Cyanobacteria research methods in water ecosystems

Cijanobakterije su najstariji fotosintetski organizmi na Zemlji. Izuzetno su zanimljivi zbog svoje građe i uloge u evolucijskim procesima stvaranja eukariotske stanice. Naseljavaju cijeli niz staništa koji su često karakterizirani ekstremnim životnim uvjetima. Popunjavaju brojne ekološke niše te značajno doprinose kruženju elemenata u prirodi. Cijanobakterije nazivamo ˝pionirima˝, jer prve nastanjuju negostoljubive okoliše. Istraživanje cijanobakterija u vodenim ekosustavima često predstavlja izazov u raznim znanstvenim područjima. Primjenjuju se brojne metode, počevši od kemijskih, bioloških i ekoloških metoda do najsuvremenijih molekularnih metoda i optičkih praćenja putem satelita. U ovom su radu prikazana dva primjera istraživanja cijanobakterija: u mikrobnom obraštaju (eng. microbial mat), gdje se javljaju kao važna fotoautotrofna komponenta te u akvakulturi, gdje svojim cvjetanjem i toksinima mogu prouzročiti negativne posljedice za zdravlje čovjeka i ekosustav te materijalne štete.Cyanobacteria are the oldest photosynthetic organisms on earth. They are exceptionally interesting due to their structure and their role in the evolutionary processes of eukaryotic cell creation. They are present in a number of habitats that are frequently characterized by extreme living conditions. They fi ll numerous ecological niches and signifi cantly contribute to the circulation of elements in nature. Cyanobacteria are called the ˝pioneers˝ because they are the fi rst to settle in inhospitable environments. Cyanobacteria research in water ecosystems frequently poses a challenge to different scientifi c areas. Numerous methods are implemented, from chemical, biological or ecological methods to the most advanced molecular methods and optical monitoring via satellites. The paper presents two examples of cyanobacteria research - in a microbial mat, where they occur as an important photoautotrophic component, and in aquaculture, where their blooms and toxins may cause adverse consequences to human and ecosystem health and material damage as well

Effects of low and high irradiation levels on growth and PSII efficiency in Lemna minor L.

Plant growth and reproduction depend on light energy that drives photosynthesis. In the present study we compared growth characteristics, photosynthetic pigments content and photosystem II (PSII) performance in Lemna minor L. grown in two different irradiation regimes: low light (LL) – 50 μmolPHOTONS m-2 s-1 and high light (HL) – 500 μmolPHOTONS m-2 s-1. The main goal was to investigate the photosynthetic regulatory mechanisms that ensure adjustment to different light conditions and integrate these observations with the data on plant multiplication and biomass production. For this purpose, we measured chlorophyll (Chls) and carotenoid (Cars) contents and analyzed the energy fluxes through the PSII by saturation pulse method as well as by Chl a transient induction and JIP test. In a comparison of the effect of LL and HL on plant multiplication and fresh biomass, it was shown that the effect on growth was primarily attributed to the biomass reduction in LL while the effect on number of plants was much smaller. Total Chl and Cars contents were decreased in plants exposed to HL which indicated long-term acclimation response to the increased irradiance. Furthermore, the HL plants revealed better capability for the utilization of absorbed light in photosynthesis accompanied by photoprotective adjustment of certain number of PSII reaction centers from active to dissipative mode of functioning. In conclusion, our study showed that duckweed plants had great adjustment potential to different irradiation conditions, which might be of great importance not only under variable light availability but also when simultaneously challenged by some other environmental disturbance (e.g. different pollutants)

Microbial mats as shelter microhabitat for amphipods in an intermittent karstic spring

Microbial mats represent complex communities where cyanobacteria and diatoms as key organisms provide shelter for diverse assemblages of aquatic invertebrates, like the small stygophilous amphipod Synurella ambulans. Studies addressing such communities in the karst springs have rarely examined springheads, and have ignored intermittent springs. During high flow conditions the stygophilic crustaceans are flushed to the surface of a temporary stream Krčić where microbial mats prevent their drift and enables their successful retreat into underground in the periods of drought. The objective of this study was to characterize the microbial mat community of the Krčić Spring as a shelter for S. ambulans during strong current and high water level. Representative samples for diatom and cyanobacterial species identification and composition, as well as the fresh mat material for potential animal activity and cyanobacterial phylogenetic analysis were collected. The most dominant diatom was Achnanthidium minutissimum, whilst Fragilaria capucina, Meridion circulare, Navicula cryptocephala and Nitzschia palea had abundance greater than 0.5%. Morphological observations of cyanobacteria revealed that Phormidium favosum was the most dominant, with Hydrocoleum muscicola as a subdominant. Cyanobacterial phylogenetic relationship revealed two distinct clusters: (i) "Phormidium cluster", confirming morphological observations in both winter and spring samples, and (ii) "Wilmottia cluster", a first report for Croatia and found exclusively in the winter sample. Laboratory observations revealed a small stygophilic amphipod S. ambulans, hiding and feeding inside the pockets of fresh microbial mat. The intermittent Krčić Spring as a predator-free and competitor-free ecosystem provides a spatiotemporal conformity between microbial mat and stygophilous amphipod

