Anatoxin-a and odorous metabolites in cyanobacteria : molecular detection of the producers

Cyanobacteria produce variety of secondary metabolites that may be toxic or cause odor problems in water environments. Taste and odor problems occur worldwide in freshwater, drinking water, and in the fish industry. Cyanobacterial mass occurrences pose a threat to users of water due to hepatotoxic or neurotoxic strains. Toxins are also a risk to recreational water use and have caused number of animal poisonings. Anatoxin-a and homoanatoxin-a are alkaloid neurotoxins produced by strains of several cyanobacterial genera. Cyanobacteria also produce strong odorous metabolites that cause musty and/or earthy odor and taste in water. The best known of these odorous metabolites are geosmin and 2-methylisoborneol (MIB). These odorous metabolites cause problems in extremely low concentrations due to the very low odor threshold of humans. Producers of bioactive metabolites and non-producing strains can occur in the same species and thus cannot be distinguished morphologically by microscopy techniques. The aims of this study were: I) to identify the ana gene cluster responsible for the biosynthesis of anatoxin-a from the strain Anabaena sp. 37; II) to develop molecular detection methods (PCR, quantitative PCR, restriction fragment length polymorphism RFLP) for the detection of potential anatoxin-a and homoanatoxin-a producers, and apply methods to environmental samples; and III) to develop a PCR-based molecular method for the detection of the producers of geosmin and MIB in cyanobacteria. Of the genomic sequence of Anabaena sp. 37, the gene cluster responsible for anatoxin-a biosynthesis (ana) was identified based on the comparison to the gene cluster from cyanobacteria Oscillatoria sp. PCC 6506. While the organization of the genes differed, the anatoxin-a synthetase genes were highly similar with Oscillatoria sp. PCC 6506 genes. PCR and qPCR detection methods were developed for anatoxin-a producing cyanobacteria. General primers were used to identify all the producer genera at the same time and genus-specific primers for Anabaena or Oscillatoria producers. Primers were designed to recognize the anaC gene and tested with anatoxin-a and/or homoanatoxin-a producing strains in PCR and qPCR. In addition, RFLP analysis of the anaC amplicons was used to simultaneously identify three anatoxin-a producer genera Anabaena, Oscillatoria, and Aphanizomenon. Molecular methods for anatoxin-a producers were also applied to environmental samples. With developed PCR and RFLP methods, the presence of Anabaena and Oscillatoria as potential anatoxin-a producers in Finnish freshwaters and the Baltic Sea was observed. The developed qPCR method was used to quantify the anaC gene copy numbers in Lake Garda during February-November 2013. The gene copy numbers correlated positively with the anatoxin-a concentration measured in lake samples. Thus, the developed method could be used for the detection of potential anatoxin-a and homoanatoxin-a producers in cyanobacterial strains and to predict the amount of anatoxin-a producers from lake water samples. A hundred cyanobacterial strains were studied with solid-phase microextraction coupled with gas chromatography/mass spectrometry (SPME GC-MS) in order to identify the producers of geosmin and MIB. Geosmin was found in 21 strains out of the 100 studied, representing Nostoc, Oscillatoria, Calothrix, Planktothrix, Aphanizomenon, and Cylindrospermum genera. MIB was found in two strains, Planktothrix and Oscillatoria, both also producing geosmin. Cyanobacteria-specific primers were designed to detect biosynthetic genes of geosmin (geoA) and MIB (MIB synthase) and tested with the odorous metabolite-producing strains. Molecular detection by PCR was consistent with chemical detection by SPME GC-MS. Molecular detection methods developed in this study to detect anatoxin-a or odorous metabolites producing strains could be used in surveys to identify possible producers among cyanobacterial