Hydrobiologia 342/343: 7178, 1997.

71

L. Kufel, A. Prejs & J. I. Rybak (eds), Shallow Lakes 95. c 1997 Kluwer Academic Publishers. Printed in Belgium.

The occurrence of heterotrophic bacteria decomposing some macromolecular compounds in shallow estuarine lakes
Zbigniew Mudryk1 & Wojciech Donderski2
1

Pedagogical University, Institute of Biology, Department of Experimental Biology, Arciszewskiego 22b, 76-200 Supsk, Poland 2 Nicolaus Copernicus University, Institute of Biology, Department of Water Microbiology and Biotechnology, Gagarina 9, 87-100 Toru n, Poland

Key words: estuary, planktonic bacteria, physiological properties, organic matter, decomposition

Abstract Planktonic bacteria participating in decomposition processes of organic macromolecular compounds were studied in shallow estuarine lakes ebsko, Gardno and Jamno.The abilities of heterotrophic bacteria to decompose proteins, lipids, starch, nucleic acids, pectin, chitin and cellulose were determined. Characteristic features among the bacteria in the lakes under study were to decompose a wide spectrum of organic macromolecular compounds. Most bacteria hydrolysed proteins, lipids and starch. Pectinolytic and chitinolytic bacteria were less numerous as well as microorganisms able to hydrolyse nucleic acids. The microora hydrolysing cellulose was represented by the least abundant group of organisms. Introduction Heterotrophic bacteria performing the processes of organic matter decomposition are the most numerous group of microorganisms in aquatic ecosystems (Donderski et al., 1984; Kim & Hoppe, 1984; Chr ost, 1991; Karner et al., 1992). These organisms are able to decompose a wide spectrum of organic compounds whose molecules differ in size from monomers to polymeres (Jacobsen & Azam, 1984; Quemeneur & Morty, 1992). Microbial hydrolysis of organic matter plays an important role in substrate turnover in aquatic environments (Kim & Hoppe, 1984). Most of the organic compounds produced in natural waters have a polymeric structure (Somville, 1984; M unster & Chr ost, 1990). They cannot be directly taken by bacteria and they must be hydrolysed through the action of exo and endoenzymes and converted into monomeric low molecular substrates (Billen & Fontigny, 1987; MeyerReil, 1987).These are rapidly assimilated by bacteria (Chr ost, 1991). As there are differences between various types of water bodies in the composition and content of organic matter, the intensity of biodegradation by bacterial microora differs as a function of the biochemical activity of the dominant physiological groups (Godlewska-Lipowa, 1974; Krstulovi c & Soli c, 1988). The present study was aimed to investigate physiological properties of bacteria in the water of estuarine lakes, their ability to carry out particular metabolic processes and to evaluate their potential possibility in degradation of certain organic macromolecular compounds.

Study area Lakes ebsko, Gardno and Jamno are situated close to the Polish Baltic Sea coast (Figure 1). Two lakes ebsko and Gardno are situated in the World Biosphere Reserve the Sowinski National Park. The investigated estuarine lakes are very shallow with an avarage depth of 1.5 m and cover large areas, ca. 2070 km2 each. They are 716 km long and 38 km wide. The emergent macroora covers 39% of their surfaces forming a wide offshore belt of 20200 m, which con-

