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Salinity-tolerant nitrogen-fixing Enterobacteriaceae in the lune estuary
Zbl. Bakt. Hyg., I. Abt, Orig. C 3, 513-518 (1982) Department of Biological Sciences, University of Lancaster, Lancaster LAI 4YQ, United Kingdom
Salinity-tolerant Nitrogen-fixing Enterobacteriaceae in the Lune Estuary KEITH JONES Received February 15, 1982
Summary Nitrogen-fixing strains of the Enterobacteriaceae are the most numerous nitrogen-fixing bacteria in the waters and sediments of the Lune estuary. However, their contribution to the nitrogen status of the sediments has been questioned as they have been shown to be intolerant of sea-water salinities. Results presented here show that salinity-tolerant strains of Klebsiella pneumoniae are common and are capable of fixing nitrogen (acetylene reduction) in the sediments. Key words: Nitrogen-fixation - Acetylene reduction - Enterobacteriaceae - Klebsiella pneumoniae - Salinity Introduction Nitrogen fixation (acetylene reduction) due to heterotrophic bacteria has been demonstrated in the intertidal sediments of the Lune estuary, England (Jones, 1982). A range of nitrogen-fixing bacteria has been isolated but the only groups present in large numbers are the sulphate-reducing bacteria and the Enterobacteriaceae. The latter are the most numerous but earlier reports (Herbert, 1975; Fogg, 1978) have indicated that while nitrogen-fixing Enterobacteriaceae are readily isolated from marine sediments in British waters they do not fix nitrogen there because they are inhibited by the salinity of sea-water (3.5 gl-l). This paper reports an investigation into the distribution and identity of nitrogen-fixing Enterobacteriaceae in the estuary of the river Lune and assesses their ability to fix nitrogen at the prevailing salinities. Methods Sediment and water samples were collected from the Lune estuary near Lancaster, U.K (Nat. Grid. Ref. SD428543) (see Jones, 1982).
514
K.Jones
Nitrogen-fixing Enterobacteriaceae were enumerated and isolated using Most Probably Number (MPN) methodology. Samples of sediment (10 g) were mixed with Ringers solution and the volume made up to 100 mI. The resulting suspension was stirred for 20 min and serially diluted. 1 ml aliquots from decade dilutions were inoculated into 3 screwcapped Universal bottles (28 rnl) containing 10 ml nitrogen-free glucose broth. The atmosphere in the bottles was replaced with nitrogen and they were incubated at 25°C. After 5 d 1.5 ml acetylene was added to each of the bottles and, after a further 2 h incubation, 1 ml samples of the bottle atmosphere were removed and analysed for ethylene production using a Varian Aerograph gas chromatograph (the column was 2.74 m in length and 2.5 mm in diameter, contained Poropak R and was run with an oven temperature of 50°C with nitrogen as the carrier gas at a flow rate of 30 ml min"), Positive results for acetylene reduction were taken as positive for nitrogen fixation (Stewart et aI., 1967). Inocula from bottles positive for acetylene reduction were grown aerobically in McConkey bile salts broth and on McConkey agar plates prior to re-testing for acetylene reduction in nitrogen-free glucose medium under anaerobic conditions. MPN tables were consulted (Collins, 1967) and the results expressed as the number of nitrogen-fixing Enterobacteriaceae g-l dry sediment. MPN estimations of river and sea-water samples were carried out similarly but with dilutions made directly from water samples. Standard presumptive coliform counts were carried out using McConkey broth as the medium with acid and gas as the measure of growth (El-Abagy et aI., 1980). Nitrogen-fixing Enterobacteriaceae were isolated from positive acetylene reduction MPN assay bottles and cultured aerobically on McConkey broth plates. Pure cultures were identified using the API 20E analytical profile index (API Laboratory Products Ltd., Basingstoke, U.K.).
Tests for the effects of salinity on acetylene reduction by strains of Klebsiella pneumoniae were done in media prepared from artificial sea-water (Provasoli et al., 1957). Aliquots (0.1 rnl) of bacterial suspension in McConkey broth were added to Universal bottles containing nitrogen-free glucose broth (10 rnl) at salinities ranging from 0 to 5 g salts/I. The atmosphere was replaced with nitrogen gas and the bottles incubated at 25°C. After 1 and 4 d triplicate bottles from each salinity were tested for acetylene reduction.
Results
Seasonal variation in nitrogen-fixing Enterobacteriaceae in the Lune estuary The results presented in Table 1 show that nitrogen-fixing Enterobacteriaceae are present in considerable numbers in the sediments, sea-water and river water throughout the year. The numbers obtained using acetylene reduction as the measure of growth in MPN experiments ranged between 10 and 100% of those for presumptive coliform counts using acid and gas as the measure.
