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Occurrence and Activity of Lipolytic Bacterioneuston and Bacterioplankton in the Estuarine Lake Gardno
Estuarine, Coastal and Shelf Science (2000) 51, 763–772 doi:10.1006/ecss.2000.0719, available online at http://www.idealibrary.com on
Occurrence and Activity of Lipolytic Bacterioneuston and Bacterioplankton in the Estuarine Lake Gardno Z. J. Mudryk and P. Sko´rczewski Department of Experimental Biology, Pedagogical University, Arciszewskiego 22, 76-200 Słupsk, Poland Received 17 March 2000 and accepted in revised form 17 September 2000 The number and lipolytic activity of neustonic and planktonic bacteria inhabiting estuarine Lake Gardno were determined. Lipolytic bacteria were very numerous in investigated layers of water, accounting for 10–88% of the total number of culturable heterotrophic bacteria. Significant differences were found in the decomposition of individual lipid substrates by bacteria. The highest percentage of neustonic and planktonic strains were able to hydrolyse tributyrin and Tween 85. The least numerous bacteria group was microflora hydrolysing Tween 20 and Tween 40. The activity of lipases synthesized by bacteria from the subsurface layer was higher than that of lipases produced by bacteria isolated from the surface layers. A significant effect of salinity on the activity of lipases has been shown. 2000 Academic Press Keywords: bacterioneuston; bacterioplankton; lipolytic activity; estuarine lake
Introduction Lipids are integral components of all living organisms and are widely distributed in the environment. These organic molecules can be actively used in biosynthesis of cellular structures and as energy storage products used in respiratory processes (Parrish, 1988; Reemtsma et al., 1990). In aquatic ecosystems lipid compounds constitute 3–55% of all organic matter (Gajewski et al., 1993; Marty et al., 1996). In water basins, the lipid concentration very much depends on trophic level, taxonomic composition of plants and animals and their physiological condition (Quemeneur & Marty, 1992; Chro´st et al., 1994). The concentration of dissolved and particulate lipids in water is 10–500 g dm 3 (Parrish, 1987; Gajewski et al., 1997). Detritus and live as well as dead phytoplankton are the main source of lipids in water basins (Kattner et al., 1983; Kattner & Brokman, 1990; Martinez et al., 1996). Considerable amounts (i.e. 2–45%) of lipids are accumulated by cyanobacteria, diatoms, and especially green algae in cells of which lipids may constitute approximately 85% of dry mass (Siuda et al., 1991). This is why particularly high concentrations of lipids in water are observed shortly after the collapse of phytoplankton blooms (Kattner & Brokman, 1990). In water basins, ciliates and zooplankton are also a very important source of lipids accounting for 30–50% of the total dry mass (Arts et al., 1992; 0272–7714/00/120763+10 $35.00/0
Lardinois et al., 1995; Albers et al., 1996; Harvey et al., 1997). For heterotrophic bacteria, which in aquatic ecosystems play a key role in the processes of decomposition of organic matter, apart from proteins and polysaccharides, lipids constitute a very important source of carbon and energy (Parrish, 1987, 1988; Arts et al., 1992; Gajewski et al., 1993; Chro´st & Gajewski, 1995). Lipids are however a complex mixture of organic compounds that are too large to be directly incorporated into bacterial cells. This explains why bacteria are capable of the synthesis of many hydrolytic enzymes, including lipases which catalyse reactions of lipid depolymerization. Lipases (glycerol ester hydrolase, EC 3.1.1.3) are nonspecific esterases of molecular mass equalling about 25 000. They are usually relatively small proteins (Lawrence et al., 1967; Gajewski et al., 1993). Lipases are actively exported from living microbial organisms, or are released as free enzymes after the lysis of bacterial cells (Chro´st et al., 1994). These biocatalysts attack emulsified lipids and hydrolyse them, yielding glycerol and fatty acids. Only in this form can the products be actively assimilated by heterotrophic bacteria to be used in respiratory processes, or in biosynthesis of cellular structures (Gajewski et al., 1997). The specific role bacteria have in the modification of lipid composition, concentration and distribution is complex and so far not fully recognized (Dachs et al., 1998). The objective of the present study was to 2000 Academic Press
764 Z. J. Mudryk and P. Sko´ rczewski
Poland
ea
N
cS
ti al
B
channel st. 3
st. 2 River Lupawa st. 1
0
km
2
54°39' N
17°07' E
F 1. Lake Gardno, northern Poland, with location of sampling stations. Station 1 is near freshwater inflow, Station 2 is in mid-lake and Station 3 is near entrance to Baltic Sea.
Material and methods
saline water penetrate into the lake. Therefore, water of the lake, or its parts acquire sea water quality, resulting in a salinity of 2–5. Consistently, with the Venetian system Lake Gardno can be classified as mixo-oligohaline type (0·5–5·0 salinity) (Dethier, 1992).
Description of studied area
Sampling
The studies were carried out in an estuarine lake, Lake Gardno (Figure 1) situated in the World Biosphere Reserve, Słowin´ ski National Park (Poland). The lake is very shallow (1·3 m average depth) but covers a large area (2500 ha). The shallow depth and large area as well as the lack of shielding winds ensures the full mixing of water in both vertical and horizontal profiles. As a result, the lake can be regarded as a polymictic basin in which no thermal or oxygen stratification is observed. The emergent macroflora covers 4% of the surface of Lake Gardno forming offshore belt 20–200 m wide, which constitutes a residence for many bird species. The main species of macrophytes are: Typha angustifolia, Phragmites australis, Scirpus lacustris and Schoenoplectus lacustris. Lake Gardno is characterized by intermediate conditions between marine and inland environment. It is supplied by the water of the river Lupawa, and also via a 1·3 km channel by the Baltic Sea whose large volumes of
Water samples were taken in May, July and September during 1998 from three stations (Figure 1) which represented different environmental conditions: one near the river inflow (fresh water zone) (station 1), one near sea entrance (seawater zone) (station 3) and at one station in mid-lake (mixed zone) (station 2). Water samples for bacteriological analyses were taken from three layers. Film layer (FL) samples (thickness of 9017 m) were taken with a 3030 cm glass plate (Harvey & Burzell, 1974), surface microlayer (SM) samples (thickness of 24240 m) were collected with a 4050 cm Garrett net (24 mesh net of 2·54 cm length) (Garrett, 1965). The glass plate and polyethylene net were rinsed with ethyl alcohol and distilled sterile water prior to sampling. Water from subsurface layer (SUB) was taken with sterile glass pipettes at the depth of about 10–15 cm. The water samples were collected into sterile glass bottles and stored in an ice-box,
determine the contribution of the neustonic and planktonic bacteria to the process of biological transformation of lipids in a dynamic water ecosystem, an estuarine lake in this case.