72 stitutes a residence for many bird species. Main species of plants are Typha angustifolia, Phragmites australis and Schoenoplectus lacustris. These lakes are supported by rivers and, at the same time, they are inuenced by Baltic Sea waters. Through narrow channel, large volumes of sea water ow into the lakes, especially in autumn and winter months. Therefore, water of these lakes often equales in salinity to that of Baltic Sea. Consistently with the venetian system, these lakes can be classied as the mixo-oligohaline type (Dethier, 1992). Lakes ebsko and Gardno are of eutrophic type, Lake Jamno is a hypereutrophic reservoir. Some physicochemical properties of water in the lakes under study are shown in Table 1 (Trojanowski et al., 1990). 1. The ability to decompose proteins, starch and lipids were assayed in IPA medium enriched with either gelatin (20 g per dm3 ) or starch (5 g per dm3 ) or tributyrin (10 ml per dm3 ). The plates were point inoculated. The ability to hydrolyse proteins was tested using Frazier reagent (Weyland et al., 1970). Amylolytic bacteria were determined using Lugols solution (Seiler et al., 1980). Lipolytic properties of the bacteria were determined on the basis of clear zones formation around bacterial colonies (Lawrence et al., 1967). 2. The ability to decompose deoxyribonucleic acid (DNA) was assayed in Oxoid medium (England) containing 2.0 g DNA per dm3 of medium. Doublelayer plates were used: DNA was added to the upper layer of medium. Hydrolysis of DNA was disclosed by pouring 1 N HCl over the plates. Clear zones around the colonies pointed to the DNA decomposition ability (Seiler et al., 1980). 3. Hydrolysis of ribonucleic acid (RNA) was determined according to Jeffries et al., (1957). The decomposition of RNA was revealed as in item 2. 4. Pectinolytic bacteria were assayed in medium prepared according to Jayasankar & Graham (1970). After incubation a 1% solution of Cetrimide (Searle Co., England) was poured over the medium. The occurrence of clear zones was accepted as a positive result. 5. Chitin hydrolysis was estimated in medium prepared according to Helmke & Weyland (1986). Colloidal chitin was prepared according to Lingappa & Lockwood (1962), using chitin from Windsor, Berkshire (England). The appearance of clear zones was accepted as a positive result. 6. The ability to decompose cellulose was investigated on a medium composed of KNO3 1.0 g, K2 HPO4 1.0 g, MgSO4
7H2 O 0.2 g, CaCl2 2H2 O 0.1 g, FeCl3 0.01 g, colloidal cellulose 7.0 g of dry mass, agar 15.0 g, tap water 1 dm3 . Colloidal cellulose was prepared from Cellulose Powder 11, Whatman according to Halliwell (1962). Double layer plates were used. The occurrence of clear zones was accepted as a positive result. pH of all media were adjusted to 7.07.4 and were sterilized at 117  C for 20 min. 4872 hours cultures from slants on the IPA medium were applied as inoculum. Results were checked after 6-day incubation at 26  C, with the exception of pectin, chitin and cellulose, where the decomposition was determined after 14-day incubation. Degree of proteolytic, lipolytic and

Materials and methods Microbiological studies were carried out during the period 19861989. In each lake there were six stations: one near the river inow (station 1), one in the near-sea part (station 6) and mid-lake stations (25) (Figure 1). All stations were situated in open water. Samples were taken four times during the year: in March, May, July and October. Water samples were taken directly into sterile bottles from the surface (ca. 20 cm below water surface). Samples were placed in a container with ice and transported to the laboratory. The time between sample collection and performance of the analyses usually did not exceed 68 h. For determining the total number of heterotrophic bacteria (CFU), the samples of water were diluted with sterile buffered water (pH 7.2) (Daubner, 1967) and inoculated by the spread plate method, in ve replicates, on iron-peptone agar medium (IPA) prepared according to Ferrer et al. (1963). After 10 day incubation at 20  C, bacterial colonies were counted and results were recalculated per 1 cm3 of water. Then, from the whole surface of the plates or from a particular sector ca. 50 bacterial colonies, were collected at random from each station and transferred to semiliquid IPA medium. After purity control, the bacteria were stored at 4  C, with inoculation on fresh medium every 3 months and, subsequently they were used for further studies. The isolated bacteria were inoculated on several test media containing various organic compounds. The following decomposition abilities of bacteria were tested:

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Figure 1. Location of the ebsko, Gardno and Jamno Lakes. Table 1. Mean values of selected physiochemical parameters of the water in lakes ebsko, Gardno and Jamno in the period 19871989. (data by Trojanowski et al., 1990) Dimension Lake ebsko

Parameter Transparency Temperature pH Oxygen BOD5 N - total P - total Salinity Total suspended matter Chlorophyll a Primary production

Lake Gardno 0.35 14.7 9.0 11.5 6.3 3.62 0.370 510 52 85.3 236 (0.101.10) (0.523.4) (7.79.7) (8.712.8) (4.38.4) (1.515.94) (0.1450.600) (302090) (2869) (183241)

Lake Jamno 0.15 13.8 9.1 7.7 9.0 5.16 0.414 550 75 120.4 881 (0.100.25) (0.122.7) (7.210.0) (1.610.9) (6.010.5) (3.826.48) (0.2350.691) (492191) (36104) (740961)

C
mg O2 dm 3 mg O2 dm 3 mg N dm 3 mg P dm 3 mg Cl dm 3 mg dm 3 mg m 3 mg C m 2 h

0.57 13.3 8.8 11.8 5.1 3.46 0.256 542 41 68.5 220

(0.200.25) (7.023.0) (7.0-10.2) (8.3-12.5) (2.66.8) (1.056.24) (0.1710.393) (212462) (55 - 61) (214229)

amylolytic activity of bacteria was determined by measured diameter clear zones around the colonies according to Lawrence et al. (1967), Strzelczyk et al. (1976).