Variation with sediment depth The highest numbers of nitrogen-fixing Enterobacteriaceae are found in the surface layers of the sediments with the number at a depth of 10 em only 2% of that at the surface (Table 2).
Identity of the nitrogen-fixing Enterobacteriaceae Isolates of bacteria from positive MPN bottles were obtained in pure culture by growing them alternatively in nitrogen-free glucose broth under nitrogen and on plates of McConkey agar in air. They were identified using the API computer-assisted
Salinity-tolerant Nitrogen-fixing Enterobacteriaceae
515
Table 1. Seasonal variation in the numbers of nitrogen-fixing Enterobacteriaceae in the Lune estuary
Month
Sediments (g-l dry wt)
MPN Bacteria River (Low tide) Sea-water (High tide) (100 ml-1 ) (100 ml- 1 )
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
1489 3648 4741 16044 14530 16320 2689 14918 1502 829 8665 4028
1100 1100* 1100" 1100* 1100 1100 1100" 1100* 1100* 1100* 1100" 1100
1100 1100 1100* 1100 460 240 1100" 240 1100 1100* 1100" 1100
* All dilutions positive for acetylene reduction therefore the number is above 1100/100 mL
Table 2. Occurrence of nitrogen-fixing Enterobacteriaceae at different depths in estuarine sediments
Depth (ern)
MPN Bacteria (g-l dry sediment)
0-1 4-5 9-10 19-20
14530 3407 308 350
method. The predominant species was Klebsiella pneumoniae with occasional isolates of Enterobacter agglomerans and Citrobacter freundii. Effects of salinity
MPN estimates on the sediments using media of differing salinities show that the highest numbers are obtained using media at low salinity and that there is a substantial population able to reduce acetylene and grow at 3 gil and 4 gil (Table 3) which are the salinities of the sea-water in the Lune estuary and surface sediments, respectively. However, many of the bacteria isolated from MPN bottles at low salinities are able to grow at sea-water salinities. For example, in one MPN experiment using low salinity media, inocula from each positive bottle (18 in all) were grown aerobically on McConkey agar and retested for acetylene reduction in nitrogen-free glucose broth at 0 gil and 3.5 gil. All the cultures were positive for acetylene reduction at both salinities. This is not always the case especially when the species isolated is not K. pneumoniae. 6 strains of K. pneumoniae, 4 from the river and 2 from the sediments, were tested
516
K.Jones
Table 3. Effects of the salinity of media on the enumeration of nitrogen-fixing Enterobacteriaceae from sediments
Salinity (gIl)
MPN bacteria (g-l dry sediment)
o
14,779 3,142 5,269 3,675
1.5 3.0 4.0
6
Acetylene reduction
(pgC2 H4
7
culture-1 6 hour-1 )
5 Day 4
4
o
1
~
~
~
M
~
~
~
~
Salinity (g/Il
Fig. 1. The effects of salinity on acetylene reduction by Klebsiella pneumoniae isolated from marine sediments.
for their ability to reduce acetylene at varying levels of salinity. The results were similar in all cases and are typified by those in Fig. 1. Initially acetylene reduction at the higher salinities was less than at low salinities but after a period of adaptation substantial activity occurred at the higher levels. Discussion Nitrogen-fixing (acetylene-reducing) Enterobacteriaceae are found throughout the Lune estuary in considerable numbers (Table 1). They occur both in the fresh
Salinity-tolerant Nitrogen-fixing Enterobacteriaceae
517
water reaches of the river Lune and in the sea-water at high tide. In the sediments they predominate in the surface layer (Table 2). Numbers in the sediments are generally higher in the spring and summer months but there is not an exact correlation with nitrogen fixation rates in the sediments (Jones, 1982). The bacteria enter the estuary from two main sources. The river Lune is contaminated with run-off from land in the fresh water reaches and the estuarine section is dosed directly with sewage. Lancaster sewage, apart from maceration for cosmetic reasons, is virtually untreated and is discharged into the river Lune 15 min after high tide. MPN experiments carried out at the sewage outfall give the number of nitrogen-fixing Enterobacteriaceae as 11,000 bacteria per ml water which is 100 times that for the river above the outfall. These bacteria are mainly lost into the Irish Sea due to the scouring action of the tide but some may reach the sediments. Nitrogen-fixing Enterobacteriaceae could only be isolated using the following sequence of media and conditions: anaerobic growth in nitrogen-free glucose broth under nitrogen followed by aerobic growth on McConkey agar plates in air. Nitrogen-fixing strains could not be isolated if the medium first used was McConkey broth. This