Lipolytic bacterioneuston and bacterioplankton 765
where the temperature did not exceed 7 C until they were taken for analysis. The time between sample collection and performance of the analyses usually did not exceed 6–8 h. Bacteriological study To determine the number of heterotrophic neustonic (FL, SM) and planktonic (SUB) bacteria (CFU), the collected samples were diluted with sterile buffered water (pH 7·2) prepared according to Daubner (1967) and inoculated on iron-peptone agar medium (IPA) (Ferrer et al., 1963) using the spread method, in three replications. After 10 days incubation at 20 C, bacterial colonies were counted and results were recalculated per 1 cm3 water. Subsequently, about 50 bacterial colonies from each water layer were picked from the plates and were transferred to semiliquid IPA medium (5·0 g of agar per dm3 of medium). The cultures maintained in this medium after purity control were kept at 4 C until used for further investigation in order to determine ability of these bacteria to decompose different lipid compounds. For enumeration of the number of lipolytic bacteria, plating techniques were used. Water samples were vortex mixed, and then serial ten-fold dilutions were prepared with sterile buffered water to reach final concentrations of 10 3 and 10 4. Diluted samples were inoculated by the spread method in three parallel replicates on CPS medium (Jones, 1971) amended with of 10·0 cm3 of emulsified 50% tributyrin (Fluka). The plates were incubated at 20 C for 10 days. Colonies with clear zones around them were classified as lipolytic bacteria. In order to determine the ability of neustonic and planktonic bacteria to decompose lipid compounds substrates were chosen as representatives of fatty acids (Tween), triglycerides (tributyrin) and phospholipids (lecithin). Five Tweens (T-20, T-40, T-60, T-80, T-85) (Sigma) were tested as substrates for bacterial lipase. Tweens are water soluble high fatty acid esters of a polyoxyalkylene derivative of sorbitan; they differ only in the particular fatty acid component (Sierra, 1957). Tween 20 (polyoxethylene-sorbitian monolaurate, lauric acid approx. 50%) is a laurate acid ester, Tween 40 (polyoxethylene-sorbitian monopalmiate, palmitic acid approx. 90%) a palmitic acid ester, Tween 60 (polyoxethylene-sorbitian monostearate, stearic acid approx. 90%), is a stearic acid ester, Tween 80 (polyoxeethylene-sorbitian monooleate, oleic acid approx. 70%), an oleic acid ester and Tween 85 (polyoxethylene-sorbitian trioleate, oleic acid approx.
70%) is a trioleic acid ester. A peptone agar medium (Kjelleberg & Hakansson, 1977) was prepared with each of the Tweens. The Tweens were sterilized separately and then 0·1 cm3 of each Tween was added aseptically to 9 cm3 of molten peptone agar medium. The Tween was evenly dispersed through the medium by hand rolling prior to pouring on the plates. After incubation, the occurrence of opaque halos around the colonies was regarded as hydrolysis of Tween by bacteria. The capacity of bacteria to decompose tributyrin (glyceryltributyrate) was assayed in CPS medium (Jones, 1971) with the addition of 10·0 cm3 of emulsified 50% tributyrin. Colonies around which clear zones appeared were classified as able to synthesize extracellular tributyrinase. Lecithin (dipalmitoyl lecithin) (Fluka) breakdown was investigated in Menkina medium (Niewolak, 1980). Lecithin was prepared as follows: 1·0 g of lecithin from eggs (Fluka AG, Ch-9470 Busch) was dissolved in 2 cm3 ethyl alcohol and diluted with 18 cm3 of dissolved water, buffered with a saturated solution of sodium carbonate at pH 7·0 and three-fold steam sterilized for 40 min. In each Petri dish 0·1 cm3 of lecithin solution was introduced and Menkina medium was added and thoroughly mixed. Clear zones around the colonies were regarded as lecithin hydrolysed by bacteria. All media were adjusted to pH 7·0–7·2 and sterilized at 117 C for 20 min. As inoculum, 48–72 h cultures from slants of the IPA medium were applied. The results were recorded after 7 days of incubation at 20 C. Study on lipase activity In order to determine the lipolytic activities of the studied bacteria, strains that had clear zones around their colonies on media with tributyrin wider than 15 mm were taken. Lipolytic activity of nine bacterial strains (three strains from each station) from each water layer was determined. The selected strains of lipolytic bacteria were grown on slants of IPA medium for 72 h at 20 C. The bacteria were then rinsed from the slants with buffered water and the optical density of the culture suspension was adjusted to E=0·3 at the wavelength of 600 nm using a spectrophotometer SPECOL. The bacterial cultures were used as inocula to prepare cell-free post-culture media (CFPM). 0·5 cm3 of a given suspension was added to 25 cm3 of fresh liquid IPA medium containing 50% tributyrin. Cultures of each strain were grown in duplicate at 20 C on a shaker for 48 h. After incubation, a 5 cm3 aliquot was taken and spun down at 12 000g
766 Z. J. Mudryk and P. Sko´ rczewski T 1. Number of heterotrophic and lipolytic bacteria in brackish Lake Gardno Heterotrophic bacteria (104 cm 3)
Lipolytic bacteria (104 cm 3)
Season
Layer
mean
range
median
mean
range
median
Spring
FL SM SUB
8·8 7·6 1·9
(0·83–22·83) (2·27–18·00) (0·27–3·83)
2·67 2·63 1·60
0·9 (10)a 1·0 (13) 1·2 (63)
(0·08–2·30) (0·24–1·85) (0·27–2·80)
0·3 1·1 0·5
Summer
FL SM SUB
6·2 6·7 0·5
(0·20–17·00) (1·60–14·60) (0·07–0·90)
1·40 3·80 0·56
2·4 (39) 3·8 (57) 0·4 (80)
(0·40–4·00) (2·50–6·00) (0·05–0·80)
3·0 2·8 0·4
Autumn
FL SM SUB
3·2 2·6 2·3
(1·00–7·66) (1·05–6·30) (0·12–6·67)
1·16 5·33 0·25
2·4 (75) 2·3 (88) 1·7 (74)
(0·10–7·00) (0·35–6·00) (0·15–4·90)
0·3 0·7 0·2
a
Number of lipolytic bacteria in percentage.
for 30 min to remove bacterial cells. The CFPMs obtained in this way were the source of lipolytic enzymes. The activity of lipases synthesized by the studied bacteria was determined by the method developed by Cherry and Crandall (Mudryk, 1998). In a test tube 1 cm3 of a given CFPM was mixed with 1 cm3 of 0·3 M phosphate buffer, pH 7·0, and 2 cm3 of emulsified 50% tributyrin. All determinations were carried out in triplicate. Enzymatic reactions were performed for 24 h at 30 C. After incubation, the reactions were stopped by the addition of 3 cm3 of 95% ethanol. To each sample, two drops of 1% phenolphthalein in ethanol was added and the samples were titrated with 0·05 N NaOH. One unit of lipase activity (ULA) was defined as the amount of enzyme that, within 24 h at a temperature of 30 C, caused the liberation of the amount of fatty acids for which 1 cm3 of 0·05 N NaOH was needed to titrate. The effect of salinity on the level of lipolytic activity was determined using CFPMs and (50% emulsified tributyrin) substrate. Cell suspensions were obtained by culturing individual isolates at 20 C for 72 h using IPA medium supplemented with 0·5 1, 2, 4, 6 or 8‰ sodium chloride. The influence of salinity on the activity of lipases was determined by the method of Cherry and Crandall as described above. Results The data obtained on heterotrophic bacteria numbers in Lake Gardno are indicative of substantial spatial differences between number of these organisms inhabiting film and surface layers (FL, SM) and subsurface water (SUB) (Table 1). Bacteria living in the surface waters were more abundant than those
inhabiting the subsurface layer. Particularly great differences were recorded in summer when the bacterioneuston abundance was tenfold greater than of bacterioplankton. The results given in Table 1 indicate that lipolytic bacteria occurred in large numbers in the surface and subsurface water in Lake Gardno, making up 10–88% of the total number of culturable heterotrophic bacteria. The number of lipid–hydrolysing bacteria reached a maximum in summer and autumn and the number of lipolytic bacteria was lowest in spring. Lipolytic bacteria were more numerous in bacterioneuston than in bacterioplankton in summer and autumn. By contrast in spring, the number of lipolytic organisms among the neustonic and planktonic bacteria were almost identical. Figure 2 presents the results concerning the ability of bacteria isolated from surface and subsurface water to enzymatically depolymerize various lipids. It is evident from these data that there were no significant differences in the number of lipid decomposition between neustonic (FL, SM) and planktonic bacteria (SUB). On the other hand significant differences were found in the decomposition of individual lipids by bacteria. In all studied water layers, the highest percentage of bacterial strains were able to hydrolyse tributyrin and Tween 85. Bacteria able to hydrolyse Tween 60 and lecithin were present at lower percentages than the above-mentioned physiological groups. The microflora hydrolysing Tween 20 and Tween 40 represented the least numerous group of neustonic and planktonic bacteria. An identical phenomenon was noted on the investigated stations in Lake Gardno (Figure 3). The collection of strains was analysed for multiple– lipid decomposition (Figure 4). Most were able to decompose of 1 to 4 lipids. Only a small percentage of
Lipolytic bacterioneuston and bacterioplankton 767 90 80
T-20 T-40 T-60 T-80 T-85 Tributyrin Lecithin
Percentage of strains
70 60 50 40 30 20 10 0
SM
FL
SUB
F 2. Decomposition of lipids by bacteria inhabiting film (FL), surface (SM) and subsurface (SUB) water layers. Lipid substrates are representatives of fatty acids (Tweens, as T-20, T-40, T-60, T-80, T-85), triglycerides (tributyrin) and phospholipids (lecithin). Vertical bars indicate standard errors.
