Seasonal Dynamics of Prokaryotic Abundance and

Seasonal Dynamics of Prokaryotic Abundance and Activities in Relation to Environmental Parameters in a Transitional Aquatic Ecosystem (Cape Peloro, Italy) R. Zaccone, M. Azzaro, F. Azzaro, A. Bergamasco, G. Caruso, M. Leonardi, R. La Ferla, G. Maimone, M. Mancuso, L. S. Monticelli, et al. Microbial Ecology ISSN 0095-3628 Microb Ecol DOI 10.1007/s00248-013-0307-z

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Author's personal copy Microb Ecol DOI 10.1007/s00248-013-0307-z

MICROBIOLOGY OF AQUATIC SYSTEMS

Seasonal Dynamics of Prokaryotic Abundance and Activities in Relation to Environmental Parameters in a Transitional Aquatic Ecosystem (Cape Peloro, Italy) R. Zaccone & M. Azzaro & F. Azzaro & A. Bergamasco & G. Caruso & M. Leonardi & R. La Ferla & G. Maimone & M. Mancuso & L. S. Monticelli & F. Raffa & E. Crisafi

Received: 24 April 2013 / Accepted: 30 September 2013 # Springer Science+Business Media New York 2013

Abstract This study examines the effects of temporal changes on microbial parameters in a brackish aquatic ecosystem. To this aim, the abundances of prokaryotes and vibrios together with the rates of enzymatic hydrolysis of proteins by leucine aminopeptidase (LAP), polysaccharides by β-glucosidase (GLU) and organic phosphates by alkaline phosphatase (AP), heterotrophic prokaryotic production (HPP), respiration (R), were seasonally investigated, during a 2-year period in the coastal area of Cape Peloro (Messina, Italy), constituted by two brackish lakes (Faro and Ganzirri). In addition, physical and chemical parameters (temperature, salinity, nutrients) and particulate organic carbon and nitrogen (POC, PN) were measured. The influence of multiple factors on prokaryotic abundances and activities was analysed. The results showed that Cape Peloro area is characterised by high seasonal variability of the microbial parameters that is higher than the spatial one. Combined changes in particulate matter and temperature (T), could explain the variability in vibrios abundance, GLU and R activities in both lakes, indicating a direct stimulation of the warm season on the heterotrophic prokaryotic metabolism. Positive correlations between T (from 13.3 to 29.6 °C) and HPP, LAP, AP, POC, PN are also observed in Ganzirri Lake. Moreover, the trophic status index and most of the microbial parameters show significant seasonal differences. This study demonstrates that vibrios abundance and microbial activities are responsive to the spatial and seasonal changes of examined area. The combined effects of temperature and trophic R. Zaccone (*) : M. Azzaro : F. Azzaro : A. Bergamasco : G. Caruso : M. Leonardi : R. La Ferla : G. Maimone : M. Mancuso : L. S. Monticelli : F. Raffa : E. Crisafi CNR-IAMC, Institute for Coastal Marine Environment, Section of Messina, Spianata S. Raineri 86, 98122 Messina, Italy e-mail: [email protected]

conditions on the microbial parameters lead us to suggest their use as potential indicators of the prokaryotic response to climate changes in temperate brackish areas.

Introduction Transitional aquatic ecosystems, being located at the sea–land interface, are among the most geochemically and biologically active areas of the biosphere playing an important role in the global biogeochemical cycles. They are often characterised by shallow depth, strong thermal excursion, high productivity and vulnerability to anthropogenic or hydro-dynamic pressure [18]. The physical variability determines biological variability and in turn biological features can be proposed as ecological descriptors [11]. Heterotrophic bacteria and organic matter (OM) interactions are of utmost importance for the functioning of all aquatic ecosystems. In coastal ecosystems, OM–enzymatic relationships are further complicated by environmental forcing. In these aquatic environments, the majority of OM is in a polymerized form and cannot be assimilated directly by bacterial cells because their permeases allow only small molecules to pass through. The degradation of OM starts with the transformation of polymers to oligomers through the hydrolytic activity. The hydrolysis and utilization of autochthonous and allochthonous biopolymers, such as proteins and carbohydrates, are a crucial step in the use of macromolecules, to sustain high microbial growth rates [2]. Several studies have focused on the regulation of hydrolytic activity by its organic substrates [20, 22]. The environmental factors such as the availability of biogenic elements, temperature, dissolved oxygen, etc. can also regulate the expression of enzymatic activities [12].

Author's personal copy R. Zaccone et al.

