neustonic versus planktonic bacteria

Agnieszka KALWASIŃSKA*, Wojciech DONDERSKI

Department of Water Microbiology and Biotechnology, Nicolaus Copernicus University,Gagarina 9, 87 – 100 Toruń, Poland, e-mail: [email protected]

* corresponding author

NEUSTONIC VERSUS PLANKTONIC BACTERIA IN EUTROPHIC LAKE

POLISH JOURNAL OF ECOLOGY(Pol. J. Ecol.)

53 4 571–577 2005

Short research contribution

ABSTRACT: This paper presents the results of research on the number, the rate of second-ary production and physiological properties of neustonic (surface microlayer SM ≈ 250 μm) and planktonic (subsurface water SSW ≈10–15 cm) bacteria of the eutrophic lake (TP 30–99 μg l–1; TN 0.94–1.76 mg l–1; chlorophyll a 26.4–56.9 mg l–1; water transparency 1.2–1.9 m). It was found that the total number of neustonic bacteria (TNB) varied from 1.28 × 106 to 1.98 × 106 cells ml–1 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 be-tween 0.75 × 106 and 1.45 × 106 cells ml–1. 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 ml–1 while the CFU of bacteria in the SSW oscil-lated between 0.35 to 0.94 × 103 cells ml–1. 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 wa-ter ranged from 0.676 to 1.265 μgC l–1 h–1 while in surface microlayer from 0.118 to 0.597 μgC l–1 h–1. In neuston the bacteria decomposing fat and DNA were more common than in plankton (P <0.05).

KEY WORDS: bacterioneuston, bacterio-plankton, surface microlayer, bacterial produc-tion, physiological properties

The interest on layer separating the atmo-sphere from the water, called the surface mi-crolayer (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 bac-teria, algae and small animals living there are called neuston. The forces of adhesion occur-ring 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. Be-low there is a layer of polysaccharide-protein complexes, and below it in turn the accumu-lation of bacteria, phyto- and zooneuston oc-cur (Fa lkowska 1996). Because of the high concentration of organic substances occur-ring in the microlayer, both autotrophic and heterotrophic bacteria find optimal condi-tions for growth (Hoppe 1986). That layer makes a very stable environment for micro-organisms in terms of nutritional reserves.

journal 4.indb 571journal 4.indb 571 2005-12-08 00:27:382005-12-08 00:27:38

572 Agnieszka Kalwasińska, Wojciech Donderski

On the other hand, due to extreme tempera-ture conditions, sunlight and anthropogenic pollution, it is not a favourable site for micro-organisms as compared to the water below (Donderski et al. 1999). Neustonic organ-isms from this environment are characterised by the presence of hydrophobic compounds (mucopolysaccharides, glucoproteids and phosphatidylcholine polymers) in their ex-ternal cellular structures, which can actively combine with the surface layer of the water (Marshal 1985). Many flagellated bacteria can move to the surface microlayer (Dahl-bäck 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 trans-formation. Both bacterioneuston and bac-terioplankton 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 un-derstanding 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 north-western part of Lake Chełmżyńskie, Central Poland (Fig. 1). This lake, which represents

a typical eutrophic, medium deep lake (Table 1), lies in the Chełmińsko-Dobrzyńskie Lake District at a distance of about 20 km from Toruń town and is a part of the Fryba-Vis-tula river basin. The areas near the shore are mainly covered with cultivated fields (72%). Remaining forms of land cover in the catch-ment 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 litto-ral 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 sum-mer (August) and in autumn (October 2002) from four stations (I–IV) located in the north-western part of studied lake. In order to collect the surface microlayer (SM), the Garrett technique was used (Garret 1965). Water samples (n = 4) were collected using a nylon Garrett net with a mesh diameter of 65 μm and an active surface of 50×50 cm, al-lowing a layer of water 250 ±50 μm thick to be collected. Subsurface water (SSW) sam-ples (n = 4) from a depth of 10–15 cm were collected with sterile glass pipettes using an automatic Pipet-Boy sampler (Biotechnol-ogy, De Ville). Water samples from the SM and SSW were poured into sterile glass bot-tles and transported to the laboratory in an ice container (the temperature inside +4°C). 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 collect-ing in formaldehyde, which final concentra-tion was equal to 4%.

Table 1. Morphometric and trophic characteristics of Lake Chełmżyńskie (Central Poland) (data for 1m depth, summer, July(1), 2000).

Characteristic Value

Area (ha) 271.1Maximal depth (m) 27.1Mean depth (m) 6.0Length of shore line (m) 20985Total phosphorus (μg l–1) 30.0 – 99.0Total nitrogen (mg l–1) 0.94 – 1.76Chlorophyll a (mg l–1) 26.4 – 56.9Water transparency (m) 1.2 – 1.9

(1) data supplied by Provincial Inspectorate of Environmental Service in Bydgoszcz.


573Neustonic versus planktonic bacteria

The total number of neustonic and plank-tonic bacteria (TNB) in 1 ml of SM and SSW was determined using the method of direct counting of bacteria on membrane filters (Z immerman 1977).

The number of heterotrophic bacteria (CFU) was determined with the spread plates method, using iron-peptone agar as the me-dium (Ferrer 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 22°C. 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 Fuhrman and Azam (1980, 1982) measuring the rate of incorporation of radioactive thymidine [3H-methyl thymi-dine] (Amersham, 60Ci × nmol–1 specific ac-tivity) to bacterial DNA. Water samples with the addition of radioactive thymidine with a final concentration of 15–20 nM l–1 were incubated for 30 minutes at a temperature of 20°C. 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 nM–1 (Chróst et al. 1994). Bacterial production was expressed as the

amount of organic carbon in the biomass of bacterial cells using conversion coeffi-cient 19.8 fgC for bacterial cell (Lee and Fuhrman 1987).