100 90 80
Percentage of strains
70 60 50 40 30 20 10 0
T-20
T-40
T-60
T-80
T-85
Tributyrin
Lecithin
F 3. Hydrolysis of lipids (as listed in Figure 2) by bacteria isolated from three sampling stations (see Figure 1). Vertical bars indicate standard errors. Station 1: open bars; Station 2: grey bars; Station 3: closed bars.
bacteria inhabiting surface and subsurface water in Lake Gardno were capable of decomposing all seven lipids studied. Multiple–lipid decomposition was
generally more common in neustonic than planktonic bacteria. About 8% of strains isolated from subsurface water did not decompose lipids at all.
768 Z. J. Mudryk and P. Sko´ rczewski
30%
Strains
20%
10%
0%
0
1
2
3 4 Number of lipids
5
6
7
F 4. Multiple–lipid (0–7) decomposition by bacterial strains inhabiting film (FL, open bars), surface (SM, grey bars) and subsurface (SUB, closed bars) water layers.
Figure 5 illustrates the activity of lipolytic enzymes synthesized by neustonic and planktonic bacteria. The average activity of the tributyrinase produced by bacterioplankton (SUB) was higher than bacterioneuston (FL, SM). There were significant differences in the activity levels of bacterial lipolytic enzymes between stations. The highest lipase production in all water layers could be observed in the central part of Lake Gardno (station 2). A minimum of lipolytic activity was recorded among bacterioneuston at station 1 close to the Lupawa river outflow into Lake Gardno. Among bacterioplankton, the least lipolytic activity was found with bacteria isolated from the zone of the channel connecting the lake to the Baltic Sea (station 3). Figure 6 presents data on the influence the salinity has on lipase activity of the bacteria isolated from various water layers and research stations. The data point to the fact that due to increasing salinity the activity of lipases was hampered more when synthesized by planktonic bacteria rather than by neustonic ones. There have also been revealed significant differences in the influence the salinity may have on bacterial lipases activity isolated from different sampling stations. At the station located at the Lupawa mouth into Lake Gardno (station 1), the bacteria were most
active at 0·5–2 salinity. Above that the bacterial lipolytic activity diminished and eventually at 8, that is at a salinity equal to that of Baltic Sea, complete lipase inactivation occurred. At station 2 located in midlake, the activity of lipases synthesized by neustonic bacteria stayed at a similarly high level within a wide range of salinity (0·5–6). However, among planktonic bacteria great lipase activity change was recorded along with the salinity change. In the channel joining Lake Gardno to the Baltic Sea (station 3), it was found that at a salinity equal to that of Baltic water (8) bacterial lipases synthesized by neustonic and planktonic bacteria were still active in terms of enzymes, which was not observed at other more fresh water stations. Discussion Data on the number of heterotrophic bacteria in Lake Gardno indicate that these organisms occur more frequently in the film layer and surface microlayer than subsurface water. These results fully confirm earlier observations carried out in Lake Gardno by Mudryk et al. (1999). Similarly, the investigations carried out in other water basins (Dahlba¨ ck et al., 1982; De Souza Lima & Chretiennot-Dinet, 1984;
Lipolytic bacterioneuston and bacterioplankton 769
st. 1 FL st. 2 st. 3
st. 1 SM st. 2 st. 3
st. 1 SUB st. 2 st. 3 0.0
0.1
0.2 –3 –1 Lipase units (cm day )
0.3
0.4
F 5. The activity of lipolytic enzymes synthesised by neustonic and planktonic bacteria, collected from film (FL), surface (SM) and subsurface (SUB) water layers from three stations (see Figure 1). Error bars indicate ranges of variability.
Mudryk et al., 1991) demonstrated that the greatest abundance of heterotrophic bacteria occurred in the surface layer, and decreased with the depth. Probably high concentrations of organic matter coming from extracted phyto- and zooplankton in surface water layer, provide optimal conditions for the development and accumulation of aerobic heterotrophic microflora forming bacterioneuston (Hoppe, 1986). Authors of the presented study are fully aware of fact that estimates of bacterial abundance based on plate count method represent only a minor fraction of bacteria inhabiting aquatic ecosystem. According to Nagata (1984) the percentage of viable count to the total bacterial numbers ranged from 0·009 to 1·8%. Lipolytic bacteria represent a dominant physiological group of microflora in estuaries and marine waters. Austin et al. (1977) in the estuarine Chesapeake Bay and Sieburth (1978) in the estuarine Narragansett found that 13–84% bacteria had lipolytic properties. In the seawater, bacteria synthesizing lipases constituted 70–100% of a total number of bacteria (Krstulovic´ & Solic´ , 1988; Mudryk et al., 1991; Martinez et al., 1996; Mudryk, 1998). Also in Lake Gardno lipolytic bacteria were a plentiful physiological group and constituted from 10 to 88% of the total number of culturable heterotrophic bacteria. In Lake Gardno lipolytic bacteria were more numerous in bacterioneuston than in bacterioplank-
ton in summer and autumn. Earlier studies on this physiological group of bacteria carried out in other basins (Kjelleberg & Hakansson, 1977; Rheinheimer, 1984; Mudryk et al., 1991) confirmed these results. According to Maki (1993) the occurrence of such high numbers of lipolytic bacteria in the surface layers of water can be explained by the fact that numerous lipids, like triglycerides, phospholipids, lipoproteins, free fatty acids, glycolipids, sterols and waxes, due to their amphiphatic properties accumulate at the water surface in an emulsified form and stimulate the optimal conditions for lipolytic bacteria growth. Studies conducted by Dahlba¨ ck et al. (1982) in the Arctic Ocean and Mudryk (1998) in the Gdan´ sk Deep, revealed that the number of lipolytic organisms among the neustonic and planktonic bacteria were almost identical. In the present study, a similar balance was found in Lake Gardno in spring. The latest research (Chro´ st & Gajewski, 1995; Wakeham, 1995; Martinez et al., 1996; Dachs et al., 1998) has pointed to the fact that lipolytic bacteria play a key role modifying and transforming processes in lipid compounds in water basins. Free fatty acids, triglycerides and phospholipids are the major components of the dissolved and particulate lipids in water basins (Parrish, 1988). The present findings suggest that neustonic and planktonic bacteria isolated from estuarine Lake
770 Z. J. Mudryk and P. Sko´ rczewski
0.4
0.3
Lipase units (cm
–3
–1
24 h )
Station 1
0.2
0.1
0
0.5
1
2
4
6
8
0.4
0.3
Lipase units (cm
–3
–1
24 h )
Station 2
0.2
0.1
0
0.5
1
2
4
8
6 Station 3
Lipase units (cm
–3
–1
24 h )
0.6
0.4
0.2
0
0.5
1
2
4 Salinity
6
8
F 6. The influence of medium salinity on the lipase activity of lipolytic bacteria isolated from the film (FL, diamonds), surface (SM, squares) and subsurface (SUB, triangles) waters from three stations in Lake Gardno. For all curves, each point is the mean of three replicates. Vertical bars represent standard errors; where error bars are not visible they are too small to be illustrated.