strains and in environmental samples.Syanobakteerit tuottavat useita erilaisia sekundaarimetabolian tuotteita. Nämä yhdisteet saattavat olla myrkyllisiä ja aiheuttaa maku- ja hajuhaittoja vesistöissä. Näiden yhdisteiden aiheuttamia maku- ja hajuhaittoja esiintyy maailmanlaajuisesti juomavesissä ja kaloissa. Syanobakteerien massaesiintymät aiheuttavat riskin juomaveden käytölle hepatotoksisten ja neurotoksisten syanobakteerien vuoksi. Syanobakteerien tuottamat myrkyt haittaavat veden virkistyskäyttöä ja ne ovat myös aiheuttaneet useita eläinkuolemia. Anatoksiini-a ja homoanatoksiini-a ovat neurotoksiineja, joita tuottavat useat eri syanobakteerien suvut ja lajit. Syanobakteerien tuottamat voimakkaat maku- ja hajuaineet aiheuttavat mudan/maan maun ja hajun veteen. Parhaiten tunnettuja hajumetaboliitteja ovat geosmiini ja 2-metyyli-isoborneoli. Nämä hajumetaboliitit aiheuttavat ongelmia jo hyvin pieninä pitoisuuksina. Hajumetaboliittien ja toksisten yhdisteiden tuottajia ei voi erottaa niitä tuottamattomista saman lajin kannoista mikroskopoinnilla. Työn tavoitteena oli identifioida anatoksiini-a:n tuotosta vastaava ana geenijoukko Anabaena sp. 37 -kannasta. Lisäksi tarkoitus oli kehittää molekuularisia havainnointimenetelmiä (PCR, qPCR, katkokirjoanalyysi RFLP) anatoksiini-a:n tuottajien tunnistamiseksi ja soveltaa niitä luonnonvesinäytteisiin. Myös geosmiinin ja 2-metyyli-isoborneolin tuottajien havainnointiin syanobakteereista kehitettiin PCR-pohjainen osoitusmenetelmä. Anabaena sp. 37 -kannan genomisekvenssistä identifioitiin ana-geenijoukko vertailemalla sitä Oscillatoria sp. PCC 6506 kannan ana-geenijoukkoon. Vertailussa havaittiin, että geenien organisaatio biosynteesigeenijoukossa on erilainen, mutta Anabaena-kannan anatoksiini-a syntetaasigeenit ovat hyvin samankaltaiset Oscillatoria-kannan kanssa sekvenssitasolla. PCR- ja qPCR-menetelmät kehitettiin anatoksiini-a:n tuottajien havainnoimiseksi. Yleiset alukkeet suunniteltiin tunnistamaan tuottajia kaikista tuottajasuvuista samanaikaisesti ja sukuspesifiset alukkeet tunnistamaan Anabaena- ja Oscillatoria-sukujen tuottajat erikseen. Alukkeet suunniteltiin osoittamaan anaC-geeniä ja niiden toimivuus testattiin anatoksiini-a:n ja homoanatoksiini-a:n tuottajakannoilla. Lisäksi RFLP-menetelmä kehitettiin tunnistamaan yhtä aikaa kolmen eri tuottajasuvun anaC-geeniä, Anabaena, Aphanizomenon ja Oscillatoria. Kehitettyjä molekulaarisia menetelmiä sovellettiin luonnonvesinäytteisiin ja Anabaena sekä Oscillatoria havaittiin mahdollisina anatoksiini-a:n tuottajina suomalaisissa järvivesissä ja Itämeressä. Kehitetyn qPCR-menetelmän avulla kvantitoitiin anaC-geenikopiolukumääriä Italian Garda-järvestä helmi-marraskuussa vuonna 2013. Menetelmän avulla havaittiin, että geenikopiolukumäärät korreloivat positiivisesti järvinäytteiden anatoksiini-a:n konsentraation kanssa. Näin ollen tässä työssä kehitetyt menetelmät toimivat erinomaisesti mahdollisten anatoksiini-a:n ja homoanatoksiini-a:n tuottajien havainnoimiseksi syanobakteereista, ja niiden avulla voidaan arvioida anatoksiini-a:n tuottajien määrää luonnonvesinäytteissä. Sata syanobakteerikantaa tutkittiin kiinteäfaasimikrouutto ja kaasukromatografia -menetelmällä (SPME GC-MS), jotta voitiin tunnistaa geosmiinin ja 2-metyyli-isoborneolin tuottajia. Geosmiinin tuottajia löydettiin 21 kannasta ja kuudesta suvusta (Nostoc, Oscillatoria, Calothrix, Planktothrix, Aphanizomenon ja Cylindrospermum). 2-metyyli-isoborneolia löydettiin yhdestä Planktothrix- ja yhdestä Oscillatoria-kannasta. Molemmat 2-metyyli-isoborneolin tuottajat tuottivat myös geosmiinia. Syanobakteeri-spesifiset alukkeet suunniteltiin tunnistamaan geosmiini- ja 2-metyyli-isoborneolisyntaasi-geenit (geoA ja MIB syntaasi). Työssä kehitetyllä PCR-menetelmällä havaittiin kaikki kemiallisella menetelmällä tunnistetut hajumetaboliittien tuottajat. Tässä työssä kehitettyjä molekulaarisia havainnointimenetelmiä voidaan käyttää tutkittaessa anatoksiini-a:n ja hajumetaboliittien potentiaalisia tuottajia syanobakteerikannoista ja ympäristöstä