Results In general the results obtained suggest that abundance of heterotrophic bacteria depended on the degree of trophy of the lakes and revealed considerable seasonal

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Table 2. Occurrence of bacteria able to decompose selected macromolecules in water of three estuarine lakes (average all stations) Lake Date of sampling 08.05.86 03.07.86 09.10.86 25.03.87 20.05.87 24.07.87 14.10.87 16.03.88 11.05.89 13.07.89 12.10.89 09.03.89 Number of strains studied 285 285 287 289 296 290 282 284 289 291 290 239 % of bacteria decomposing indicated substrates protein lipid starch DNA RNA pectin 51 75 76 45 52 53 60 38 73 79 74 54 82 84 85 61 46 61 49 42 67 61 85 63 53 52 50 50 46 33 35 29 55 51 63 48 22 17 33 23 29 24 28 31 12 14 14 9 14 21 15 7 21 30 38 27 7 11 10 6 16 21 25 23 20 25 27 28 11 13 13 10

chitin 12 10 14 5 32 26 43 23 5 10 4 5

cellulose 2 5 8 2 10 6 15 8 0 2 4 1

Gardno

ebsko

Jamno

variations (Figure 2). On the whole the investigations showed that planktonic bacteria occurred in the largest number in the hypereutrophic Lake Jamno and they were the least numerous in the eutrophic lakes ebsko and Gardno. Figure 2 showed that in all three lakes the greatest number of bacteria in water appeared in summer, reaching its minimum in winter. This regularity was noticed during three years of investigation. The occurrence of bacteria with particular physiological properties among the microorganisms isolated from the water of the lakes ebsko, Gardno and Jamno are presented in Table 2. It is evident from these data that most strains showed the ability to decompose different macromolecular compounds. Organisms hydrolysing proteins and lipids were the most numerous group among the strains studied (Table 2). Proteolytic bacteria constituted 3879%, lipolytic bacteria accounted for 4285% of the total number of heterotrophic bacteria. The most rapid development of bacteria hydrolysing proteins and lipids occurred in summer and autumn. Both these physiological groups usually showed the lowest percentage in winter, in all studies lakes. The organisms capable of performing the process of starch hydrolysis also were rather numerous The largest numbers of amylolytic organisms were found in Lake Jamno, the smallest in Lake ebsko. Their maximum development was usually noted in spring and autumn. Bacteria able to hydrolyse DNA and RNA were present at lower percentages than the above-mentioned physiological groups (Table 2) and

they were more abundant in Lakes ebsko and Gardno than in Lake Jamno. In Lakes ebsko and Gardno throughout the investigation period, pectinolytic and chitinolytic organisms showed a higher percentage of occurrence than in the Lake Jamno were they only made up to 413% of the total amount of heterotrophic bacteria. The microora hydrolysing cellulose represented the least numerous physiological group of organisms isolated from three estuarine lakes. They were somewhat more abundant in the Lake ebsko where they constituted from 6% to 15% of the total number of bacteria. No regular pattern in horizontal distribution of studied physiological groups were established (Figure 3). In all parts of investigated lakes the abundance of planktonic bacteria with ability to decompose organic macromolecular compounds showed a similar level with the exception for lipolytic bacteria which showed maximum density in seawater zone (st. 6). Figure 4 illustrated the degree of proteolytic, lipolytic and amylolytic activity of bacteria isolated in the highest percentage from water of lakes ebsko, Gardno and Jamno. About 3040% of strains have high proteolytic and lipolytic activity. The amylolytic activity of planktonic bacteria was the lowest.

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Figure 2. Seasonal uctuations of heterotrophic bacteria

Figure 3. Spatial distribution of the studied bacterial physiological groups.