Lipolytic bacterioneuston and bacterioplankton 771
Gardno were most active when hydrolysing triglycerides (tributyrin). On the other hand, they were least active with fatty acid esters. In water basins, triglycerides make up 25% of all lipids and their concentration is about 0·5 M C dm 3 (Kattner et al., 1983). These lipids originate mainly from organic matter decomposition and are synthesized in great amounts by phytoplankton and zooplankton (Arts et al., 1992). Intensity of decomposition of lipids in water basins usually is determined not only by the number of microorganisms capable of carrying out the decomposition, but also by the actual activity of their enzymes (Mudryk, 1998). The results of the study presented here indicate that there were differences in the activity level between lipases produced by bacterioneuston and bacterioplankton. Average activity of lipases produced by planktonic bacteria was higher than that of lipases synthesized by neustonic bacteria. Similar results were obtained by Sieburth (1971) and Kjelleberg and Hakansson (1977). Lower activity of lipases synthesized by estuarine neustonic bacteria could have been caused by the stressful effect of many environmental factors; mainly solar radiation and considerable fluctuations of water temperature and salinity. Also a relatively high accumulation of heavy metals, polychlorinated biophenyls and pesticides in the surface microlayer could have an inhibiting effect on the synthesis and activity of lipases (Maki, 1993). The rate of depolymerization of lipids depends not only on the concentration and activity of enzymes, but also on various environmental factors affecting the kinetics of enzymatic reactions (Karner & Rassoulzadegan, 1995). The results of a number of studies (Nitkowski et al., 1977; Meyer-Reil, 1991; Mudryk, 1998) indicate that salinity, besides temperature and pH, play an important role as a factor controlling the synthesis and activity of lipases of marine and estuarine bacteria. Meyer-Reil (1991) draws attention to the fact that hydrolytic enzymes can be active in a wide range of salinities. Lipases synthesized by the bacteria isolated from Lake Gardno were active in a range of salinity (0·5–8), although the level of activity depended on the water salinity. References Albers, C. S., Kattner, G. & Hagen, W. 1996 The compositions of wax esters, triacylglycerols and phospholipids in Arctic and Antarctic copepodes: evidence of energetic adaptations. Marine Chemistry 55, 347–358. Arts, M. T., Evans, M. S. & Robarts, R. D. 1992 Seasonal patterns of total and energy reserve lipids of dominant zooplanktonic crustaceans from a hyper-eutrophic lake. Oecologia 90, 560–571. Austin, C., Allen, S. D., Mills, A. & Colwell, R. 1977 Numerical taxonomy of heavy metal–tolerant bacteria isolated from an estuary. Canadian Journal of Microbiology 23, 1433–1447.
Chro´ st, R. J., Gajewski, A. & Lalke, E. 1994 Mechanisms and control of microbiological processes of degradation and utilization of organic matter in lacustrine ecosystems of different degree of eutrophication (in Polish). Biotechnology 3, 82–95. Chro´ st, R. J. & Gajewski, A. 1995 Microbial utilization of lipids in lake water. FEMS Microbiology Ecology 18, 45–50. Dachs, J., Bayona, J. M., Fowler, S. W., Miquel, J. C. & Albaiges, J. 1998 Evidence for cyanobacterial inputs and heterotrophic alternation of lipids in sinking particles in the Alboran Sea (SW Mediterranean). Marine Chemistry 60, 189–201. Dahlba¨ ck, B., Gunnarsson, L. A., Hermansson, M. & Kjelleberg, S. 1982 Microbial investigations of surface microlayers, water column, ice and sediment in the Arctic Ocean. Marine Ecology Progress Series 9, 101–109. Daubner, J. 1967 Mikrobiologia Vody. Slov. Akad. Vied., Bratislava, 345 pp. De Souza Lima, Y. & Chretiennot-Dinet, M. J. 1984 Measurement of biomass and activity of neustonic microorganisms. Estuarine, Coastal and Shelf Science 19, 167–180. Dethier, M. N. 1992 Classifying marine and estuarine natural communities. An alternative to the coward in system. Journal Natural Areas 12, 90–100. Ferrer, E. B., Stapert, E. M. & Sokalski, W. T. 1963 A medium for improved recovery of bacteria from water. Canadian Journal of Microbiology 9, 420–422. Gajewski, A., Chro´ st, R. J. & Siuda, W. 1993 Bacterial lipolytic activity in an eutrophic lake. Archiv fu¨ r Hydrobiologie 128, 107– 126. Gajewski, A., Kirschner, A. K. T. & Velimirov, B. 1997 Bacterial lipolytic activity in a hypertrophic dead arm of the river Danube in Vienna. Hydrobiologia 344, 1–10. Garrett, W. D. 1965 Collection of slick-forming materials from the sea surface. Limnology and Oceanography 10, 602–605. Harvey, G. W. & Burzell, L. A. 1974 A simple microlayer method for small samples. Limnology and Oceanography 17, 156–157. Harvey, H. R., Ederington, M. C. & Mcmanus, G. B. 1997 Lipid composition of the marine ciliates Pleuronema sp. and Fabera salina: shifts in response to changes in diet. Journal Eucaryote Microbiology 44, 189–193. Hoppe, H. G. 1986 Degradation in sea water. In Biotechnology a Comprehensive Treatise (Rehm, U. J. & Reed, G., eds). Verlag Chemie, Weinheim, no. 8, pp. 453–474. Jones, J. G. 1971 Studies on freshwater bacteria: factors which influence the population and its activity. Journal of Ecology 59, 593–613. Karner, M. & Rassoulzadegan, F. 1995 Extracellular enzyme activity: Indications for high short-term variability in a marine ecosystem. Microbial Ecology 30, 143–156. Kattner, G., Gercken, G. & Hammer, K. D. 1983 Development of lipids during a spring plankton bloom in the northern North Sea. II. Dissolved lipids and fatty acids. Marine Chemistry 14, 163– 173. Kattner, B. & Brockman, U. H. 1990 Particulate and dissolved fatty acids in an enclosure containing a unialgal Skeletonema costatum (Greve.) Cleve culture. Journal of Experimental Biology and Ecology 141, 1–13. Kjelleberg, S. & Hakansson, N. 1977 Distribution of lipolytic, proteolytic and amylolytic marine bacteria between the film and subsurface water. Marine Biology 39, 103–109. Krstulovic´ , N. & Solic´ , M. 1988 Distribution of proteolytic, amylolytic and lipolytic bacteria in the Kastela Bay. Acta Adriatica 29, 75–83. Lardinois, D., Eisma, D. & Chen, S. 1995 Seasonal differences in concentrations of particulate lipids, proteins and chitin in the North Sea. Netherlands Journal of Sea Research 33, 147–161. Lawrence, R. C., Fryer, T. F. & Reiter, B. 1967 The production and characterization of lipases from a Microccocus and Pseudomonas. Journal of General Microbiology 48, 401–418. Maki, J. S. 1993 The air–water interface as an extreme environment. In Aquatic Microbiology. An Ecological Approach (Ford,
