POLISH JOURNAL OF ECOLOGY (Pol. J. Ecol.

53

571577

2005

Short research contribution

Agnieszka KALWASISKA*, Wojciech DONDERSKI

Department of Water Microbiology and Biotechnology, Nicolaus Copernicus University, Gagarina 9, 87 100 Toru, Poland, e-mail: kala@biol.uni.torun.pl * corresponding author

NEUSTONIC VERSUS PLANKTONIC BACTERIA IN EUTROPHIC LAKE

ABSTRACT: This paper presents the results of research on the number, the rate of secondary production and physiological properties of neustonic (surface microlayer SM 250 m) and planktonic (subsurface water SSW 1015 cm) bacteria of the eutrophic lake (TP 3099 g l1; TN 0.941.76 mg l1; chlorophyll a 26.456.9 mg l1; water transparency 1.21.9 m). It was found that the total number of neustonic bacteria (TNB) varied from 1.28 106 to 1.98 106 cells ml1 and was from 1.4 to 2.0 times higher than the number of planktonic bacteria (P <0.001). TNB range for planktonic bacteria oscillated between 0.75 106 and 1.45 106 cells ml1. The number of heterotrophic neustonic (SM) bacteria (CFU 22 C) was also higher by 2.0 to 13.3 times (P <0.001) being between 1.48 and 12.5 103 cells ml1 while the CFU of bacteria in the SSW oscillated between 0.35 to 0.94 103 cells ml1. Both the values of TNB and CFU displayed a distinct seasonal variation (P <0.001). However, the rate of secondary production of planktonic bacteria was higher (from 1.1 to 6.0 times) than the rate of production of neustonic bacteria (P <0.05) and displayed seasonal variability (P <0.001). The rate of secondary production in subsurface water ranged from 0.676 to 1.265 gC l1 h1 while in surface microlayer from 0.118 to 0.597 gC l1 h1. In neuston the bacteria decomposing fat and DNA were more common than in plankton (P <0.05).

KEY WORDS: bacterioneuston, bacterioplankton, surface microlayer, bacterial production, physiological properties The interest on layer separating the atmosphere from the water, called the surface microlayer (SM) has increased in recent years. It is a specific type of environment, differing clearly from subsurface water (SSW) both in physical properties and in the chemical and biological composition. Organisms like bacteria, algae and small animals living there are called neuston. The forces of adhesion occurring at the border of the two environments water and air contribute to existence of this microlayer. The first few nanometres of the surface layer constitute a lipid layer which is formed by free fatty acids, glycerides and phospholipids with hydrocarbons. Below there is a layer of polysaccharide-protein complexes, and below it in turn the accumulation of bacteria, phyto- and zooneuston occur (Fa l kowska 1996). Because of the high concentration of organic substances occurring in the microlayer, both autotrophic and heterotrophic bacteria find optimal conditions for growth (Hopp e 1986). That layer makes a very stable environment for microorganisms in terms of nutritional reserves.

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Agnieszka Kalwasiska, Wojciech Donderski

Table 1. Morphometric and trophic characteristics of Lake Chemyskie (Central Poland) (data for 1m depth, summer, July(1), 2000).
Characteristic Area (ha) Maximal depth (m) Mean depth (m) Length of shore line (m) Total phosphorus (g l1) Total nitrogen (mg l1) Chlorophyll a (mg l1) Water transparency (m)
(1)

Value 271.1 27.1 6.0 20985 30.0 99.0 0.94 1.76 26.4 56.9 1.2 1.9

data supplied by Provincial Inspectorate of Environmental Service in Bydgoszcz.