76 Discussion Aquatic ecosystems are inhabited by bacteria able to carry out various metabolic processes which facilitates matter and energy transformation (Riemann, 1983; Donderski & Lalke, 1993). Due to their abilities, heterotrophic bacteria participate in the decomposition of dead bodies of plants and animals or their remains (Donderski & Stopinski, 1993). A considerable part of heterotrophic bacteria inhabiting water of studied lakes was capable to hydrolyse different macromolecular compounds being a source of food and energy for them. In general proteolytic and lipolytic bacteria were numerous among the studied physiological groups. The occurrence of large quantities of those bacteria in other estuarine reservoirs has also been observed earlier by Sieburth (1978); Austin et al. (1977); Hoshimoto et al. (1983); Ellis-Evans (1985). According to Little et al. (1979), the protein decomposing bacteria occur in such a great number because in water bodies proteins, peptides and amino acids are the main components of organic matter. Excretions and dead bodies of the phyto and zooplankton are their basic source (Billen & Fontigny, 1987). Hoppe et al. (1988) reported that bacteria of brackish waters display a higher metabolic activity against proteins and amino acids than against carbohydrates. In examined lakes proteolytic activity was the lowest in winter period, probably on account of low temperatures; namely according to Little et al. (1979), temperatures below 15  C abruptly inhibit the activity and synthesis of proteases. From our investigations it follows that lipolytic organisms also occurred in great number among bacterioplankton. Studies carried out in other brackish waters conrm the presence of a large number of lipolytic bacteria. Sieburth (1978) found in the estuarine reservoir Narragansett 1384% of lipolytic bacteria and Austin et al. (1977) in the Cheaspeake Bay more than 50%. In the sea water lipolytic bacteria constituted 80100% of the total number of microora (B olter & Rheinheimer, 1987; Prieur, 1989; Mudryk et al., 1991). Kjelleberg & Hakansson (1977) and Quemeneur & Morty (1992) explain this phenomenon by the accumulation of many lipids on water surface, like the triglycerides, phospolipides, free fatty acids, lipoproteins or wax esters, whose presence creates optimal conditions for the development of lipolytic bacteria. Particularly large amounts of lipids are accumulated in the cells of bluegreen algae, green algae as well as Copepoda in the

Figure 4. Degree of proteolytic, lipolytic and amylolytic activity of planktonic bacteria.

77 case zooplankton (Jacobsen & Azam, 1984; Arts et al., 1992). According to Nitkowski et al. (1977) a rise of salinity of the aquatic environment leads to an increase in the lipase activity. The present results conrm this regularity. Namely, in the water of investigated lakes the percentage of lipolytic bacteria was the highest in seawater zone where the salinity was higher than that at the remaining stations. Amylolytic bacteria represent a relatively numerous group of heterotrophic bacteria in the investigated estuarine reservoirs. They accounted for ca 40% of the total numbers of planktonic microora. Similar results were observed earlier in other lakes by Ellis-Evans (1985); Sugita et al. (1987); Donderski & Stopi nski (1993). The results of the present work point out that organisms capable to hydrolyse starch occur in the highest numbers during spring and autumn. This corresponds with the results obtained by Krstulovi c & Soli c (1988) in the Kastela Bay. Degradation of nucleic acids from decaying plant and animal tissues is the essential function of heterotrophic bacteria. As a result, bacteria able to decompose DNA and RNA are numerous both in inland basins and in the marine environment (Niewolak, 1980; Strzelczyk et al., 1972; Maeda & Taga, 1974; Stewart et al., 1991). On the other hand, in our investigations these organisms were present in lower percentage than other physiological groups of bacteria. The ability of many bacteria to hydrolyse pectin and chitin and the high activity of their enzymes indicate the considerable importance of these groups of organisms for the processes of plant and animal remains decomposition in aquatic ecosystems. Occurrence of large number of chitinolytic bacteria in lakes Lebsko and Gardno can be accounted for by rapid mass growth of zooplankton (1000 ind. cm3 ) mainly Clado_ cera and Copepoda (Zmudzi nski et al., 1992). Their shells may stimulate the development of chitinoclastic bacteria. Relatively small number of bacteria decomposing pectin and chitin in the water of hypereutrophic Lake Jamno conrms earlier studies by Mudryk, (1994). This is probably related to its content of very diverse food substances, many of which may be metabolized more easily and more readily than chitin and pectin. The least numerous group among planktonic bacteria in studied lakes were microorganisms decomposing cellulose. The results agree with those published earlier by other authors (B olter, 1977; Sugita et al., 1987; Petrycka et al., 1990). References
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