In this context, coupled studies of microbial activities and particulate organic matter quality may give useful insights about changes in microbial metabolism in response to changes in the trophic conditions, as already stated by Chróst and Siuda [13]. Direct relationships have also been observed between enzymatic activities and trophic parameters (heterotrophic bacteria, chlorophyll, POC, PN) [11, 33, 58]. Within the microflora inhabiting marine and estuarine ecosystems, Vibrio spp. have gained increased attention due to their potential pathogenicity for humans when transmitted via raw seafood [23, 36, 40]. Some Vibrio spp. are responsible for important diseases in marine organisms [27, 34, 35]. As a part of their strategy for survival in aquatic habitats, Vibrio spp. can interact with all kinds of aquatic organisms or particulate matter releasing dissolved compounds and nutrients [16, 19, 31]. This group of bacteria can also secrete a variety of enzymes involved in organic matter degradation, e.g., protease, chitinase, lipase, mucinase [38, 41, 50]. Some ecological studies have suggested that nutrients availability as well as water temperature and salinity affect survival and persistence of Vibrio population in aquatic environments [21, 24]. In particular the possible changes in virulence and pathogenicity of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus as consequence of waters warming have been observed [5, 27]. The present study aimed at estimating the relative significance of physical, chemical parameters and particulate matter quality on prokaryotic community distribution in the brackish area of Cape Peloro (Messina, Italy). In particular, the prokaryotic abundance and biomass, vibrios, heterotrophic cultivable bacteria, faecal indicators and microbial activities (enzymatic hydrolysis of proteins—leucine aminopeptidase (LAP), polysaccharides—β-glucosidase (GLU), and organic phosphates— alkaline phosphatase (AP); heterotrophic prokaryotic production—HPP, respiration—R) were considered in relation to particulate organic carbon and nitrogen (POC, PN) and environmental parameters (temperature, salinity, oxygen, nutrients). The specific objectives of the research were: (1) to investigate the spatial and temporal heterogeneity of the Cape Peloro area, highlighting the differences between the two lakes according to both physical and biological parameters; (2) to define the variables that control and regulate the prokaryotic abundances and activities; (3) to focus on the response of an autochthonous component (Vibrio spp.) of the heterotrophic bacterial community to temperature and trophic conditions.

lakes, named Ganzirri and Faro, connected to each other by a narrow channel. Both of them are also connected with the Ionian Sea. The Faro Lake is a meromictic environment and hosts mussel cultures. The Ganzirri Lake is colonized by macroalgae and occasionally suffers dystrophic crisis. Functional differences between the two lakes are due to their different morphology, depth and salinity ranges. Moreover, the lakes are usually affected by nutrient enrichment from the terrestrial runoff, that in presence of rapid temperature increase, high pressure, lack of wind, etc. may lead to occasional and sometimes dramatic anoxia crisis [4, 33]. Sampling In the frame of VECTOR Line 4 -DIVCOST project, water samples were seasonally collected from the surface of Faro and Ganzirri Lakes (one central station in Faro Lake and five stations in Ganzirri Lake) over a 2-year period (2006-08) (Fig. 1). Temperature (T), salinity (S ), chlorophyll fluorescence (CHL) and pH measurements were obtained by an oceanographic multi-parametric probe (SBE 19 Plus). Environmental Parameters

Methods

Water samples for the determination of dissolved oxygen (OX) content were fixed immediately after collection, and analysed by Winkler’s method [8]. Samples for nutrient determinations, ionized ammonia (NH4), nitrite (NO2), nitrate (NO3), orthosphate (PO4), were filtered using GF/F glass-fibre filters and kept frozen (-20 °C). Analytical determinations were performed according to Strickland and Parsons [49], while NH 4 was measured according to Aminot and Chaussepied method [1]. All nutrient concentrations were determined by a Varian spectrophotometer Mod. Cary 50. Trophic State Index (TSI) was calculated from CHL data according to Carlson and Simpson [7]. Total suspended matter (TSM) was evaluated by gravimetric method using a Mettler AT261 electronic microbalance (accuracy±1.0 μg). The particle material was collected by filtering variable volumes of water on pre-combusted (480 °C for 4 h) pre-weighted glassfibre filters (Whatman GF/F), subsequently oven-dried at 60 °C for 24 h. To estimate the concentration of particulate organic carbon and nitrogen (POC and PN), 500 ml of water samples were concentrated on pre-combusted Whatman GF/F glass-fibre filters and processed at 980 °C in a Perkin-Elmer CHN-Autoanalyzer 2400 [33]. C/N molar ratio was also calculated from POC and PN determinations.

Study Area

Microbiological Variables

Capo Peloro is a brackish aquatic system located in the northeastern corner of Sicily (Italy). It consists of two neighbouring

Total prokaryotic abundance (PA) was determined after DAPI staining [44] by a Zeiss AXIOPLAN 2 Imaging microscope

Author's personal copy Seasonal changes of microbial parameters in brackish waters

