Structure and function of bacterioplankton in the rehabilitated Lake Trzesiecko

Ecohydrology & Hydrobiology 14 (2014) 96–105

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Original Research Article

Structure and function of bacterioplankton in the rehabilitated Lake Trzesiecko Katarzyna Lewicka-Rataj *, Magdalena Kaczorkiewicz, Tomasz Heese, Marzena Wasiniewska, Malwina Miszczyszyn Department of Environmental Biology, Koszalin University of Technology, S´niadeckich 2, 75-453 Koszalin, Poland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 December 2013 Received in revised form 18 February 2014 Accepted 19 February 2014 Available online 2 March 2014

Changes in bacterioplankton in relation to the temperature and water chemistry in Lake Trzesiecko were studied from 2008 to 2013. The lake was highly eutrophic until 2005 when measures were begun to precipitate phosphate, to increase oxygen concentrations by re-aeration and to reduce algal crops through biomanipulation of the fish community. Some improvements were also made to reduce phosphorus levels in the inflows. Conductivity increased, redox potential remained largely unchanged, and pH decreased during the period. The abundance, biomass, and morphological structure of the bacterioplankton suggest Lake Trzesiecko remains eutrophic but bacterial numbers and biomass have declined since 2008. The decline was significantly related to decreases in concentration of nitrogen and phosphorus compounds. The bacterial community comprised mostly small cells (<0.05 mm3), with a lower proportion of cells >0.1 mm3. Most were cocci. A negative correlation between conductivity and bacterial numbers and biomass suggests that mineral salts from the phosphorus coagulant inhibited the bacterioplankton. Key nutrients (nitrogen and phosphorus) and DOC were positively related to bacterial biomass and numbers. ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Keywords: Rehabilitation Bacterial numbers Bacterial biomass Cell volume

1. Introduction Bacteria are numerous and important in the functioning of plankton systems and their nature reflects the trophic status of lakes (Chro´st and Siuda, 2006). Physical and chemical (organic and inorganic) conditions regulate the growth of aquatic bacteria (Chro´st et al., 1986). Grazing by protozoa and other animals is important in determining population size (Hahn and Ho¨fle, 2001). Lake Trzesiecko in 2004 was strongly hypertrophic (Heese et al., 2004) with high loads of urban and agricultural phosphorus from the catchment. The hypolimnion was oxygen-depleted below 4–5 m and cyanobacterial blooms were observed, linked to

* Corresponding author. Tel.: +48 943478554; fax: +48 943427652. E-mail address: [email protected] (K. Lewicka-Rataj).

low ratios of nitrogen to phosphorus of around 4:1 (by weight). The plankton was dominated by Microcystis aeruginosa, Microcystis viridis, Microcystis wesenbergii, and Anabaena flos aquae. Concentrations of microcystins (total isomorphs: RR, YR, LR) varied from 4.5 to 9.25 mg dm3, in excess of WHO (2003) bathing water norms of 2 mg dm3, and excluded recreational use of the lake. Rehabilitation measures were undertaken on the lake in 2005. They included aeration and chemical coagulation and the introduction of predatory fish, (pike-perch, pike, catfish, and eel), to increase pressure on planktivorous fish. In the catchment, the storm water drainage system was equipped with separators to divert waste water for treatment from rain water and reduce discharge of untreated sewage in wet periods. A dam was constructed on the lake’s outflow, regulating the water level, and therefore ensuring precision of coagulant dosage. Water

http://dx.doi.org/10.1016/j.ecohyd.2014.02.002 1642-3593/ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

K. Lewicka-Rataj et al. / Ecohydrology & Hydrobiology 14 (2014) 96–105

level control can also be important during spawning of planktivorous fish by limiting their spawning sites and density, and thus increasing zooplankton grazing and reducing algal crops (Zalewski, 1999). Undertaking such activities is in accord with the ecohydrological concept of integration of hydrological, biological, and biochemical processes (Zalewski, 2000). Reactions of aquatic bacteria to such rehabilitation can be varied. The density of planktonic bacteria may increase immediately after application of coagulant and then fall. There can also be changes in the size of bacterial cells (Go´rniak et al., 2003a,b; S´wia˛tecki et al., 2001). Here we have focused on the characteristics (abundance, biomass, size and morphological structure) of the bacterioplankton in relation to changing physical and chemical variables of Lake Trzesiecko following rehabilitation. Our hypothesis was that rehabilitation would lead to reduced bacterial activity.