Cyanobacteria and Their Metabolites in Mono- and Polidominant Shallow Eutrophic Temperate Lakes

Monodominant (one species dominates) or polidominant (multiple species dominate) cyanobacterial blooms are pronounced in productive freshwater ecosystems and pose a potential threat to the biota due to the synthesis of toxins. Seasonal changes in cyanobacteria species and cyanometabolites composition were studied in two shallow temperate eutrophic lakes. Data on cyanobacteria biomass and diversity of dominant species in the lakes were combined with chemical and molecular analyses of fifteen potentially toxin-producing cyanobacteria species (248 isolates from the lakes). Anatoxin-a, saxitoxin, microcystins and other non-ribosomal peptides formed the diverse profiles in monodominant (Planktothrix agardhii) and polidominant (Aphanizomenon gracile, Limnothrix spp. and Planktolyngbya limnetica) lakes. However, the harmfulness of the blooms depended on the ability of the dominant species to synthesize cyanometabolites. It was confirmed that P. agardhii produced a greater amount and diverse range of MCs and other NRPs. In the polidominant lake, isolates of the co-dominant A. gracile, L. planctonica and P. limnetica synthesized no or only small amounts of cyanometabolites. In general, the profile of cyanometabolites was greater in cyanobacteria isolates than in environmental samples, indicating a high potential for toxic cyanobacteria bloom

The Biosynthesis of Rare Homo-Amino Acid Containing Variants of Microcystin by a Benthic Cyanobacterium

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic cyanobacterium Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins

Runkotilavuusyhtälöt panamalaiselle tiikille

Puun tilavuus on yksi metsänmittauksen perustunnuksia. Tilavuutta tarvitaan hakkuiden suunnitteluun ja metsän arvon määrittämiseen. Plantaasiviljelmillä metsän tilavuus määritetään yksitäisen puun tilavuusyhtälön avulla. Plantaasiviljelmillä koealalta mitatut yksit-täisten puiden tilavuudet summataan ja koealan tilavuudesta saadaan muuntokertoimen avulla hehtaarikohtainen tilavuus. Opinnäytetyön tarkoituksena oli kehittää toimeksiantajalle tiikin (Tectona grandis) runkotilavuusyhtälöitä metsäninventointiin Panamaan. Työn tarkoitus oli mallintaa kolme tilavuusyhtälöä: kaksi kokonaistilavuuden yhtälöä, joissa toisessa on muuttujana rinnankorkeusläpimitta ja toisessa rinnankorkeusläpimitta ja puun pituus, sekä kaupallisen tilavuu-den yhtälö, jossa muuttujina ovat rinnankorkeusläpimitta, pituus ja tukin yläläpimitta. Havaintoaineistona työssä oli toimeksiantajan useamman vuoden aikana keräämä mitta-aineisto, joka koostui 444 rungon mittatiedoista. Aineisto sisälsi jokaisesta puusta rinnankorkeusläpimitan, kokonaispituuden ja läpimitta ja pituus tiedot suhteellisilta etäisyksiltä pitkin runkoa. Kaikki aineiston puut ovat siemenlähtöisiä, mukana ei ole kloonattuja puita. Läpimitat on mitattu kuorellisina läpimittoina. Samoin kaikki tilavuusyhtälöt on laskettu kuorellisina tilavuuksina. Koko aineisto jaettiin kahtia mallinnusta varten: varsinaiseen mallinnusaineistoon (n=399) ja estimoitujen mallien testausaineistoon (n=40). Objektiiviseen testiaineistoon puut valittiin ositetulla satunnaisotannalla. Kokonaisuudessaan 21 tilavuusmallia estimoitiin tilasto-ohjelmalla. Testausvaiheessa tässä tutkimuksessa mallinnettujen yhtälöiden lisäksi testattiin myös muissa tutkimuksissa parhaiksi todettuja tiikin tilavuusyhtälöitä. Testaus osoitti että tässä tutkimuksessa mallinnetut ja parhaimmiksi osoittautuneet tilavuusyhtälöt sopivat paremmin havaintoaineiston kaltaiseen puustoon kuin muiden tutkimusten tilavuusmallit. Kokonaistilavuuden yhtälöissä pelkkää rinnankorkeusläpimittaa käyttävä malli on helpompi käyttää käytännössä ja läpimitan mittaustulokset ovat kohtalaisen tarkkoja ja luotettavia. Puun pituuden mittaaminen on läpimittaan verrattuna epätarkempaa ja enemmän aikaa vievää. Yhtälö, jossa tilavuuden selittävinä muuttujina ovat sekä läpimitta että pituus, antaa kuitenkin laskennallisesti tarkempia tuloksia kuin pelkkään läpimittaan perustuva yhtälö. Kaupallisen tilavuuden yhtälöllä pystytään asetetun minimiläpimitan avulla laskemaan saatavan käyttöosan tilavuus. Toimeksiantajalla on tutkimuksen ansioista nyt käytössään omaan aineistoon perustuvat tilavuusyhtälöt, joilla pystytään luotettavasti ja aikaisempaa tarkemmin ennustamaan puiden tilavuus panamalaisilla viljelmillä.Determination of the timber volume is often the most important task in forest inventory. Estimating the volume of trees is very important for the plantations’ forest management planning and evaluation of their biological asset. In plantation forests individual-tree volume equations are commonly used to calculate the total and merchantable volume of trees in the sample plot. By aggregating the individual tree volumes, the plot level volume is obtained. This plot level volume is then extrapolated to an area unit by an expansion factor. The aim of the study was to develop equations which would best predict the total volume and merchantable volume of an individual tree of Tectona grandis in planted forests in Panama. The total volume equation was developed for one and two input variables: the diameter at the breast height (1.3 m) (dbh) and dbh and total height. The merchantable volume equation was developed for variable-top diameters. The material for the study was collected from the commissioner’s plantations in Panama during several years. The data included measurements from a total of 444 felled trees. For each tree diameter at the breast height, total height of the tree and diameter measure-ments at relative heights from a tree at ten percent intervals were measured. All the trees in the data came from seedling-based plantations. There were no cloned trees. Diameters were measured with bark and all the modeled equations predicted volume with bark. The total data was divided into two groups: one was used to model the equations and one to test the quality of the modelled equations. Stratified random sampling was used to select trees for the testing dataset. A total of 21 equations was modelled with a statistic programme. Equations from other studies for teak’s volume were included in the testing phase with the independent dataset. The testing showed that the equations tested in the study proved to fit better to estimate volumes in this kind of data than the equations in other studies. Calculating the total volume with the equation with dbh as only variable is easier and quicker to use and the measurement of dbh is an accurate task. The measurement of height can be difficult to measure consistently in standing trees and it is more time-consuming. Equations with dbh and height as input variables give more accurate results than equations where dbh is the only variable. Due to this study, the commissioner has volume equations that base on its own data. The equations can predict the volumes of teak in Panama reliably and more precisely than before

Increased sulfate availability in saline water promotes hydrogen sulfide production in fish organic waste

The risk of hydrogen sulfide (H2S) production can be a challenge in marine land-based recirculating aquaculture systems (RAS). Hydrogen sulfide is a toxic gas that can cause massive fish mortality even at low concentrations, and in addition, serious odour problems in the surroundings. It is a bacterial by-product originating from the degradation of organic matter in sulfur-rich waters such as marine waters. In order to hinder H2S production in marine land-based RAS, more information on the H2S production conditions and the associated microbiology is needed. In this study, the production of H2S from rainbow trout (Oncorhynchus mykiss) organic waste was examined using a novel H2S measurement method under a range of salinities (0, 5, 10, 15, 25 and 35 g/L) in anaerobic mixed reactors, and the microbial communities as well as abundance of sulfate reducing bacteria (SRB) were characterized. The maximum H2S concentration increased from 23.1 ± 8.2 mg H2S/L at 0 g/L salinity to 153.9 ± 34.1 mg H2S/L at 35 g/L salinity. Similarly, the H2S production rate increased from 5.6 ± 0.2 at 0 g/L salinity to 26.4 ± 12.7 mg of H2S produced per day at 35 g/L salinity. The highest H2S production was recorded after increased availability of volatile fatty acids, which were produced by fermentative bacteria from phyla Firmicutes and Bacteroidetes that dominated the microbial communities after day 5. The traditional sulfate reducing bacteria (SRB) were found only at 0 and 5 g/L salinity, while at higher salinities, H2S production was carried out by novel unquantifiable SRB. The results demonstrate that H2S can be a pronounced problem in marine RAS, although it can be controlled through preventing anaerobic conditions within the system.peerReviewe