On the other hand, due to extreme temperature conditions, sunlight and anthropogenic pollution, it is not a favourable site for microorganisms as compared to the water below (D onderski et al. 1999). Neustonic organisms from this environment are characterised by the presence of hydrophobic compounds (mucopolysaccharides, glucoproteids and phosphatidylcholine polymers) in their external cellular structures, which can actively combine with the surface layer of the water (Marsha l 1985). Many flagellated bacteria can move to the surface microlayer (D a h l b ck 1983) displaying the ability to adhere to various organic particulate compounds, mainly grains of starch or drops of fat. The neuston community is the link through which organic matter flows from the atmosphere and microlayer to the water column and vice versa, undergoing transformation. Both bacterioneuston and bacterioplankton play an important role in the processes of biotransformation of organic, auto- and allochtonic substances. Hence, the investigation of the distribution, numbers, secondary production and physiological and biochemical properties of bacteria inhabiting the surface microlayer in comparison with bacterioplankton is important for better understanding of an aquatic environment. The aim of this study was to determine the occurrence, numbers, secondary production and physiological and biochemical properties of bacteria inhabiting the surface microlayer in comparison with bacterioplankton from subsurface water in a typical eutrophic lake. The research was carried out in the northwestern part of Lake Chemyskie, Central Poland (Fig. 1). This lake, which represents

a typical eutrophic, medium deep lake (Table 1), lies in the Chemisko-Dobrzyskie Lake District at a distance of about 20 km from Toru town and is a part of the Fryba-Vistula river basin. The areas near the shore are mainly covered with cultivated fields (72%). Remaining forms of land cover in the catchment are rest plots (4.9%), orchards (2.8%), grasslands (2.4%) and forests (2.0%). Around the lake, flat shores are predominant with 60% of them providing access to bathing. The littoral zone of the lake is well developed. The lake is a recreational and water sports centre. Samples of water were collected from the pelagic zone in spring (May), in summer (August) and in autumn (October 2002) from four stations (IIV) located in the north-western part of studied lake. In order to collect the surface microlayer (SM), the Garrett technique was used (G ar ret 1965). Water samples (n = 4) were collected using a nylon Garrett net with a mesh diameter of 65 m and an active surface of 5050 cm, allowing a layer of water 250 50 m thick to be collected. Subsurface water (SSW) samples (n = 4) from a depth of 1015 cm were collected with sterile glass pipettes using an automatic Pipet-Boy sampler (Biotechnology, De Ville). Water samples from the SM and SSW were poured into sterile glass bottles and transported to the laboratory in an ice container (the temperature inside +4C). The time from the moment of taking the sample to conducting the analyses did not exceed 6 hours. The subsamples of water meant for determining the total number of bacteria were fixed immediately after collecting in formaldehyde, which final concentration was equal to 4%.

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Neustonic versus planktonic bacteria

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The total number of neustonic and planktonic bacteria (TNB) in 1 ml of SM and SSW was determined using the method of direct counting of bacteria on membrane filters (Z immer man 1977). The number of heterotrophic bacteria (CFU) was determined with the spread plates method, using iron-peptone agar as the medium (Fer rer et al. 1963). Seedings were carried out in three parallel repetitions. The bacterial colonies that grew up were counted after 10 days of incubation at a temperature of 22C. The result was calculated per 1 ml of lake water. Secondary production of bacteria in the water samples was determined according to the method of Fu hr man and Azam (1980, 1982) measuring the rate of incorporation of radioactive thymidine [3H-methyl thymidine] (Amersham, 60Ci nmol1 specific activity) to bacterial DNA. Water samples with the addition of radioactive thymidine with a final concentration of 1520 nM l1 were incubated for 30 minutes at a temperature of 20C. The results were calculated using a Liquid Scintillation Counter, Wallace 1409. The amount of incorporated thymidine in the samples was calculated as the rate of cell division using the conversion coefficient 1.24 109 cells nM1 (C hrst et al. 1994). Bacterial production was expressed as the