2. Materials and methods 2.1. Study area Lake Trzesiecko (altitude 134 m a.s.l) is located in the Zachodniopomorskie province in the Szczecinek commune in the river basin that contains the Nizica (Niezdobna),

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Gwda, Notec´, Warta, and Oder rivers that discharge to the Baltic Sea. Lake Trzesiecko is located (Kondracki, 2002) in the northern regions of the Południowopomorskie and Szczecineckie lakelands. It lies in a once glaciated channel, has an area of 295 ha and volume of 16.1 million m3. Its maximum depth reaches 12.6 m, with an average depth of 5.4 m. The bottom is uneven in topography with three troughs in an elongated basin. The lake is polymictic with periodically occurring thermal stratification. The catchment area covers 15,800 ha (including the lake’s surface area of 295 ha and is dominated by forests (47%). Urban development covers 34% of the area, and the rest includes meadows and fields. Lake Trzesiecko is fed by the Radacki Canal, and the Mulisty Strumien´, S´wie˛ty, and Lipowy Potok streams. It is the source of the Niezdobna River. Rehabilitation began in 2005 with two pulverising aerators installed in the lake’s deepest parts, each supplying approximately 20 kg of iron III sulphate to the near-bottom zone per month. Aeration with the application of iron III sulphate under the trade name PIX 112 (manufactured by Kemipol) has been carried out three times per year in April, May and July since 2005. Legal permits allow the annual application of 9 kg of coagulant per hectare.

Fig. 1. Location of the sampling positions in Lake Trzesiecko. Grey scale indicates depth in m. Inset graph shows location within Poland.

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2.2. Sampling

2.5. Data analysis

Water samples were collected from 2008 to 2013 at 0.5 m, 5 m, and 10 m depths at three sites located in the lake’s three main sub-basins (Fig. 1). The samples were taken after the application of coagulant. Water samples were collected using a Ruttner sampler and kept in polyethylene bottles for transport to the laboratory.

Statistical analyses were conducted using Statistica 9.0 software. The significance of differences between samples was verified by means of one-way analysis of variance. Relationships between the microbiological and physical and chemical variables were determined by correlation analysis.

2.3. Physical and chemical analyses

3. Results

Conductivity, pH, redox potential, oxygen saturation, and temperature were measured in situ with a multiparameter sonde by YSI Incorporated, but only in 2012 and 2013. In previous years, they were measured by three different probes. Chemical analyses (N-NO3, total P, and total N) were carried out according to Hermanowicz et al. (1999). Chlorophyll a concentration was determined by standard methods (PN-86). The dissolved organic carbon (DOC) analyses were made with a Sievers InnovOx (GE Analytical Instruments) analyser. Before determination the samples were filtered through a glass fibre filter (Whatman, GF/F type, 47 mm). The analyser uses supercritical water oxidation, by blowing gas through to remove inorganic carbon (e.g. CO2). Non-purgeable organic carbon is then determined. Eight replicate measurements were made per sample.

3.1. Physical and chemical variables In spring 2008, the oxycline began at 5 m. From 2 to 4 m, oxygen supersaturation was observed, suggesting high activity of phytoplankton. In the springs of 2009 and 2012, there was oxygenation of the entire water column with up to 9 mg O2 dm3 at temperatures of 7–8 8C. In summer, oxygen decreased to 3 mg O2 dm3 in the near-bottom layer. At some locations, complete oxygen depletion occurred. Hypolimnion temperature was 12 8C, and at the surface it was up to 24 8C. In autumn, complete oxygenation of the lake was observed and the water column was isothermal. An exception was autumn 2008, when only 0.32 mg O2 dm3 at a temperature of 16.7 8C was observed in the near-bottom layer. Mean redox potential varied little and was relatively high overall with a decline in 2013 caused by low values in the near-bottom layer (Fig. 2). Conductivity varied from 282 mS cm1 in 2008 to 513 mS cm1 in 2013 and increased each year by approximately 30% between 2008 and 2012, probably owing to addition of coagulant. Mean pH decreased in consecutive study years from 8.68 in 2008 to 6.60 in autumn 2012. BOD5 values were steady, not exceeding 7.0 mg O2 dm3. Values were highest in spring. Chlorophyll a concentrations ranged from indetectable to 18 mg dm3, with peaks in the summer (Fig. 2). Nitrogen compounds were dominated by nitrate and were relatively steady and low (maximally 0.886 mg NNO3 dm3), reflecting the small proportion of the catchment that was cultivated. Few winter values, when concentrations are likely to be much higher, were available, however (Fig. 2). Total nitrogen was also relatively low (<1.7 mg N dm3), whilst total phosphorus was very high, reaching 1.20 mg P-PO4 dm3, and with a