Skin Culturable Microbiota in Farmed European Seabass (Dicentrarchus labrax) in Two Aquacultures with and without Anibiotic Use

This study examined culturable skin microbiota that was associated with farmed European seabass (Dicentrarchus labrax). Healthy European seabass were sampled during summer commercial harvest from one conventional fish farm where antibiotics are used, and from another practicing a certified antibiotic-free fish aquaculture. Physicochemical and microbiological analysis of seawater and sediment were performed, as well as determination of culturable bacteria, including Vibrio, from skin swabs of European seabass and seawater and sediment at both farms. Samples were processed for isolation of bacteria and their characterization by molecular and antibiotic susceptibility tests. In both fish farms, most of the bacteria that were identified in the skin belonged to the genera Pseudomonas and Vibrio. Some of the microbiota that were identified are known to be pathogenic to fish: V. alginolyticus, V. anguillarum, and V. harveyi. Vibrio strains showed higher resistance to certain antibiotics compared to previous studies. This study provides, for the first time, information on the culturable skin bacteria that is associated with healthy European seabass under culture conditions with and without the use of antibiotics. This information will be useful in assessing how changes in culturable microbiota may affect the health of farmed European seabass, indicating a potential problem for fish health management during disease outbreaks

Ekologija i raznolikost cijanobakterija u promjenjivim ekosustavima u području istočnog Jadrana

Cyanobacteria are ubiquitous bacteria that evolved photosynthesis and therefore made life on Earth possible by creating aerobic atmosphere more than 2 billion years ago. As primary colonizers, they have adapted to diverse types of environments, and primordial as well as actual extreme conditions. In that respect, they are considered indicators of the ecological properties of the environment in which they are found. This thesis aims at exploring the vast diversity and ecology of the Cyanobacteria, from ecosystems under different selective pressures: extreme drought and flood conditions as well anthropogenic pressures such as aquaculture, agriculture and urbanization Modern molecular-based methods, with emphasis on the highthroughput sequencing, were the main methods used in this study, thus providing an accurate picture of their diversity and community composition in different ecological niches. In both freshwater and marine ecosystems, habitats such as microbial mats, sediment and water column were explored to give an overview of diversity and adaptation of Cyanobacteria to different environmental conditions. These conditions are ever shifting under the climate change, whose impact is actually observed in the semi-enclosed Adriatic Sea. As a matter of fact, the relevance of the Cyanobacteria is especially highlighted in the vulnerable geographical and geological area of the karstic Adriatic, where the effects of climate change and land-based pollution are acting much faster than in other areas.Cijanobakterije su sveprisutne bakterije, koje su razvile oksigenu fotosintezu te omogućile život na Zemlji stvorivši aerobnu atmosferu prije više od 2 milijarde godina. Kao primarni kolonizatori, prilagodili su se različitim vrstama okoliša te ekstremnim uvjetima iz Zemljine prošlosti, kao i današnjim ektremnim uvjetima. Zahvaljujući tim prilagodbama, one su indikatori ekoloških svojstava okoliša u kojem se nalaze. Cilj ovog doktorskog rada je istražiti raznolikost i ekologiju cijanobakterija u ekosustavima koji su pod različitim pritiscima selekcije: ekstremnim sušama i poplavama te antropogenim pritiscima kao što su akvakultura, poljoprivreda i urbanizacija. Molekularne metode, s naglaskom na sekvenciranju druge generacije, su glavne metode korištene u ovom istraživanju. Njihovim implementacijom dobiva se potpunija slika raznolikosti i sastava zajednice cijanobakterija u različitim ekološkim nišama. U slatkovodnim i morskim ekosustavima istraživana su staništa poput mikrobnih obraštaja, sedimenata i vodenog stupca, upravo kako bi se dobio uvid u raznolikost i prilagodbe cijanobakterija različitim uvjetima okoliša. Ti se uvjeti neprestano mijenjaju u skladu s klimatskim promjenama, čiji je utjecaj očit u poluzatvorenom ekosustavu kao što je Jadransko more. Zapravo, važnost istraživanja ekologije cijanobakterija je posebno primjerena u osjetljivom geografskom i geološkom području krškog Jadrana, u kojem su učinci klimatskih promjena i onečišćenja s kopna evidentni mnogo brže nego na drugim područjima