amount of organic carbon in the biomass of bacterial cells using conversion coefficient 19.8 fgC for bacterial cell (L e e and Fu hr man 1987). After incubation and counting the bacterial colonies, 25 bacterial strains were separated at random each time and transferred to semi-liquid iron-peptonic agar (5g agar l1) and incubated for 6 days at a temperature of 22C. After checking the purity of the bacterial culture, the strains were kept in the refrigerator at a temperature of +4C for further tests. Every two months these strains were transplanted to fresh iron-peptone agar. The physiological properties of the bacteria were determined by seeding the strains on a series of test media containing appropriate substrates. In the tests, the ability of bacteria to hydrolyse protein, urea, starch, fat, DNA, pectin, cellulose and chitin was taken into account. The media used for the tests were prepared after D ondersk i (1971) and D ondersk i and St r z elc z y k (1992). Statistical analyses were done using STATISTICA 597 software for Windows. In order to estimate the variation of abundance (TNB and CFU) and rate of secondary production (BP) of neustonic and planktonic bacteria, multifactor analysis of variance (ANOVA) was used. Percentage of strains from different physiological groups of neustonic and

Fig. 1. Outline of studied Lake Chemyskie and sampling stations (IIV).

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planktonic bacteria, the total number (TNB) and number of heterotrophic bacteria (CFU) and bacterial production (BP) of neustonic and planktonic bacteria were compared using t test. The total number (TNB) of neustonic bacteria varied from 1.28 106 to 1.98 106 cells ml1, while TNB of planktonic bacteria varied from 0.75 106 to 1.45 106 cells ml1 (Fig. 2). The number of bacteria coming from the SM was on average 1.4 to 2.0 times higher than from the SSW (P <0.001). The number of heterotrophic neustonic
N ( P < 0.001); P ( P <0.001) 2.5 TNB x 10 6cells ml -1

bacteria (CFU 22C) was also by 2.0 to 13.3 times on average higher than the number of heterotrophic bacteria in SSW (P <0.001) being 1.48 and 12.52 103 cells ml1, while the CFU of bacteria in SSW oscillated between 0.35 to 0.94 103 cells ml1 (Fig. 3). The values of TNB and CFU 22C of neustonic and planktonic bacteria were all characterised by seasonal variation (P < 0.001; Figs 2, 3). The higher number of bacteria (TNB and CFU 22C) in SM and in SSW was found in summer. The lower total number of plankN (P <0.001); P (P <0.001) 20 CFU x 103cells ml-1 N P

2.0 1.5 1.0 0.5 0.0 SPRING SUMMER

N P

15 10 5 0 SPRING SUMMER

AUTUMN

AUTUMN

Fig. 2. Average (IIV sampling stations) total number of neustonic and planktonic bacteria (TNB) in studied lake in spring, summer and autumn. Vertical bars represents SD. N neustonic bacteria, P planktonic bacteria, P significance level.

Fig. 3. Average (IIV sampling stations) number of heterotrophic neustonic and planktonic bacteria (CFU) in studied lake in spring, summer and autumn. Vertical bars represents SD. N neustonic bacteria, P planktonic bacteria, P significance level.

N (P <0,001); P (P <0.140) 2.5

Bacteria in %

70

* N * P

2.0 BP gC l -1h -1 1.5 1.0 0.5 0.0 SPRING SUMMER

N P

60 50 40 30 20 10 0

AUTUMN

LI

PR

AM

CE DNA CH

UR

PE

Fig. 4. Average (IIV sampling stations) bacterial production of neustonic and planktonic bacteria (BP) in studied lake in spring, summer and autumn. Vertical bars represents SD. N neustonic bacteria, P planktonic bacteria, P significance level.