is broadly in agreement with results obtained by Wright and Weaver (1981) working with forage grass roots. They obtained many fewer nitrogen-fixing colonies when McConkey medium rather than nitrogen-free medium was first used to isolate Enterobacteriaceae. The predominant nitrogen-fixing species of the Enterobacteriaceae in the waters and the sediments is Klebsiella pneumoniae. Herbert (1975), who carried out a study on marine sediments off the coast of Scotland, also isolated nitrogen-fixing strains of K. pneumoniae. However, he found that they were of fresh water origin and were unable to grow at the salinity of sea-water. Nitrogen fixation has not been detected in the water column of the Lune estuary (Jones, 1982) but it has been quantified for the sediments where the salinity of the surface layers can be as high as 4.1 gil. Therefore if K. pneumoniae is a contributor of fixed nitrogen it should be able to grow at this salinity. Most isolates from MPN experiments on the sediments, whether from low or high salinity media, are able to reduce acetylene at seawater salinities (3.5 gil) and 6 strains of K. pneumoniae tested for growth in a range of salinities could reduce acetylene at 5 gil after a period of adaptation (Fig. 1). It seems likely, therefore, that salinity is not a major factor in limiting nitrogen fixation by K. pneumoniae in estuarine sediments although it may cause a slow down in the rate of growth. Werner et al. (1974) have isolated nitrogen-fixing Enterobacteriaceae from marine habitats in North America which reduced acetylene at salinities 66% that of sea-water. Acknowledgements. I wish to thank Mr.]. V.Davies for help with the preparation of
media.
References El-Abagy, M. M., El-Zanialy, H. T., EI-Hawaary, S.: Direct MPN for faecal coliform. Zbl.
Bakt., II. Abt. 135, 396-401 (1980)
Collins, C.H.: Microbial methods, 2nd Ed. London, Butterworth 1967 Fogg, G.E.: Nitrogen fixation in the oceans. Ecol. Bull. (Stockh.) 26, 11-29 (1978) Herbert, R. A.: Heterotrophic nitrogen fixation in shallow estuarine sediments. J. Exp. Mar.
BioI. 18, 215-225 (1975)
518
K.Jones
Jones, K.: Nitrogen fixation in the temperate estuarine intertidal sediments of the river Lune. Limno!' Oceanogr. 27, 455-460 (1982) Prouasoli, L., McLaughlin, j., Droop, D. R.: The development of artificial media for marine algae. Arch. Microbiol. 25, 392-428 (1957) Stewart, W.D.P., Fitzgerald, G.P., Burris, R.H.: In situ studies of nitrogen fixation using the acetylene reduction technique. Proc. nat. Acad. Sci. (Wash.) 58,2071-2078 (1967) Werner, D., Evans, H.j., Seidler, R.J.: Facultatively anaerobic nitrogen-fixing bacteria from the marine environment. Canad. J. Microbiol. 20, 59-64 (1974) Wright, S.F., Weaver, R. W.: Enumeration and identification of nitrogen-fixing bacteria from forage grass roots. Appl, Environ. Microbiol. 22, 97-101 (1981) Dr. Keith Jones, Dept. of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, U.K.
Salinity-tolerant Nitrogen-fixing Enterobacteriaceae in the Lune Estuary KEITH JONES Received February 15, 1982
Summary Nitrogen-fixing strains of the Enterobacteriaceae are the most numerous nitrogen-fixing bacteria in the waters and sediments of the Lune estuary. However, their contribution to the nitrogen status of the sediments has been questioned as they have been shown to be intolerant of sea-water salinities. Results presented here show that salinity-tolerant strains of Klebsiella pneumoniae are common and are capable of fixing nitrogen (acetylene reduction) in the sediments. Key words: Nitrogen-fixation - Acetylene reduction - Enterobacteriaceae - Klebsiella pneumoniae - Salinity Introduction Nitrogen fixation (acetylene reduction) due to heterotrophic bacteria has been demonstrated in the intertidal sediments of the Lune estuary, England (Jones, 1982). A range of nitrogen-fixing bacteria has been isolated but the only groups present in large numbers are the sulphate-reducing bacteria and the Enterobacteriaceae. The latter are the most numerous but earlier reports (Herbert, 1975; Fogg, 1978) have indicated that while nitrogen-fixing Enterobacteriaceae are readily isolated from marine sediments in British waters they do not fix nitrogen there because they are inhibited by the salinity of sea-water (3.5 gl-l). This paper reports an investigation into the distribution and identity of nitrogen-fixing Enterobacteriaceae in the estuary of the river Lune and assesses their ability to fix nitrogen at the prevailing salinities. Methods Sediment and water samples were collected from the Lune estuary near Lancaster, U.K (Nat. Grid. Ref. SD428543) (see Jones, 1982).