772 Z. J. Mudryk and P. Sko´ rczewski T. D., ed.). Blackwell Science Publications, Boston, Oxford, London, Eidenburg, Melbourne, Paris, Berlin, pp. 409–439. Martinez, J., Smith, D. C., Steward, D. F. & Azam, F. 1996 Variability in ectohydrolytic enzyme actives of pelagic marine bacteria and its significance for substrate processing in the sea. Aquatic Microbial Ecology 10, 223–230. Marty, Y., Quemeneur, M., Aminot, A. & Corre, P. 1996 Laboratory study on degradation of fatty acids and sterols from urban wastes in seawater. Water Research 30, 1127–1136. Meyer-Reil, L. A. 1991 Ecological aspects of enzymatic activity in marine sediments. In Microbial Enzymes in Aquatic Environments (Chro´ st, R. J., ed.). Springer-Verlag, New York, Berlin, Heidelberg, Paris, Tokyo, Hong Kong, Barcelona, pp. 84–95. Mudryk, Z. 1998 Numbers and activity of lipolytic and amylolytic marine inhabiting surface microlayer and subsurface water. Polskie Archiwum Hydrobiologii 45, 489–500. Mudryk, Z., Korzeniewski, K. & Falkowska, L. 1991 Bacteriological investigation of the surface microlayer of the Gulf of Gdan´ sk. Oceanologia 30, 93–103. Mudryk, Z., Donderski, W., Sko´ rczewski, P. & Walczak, M. 1999 Neustonic and planktonic bacteria isolated from brackish lake Gardno. Polskie Archiwum Hydrobiologii 46, 121–129. Nagata, T. 1984 Bacterioplankton in Lake Biwa: Annual fluctuations of bacterial numbers and their possible relationship with environmental variables. Japan Journal of Limnology 45, 126–133. Niewolak, S. 1980 Occurrence of microorganisms in fertilized lakes. II. Lecithin-mineralizing microorganisms. Polskie Archiwum Hydrobiologii 27, 53–71. Nitkowski, N. F., Dudly, S. & Graikowski, J. T. 1977 Identification and characterization of lipolytic and proteolytic bacteria isolated from marine sediments. Marine Pollution Bulletin 8, 276–279.
Quemeneur, M. & Marty, Y. 1992 Sewage influence in a macrotidal estuary: fatty acid and sterol distribution. Estuarine, Coastal and Shelf Science 34, 347–363. Parrish, Ch. C. 1987 Time series of particulate and dissolved lipid classes during spring phytoplankton blooms in Bedford Basin, a marine inlet. Marine Ecology Progress Series 35, 129–139. Parrish, Ch. C. 1988 Dissolved and particulate marine lipid classes: A review. Marine Chemistry 23, 17–40. Reemtsma, T., Haake, B., Ittekkot, V., Nair, R. R. & Brockmann, U. H. 1990 Downward flux of particulate fatty acids in the Central Arabian Sea. Marine Chemistry 29, 183–202. Rheinheimer, G. 1984 Bacterial ecology of the North and Baltic Seas. Botany Marine 27, 277–299. Sieburth, J. N. 1971 Distribution and activity of oceanic bacteria. Deep-Sea Research 18, 1111–1121. Sieburth, J. N. 1978 Bacterioplankton: nature, biomass, activity and relationship to the protist plankton. Journal of Phycology 14, 31–41. Sierra, G. 1957 A simple method for the detection of lipolytic activity of microorganisms and some observations on the influence of the contact between cells and fatty substrates. Journal of Microbiology and Serology 23, 15–22. Siuda, W., Wcisło, R. & Chro´ st, R. J. 1991 Composition and bacterial utilization of photosynthetically produced organic matter in an eutrophic lake. Archiv fu¨ r Hydrobiologie 121, 473– 484. Wakeham, S. G. 1995 Lipid markers for heterotrophic alternation of suspended particulate organic matter in oxygenated and anoxic water columns of the ocean. Deep Sea Research 42, 1749–1771.
Occurrence and Activity of Lipolytic Bacterioneuston and Bacterioplankton in the Estuarine Lake Gardno Z. J. Mudryk and P. Sko´rczewski Department of Experimental Biology, Pedagogical University, Arciszewskiego 22, 76-200 Słupsk, Poland Received 17 March 2000 and accepted in revised form 17 September 2000 The number and lipolytic activity of neustonic and planktonic bacteria inhabiting estuarine Lake Gardno were determined. Lipolytic bacteria were very numerous in investigated layers of water, accounting for 10–88% of the total number of culturable heterotrophic bacteria. Significant differences were found in the decomposition of individual lipid substrates by bacteria. The highest percentage of neustonic and planktonic strains were able to hydrolyse tributyrin and Tween 85. The least numerous bacteria group was microflora hydrolysing Tween 20 and Tween 40. The activity of lipases synthesized by bacteria from the subsurface layer was higher than that of lipases produced by bacteria isolated from the surface layers. A significant effect of salinity on the activity of lipases has been shown. 2000 Academic Press Keywords: bacterioneuston; bacterioplankton; lipolytic activity; estuarine lake
Introduction Lipids are integral components of all living organisms and are widely distributed in the environment. These organic molecules can be actively used in biosynthesis of cellular structures and as energy storage products used in respiratory processes (Parrish, 1988; Reemtsma et al., 1990). In aquatic ecosystems lipid compounds constitute 3–55% of all organic matter (Gajewski et al., 1993; Marty et al., 1996). In water basins, the lipid concentration very much depends on trophic level, taxonomic composition of plants and animals and their physiological condition (Quemeneur & Marty, 1992; Chro´st et al., 1994). The concentration of dissolved and particulate lipids in water is 10–500 g dm 3 (Parrish, 1987; Gajewski et al., 1997). Detritus and live as well as dead phytoplankton are the main source of lipids in water basins (Kattner et al., 1983; Kattner & Brokman, 1990; Martinez et al., 1996). Considerable amounts (i.e. 2–45%) of lipids are accumulated by cyanobacteria, diatoms, and especially green algae in cells of which lipids may constitute approximately 85% of dry mass (Siuda et al., 1991). This is why particularly high concentrations of lipids in water are observed shortly after the collapse of phytoplankton blooms (Kattner & Brokman, 1990). In water basins, ciliates and zooplankton are also a very important source of lipids accounting for 30–50% of the total dry mass (Arts et al., 1992; 0272–7714/00/120763+10 $35.00/0
Lardinois et al., 1995; Albers et al., 1996; Harvey et al., 1997). For heterotrophic bacteria, which in aquatic ecosystems play a key role in the processes of decomposition of organic matter, apart from proteins and polysaccharides, lipids constitute a very important source of carbon and energy (Parrish, 1987, 1988; Arts et al., 1992; Gajewski et al., 1993; Chro´st & Gajewski, 1995). Lipids are however a complex mixture of organic compounds that are too large to be directly incorporated into bacterial cells. This explains why bacteria are capable of the synthesis of many hydrolytic enzymes, including lipases which catalyse reactions of lipid depolymerization. Lipases (glycerol ester hydrolase, EC 3.1.1.3) are nonspecific esterases of molecular mass equalling about 25 000. They are usually relatively small proteins (Lawrence et al., 1967; Gajewski et al., 1993). Lipases are actively exported from living microbial organisms, or are released as free enzymes after the lysis of bacterial cells (Chro´st et al., 1994). These biocatalysts attack emulsified lipids and hydrolyse them, yielding glycerol and fatty acids. Only in this form can the products be actively assimilated by heterotrophic bacteria to be used in respiratory processes, or in biosynthesis of cellular structures (Gajewski et al., 1997). The specific role bacteria have in the modification of lipid composition, concentration and distribution is complex and so far not fully recognized (Dachs et al., 1998). The objective of the present study was to 2000 Academic Press
764 Z. J. Mudryk and P. Sko´ rczewski
Poland
ea
N
cS
ti al
B
channel st. 3
st. 2 River Lupawa st. 1
0
km
2
54°39' N
17°07' E
F 1. Lake Gardno, northern Poland, with location of sampling stations. Station 1 is near freshwater inflow, Station 2 is in mid-lake and Station 3 is near entrance to Baltic Sea.