2.4. Microbiological analyses Water samples were fixed with pre-filtered formaldehyde to a final concentration of 2%. DAPI (40 ,6-diamidino2-phenylindole)-stained water samples were retained on black polycarbonate (Nucleopore) filters with 0.2 mm pore size (Porter and Feig, 1980). Bacterial cells were counted and measured directly in a fluorescence microscope (Nikon Eclipse 80i) equipped with a camera Nikon DS.-Ri1 and the NIS-Elements BR laboratory image analysis system. Bacterial biomass was calculated based on the conversion rate specified by Norland (1993). The application of this method, modified following S´wia˛tecki (1997), permitted the determination of the following: TBN – total bacterial numbers, BB – bacterial biomass, MCV – mean cell volume, and morphological structure of the community.

Table 1 Relationships between environmental variables and total bacterial numbers (TBN), bacterial biomass (BB) and mean bacterial cell volume (MCV) in Lake Trzesiecko. r, correlation coefficient, p, probability. Values shown in bold are significant at p < 0.05. Variables

Conductivity pH Temperature Redox N-NO3 Total phosphorus Total nitrogen DOC Chlorophyll a

TBN

BB

MCV

r

p

r

p

r

p

0.53 0.6 0.33 0.22 0.77 0.29 0.39 0.52 0.06

0.001 0.001 0.001 0.05 0.001 0.001 0.001 0.001 0.499

0.58 0.65 0.01 0.07 0.64 0.2 0.44 0.65 0.06

0.001 0.001 0.956 0.431 0.001 0.05 0.001 0.001 0.466

0.09 0.11 0.3 0.52 0.13 0.2 0.08 0.23 0.34

0.295 0.192 0.001 0.001 0.117 0.05 0.375 0.05 0.001

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Fig. 2. Physicochemical variables in Lake Trzesiecko. Data for each date are the mean value and SD of all water samples for each date. Black arrows on the Figure indicate time of coagulant (PIX 112) application. Numbers next to the arrows indicate application time: 1 in April, 2 in May and 3 in July.

mean around 0.2 mg P-PO4 dm3. They showed no consistent trend in our data over the period, despite remedial measures taken to reduce them, but fuller independent data suggest there has been some improvement. DOC, however tended to decline during the period, though rather irregularly. Dissolved organic carbon concentration decreased towards the end of the growth season with the lowest values recorded in autumn.

3.2. Bacteria The numbers and biomass of the bacterioplankton of Lake Trzesiecko varied among years. The highest values of the total numbers of bacteria were observed in 2008, varying from 9.63  106 cm3 to 20.3  106 cm3, and averaging 14.5  106 cm3. In subsequent years numbers decreased significantly to between 5.89  106 cm3 and

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the lowest in autumn, with 5.18  106 cm3 and 87.7 mg C dm3. The total numbers and biomass of bacteria were significantly positively correlated with pH, N-NO3 concentration, and DOC (Fig. 5B–D), total P and total N (Table 1). Negative correlations were observed between bacterial numbers and biomass, and conductance (Table 1 and Fig. 5A), as well as a positive one between bacterial numbers and water temperature (Table 1). The size of bacterioplankton cells in Lake Trzesiecko also varied by year. The range of cell volume was 0.029– 0.105 mm3 (Fig. 6A and B). Cells with a volume of less than 0.05 mm3 constituted 50% of the total numbers (Fig. 7). The mean contribution of cells sized over 0.1 mm3 was 35%. The lowest contribution of 15% was reached by cells from 0.05 to 0.1 mm3. Cocci contributed 51%, rods 29% and curved cells 20%. The contribution of the remaining cylindrical forms amounted to 29% for rods and 20% for curved cells (Fig. 7). 4. Discussion Fig. 3. Variation in total abundance (TBN) and biomass of bacterioplankton (BB) in Lake Trzesiecko between 2008 and 2013. Error bars are standard deviations; values between 2008 and 2009–2013 are significant at p < 0.001, n = 36 per year.

