Jarkko Rapala

Persistence of cyanobacterial hepatotoxin, microcystin-LR in particulate material and dissolved in lake water

Abstract The persistence of cyanobacterial hepatotoxin, microcystin-LR, was investigated in Lake Tuusulanjarvi in southern Finland from August to October, 1993 and 1994. The amount of toxin in particulate material and dissolved in water were determined by HPLC from samples collected from mesocosm enclosures and from the surrounding lake water. In the beginning of the experiments over 80% of the phytoplankton biomass consisted of cyanobacteria. The main species were Microcystis wesenbergii (Chroococcales, Cyanobacteria), M. viridis and M. aeruginosa. The microcystin-LR concentration in particulate material varied from 2.7 to 3.2 μg l−1 and the corresponding concentration of microcystin LR dissolved in water from 0.06 to 0.21 μg l−1. The cyanobacterial biomass decreased towards the middle of September and simultaneously the microcystin concentration in freeze dried particulate material decreased below the detection limit of 10 μg g−1, corresponding 0.01 μg l−1. Dissolved microcystin-LR was detected in a concentration range of 1 to 5 ng l−1 even at the end of the experiments in October, when the cyanobacterial biomass was less than 1 mg l−1. Thus, dissolved microcystin was more persistent compared to microcystin in particulate material: the decimal reduction time for dissolved toxin was 30 d and for toxin in particulate material about 15 d.

High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms

Cyanobacterial mass occurrences (water blooms) cause ecological, economic and health problems worldwide. Still, little is known about heterotrophic bacteria associated with cyanobacteria and the interactions between those organisms. We isolated 460 bacterial strains from more than 40 lakes and rivers (151 samples), Baltic Sea (32 samples) and treated drinking water of seven treatment plants (29 samples). The water bodies and the raw water of the treatment plants were frequently dominated by high numbers of cyanobacteria. Various growth media were used to isolate the strains. Analysis of partial 16S rRNA gene fragments (701–905 bp for 358 strains and 413–497 bp for 102 strains) classified the isolated bacteria as Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Deinococcus-Thermus. Some of these isolates represented possible new bacterial orders, families, genera or species. We isolated various potentially pathogenic bacteria, such as Aeromonas, Vibrio, Acinetobacter and Pseudomonas, that may cause adverse health effects in humans and animals and should be taken into consideration when assessing the risks caused by cyanobacterial blooms. Several strains also inhibited or enhanced the growth of cyanobacteria. Most of such strains had an enhancing effect on the cyanobacterial growth. Other isolates were affiliated with genera such as Sphingomonas or Flavobacterium, which include strains that are capable of degrading cyanobacterial toxins or other recalcitrant and problematic organic compounds. The isolated strains provide a large group of bacteria that could be used in assessing and controlling the harmful effects of cyanobacteria.

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N2 fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.

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.

Endotoxins associated with cyanobacteria and their removal during drinking water treatment

The aim of this study was to investigate endotoxin concentrations in cyanobacterial water blooms and strains, and to assess the removal of endotoxins during drinking water treatment. Endotoxin concentrations were measured from 151 hepatotoxic, neurotoxic and non-toxic cyanobacterial water blooms by using Limulus amebocyte lysate (LAL) assay, and the results were compared to bacterial data. Endotoxin activities ranged from 20 to 3.8 x 10(4) endotoxin units (EU) per ml. Endotoxicity of the samples correlated with phycobiliprotein concentration that was used to assess cyanobacterial abundance, heterotrophic plate count, and Aeromonas spp. but it did not correlate with the number of coliforms or streptococci. The high endotoxin concentrations occasionally detected in the water bloom samples were probably due to Gram negative bacteria that existed together with cyanobacteria since the 26 axenic cyanobacterial strains from different genera that were studied showed very low endotoxin activity. No differences in endotoxin activity were detected between hepatotoxic, neurotoxic and non-toxic strains. Removal of endotoxins during drinking water treatment was studied at nine waterworks that previously had been associated with high numbers of cyanobacteria and that used different processes for water purification. Endotoxin concentration in raw waters ranged from 18 to 356 EU ml(-1). The treatment processes reduced 59-97% of the endotoxin activity; in the treated water the concentration ranged from 3 to 15 EU ml(-1). The most significant reduction occurred at the early stages of water treatment, during coagulation, settling and sand filtration. Activated carbon filtration either increased or had no effect on endotoxin concentration. Ozonation and chlorination had little effect on the endotoxin concentrations.

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

Abstract 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.

Previously uncultured β-Proteobacteria dominate in biologically active granular activated carbon (BAC) filters.

Bacteria colonizing BAC filters used in drinking water purification from lake water were characterized by morphology, physiological tests, whole cell protein profiles and PLFA (phospholipid fatty acid) composition, and identified by partial 16S rRNA gene sequencing. Epifluorescence revealed prothecate bacteria to dominate in BAC. The majority of the isolates belonged to order Burkholderiales of beta-Proteobacteria, a few to Comamonadaceae but the majority to an undescribed family and the related sequences belonged mainly to uncultured bacteria. Among the less common alpha-Proteobacteria the genus Sphingomonas and the genera Afipia, Bosea or Bradyrhizobium of the Bradyrhizobiaceae family were detected. The majority of cultured bacteria persisting in the BAC biofilter were Burkholderiales, which according to ecological information are efficient in the mineralisation of dissolved organic matter in BAC. The biotechnical potential of the previously uncultured dominant bacteria warrants to be further studied.

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.

Biosynthesis of anatoxin-a in Anabaena flos-aquae and homoanatoxin-a in Oscillatoria formosa

The carbon skeleton of anatoxin-a is derived from acetate and glutamate with retention of C-1 of the amino acid.

Virulence genes of Aeromonas isolates, bacterial endotoxins and cyanobacterial toxins from recreational water samples associated with human health symptoms

Exposure to cyanobacterial water blooms has been associated with various kinds of adverse health effects. In addition to cyanobacteria and their toxins, the bacteria associated with cyanobacteria could also be the etiological agents. We isolated Aeromonas strains (n = 176) from water samples (n = 38) taken from sites where cyanobacteria were suspected to have caused human health symptoms, of which fever and gastrointestinal symptoms were the most common. The isolates were screened by PCR for six virulence gene types (12 genes). The majority (90%) of the strains contained at least one of the virulence genes. Most common amplification products were those of genes (act/aerA/hlyA) that encode cytotoxic enterotoxin and haemolytic products. The genes encoding cytotonic enterotoxins (ast and alt), phospholipase (lip/pla/lipH3/alp-1), elastase (ahyB) and flagellin subunits (flaA/flaB) were also present in 5-37% of the Aeromonas strains. Analysed toxins (cyanobacterial hepatotoxins and neurotoxins, and bacterial endotoxins) were not detectable or were present in only low concentrations in the majority of the samples. The results indicated that the toxins were unlikely to be the main cause of the reported adverse health effects, whereas more attention should be paid to bacteria associated with cyanobacteria as a source of health effects.