After incubation and counting the bacte-rial colonies, 25 bacterial strains were sepa-rated at random each time and transferred to semi-liquid iron-peptonic agar (5g agar l–1) and incubated for 6 days at a temperature of 22°C. After checking the purity of the bacte-rial culture, the strains were kept in the re-frigerator at a temperature of +4°C for further tests. Every two months these strains were transplanted to fresh iron-peptone agar.

The physiological properties of the bac-teria were determined by seeding the strains on a series of test media containing appropri-ate substrates. In the tests, the ability of bac-teria to hydrolyse protein, urea, starch, fat, DNA, pectin, cellulose and chitin was taken into account. The media used for the tests were prepared after Donderski (1971) and Donderski and Strzelczyk (1992).

Statistical analyses were done using STA-TISTICA 5’97 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 differ-ent physiological groups of neustonic and

Fig. 1. Outline of studied Lake Chełmżyńskie and sampling stations (I–IV).



planktonic bacteria, the total number (TNB) and number of heterotrophic bacteria (CFU) and bacterial production (BP) of neustonic and planktonic bacteria were compared us-ing t – test.

The total number (TNB) of neustonic bacteria varied from 1.28 × 106 to 1.98 × 106 cells ml–1, while TNB of planktonic bacteria varied from 0.75 × 106 to 1.45 × 106 cells ml–1 (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

bacteria (CFU 22°C) was also by 2.0 to 13.3 times on average higher than the number of heterotrophic bacteria in SSW (P <0.001) be-ing 1.48 and 12.52 × 103 cells ml–1, while the CFU of bacteria in SSW oscillated between 0.35 to 0.94 × 103 cells ml–1 (Fig. 3).

The values of TNB and CFU 22°C 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 22°C) in SM and in SSW was found in summer. The lower total number of plank-

N (P < 0.001); P ( P <0.001)

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Fig. 2. Average (I–IV sampling stations) total number of neustonic and planktonic bacteria (TNB) in studied lake in spring, summer and au-tumn. Vertical bars represents SD. N – neustonic bacteria, P – planktonic bacteria, P – significance level.

Fig. 3. Average (I–IV sampling stations) number of heterotrophic neustonic and planktonic bacte-ria (CFU) in studied lake in spring, summer and autumn. Vertical bars represents SD. N – neus-tonic bacteria, P – planktonic bacteria, P – sig-nificance level.

Fig. 4. Average (I–IV sampling stations) bacterial production of neustonic and planktonic bacteria (BP) in studied lake in spring, summer and au-tumn. Vertical bars represents SD. N – neustonic bacteria, P – planktonic bacteria, P – significance level.

Fig. 5. The contribution (%) of different physi-ological strains of bacterioneuston (N) and bac-terioplankton (P) in total number of strains of studied lake.Explanations: LI – lipolytic bacteria; PR – pro-teolytic; AM – amylolytic; CE – cellulolytic; DNA – degrading DNA; CH – chitynolytic; UR – ureo-lytic; PE – pectinolytic bacteria. * – significance level P <0.001



tonic bacteria and the number of heterotro-phic, 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 second-ary production in subsurface water ranged from 0.676 to 1.265 μg C l–1 h–1 while in sur-face microlayer from 0.118 to 0.597 μg C l–1 h–1. The highest rate of secondary production of bacterioplankton was found in spring and of bacterioneuston in summer. The lowest secondary production of planktonic bacte-ria 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 aim-ing 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 stud-ied 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), Maki and Her wig (1991), Donderski et al. (1998) and Mudr yk et al. (1999). The higher number of bacteria from the surface microlayer is probably caused by the high-er concentration of organic compounds in this layer. It follows from the data obtained by Hi l lbr icht-I lkowska and Kostrze-wska-Szlakowska (2004) that the con-centration of N and P in surface microlayer of eutrophic lake is at least 1.5 times grater than in subsurface water. Moreover, as fol-lows from Parson and Takahashi (1973) results, the surface microlayer is character-ised by a high concentration of organic car-bon, the main stimulator of bacterial growth,

which was in their research as much as 17 times higher. According to Hermans-son et al . (1987) and Maki and Her-wig (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 Chełmżyńskie in the subsurface layer than in the surface microlayer. Similar results were obtained by Bay ley et al. (1983), Donderski et al. (1998) and Mudr yk et al. (1999). Accord-ing to Wil l iams 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 im-peded by a relatively high accumulation in the surface microlayer of heavy metals, bio-phenol, polyvinyl chloride and pesticides, which may inhibit the metabolic activity of bacterioneuston (Maki and Hermansson 1994).

It follows from the data obtained in this study regarding the physiological proper-ties 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 (Jorgensen and Jacob-sen 1996) and also N and C (Jorgensen et al. 1993), is greater than in subsurface water. Earlier studies of this physiological group of bacteria carried out in lakes (Donderski et al. 1998; Mudr yk et al. 1999) confirmed these results. The reason of the higher num-ber of strains able to hydrolyse the deoxyri-bonucleic acid in surface microlayer is prob-ably 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, men-tioned above.

A large number of bacteria able to de-compose fat were observed in lake by Donderski et al. (1998). Mudr yk et al. (1999) observed 78–80% of these bacteria in the lake littoral. According to Dahl-bäck (1983) and Maki and Hermansson(1994), the occurrence of such a high num-



ber of lipolytic bacteria in the biofilm of the lake water is caused by an accumula-tion of many lipids (such as triglycerides, phospholipids, lipoproteins, free fatty acids, glycolipids, sterols and wax in emulsified form) in this layer, creating optimal condi-tions for the development of lipolytic bac-teria.

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(Received after revising July 2005)


neustonic versus planktonic bacteria

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