8.93  106 cm3 (Fig. 3). The biomass of bacterioplankton in Lake Trzesiecko showed a similar change with 298 mg C dm3 in 2008 and 128 mg C dm3 to 140 mg C dm3 subsequently. Numbers and biomass of bacterioplankton in Lake Trzesiecko showed seasonal variability in 2012, when seasonal samples were taken (Fig. 4). The highest density and biomass of bacteria were observed in spring, when mean values were 7.99  106 cm3 and 175 mg C dm3, respectively, and

Fig. 4. Seasonal variation in total abundance (TBN) and biomass of bacterioplankton (BB) in Lake Trzesiecko in 2012. Error bars are standard deviations.

Lake Trzesiecko maintains only moderate ecological status according to the criteria specified in the Regulation of the Minister of the Environment in 2011. Such a situation is determined by high phosphorus concentrations, and oxygen depletion in the near-bottom layer. The remaining variables do not exceed values typical of class II of water cleanliness under the Polish system. According to Heese et al. (2013), the lake’s condition has been continuously improving since the start of the rehabilitation treatments. The lake’s tributaries, and particularly the Radacki Canal, continue to supply high amounts of nutrients, including ammonium nitrogen, which constitutes a serious issue (Cierpiszewski, 2002 and unpublished data). Low redox potential values below 200 mV are also disturbing. They probably contribute to the release of phosphorus from bottom sediments (Carman et al., 2000; Ahlgren et al., 2006; Søndergaard et al., 1996; Jin et al., 2006). In the hypolimnion, deprived of oxygen, phosphorus bound to iron is released to the water, and may eventually support phytoplankton production (Wis´niewski, 1995). Chlorophyll a concentration in Lake Trzesiecko showed only a modestly decreasing tendency in our data, possibly resulting from the implemented treatments. After the determination in August 2004 of very high values of 38 mg dm3 in the surface layer (Heese et al., 2004), chlorophyll a concentration decreased to 13 mg dm3 in successive years (Heese et al., 2007). Our data show a much less marked trend. Chlorophyll a concentration depended not only on the season, but also on aeration and coagulation carried out in the lake, which also precipitated some phytoplankton. Lurling and Oosterhout (2013) obtained a decrease in the chlorophyll concentration from 27 mg dm3 to 5 mg dm3 after four years of addition of modified bentonite clay and polyaluminium chloride, applied in a combined method known as Flock and Lock. Using H2O2, clay, and polymeric iron sulphate, Wang et al. (2012) obtained a reduction of chlorophyll concentration from 1.37 mg dm3 to 0.03 mg dm3. A

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r=-0.53, p<0.001

B

22

20

20

18

18

16

16

TBN 106 cell cm-3

TBN 106 cell cm-3

A 22

14 12 10 8

r=0.60, p<0.001

14 12 10 8

6

6

4

4

2 280

320

360

400

440

480

2 6.4

520

6.8

7.2

-3

Conductivity (µS cm )

C 22

101

7.6

8.0

8.4

8.8

20

24

pH

D

r=0.77, p<0.001

20

16

r=0.52, p<0.001 14

18 12

TBN 106 cell cm-3

TBN 106 cell cm-3

16 14 12 10

10

8

6

8 4

6 4

2

2 0.0

0.2

0.4

0.6

0.8

1.0

N-NO3 (mg N-NO3 dm-3)

4

8

12

16

DOC (mg dm-3)

Fig. 5. Relationships between total bacteria number and conductivity (A), pH (B), and N-NO3 (C) and DOC (D) concentrations in water of Lake Trzesiecko.

decrease in chlorophyll concentration in Lake Trzesiecko could also be partly caused by the simultaneous application of biomanipulation measures. Søndergaard et al. (2008) observed the combination of biomanipulation with the use of chemical agents may have increased the efficiency of both activities. Lakes subject to such rehabilitation, however, often return to the state before reclamation after 6–10 years. The numbers, biomass, and morphological structure of the bacterioplankton are closely related to the trophic status of lakes (Billen et al., 1990; Porter et al., 2004; Chro´st and Siuda, 2006). Variability of bacterial numbers and biomass in the water of Lake Trzesiecko was probably determined by the improvement of its ecological status resulting from the rehabilitation treatments. Very high numbers and biomass of bacteria observed in 2008 were typical of strongly eutrophicated waters. Similar values were observed in the highly eutrophic Lake Tałtowisko in

