor causing the release of the toxins into the growth medium. Time, nitrogen added into the growth medium, and light fluxes above 25 mumol m-2 s-1 significantly increased the concentrations of the dissolved toxins. According to the results, it thus seems that the reduction of phosphorus loads in bodies of water might play a role in preventing the health hazards that toxic cyanobacterial water blooms pose, not only by decreasing the cyanobacteria but also by decreasing their toxin content.

Assessment of rapid bioassays for detecting cyanobacterial toxicity.

Simple and easy-to-use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato- and neurotoxins. The hepatotoxins and a neurotoxin, anatoxin-a, were extracted from laboratory-grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays. The toxin concentration of cyanobacterial extracts was determined with HPLC. The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3-17 mg l-1. The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin-a. The fractionated extract of anatoxin-a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards. Filtered cyanobacterial cultures with anatoxin-a, on the other hand, caused mortality of Artemia larvae at concentrations of 2-14 mg l-1. With the solid phase fractionation of cyanobacterial samples, no non-specific toxicity due to compounds other than hepato- and neurotoxins was observed. In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin-a. The growth of Ps. putida was enhanced, rather than inhibited by cyanobacterial toxin fractions.

Biodegradability and adsorption on lake sediments of cyanobacterial hepatotoxins and anatoxin-a.

Cyanobacterial hepatotoxins and anatoxin-a, a neurotoxin, were shown to be degraded when crude extracts of lysed toxic laboratory strains of cyanobacteria were exposed to natural populations of micro-organisms from lakes. While anatoxin-a decayed equally fast with all the inocula from lake sediment and water, the degradation rate of hepatotoxins was higher with inocula from places at which cyanobacterial water blooms had occurred than with inocula from places with no known mass occurrences of cyanobacteria. Degradation was slowest when an inoculum from a humic lake was used. A part of the loss of the toxins was shown to be due to adsorption on lake sediments.