Fig. 1 Study area with the sampling stations

equipped with the Axiocam digital camera and the AXIOVISION 3.1 software. Prokaryotic biomass (PB) was estimated from cell counts and volumetric measurements according to La Ferla et al. [30]. Heterotrophic cultivable bacteria (HB) were counted on Marine Agar plates incubated at 20 °C for 7 days [54]. Vibrios abundance was determined on plates of the selective TCBS agar added with 1.5 % of NaCl and incubated at 35 °C for 24 h; this temperature has been chosen since it allows detection of those species related to human infections [53]. Presumptive Vibrio spp. were characterised by biochemical tests (API 20E) and identified according to Noquerela and Blanch keys [39]. The faecal coliforms (FC) and enterococci (ENT) as indicators of anthropogenic input were evaluated by membrane filtering methods, followed by incubation on appropriate cultural media (m-FC agar and m-Enterococcus agar for 24 and 48 h, respectively). Total extracellular enzymatic activity (EEA) rates were immediately measured after sample collection, by using methylumbelliferyl (MUF) substrates (MUF-phosphate and MUF-β-glucopyranoside, Sigma Aldrich) for AP and GLU a c t i v i t i e s , r e s p e c t i v e l y. L - l e u c i n e - 4 m e t h y l - 7 coumarinylamide (Leu-MCA, Sigma Aldrich) was the substrate used to measure leucine aminopeptidase activity (LAP). The increase of fluorescence over time (measured with a Turner TD 700 fluorimeter) was converted into hydrolysis velocity using a standard curve of MUF and MCA. [54, 59]. Net HPP was estimated from the rate of [3H] leucine incorporation using the micro centrifugation method according to Smith and Azam [47]. Triplicate 1.0 ml samples and two blanks were incubated in the dark, for 1 h at in situ ±1 °C temperatures with L -[4,53H] leucine (Amershan Biosciences

UK Limited) (25 nM final concentration). HPP was calculated according to Kirchman [26] using in situ determination of leucine isotopic dilutions (ID) performed using a kinetic approach [43]. The respiration rates and the consequent metabolic production of CO2 (R) were measured by the Electron Transport System activity (ETS); the assay is based on the conversion of tetrazolium salt into formazan [3, 29, 42]. Statistical Analysis Differences in each variable of the data set were established by means of the analysis of variance (ANOVA and Kruskal– Wallis). Pearson correlation coefficients were calculated among log-transformed microbiological data and environmental parameters using the SigmaStat software V3.0. In order to evaluate the degree of similarity among the sampling times the Cluster analysis was performed using the group average linkage method and by calculating the Euclidean distance coefficient. A non-metric multi dimensional scaling (MDS) analysis was also performed to elucidate the relationships among microbial and environmental variables. Both Cluster and MDS analyses were carried out with the statistical package PRIMER v 6.0.

Results Environmental Parameters The mean temperature values recorded in the surface waters of Ganzirri and Faro Lakes during the 2-year study ranged from 13.3 to 29.6 °C in Ganzirri and from 14.23 to 28.79 °C in

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Salinity

Fig. 2 Seasonal patterns of temperature (solid lines) and salinity (dotted lines) in the examined lakes (mean surface data)

Temperature (°C)

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Faro; similar seasonal trends were observed in the two lakes. Salinity ranged from 30.04 to 35.24 in Ganzirri Lake and from 33.56 to 37.83 in Faro Lake (Fig. 2). The mean values of abiotic, trophic and biological parameters are reported in Table 1. The OX expressed in terms of saturation values was beyond or close to the saturation level, with maximum values in March 2008 in Faro Lake and in January 2008 in Ganzirri Lake, while minimum values were reached in March 2007 in Faro Lake and in September 2006 in Ganzirri Lake. Within the inorganic N ions, NO3 was generally predominant, with concentrations always higher than 1.0 μM in Ganzirri and Faro Lakes. TSM showed seasonal trends with highest values in September 2006 in both lakes. POC and PN concentrations were higher in Ganzirri than in Faro Lake; their distribution was characterised by higher values in the warm months (June–October), and lower in the cold ones with significant differences (P = 0.00012). The occurrence of seasonal trends was confirmed by the significant correlations found with temperature (Table 2). The C/N ratios were generally less than 5, with lower values in Faro (2.39–5.20) than in Ganzirri Lake (4.06–5.98). Prokaryotic Abundance and Biomass In Ganzirri Lake, PA were in the order of 106–107 cell mL−1. The highest concentrations occurred in summer months when they reached mean values of 3.3 and 5.4×107 ml−1 in 2007 and 2008, respectively, while the lowest values were observed in September 2006. In Faro Lake, lower values than in Ganzirri Lake were always registered (Table 1). The values of PB were higher in Ganzirri than in Faro Lake (Fig. 3a). The pattern of PB depended closely on that of PA, with the exception of June 2007, when the cell volumes rather than the abundances modulated the biomass. Wide variations in PA and PB occurred among the sampling sites in Ganzirri Lake, as shown by calculation of the

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Coefficient of Variation (CV=standard deviation/mean×100). High variability was found in the sites 7 and 9 (PA: CV=66.3 and 74.7 %; PB: CV=73.3 and 97.5 %, respectively). No significant correlations between T and PA or PB were observed. Conversely, highly significant negative correlations of S with PA and PB (n =35, r =−0.50, r =−0.54, P