Material and methods
saline water penetrate into the lake. Therefore, water of the lake, or its parts acquire sea water quality, resulting in a salinity of 2–5. Consistently, with the Venetian system Lake Gardno can be classified as mixo-oligohaline type (0·5–5·0 salinity) (Dethier, 1992).
Description of studied area
Sampling
The studies were carried out in an estuarine lake, Lake Gardno (Figure 1) situated in the World Biosphere Reserve, Słowin´ ski National Park (Poland). The lake is very shallow (1·3 m average depth) but covers a large area (2500 ha). The shallow depth and large area as well as the lack of shielding winds ensures the full mixing of water in both vertical and horizontal profiles. As a result, the lake can be regarded as a polymictic basin in which no thermal or oxygen stratification is observed. The emergent macroflora covers 4% of the surface of Lake Gardno forming offshore belt 20–200 m wide, which constitutes a residence for many bird species. The main species of macrophytes are: Typha angustifolia, Phragmites australis, Scirpus lacustris and Schoenoplectus lacustris. Lake Gardno is characterized by intermediate conditions between marine and inland environment. It is supplied by the water of the river Lupawa, and also via a 1·3 km channel by the Baltic Sea whose large volumes of
Water samples were taken in May, July and September during 1998 from three stations (Figure 1) which represented different environmental conditions: one near the river inflow (fresh water zone) (station 1), one near sea entrance (seawater zone) (station 3) and at one station in mid-lake (mixed zone) (station 2). Water samples for bacteriological analyses were taken from three layers. Film layer (FL) samples (thickness of 9017 m) were taken with a 3030 cm glass plate (Harvey & Burzell, 1974), surface microlayer (SM) samples (thickness of 24240 m) were collected with a 4050 cm Garrett net (24 mesh net of 2·54 cm length) (Garrett, 1965). The glass plate and polyethylene net were rinsed with ethyl alcohol and distilled sterile water prior to sampling. Water from subsurface layer (SUB) was taken with sterile glass pipettes at the depth of about 10–15 cm. The water samples were collected into sterile glass bottles and stored in an ice-box,
determine the contribution of the neustonic and planktonic bacteria to the process of biological transformation of lipids in a dynamic water ecosystem, an estuarine lake in this case.
Lipolytic bacterioneuston and bacterioplankton 765
where the temperature did not exceed 7 C until they were taken for analysis. The time between sample collection and performance of the analyses usually did not exceed 6–8 h. Bacteriological study To determine the number of heterotrophic neustonic (FL, SM) and planktonic (SUB) bacteria (CFU), the collected samples were diluted with sterile buffered water (pH 7·2) prepared according to Daubner (1967) and inoculated on iron-peptone agar medium (IPA) (Ferrer et al., 1963) using the spread method, in three replications. After 10 days incubation at 20 C, bacterial colonies were counted and results were recalculated per 1 cm3 water. Subsequently, about 50 bacterial colonies from each water layer were picked from the plates and were transferred to semiliquid IPA medium (5·0 g of agar per dm3 of medium). The cultures maintained in this medium after purity control were kept at 4 C until used for further investigation in order to determine ability of these bacteria to decompose different lipid compounds. For enumeration of the number of lipolytic bacteria, plating techniques were used. Water samples were vortex mixed, and then serial ten-fold dilutions were prepared with sterile buffered water to reach final concentrations of 10 3 and 10 4. Diluted samples were inoculated by the spread method in three parallel replicates on CPS medium (Jones, 1971) amended with of 10·0 cm3 of emulsified 50% tributyrin (Fluka). The plates were incubated at 20 C for 10 days. Colonies with clear zones around them were classified as lipolytic bacteria. In order to determine the ability of neustonic and planktonic bacteria to decompose lipid compounds substrates were chosen as representatives of fatty acids (Tween), triglycerides (tributyrin) and phospholipids (lecithin). Five Tweens (T-20, T-40, T-60, T-80, T-85) (Sigma) were tested as substrates for bacterial lipase. Tweens are water soluble high fatty acid esters of a polyoxyalkylene derivative of sorbitan; they differ only in the particular fatty acid component (Sierra, 1957). Tween 20 (polyoxethylene-sorbitian monolaurate, lauric acid approx. 50%) is a laurate acid ester, Tween 40 (polyoxethylene-sorbitian monopalmiate, palmitic acid approx. 90%) a palmitic acid ester, Tween 60 (polyoxethylene-sorbitian monostearate, stearic acid approx. 90%), is a stearic acid ester, Tween 80 (polyoxeethylene-sorbitian monooleate, oleic acid approx. 70%), an oleic acid ester and Tween 85 (polyoxethylene-sorbitian trioleate, oleic acid approx.