Salinity affects nitrate removal and microbial composition of denitrifying woodchip bioreactors treating recirculating aquaculture system effluents

This study investigated the effect of salinity on microbial composition and denitrification capacity of woodchip bioreactors treating recirculating aquaculture system (RAS) effluents. Twelve laboratory-scale woodchip bioreactors were run in triplicates at 0, 15, 25, and 35 ppt salinities, and water chemistry was monitored every third day during the first 39 days of operation. Microbial communities of the woodchips bioreactors were analyzed at the start, after one week, and at the end of the trial. Woodchip bioreactors removed nitrate at all salinities tested. The highest NO3-N removal rate of 22.0 ± 6.9 g NO3-N/m3/d was obtained at 0 ppt, while 15.3 ± 4.9, 12.5 ± 5.4 and 11.8 ± 4.0 g NO3-N/m3/d were obtained at salinities of 15, 25 and 35 ppt, respectively. Nitrate removal rates thus decreased with salinity, being 54–69% lower than at 0 ppt. Leaching of total ammonia nitrogen (TAN) and orthophosphate (PO4-P) from woodchips was initially higher at saline treatments compared to 0 ppt, while initial leaching of BOD5 appeared to be similar across all treatments. Production of alkalinity per g NO3-N removed was higher at 0 (3.6 ± 0.5 gCaCO3/gNO3-N) and 15 ppt (3.5 ± 0.8) than at the more saline treatments (25 ppt: 2.0 ± 0.9, 35 ppt: 1.12 ± 0.5 gCaCO3/gNO3-N), indicating that heterotrophic denitrification was the dominant nitrate removing process at 0 and 15 ppt, while autotrophic denitrification processes probably interfered with the alkalinity balance at 25 and 35 ppt. In the woodchip reactors, Gammaproteobacteria was the most abundant taxa. However, salinity shaped the woodchip microbiome, resulting in an increase in the abundance of sulfide oxidizing autotrophic denitrifiers, but decrease in the overall abundance of denitrifying microbes at higher salinities, which presumably explained the reduced nitrate removal rates at elevated salinities. This study demonstrates that woodchip bioreactors can be applied to remove nitrate from saline RAS effluents albeit at lower nitrate removal rates compared to freshwater installations.peerReviewe

Microbial communities in full-scale woodchip bioreactors treating aquaculture effluents

Woodchip bioreactors are being successfully applied to remove nitrate from commercial land-based recirculating aquaculture system (RAS) effluents. In order to understand and optimize the overall function of these bioreactors, knowledge on the microbial communities, especially on the microbes with potential for production or mitigation of harmful substances (e.g. hydrogen sulfide; H2S) is needed. In this study, we quantified and characterized bacterial and fungal communities, including potential H2S producers and consumers, using qPCR and high throughput sequencing of 16S rRNA gene. We took water samples from bioreactors and their inlet and outlet, and sampled biofilms growing on woodchips and on the outlet of the three full-scale woodchip bioreactors treating effluents of three individual RAS. We found that bioreactors hosted a high biomass of both bacteria and fungi. Although the composition of microbial communities of the inlet varied between the bioreactors, the conditions in the bioreactors selected for the same core microbial taxa. The H2S producing sulfate reducing bacteria (SRB) were mainly found in the nitrate-limited outlets of the bioreactors, the main groups being deltaproteobacterial Desulfobulbus and Desulfovibrio. The abundance of H2S consuming sulfate oxidizing bacteria (SOB) was 5–10 times higher than that of SRB, and SOB communities were dominated by Arcobacter and other genera from phylum Epsilonbacteraeota, which are also capable of autotrophic denitrification. Indeed, the relative abundance of potential autotrophic denitrifiers of all denitrifier sequences was even 54% in outlet water samples and 56% in the outlet biofilm samples. Altogether, our results show that the highly abundant bacterial and fungal communities in woodchip bioreactors are shaped through the conditions prevailing within the bioreactor, indicating that the bioreactors with similar design and operational settings should provide similar function even when conditions in the preceding RAS would differ. Furthermore, autotrophic denitrifiers can have a significant role in woodchip biofilters, consuming potentially produced H2S and removing nitrate, lengthening the operational age and thus further improving the overall environmental benefit of these bioreactors.peerReviewe