Antonie van Leeuwenhoek (2018) 111:2425–2440 https://doi.org/10.1007/s10482-018-1132-7 (0123456789().,-volV)(0123456789().,-volV) ORIGINAL PAPER Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake Tisza A. S. Bell . Emel Sen-Kilic . Tama´s Felfo¨ldi . Gabor Vasas . Matthew W. Fields . Brent M. Peyton Received: 19 December 2017 / Accepted: 13 July 2018 / Published online: 1 August 2018 Ó This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2018 Abstract The Carpathian Basin is a lowland plain were analyzed to identify potential correlations with located mainly in Hungary. Due to the nature of the other community members and environmental param- bedrock, alluvial deposits, and a bowl shape, many eters at six timepoints over 6 weeks in the Spring of lakes and ponds of the area are characterized by high 2012. Correlations between community members alkalinity. In this study, we characterized temporal suggest a positive relationship between certain algal changes in eukaryal and bacterial community dynam- and bacterial populations (e.g. Chlamydomondaceae ics with high throughput sequencing and relate the with Actinobacteria and Acidobacteria), while other changes to environmental conditions in Lake Velence correlations allude to changes in these relationships located in Feje´r county, Hungary. The sampled Lake over time. During the study, high nitrogen availability Velence microbial populations (algal and bacterial) may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10482-018-1132-7) con- high phosphorus availability as well as the high tains supplementary material, which is available to authorized calcium and magnesium content of the Carpathian users. T. A. S. Bell (&) Á E. Sen-Kilic Á M. W. Fields Á B. M. Peyton M. W. Fields Á B. M. Peyton (&) Energy Research Institute, Montana State University, Department of Chemical and Biological Engineering, Bozeman, MT, USA Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA E. Sen-Kilic Á B. M. Peyton e-mail: [email protected] Department of Chemical and Biological Engineering, Montana State University, Bozeman, MT, USA B. M. Peyton e-mail: [email protected] G. Vasas Department of Botany, University of Debrecen, Debrecen, T. A. S. Bell Á M. W. Fields Hungary Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA T. A. S. Bell Genome Core and Bioscience Division, Los Alamos T. Felfo¨ldi National Laboratory, Los Alamos, NM, USA Department of Microbiology, Eo¨tvo¨s Lora´nd University, Budapest, Hungary 123 2426 Antonie van Leeuwenhoek (2018) 111:2425–2440 Basin bedrock, potentially fostering exopolymer pro- Submerged macrophytes differentiate the western duction and cell aggregation. Cyanobacterial bloom portion of the lake from the open water of the eastern formation could have a negative environmental impact portion with close proximity to Budapest (50 km on other community members and potentially affect southwest), making the lake a popular holiday desti- overall water quality as well as recreational activities. nation and recreational resource (Boromisza et al. To our knowledge, this is the first prediction for 2014). Due to agricultural runoff, the lake has become relationships between photoautotrophic eukaryotes eutrophic over the last century and highly susceptible and bacteria from an alkaline, Hungarian lake. to large cyanobacterial blooms (Water Management Directorate of VITUKI Environmental Protection and Keywords Lake ecology Á Alkaline lake Á Water Management Research Institute on behalf of the Phytoplankton Á Algae Á Eutrophication Á Ministry for Environment and Water 2006). Cyanobacteria bloom Á Microcystin Á Microcystis Certain non-nitrogen fixing cyanobacteria, such as Microcystis aeruginosa isolated from Lake Velence, capitalize on high nitrogen availability and form large visible blooms capable of producing the toxin micro- Introduction cystin (MC) (Habermehl et al. 1997; Resko´ne´ and Torokne 2000). MC occurs in different forms, of The shallow lakes of the Carpathian Basin in Hungary which MC-LR is the most toxic to lake organisms, are some of the most alkaline in the world (Boros et al. wildlife, livestock, and humans inhibiting liver protein 2014). The basin is surrounded by slowly eroding phosphatases leading to hemorrhaging (Runnegar mountains composed of limestone and carboniferous et al. 1995; Gupta et al. 2003; Paerl and Otten 2013). granite with high magnesium and calcium carbonate Blooms may be augmented by the high availability of content deposited into the lakes and ponds that have soluble calcium and magnesium, enhancing lectin formed on top of an ancient alluvial fan formed by the activity, a key constituent of extracellular polymeric Danube, Drava, and Tisza Rivers (Horva´th et al. 2004; substances (EPS) important in bloom formation (Zhao Borics et al. 2016). Despite the extreme alkaline et al. 2011). conditions, high carbon availability supports produc- Large cyanobacterial blooms threaten biodiversity tive