Fig. 5. The contribution (%) of different physiological strains of bacterioneuston (N) and bacterioplankton (P) in total number of strains of studied lake. Explanations: LI lipolytic bacteria; PR proteolytic; AM amylolytic; CE cellulolytic; DNA degrading DNA; CH chitynolytic; UR ureolytic; PE pectinolytic bacteria. * significance level P <0.001

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Neustonic versus planktonic bacteria

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tonic bacteria and the number of heterotrophic, both neustonic and planktonic, bacteria was observed in autumn. In spring the lower TNB of neustonic bacteria was recorded (Figs 2, 3). It follows from the data on the bacterial production (BP) (Fig. 4) that higher rate of secondary production was observed in SM than in SSW (P <0.001). The rate of secondary production in subsurface water ranged from 0.676 to 1.265 g C l1 h1 while in surface microlayer from 0.118 to 0.597 g C l1 h1. The highest rate of secondary production of bacterioplankton was found in spring and of bacterioneuston in summer. The lowest secondary production of planktonic bacteria was found in summer and of neustonic bacteria in autumn. There were significant differences between the rates of secondary production in spring, summer and autumn (P <0.05; Fig. 4). Fig. 5 shows the results of the t-test aiming to determine the statistically significant differences between the number of strains as % contribution of neustonic and planktonic bacteria belonging to different physiological groups. It follows that SM was more abundant with strains that hydrolyse fat (P <0.016) and the deoxyribonucleic acid (P <0.046) than SSW. There were not statistically significant differences between the percentage of strains belonging to the other physiological groups of neustonic and planktonic bacteria. It follows from the research that in studied lake the number of neustonic bacteria was higher than the number of bacteria occurring in the subsurface layer. This is in accordance with the results obtained by Apine (1989), Ma k i and Her w ig (1991), D ondersk i et al. (1998) and Mudr y k et al. (1999). The higher number of bacteria from the surface microlayer is probably caused by the higher concentration of organic compounds in this layer. It follows from the data obtained by Hi l l br icht -Il kowsk a and Kos trz ew s k a - S z l a kows k a (2004) that the concentration of N and P in surface microlayer of eutrophic lake is at least 1.5 times grater than in subsurface water. Moreover, as follows from Pars on and Ta ka hashi (1973) results, the surface microlayer is characterised by a high concentration of organic carbon, the main stimulator of bacterial growth,

which was in their research as much as 17 times higher. According to Her mans s on et al . (1987) and Ma k i and Herw i g (1991) pigmentation and plasmids with a coded UV resistance, which are present in cells, protect bacterioneuston against the harmful influence of UV radiation in surface microlayer of water. A higher rate of secondary production of bacteria was found in Lake Chemyskie in the subsurface layer than in the surface microlayer. Similar results were obtained by B ay le y et al. (1983), D ondersk i et al. (1998) and Mud r y k et al. (1999). According to Wi l l i ams et al. (1986), the lower secondary production of bacterioneuston is probably caused by the stressogenic action of environmental factors, mainly sunlight radiation and temperature. Moreover, the rate of secondary production can be impeded by a relatively high accumulation in the surface microlayer of heavy metals, biophenol, polyvinyl chloride and pesticides, which may inhibit the metabolic activity of bacterioneuston (Ma k i and Her manss on 1994). It follows from the data obtained in this study regarding the physiological properties of isolated strains from SM and SSW, that in surface microlayer the percentage of strains able to decompose DNA, which is a good source of P (Jorgens en and Jac obs en 1996) and also N and C (Jorgens en et al. 1993), is greater than in subsurface water. Earlier studies of this physiological group of bacteria carried out in lakes (D ondersk i et al. 1998; Mud r y k et al. 1999) confirmed these results. The reason of the higher number of strains able to hydrolyse the deoxyribonucleic acid in surface microlayer is probably the relatively high amount of available substrate i.e. free DNA coming there from decaying cells of algae and bacteria and also viruses which are abundant in this layer due to unfavourable environment factors, mentioned above. A large number of bacteria able to decompose fat were observed in lake by D onderski et al. (1998). Mud r y k et al. (1999) observed 7880% of these bacteria in the lake littoral. According to D a h lb ck (1983) and Ma k i and Her manss on (1994), the occurrence of such a high num-

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(Received after revising July 2005)

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