the Masurian Lakeland (Chro´st et al., 2009), and in the Warnow River in north-eastern Germany (Freese et al., 2006). The numbers and biomass of bacteria in consecutive years in Lake Trzesiecko decreased, but they are still typical of eutrophic waters as found elsewhere (Gołdyn and Szela˛g-Wasielewska, 2005; Chro´st and Siuda, 2006; S´wia˛tecki et al., 2007). A decrease in the numbers and biomass of bacterioplankton in Lake Trzesiecko can be related to the decrease in concentrations of certain forms of nitrogen and phosphorus (N-NO3, total nitrogen, and total phosphorus). Similar findings include Szela˛g-Wasielewska et al. (2009) and Lindstro¨m (2000). Nitrogen and phosphorus play key roles in the regulation of biomass and activity of bacterioplankton (Morris and Lewis, 1992; Le et al., 1994) and can regulate bacterial populations through direct stimulation of their growth and activity, or indirectly through the stimulation of phytoplanktonic organisms for

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Fig. 6. Variation in mean cell volume of bacterioplankton in Lake Trzesiecko in 2008–2013 (A) and by season in 2012 (B). Error bars are standard deviations, values are significantly lower in 2013 (p < 0.001, n = 36 sample counts).

intensive primary production and secretion of soluble organic carbon easily available to planktonic bacteria (Vrede, 1999). The numbers and biomass of bacterioplankton in Lake Trzesiecko were also strongly related to the concentration of DOC, as found elsewhere by Szela˛gWasielewska et al. (2009) and Chro´st et al. (2000). A special role is played by the low molecular weight fraction including amino acids, short peptides, organic acids, carboxylic acids, and monosaccharides that are easily

Fig. 7. Percentage contributions of morphological forms (A) and size groups (B) to total bacteria number in Lake Trzesiecko in 2008–2013.

available for microorganisms (Jonsson et al., 2007). A significant part of this fraction comes from organic carbon produced by autotrophs (Sundh and Bell, 1992; Sundh, 1992; Chro´st and Faust, 1983). One of the indicators of primary production in a water body is chlorophyll a concentration, frequently strongly positively correlated with the numbers or biomass of bacterioplankton (Szela˛g-Wasielewska et al., 2009; Gołdyn and Szela˛g-Wasielewska, 2005) and bacterial production (Chro´st et al., 2000; Cole et al., 1988). The strength of correlation between primary production and bacterial production changes along the trophic gradient (Chro´st and Siuda, 2006), and is strongest in oligotrophic ecosystems compared with hypertrophic ones (Jeppesen et al., 1997). Lack of significant correlation between bacterial numbers and biomass and chlorophyll a concentration in Lake Trzesiecko could result from the application of the iron (III) sulphate coagulant through differential loss of either component, as found by Go´rniak et al. (2003a) in rehabilitated Lake Długie. The lack of correlation in Lake Trzesiecko suggests that planktonic bacteria used organic carbon originating from sources other than primary production, as confirmed by the lack of correlation (or very weak and not significant correlation) between chlorophyll a concentration and DOC concentration (r = 0.16; p = 0.100). Another factor which may have an impact on bacterial communities is conductivity (Lindstro¨m, 2000; Szela˛gWasielewska et al., 2009). We found a negative correlation between conductivity and bacterial numbers and biomass in the lake. The increase in conductivity could be a consequence of the application of the iron (III) sulphate coagulant or inflow of mineral contaminants from the catchment. Bacterial communities are often determined by pH (Percent et al., 2008). This factor also determines the distribution of particular groups of microorganisms in aquatic ecosystems (Lindstro¨m et al., 2005) with a decrease in the numbers, biomass, and activity of bacteria