microbial communities in the region’s lakes and by limiting light, oxygen, and nutrient availability, and ponds (A´ cs et al. 1994, 2003;Vo¨ro¨s et al. 2006; Grant although cyanobacteria release oxygen as a byproduct 2006; Felfo¨ldi et al. 2009;Pa´lffy et al. 2014; Szabo´ of photosynthesis, oxygen is consumed when these et al. 2017). large blooms begin to decompose (Paerl and Otten Lake Velence, located in Feje´r county, is the third 2013). Elevated global temperatures and increased largest lake in Hungary (24.17 km2). Shallow, with a eutrophication may additively increase toxic bloom mean depth ranging from 0.5 to 2 m, Lake Velence is frequency and extent (Davis et al. 2009; De Senerpont an endorheic or terminal lake supplied by two streams Domis et al. 2012; Paerl and Otten 2013; Harke et al. and catchment runoff (Horva´th et al. 2004;Za´ray et al. 2016; Wood et al. 2016; Hamilton et al. 2016). 2005; Boromisza et al. 2014). Adjacent basement Fluctuations in community members in response to springs make limited contributions to the lake, evident bloom frequency can reduce biodiversity, ecosystem in several recorded desiccation periods (Horva´th et al. function, and be indicative of larger global trends such 2004). The lake has a unique limnogeology charac- as climate change (Strayer and Dudgeon 2010; Dodds terized by concentrated calcium carbonate inputs that et al. 2012; Paerl and Paul 2012). reflect the region’s carbonaceous bedrock. These Here, we investigated fluctuations in microbial factors contribute to characteristically high pH community composition and diversity by observing (8.5–9.9), sodium (* 300 mg L-1), bicarbonate the environmental conditions of Lake Velence during (* 680 mg L-1), magnesium (* 420 mg L-1) and a toxic M. aeruginosa bloom. Over several weeks, sulfate (* 670 mg L-1) concentrations (Horva´th increases in water temperature and nutrient availabil- et al. 2004; Borsodi et al. 2005; Haas 2012; Boromisza ity contributed to optimal conditions for large and et al. 2014). toxic bloom formation. By constructing a predictive inter-domain population network, our findings depict 123 Antonie van Leeuwenhoek (2018) 111:2425–2440 2427 potential relationships within the microbial commu- Sample collection nity. Abiotic variables, such as cations present in the bedrock, may have exacerbated bloom formation To analyze biotic and abiotic microbial community facilitating cell aggregation through EPS synthesis. dynamics in this portion of Lake Velence, limnolog- By investigating these connections we can improve ical chemistry and biological samples were collected our understanding of toxic bloom formation and at six timepoints (1—5/18, 2—5/25, 3—6/1, 4—6/8, develop effective mitigation strategies. 5—6/13, and 6—6/21) approximately 1 week apart (every 5–8 days) at mid-day from the same location during the spring of 2012 (May 18th–June 21st). Due Materials and methods to the shallow depth of this portion of the lake (0.5 m), only surface water samples (0–0.25 m) were collected Site description using sterile polypropylene bottles (Nalgene Roche- ster, NY, USA). Samples for chemical and community Lake Velence, located in Feje´r county, Hungary, is analyses (1 L) were kept at 4 °C for transport back to approximately 50 km southwest of the capital, the laboratory for processing described in detail Budapest. The lake is considered endorheic, with no below. Biomass was also collected on June 8th from outlets and a shallow depth (0.5–1.6 m) that impedes a large bloom that persisted for approximately seasonal nutrient exchange between the epilimnion 10 days. Samples were kept frozen until being and hypolimnion characteristic of deeper lakes. Sam- lyophilized for mass spectrometer analysis described ples were collected from a single location and are only below. representative of a small portion of the larger system. Figure 1 illustrates the sampling location MALDI-TOF MS analysis (47°1403.48100N, 18°37051.45600E) on a dock located in the northwestern corner of the lake, approximately Biomass was collected with a 50 lm mesh size 10 m southeast of the swimming access. The north- plankton net, lyophilized, and assayed for microcystin west area of the lake is one of the most developed, with matrix-assisted desorption/ionization time-of- providing easy access for recreation, and highly flight mass spectrometry (MALDI-TOF MS) accord- susceptible to toxic bloom formation. ing to methods described by Farkas and others (Farkas Fig. 1 Location of Lake Velence within Hungary and the sampling location (indicated by the star) 123 2428 Antonie van Leeuwenhoek (2018) 111:2425–2440 et al. 2014). Five milligrams of lyophilized biomass analysis, samples were acidified with nitric acid to a and 200 lL of 50% aqueous methanol were homog- 0.15% final concentration. Hardness was determined enized, sonicated for 5 mins, and incubated at room with the standard ethylenediamine-tetraacetic acid temperature for 1 h.