Ekologija i raznolikost cijanobakterija u promjenjivim ekosustavima u području istočnog Jadrana

Cyanobacteria are ubiquitous bacteria that evolved photosynthesis and therefore made life on Earth possible by creating aerobic atmosphere more than 2 billion years ago. As primary colonizers, they have adapted to diverse types of environments, and primordial as well as actual extreme conditions. In that respect, they are considered indicators of the ecological properties of the environment in which they are found. This thesis aims at exploring the vast diversity and ecology of the Cyanobacteria, from ecosystems under different selective pressures: extreme drought and flood conditions as well anthropogenic pressures such as aquaculture, agriculture and urbanization Modern molecular-based methods, with emphasis on the highthroughput sequencing, were the main methods used in this study, thus providing an accurate picture of their diversity and community composition in different ecological niches. In both freshwater and marine ecosystems, habitats such as microbial mats, sediment and water column were explored to give an overview of diversity and adaptation of Cyanobacteria to different environmental conditions. These conditions are ever shifting under the climate change, whose impact is actually observed in the semi-enclosed Adriatic Sea. As a matter of fact, the relevance of the Cyanobacteria is especially highlighted in the vulnerable geographical and geological area of the karstic Adriatic, where the effects of climate change and land-based pollution are acting much faster than in other areas.Cijanobakterije su sveprisutne bakterije, koje su razvile oksigenu fotosintezu te omogućile život na Zemlji stvorivši aerobnu atmosferu prije više od 2 milijarde godina. Kao primarni kolonizatori, prilagodili su se različitim vrstama okoliša te ekstremnim uvjetima iz Zemljine prošlosti, kao i današnjim ektremnim uvjetima. Zahvaljujući tim prilagodbama, one su indikatori ekoloških svojstava okoliša u kojem se nalaze. Cilj ovog doktorskog rada je istražiti raznolikost i ekologiju cijanobakterija u ekosustavima koji su pod različitim pritiscima selekcije: ekstremnim sušama i poplavama te antropogenim pritiscima kao što su akvakultura, poljoprivreda i urbanizacija. Molekularne metode, s naglaskom na sekvenciranju druge generacije, su glavne metode korištene u ovom istraživanju. Njihovim implementacijom dobiva se potpunija slika raznolikosti i sastava zajednice cijanobakterija u različitim ekološkim nišama. U slatkovodnim i morskim ekosustavima istraživana su staništa poput mikrobnih obraštaja, sedimenata i vodenog stupca, upravo kako bi se dobio uvid u raznolikost i prilagodbe cijanobakterija različitim uvjetima okoliša. Ti se uvjeti neprestano mijenjaju u skladu s klimatskim promjenama, čiji je utjecaj očit u poluzatvorenom ekosustavu kao što je Jadransko more. Zapravo, važnost istraživanja ekologije cijanobakterija je posebno primjerena u osjetljivom geografskom i geološkom području krškog Jadrana, u kojem su učinci klimatskih promjena i onečišćenja s kopna evidentni mnogo brže nego na drugim područjima

Microbial mats as shelter microhabitat for amphipods in an intermittent karstic spring

Microbial mats represent complex communities where cyanobacteria and diatoms as key organisms provide shelter for diverse assemblages of aquatic invertebrates, like the small stygophilous amphipod Synurella ambulans. Studies addressing such communities in the karst springs have rarely examined springheads, and have ignored intermittent springs. During high flow conditions the stygophilic crustaceans are flushed to the surface of a temporary stream Krčić where microbial mats prevent their drift and enables their successful retreat into underground in the periods of drought. The objective of this study was to characterize the microbial mat community of the Krčić Spring as a shelter for S. ambulans during strong current and high water level. Representative samples for diatom and cyanobacterial species identification and composition, as well as the fresh mat material for potential animal activity and cyanobacterial phylogenetic analysis were collected. The most dominant diatom was Achnanthidium minutissimum, whilst Fragilaria capucina, Meridion circulare, Navicula cryptocephala and Nitzschia palea had abundance greater than 0.5%. Morphological observations of cyanobacteria revealed that Phormidium favosum was the most dominant, with Hydrocoleum muscicola as a subdominant. Cyanobacterial phylogenetic relationship revealed two distinct clusters: (i) "Phormidium cluster", confirming morphological observations in both winter and spring samples, and (ii) "Wilmottia cluster", a first report for Croatia and found exclusively in the winter sample. Laboratory observations revealed a small stygophilic amphipod S. ambulans, hiding and feeding inside the pockets of fresh microbial mat. The intermittent Krčić Spring as a predator-free and competitor-free ecosystem provides a spatiotemporal conformity between microbial mat and stygophilous amphipod