70%) is a trioleic acid ester. A peptone agar medium (Kjelleberg & Hakansson, 1977) was prepared with each of the Tweens. The Tweens were sterilized separately and then 0·1 cm3 of each Tween was added aseptically to 9 cm3 of molten peptone agar medium. The Tween was evenly dispersed through the medium by hand rolling prior to pouring on the plates. After incubation, the occurrence of opaque halos around the colonies was regarded as hydrolysis of Tween by bacteria. The capacity of bacteria to decompose tributyrin (glyceryltributyrate) was assayed in CPS medium (Jones, 1971) with the addition of 10·0 cm3 of emulsified 50% tributyrin. Colonies around which clear zones appeared were classified as able to synthesize extracellular tributyrinase. Lecithin (dipalmitoyl lecithin) (Fluka) breakdown was investigated in Menkina medium (Niewolak, 1980). Lecithin was prepared as follows: 1·0 g of lecithin from eggs (Fluka AG, Ch-9470 Busch) was dissolved in 2 cm3 ethyl alcohol and diluted with 18 cm3 of dissolved water, buffered with a saturated solution of sodium carbonate at pH 7·0 and three-fold steam sterilized for 40 min. In each Petri dish 0·1 cm3 of lecithin solution was introduced and Menkina medium was added and thoroughly mixed. Clear zones around the colonies were regarded as lecithin hydrolysed by bacteria. All media were adjusted to pH 7·0–7·2 and sterilized at 117 C for 20 min. As inoculum, 48–72 h cultures from slants of the IPA medium were applied. The results were recorded after 7 days of incubation at 20 C. Study on lipase activity In order to determine the lipolytic activities of the studied bacteria, strains that had clear zones around their colonies on media with tributyrin wider than 15 mm were taken. Lipolytic activity of nine bacterial strains (three strains from each station) from each water layer was determined. The selected strains of lipolytic bacteria were grown on slants of IPA medium for 72 h at 20 C. The bacteria were then rinsed from the slants with buffered water and the optical density of the culture suspension was adjusted to E=0·3 at the wavelength of 600 nm using a spectrophotometer SPECOL. The bacterial cultures were used as inocula to prepare cell-free post-culture media (CFPM). 0·5 cm3 of a given suspension was added to 25 cm3 of fresh liquid IPA medium containing 50% tributyrin. Cultures of each strain were grown in duplicate at 20 C on a shaker for 48 h. After incubation, a 5 cm3 aliquot was taken and spun down at 12 000g
766 Z. J. Mudryk and P. Sko´ rczewski T 1. Number of heterotrophic and lipolytic bacteria in brackish Lake Gardno Heterotrophic bacteria (104 cm 3)
Lipolytic bacteria (104 cm 3)
Season
Layer
mean
range
median
mean
range
median
Spring
FL SM SUB
8·8 7·6 1·9
(0·83–22·83) (2·27–18·00) (0·27–3·83)
2·67 2·63 1·60
0·9 (10)a 1·0 (13) 1·2 (63)
(0·08–2·30) (0·24–1·85) (0·27–2·80)
0·3 1·1 0·5
Summer
FL SM SUB
6·2 6·7 0·5
(0·20–17·00) (1·60–14·60) (0·07–0·90)
1·40 3·80 0·56
2·4 (39) 3·8 (57) 0·4 (80)
(0·40–4·00) (2·50–6·00) (0·05–0·80)
3·0 2·8 0·4
Autumn
FL SM SUB
3·2 2·6 2·3
(1·00–7·66) (1·05–6·30) (0·12–6·67)
1·16 5·33 0·25
2·4 (75) 2·3 (88) 1·7 (74)
(0·10–7·00) (0·35–6·00) (0·15–4·90)
0·3 0·7 0·2
a
Number of lipolytic bacteria in percentage.
for 30 min to remove bacterial cells. The CFPMs obtained in this way were the source of lipolytic enzymes. The activity of lipases synthesized by the studied bacteria was determined by the method developed by Cherry and Crandall (Mudryk, 1998). In a test tube 1 cm3 of a given CFPM was mixed with 1 cm3 of 0·3 M phosphate buffer, pH 7·0, and 2 cm3 of emulsified 50% tributyrin. All determinations were carried out in triplicate. Enzymatic reactions were performed for 24 h at 30 C. After incubation, the reactions were stopped by the addition of 3 cm3 of 95% ethanol. To each sample, two drops of 1% phenolphthalein in ethanol was added and the samples were titrated with 0·05 N NaOH. One unit of lipase activity (ULA) was defined as the amount of enzyme that, within 24 h at a temperature of 30 C, caused the liberation of the amount of fatty acids for which 1 cm3 of 0·05 N NaOH was needed to titrate. The effect of salinity on the level of lipolytic activity was determined using CFPMs and (50% emulsified tributyrin) substrate. Cell suspensions were obtained by culturing individual isolates at 20 C for 72 h using IPA medium supplemented with 0·5 1, 2, 4, 6 or 8‰ sodium chloride. The influence of salinity on the activity of lipases was determined by the method of Cherry and Crandall as described above. Results The data obtained on heterotrophic bacteria numbers in Lake Gardno are indicative of substantial spatial differences between number of these organisms inhabiting film and surface layers (FL, SM) and subsurface water (SUB) (Table 1). Bacteria living in the surface waters were more abundant than those
inhabiting the subsurface layer. Particularly great differences were recorded in summer when the bacterioneuston abundance was tenfold greater than of bacterioplankton. The results given in Table 1 indicate that lipolytic bacteria occurred in large numbers in the surface and subsurface water in Lake Gardno, making up 10–88% of the total number of culturable heterotrophic bacteria. The number of lipid–hydrolysing bacteria reached a maximum in summer and autumn and the number of lipolytic bacteria was lowest in spring. Lipolytic bacteria were more numerous in bacterioneuston than in bacterioplankton in summer and autumn. By contrast in spring, the number of lipolytic organisms among the neustonic and planktonic bacteria were almost identical. Figure 2 presents the results concerning the ability of bacteria isolated from surface and subsurface water to enzymatically depolymerize various lipids. It is evident from these data that there were no significant differences in the number of lipid decomposition between neustonic (FL, SM) and planktonic bacteria (SUB). On the other hand significant differences were found in the decomposition of individual lipids by bacteria. In all studied water layers, the highest percentage of bacterial strains were able to hydrolyse tributyrin and Tween 85. Bacteria able to hydrolyse Tween 60 and lecithin were present at lower percentages than the above-mentioned physiological groups. The microflora hydrolysing Tween 20 and Tween 40 represented the least numerous group of neustonic and planktonic bacteria. An identical phenomenon was noted on the investigated stations in Lake Gardno (Figure 3). The collection of strains was analysed for multiple– lipid decomposition (Figure 4). Most were able to decompose of 1 to 4 lipids. Only a small percentage of
Lipolytic bacterioneuston and bacterioplankton 767 90 80
T-20 T-40 T-60 T-80 T-85 Tributyrin Lecithin
Percentage of strains
70 60 50 40 30 20 10 0
SM
FL
SUB
F 2. Decomposition of lipids by bacteria inhabiting film (FL), surface (SM) and subsurface (SUB) water layers. Lipid substrates are representatives of fatty acids (Tweens, as T-20, T-40, T-60, T-80, T-85), triglycerides (tributyrin) and phospholipids (lecithin). Vertical bars indicate standard errors.
100 90 80
Percentage of strains
70 60 50 40 30 20 10 0
T-20
T-40
T-60
T-80
T-85
Tributyrin
Lecithin
F 3. Hydrolysis of lipids (as listed in Figure 2) by bacteria isolated from three sampling stations (see Figure 1). Vertical bars indicate standard errors. Station 1: open bars; Station 2: grey bars; Station 3: closed bars.
bacteria inhabiting surface and subsurface water in Lake Gardno were capable of decomposing all seven lipids studied. Multiple–lipid decomposition was
generally more common in neustonic than planktonic bacteria. About 8% of strains isolated from subsurface water did not decompose lipids at all.
768 Z. J. Mudryk and P. Sko´ rczewski
30%
Strains
20%
10%
0%
0
1
2
3 4 Number of lipids
5
6
7
F 4. Multiple–lipid (0–7) decomposition by bacterial strains inhabiting film (FL, open bars), surface (SM, grey bars) and subsurface (SUB, closed bars) water layers.