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with a decrease in pH (Lindstro¨m, 2000, Scully et al., 2003). The correlation between bacterial numbers and biomass and water pH found here suggests that an increase in pH would stimulate bacterial production. Temperature was another factor determining the numbers of the bacterioplankton of Lake Trzesiecko. This is confirmed by a significant correlation that has also been found elsewhere (S´wia˛tecki et al., 2007; Szela˛g-Wasielewska et al., 2009) involving an increase in the numbers of bacteria with an increase in temperature. More bacterial cells were produced in the spring and summer with a substantial decrease in autumn. Increase in temperature also contributed to a decrease in the mean volume of cells, as confirmed by the negative correlation between those variables (Table 1). The situation was particularly evident in 2013, when a substantial decrease in the mean volume of bacterial cells was observed, with a simultaneous increase in the density of bacterioplankton, which however did not result in an increase in bacterial biomass (Fig. 2). This suggests the adoption of a ‘‘K’’ strategy, involving a lower rate of cell division, and generating higher density of cells with smaller sizes, more resistant to feeding on them by protozoa (Weinbauer and Ho¨fle, 1998). Moreover, the low contribution of cells sized 0.05–0.1 mm3 observed in the bacterioplankton of Lake Trzesiecko, with simultaneous high contribution of small forms with a size of <0.05 mm3 and very large cells of >0.1 mm3 suggests a response of bacteria to consumer feeding pressure. Epstein and Shiaris (1992) suggest that bacterial cells sized from 0.03 to 0.1 mm3 are the most preferred by bacteriovores, and an increase as well as a decrease in size constitutes a strategy of avoiding bacterial consumption by protozoa (Ju¨rgens and Gu¨de, 1994; Hahn and Ho¨fle, 2001). According to Sanders et al. (1992), top-down control plays a large role in eutrophic ecosystems. However predominance of cells with volumes from 0.01 to 0.1 mm3 is common in freshwater environments (Pernthaler et al., 1996; Ju¨rgens and Gu¨de, 1994). The range of mean volumes of cells of planktonic bacteria in Lake Trzesiecko was similar to the sizes of cells observed in oligo-mesotrophic lakes (Coveney and Wetzel, 1992; Psenner and Sommaruga, 1992). The occurrence of smaller cells frequently constitutes a response of microorganisms to a shortage of nutritional substrates (Vrede et al., 2002; Letarte and Pinel-Alloul, 1991). Moreover, the mean volumes of cells of bacterioplankton in Lake Trzesiecko may have been linked to changing redox potential values in the study period. An increase in redox potential was correlated with an increase in the mean sizes of cells, with a simultaneous decrease in their population density (Table 1). This suggests that in conditions of better oxygenation, the bacterial community showed a strategy involving the predomination of anabolic processes over catabolic ones, resulting in the incorporation of a higher amount of organic carbon into the cell biomass (Smith and Prairie, 2004). The predominance of cocci corresponds with findings elsewhere (Szela˛g-Wasielewska et al., 2009; S´wia˛tecki et al., 2001; Psenner and Sommaruga, 1992), although findings of a higher contribution of cylindrical cells are also frequent (S´wia˛tecki et al., 2004; Billen et al., 1990). Posch et al. (1997) observed

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high respiratory activity of spherical cells among aquatic bacteria. Therefore, a significant proportion (about 50% of total numbers) of cocci found in the bacterioplankton of Lake Trzesiecko might suggest intensive transformation of organic matter. The changes observed in the size structure of bacterioplankton in Lake Trzesiecko may also constitute a specific adjustment of bacteria to variable environmental conditions induced by the application of the iron (III) sulphate coagulant and biomanipulation in the study period though the relationships are so complex that this is only speculative. 5. Conclusions The implemented rehabilitation treatments have resulted in a substantial decrease in chlorophyll a concentration, restricting the occurrence of cyanobacterial blooms in Lake Trzesiecko. They may also have caused a decrease in the numbers and biomass of planktonic bacteria. The decline in the numbers and biomass of bacterioplankton was correlated with decrease in concentration of certain forms of nitrogen and phosphorus. The reduction of density and biomass of bacterioplankton in Lake Trzesiecko may have been a result of the application of coagulant and biomanipulation over eight years. The functioning of bacterioplankton in the lake could have been regulated by a bottom-up mechanism by means of concentrations and availability of nitrogen and phosphorus as well as DOC, as confirmed by positive correlations. The restriction of development of algae by coagulation with iron (III) sulphate may have caused disturbances in the functioning of the microbial loop as suggested by the lack of correlation between numbers and biomass of bacteria and chlorophyll a concentration. The lack of correlation of DOC with the chlorophyll a concentration suggests that bacterioplankton in Lake Trzesiecko used organic carbon originating from sources other than primary production. Another consequence of coagulation was probably an increase in the concentration of mineral ions, which, as evidenced by the determined negative correlation between conductivity and bacterial community, may have restricted the development of bacterioplankton. Despite several years of rehabilitation, the abundance of bacteria in Lake Trzesiecko is still typical for eutrophic waters, but the mean volumes of cells are characteristic for oligo-mesotrophic lakes. Rehabilitation is still underway and further changes may be expected. Conflict of interest None declared. Financial disclosure None declared. References Ahlgren, J., Reitzel, K., Danielsson, R., Gogoll, A., Rydin, E., 2006. Biogenic phosphorus in oligotrophic mountain lake sediments: differences in composition measured with NMR spectroscopy. Water Res. 40, 3705– 3712.

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