Profiling of bacterial assemblages in the marine cage farm environment, with implications on fish, human and ecosystem health

This research presents a comprehensive study of bacterial assemblages within the water column and in the surface sediments in the zone of two European sea bass cage farms. By the application of the high-throughput amplicon sequencing of 16S rRNA gene, and further implementing microbial ecology tools, a bacterial segment from cage culturing systems and their respective controls were analyzed, with special reference to potential impact on animal, human and environmental health. Samples of seawater and sediments were collected seasonally, at locations situated in the central and southern Adriatic Sea. Bacterial composition was significantly different in the seawater vs. sediment. No significant differences in alpha diversity in sediments were indicated between aquaculture and control sites, and it appears that it is not affected by farming practices. Control sediments have higher relative abundance of aerobic and facultative anaerobic bacteria, while aquaculture sediments are markedly anaerobic. Sediments largely contain functional groups for respiration of sulfate and sulfur compounds, though doubly more in aquaculture sites. Seasonal groupings of bacterial assemblages were confirmed in the seawater, with higher relative abundance of known aquaculture pathogens (except Photobacterium in the winter samples) detected in the winter and summer, opposed to other two seasons. Rare taxa were analyzed in the sediment and in the water column in the search for known fish pathogens, with five genera detected: Vibrio, Pseudomonas, Photobacterium, Tenacibaculum and Mycobacterium. Biomarkers important for the impact of aquaculture on the environment were identified, e.g. Blastopirullela, Sva0081, Suflurovum, Spirochaeta 2, etc., as well as human and fish potential pathogens: Vibrio ichtyoentery, V. harvey, Acinetobacter lwoffi, A. johnsonii, Clostridium perfringens, etc. Chemoheterotrophy has emerged as the dominant functional group in both environments. Regarding priorities for aquaculture microbial management, seawater seems to contain a higher percentage of taxa connected to health-related functional groups

Profiling of bacterial assemblages in the marine cage farm environment, with implications on fish, human and ecosystem health

This research presents a comprehensive study of bacterial assemblages within the water column and in the surface sediments in the zone of two European sea bass cage farms. By the application of the high-throughput amplicon sequencing of 16S rRNA gene, and further implementing microbial ecology tools, a bacterial segment from cage culturing systems and their respective controls were analyzed, with special reference to potential impact on animal, human and environmental health. Samples of seawater and sediments were collected seasonally, at locations situated in the central and southern Adriatic Sea. Bacterial composition was significantly different in the seawater vs. sediment. No significant differences in alpha diversity in sediments were indicated between aquaculture and control sites, and it appears that it is not affected by farming practices. Control sediments have higher relative abundance of aerobic and facultative anaerobic bacteria, while aquaculture sediments are markedly anaerobic. Sediments largely contain functional groups for respiration of sulfate and sulfur compounds, though doubly more in aquaculture sites. Seasonal groupings of bacterial assemblages were confirmed in the seawater, with higher relative abundance of known aquaculture pathogens (except Photobacterium in the winter samples) detected in the winter and summer, opposed to other two seasons. Rare taxa were analyzed in the sediment and in the water column in the search for known fish pathogens, with five genera detected: Vibrio, Pseudomonas, Photobacterium, Tenacibaculum and Mycobacterium. Biomarkers important for the impact of aquaculture on the environment were identified, e.g. Blastopirullela, Sva0081, Suflurovum, Spirochaeta 2, etc., as well as human and fish potential pathogens: Vibrio ichtyoentery, V. harvey, Acinetobacter lwoffi, A. johnsonii, Clostridium perfringens, etc. Chemoheterotrophy has emerged as the dominant functional group in both environments. Regarding priorities for aquaculture microbial management, seawater seems to contain a higher percentage of taxa connected to health-related functional groups