Figure 5 illustrates the activity of lipolytic enzymes synthesized by neustonic and planktonic bacteria. The average activity of the tributyrinase produced by bacterioplankton (SUB) was higher than bacterioneuston (FL, SM). There were significant differences in the activity levels of bacterial lipolytic enzymes between stations. The highest lipase production in all water layers could be observed in the central part of Lake Gardno (station 2). A minimum of lipolytic activity was recorded among bacterioneuston at station 1 close to the Lupawa river outflow into Lake Gardno. Among bacterioplankton, the least lipolytic activity was found with bacteria isolated from the zone of the channel connecting the lake to the Baltic Sea (station 3). Figure 6 presents data on the influence the salinity has on lipase activity of the bacteria isolated from various water layers and research stations. The data point to the fact that due to increasing salinity the activity of lipases was hampered more when synthesized by planktonic bacteria rather than by neustonic ones. There have also been revealed significant differences in the influence the salinity may have on bacterial lipases activity isolated from different sampling stations. At the station located at the Lupawa mouth into Lake Gardno (station 1), the bacteria were most
active at 0·5–2 salinity. Above that the bacterial lipolytic activity diminished and eventually at 8, that is at a salinity equal to that of Baltic Sea, complete lipase inactivation occurred. At station 2 located in midlake, the activity of lipases synthesized by neustonic bacteria stayed at a similarly high level within a wide range of salinity (0·5–6). However, among planktonic bacteria great lipase activity change was recorded along with the salinity change. In the channel joining Lake Gardno to the Baltic Sea (station 3), it was found that at a salinity equal to that of Baltic water (8) bacterial lipases synthesized by neustonic and planktonic bacteria were still active in terms of enzymes, which was not observed at other more fresh water stations. Discussion Data on the number of heterotrophic bacteria in Lake Gardno indicate that these organisms occur more frequently in the film layer and surface microlayer than subsurface water. These results fully confirm earlier observations carried out in Lake Gardno by Mudryk et al. (1999). Similarly, the investigations carried out in other water basins (Dahlba¨ ck et al., 1982; De Souza Lima & Chretiennot-Dinet, 1984;
Lipolytic bacterioneuston and bacterioplankton 769
st. 1 FL st. 2 st. 3
st. 1 SM st. 2 st. 3
st. 1 SUB st. 2 st. 3 0.0
0.1
0.2 –3 –1 Lipase units (cm day )
0.3
0.4
F 5. The activity of lipolytic enzymes synthesised by neustonic and planktonic bacteria, collected from film (FL), surface (SM) and subsurface (SUB) water layers from three stations (see Figure 1). Error bars indicate ranges of variability.
Mudryk et al., 1991) demonstrated that the greatest abundance of heterotrophic bacteria occurred in the surface layer, and decreased with the depth. Probably high concentrations of organic matter coming from extracted phyto- and zooplankton in surface water layer, provide optimal conditions for the development and accumulation of aerobic heterotrophic microflora forming bacterioneuston (Hoppe, 1986). Authors of the presented study are fully aware of fact that estimates of bacterial abundance based on plate count method represent only a minor fraction of bacteria inhabiting aquatic ecosystem. According to Nagata (1984) the percentage of viable count to the total bacterial numbers ranged from 0·009 to 1·8%. Lipolytic bacteria represent a dominant physiological group of microflora in estuaries and marine waters. Austin et al. (1977) in the estuarine Chesapeake Bay and Sieburth (1978) in the estuarine Narragansett found that 13–84% bacteria had lipolytic properties. In the seawater, bacteria synthesizing lipases constituted 70–100% of a total number of bacteria (Krstulovic´ & Solic´ , 1988; Mudryk et al., 1991; Martinez et al., 1996; Mudryk, 1998). Also in Lake Gardno lipolytic bacteria were a plentiful physiological group and constituted from 10 to 88% of the total number of culturable heterotrophic bacteria. In Lake Gardno lipolytic bacteria were more numerous in bacterioneuston than in bacterioplank-
ton in summer and autumn. Earlier studies on this physiological group of bacteria carried out in other basins (Kjelleberg & Hakansson, 1977; Rheinheimer, 1984; Mudryk et al., 1991) confirmed these results. According to Maki (1993) the occurrence of such high numbers of lipolytic bacteria in the surface layers of water can be explained by the fact that numerous lipids, like triglycerides, phospholipids, lipoproteins, free fatty acids, glycolipids, sterols and waxes, due to their amphiphatic properties accumulate at the water surface in an emulsified form and stimulate the optimal conditions for lipolytic bacteria growth. Studies conducted by Dahlba¨ ck et al. (1982) in the Arctic Ocean and Mudryk (1998) in the Gdan´ sk Deep, revealed that the number of lipolytic organisms among the neustonic and planktonic bacteria were almost identical. In the present study, a similar balance was found in Lake Gardno in spring. The latest research (Chro´ st & Gajewski, 1995; Wakeham, 1995; Martinez et al., 1996; Dachs et al., 1998) has pointed to the fact that lipolytic bacteria play a key role modifying and transforming processes in lipid compounds in water basins. Free fatty acids, triglycerides and phospholipids are the major components of the dissolved and particulate lipids in water basins (Parrish, 1988). The present findings suggest that neustonic and planktonic bacteria isolated from estuarine Lake
770 Z. J. Mudryk and P. Sko´ rczewski
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F 6. The influence of medium salinity on the lipase activity of lipolytic bacteria isolated from the film (FL, diamonds), surface (SM, squares) and subsurface (SUB, triangles) waters from three stations in Lake Gardno. For all curves, each point is the mean of three replicates. Vertical bars represent standard errors; where error bars are not visible they are too small to be illustrated.
Lipolytic bacterioneuston and bacterioplankton 771
Gardno were most active when hydrolysing triglycerides (tributyrin). On the other hand, they were least active with fatty acid esters. In water basins, triglycerides make up 25% of all lipids and their concentration is about 0·5 M C dm 3 (Kattner et al., 1983). These lipids originate mainly from organic matter decomposition and are synthesized in great amounts by phytoplankton and zooplankton (Arts et al., 1992). Intensity of decomposition of lipids in water basins usually is determined not only by the number of microorganisms capable of carrying out the decomposition, but also by the actual activity of their enzymes (Mudryk, 1998). The results of the study presented here indicate that there were differences in the activity level between lipases produced by bacterioneuston and bacterioplankton. Average activity of lipases produced by planktonic bacteria was higher than that of lipases synthesized by neustonic bacteria. Similar results were obtained by Sieburth (1971) and Kjelleberg and Hakansson (1977). Lower activity of lipases synthesized by estuarine neustonic bacteria could have been caused by the stressful effect of many environmental factors; mainly solar radiation and considerable fluctuations of water temperature and salinity. Also a relatively high accumulation of heavy metals, polychlorinated biophenyls and pesticides in the surface microlayer could have an inhibiting effect on the synthesis and activity of lipases (Maki, 1993). The rate of depolymerization of lipids depends not only on the concentration and activity of enzymes, but also on various environmental factors affecting the kinetics of enzymatic reactions (Karner & Rassoulzadegan, 1995). The results of a number of studies (Nitkowski et al., 1977; Meyer-Reil, 1991; Mudryk, 1998) indicate that salinity, besides temperature and pH, play an important role as a factor controlling the synthesis and activity of lipases of marine and estuarine bacteria. Meyer-Reil (1991) draws attention to the fact that hydrolytic enzymes can be active in a wide range of salinities. Lipases synthesized by the bacteria isolated from Lake Gardno were active in a range of salinity (0·5–8), although the level of activity depended on the water salinity. References Albers, C. S., Kattner, G. & Hagen, W. 1996 The compositions of wax esters, triacylglycerols and phospholipids in Arctic and Antarctic copepodes: evidence of energetic adaptations. Marine Chemistry 55, 347–358. Arts, M. T., Evans, M. S. & Robarts, R. D. 1992 Seasonal patterns of total and energy reserve lipids of dominant zooplanktonic crustaceans from a hyper-eutrophic lake. Oecologia 90, 560–571. Austin, C., Allen, S. D., Mills, A. & Colwell, R. 1977 Numerical taxonomy of heavy metal–tolerant bacteria isolated from an estuary. Canadian Journal of Microbiology 23, 1433–1447.
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