514
K.Jones
Nitrogen-fixing Enterobacteriaceae were enumerated and isolated using Most Probably Number (MPN) methodology. Samples of sediment (10 g) were mixed with Ringers solution and the volume made up to 100 mI. The resulting suspension was stirred for 20 min and serially diluted. 1 ml aliquots from decade dilutions were inoculated into 3 screwcapped Universal bottles (28 rnl) containing 10 ml nitrogen-free glucose broth. The atmosphere in the bottles was replaced with nitrogen and they were incubated at 25°C. After 5 d 1.5 ml acetylene was added to each of the bottles and, after a further 2 h incubation, 1 ml samples of the bottle atmosphere were removed and analysed for ethylene production using a Varian Aerograph gas chromatograph (the column was 2.74 m in length and 2.5 mm in diameter, contained Poropak R and was run with an oven temperature of 50°C with nitrogen as the carrier gas at a flow rate of 30 ml min"), Positive results for acetylene reduction were taken as positive for nitrogen fixation (Stewart et aI., 1967). Inocula from bottles positive for acetylene reduction were grown aerobically in McConkey bile salts broth and on McConkey agar plates prior to re-testing for acetylene reduction in nitrogen-free glucose medium under anaerobic conditions. MPN tables were consulted (Collins, 1967) and the results expressed as the number of nitrogen-fixing Enterobacteriaceae g-l dry sediment. MPN estimations of river and sea-water samples were carried out similarly but with dilutions made directly from water samples. Standard presumptive coliform counts were carried out using McConkey broth as the medium with acid and gas as the measure of growth (El-Abagy et aI., 1980). Nitrogen-fixing Enterobacteriaceae were isolated from positive acetylene reduction MPN assay bottles and cultured aerobically on McConkey broth plates. Pure cultures were identified using the API 20E analytical profile index (API Laboratory Products Ltd., Basingstoke, U.K.).
Tests for the effects of salinity on acetylene reduction by strains of Klebsiella pneumoniae were done in media prepared from artificial sea-water (Provasoli et al., 1957). Aliquots (0.1 rnl) of bacterial suspension in McConkey broth were added to Universal bottles containing nitrogen-free glucose broth (10 rnl) at salinities ranging from 0 to 5 g salts/I. The atmosphere was replaced with nitrogen gas and the bottles incubated at 25°C. After 1 and 4 d triplicate bottles from each salinity were tested for acetylene reduction.
Results
Seasonal variation in nitrogen-fixing Enterobacteriaceae in the Lune estuary The results presented in Table 1 show that nitrogen-fixing Enterobacteriaceae are present in considerable numbers in the sediments, sea-water and river water throughout the year. The numbers obtained using acetylene reduction as the measure of growth in MPN experiments ranged between 10 and 100% of those for presumptive coliform counts using acid and gas as the measure.
Variation with sediment depth The highest numbers of nitrogen-fixing Enterobacteriaceae are found in the surface layers of the sediments with the number at a depth of 10 em only 2% of that at the surface (Table 2).
Identity of the nitrogen-fixing Enterobacteriaceae Isolates of bacteria from positive MPN bottles were obtained in pure culture by growing them alternatively in nitrogen-free glucose broth under nitrogen and on plates of McConkey agar in air. They were identified using the API computer-assisted
Salinity-tolerant Nitrogen-fixing Enterobacteriaceae
515
Table 1. Seasonal variation in the numbers of nitrogen-fixing Enterobacteriaceae in the Lune estuary
Month
Sediments (g-l dry wt)
MPN Bacteria River (Low tide) Sea-water (High tide) (100 ml-1 ) (100 ml- 1 )
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
1489 3648 4741 16044 14530 16320 2689 14918 1502 829 8665 4028
1100 1100* 1100" 1100* 1100 1100 1100" 1100* 1100* 1100* 1100" 1100
1100 1100 1100* 1100 460 240 1100" 240 1100 1100* 1100" 1100
* All dilutions positive for acetylene reduction therefore the number is above 1100/100 mL
Table 2. Occurrence of nitrogen-fixing Enterobacteriaceae at different depths in estuarine sediments
Depth (ern)
MPN Bacteria (g-l dry sediment)
0-1 4-5 9-10 19-20
14530 3407 308 350
method. The predominant species was Klebsiella pneumoniae with occasional isolates of Enterobacter agglomerans and Citrobacter freundii. Effects of salinity
MPN estimates on the sediments using media of differing salinities show that the highest numbers are obtained using media at low salinity and that there is a substantial population able to reduce acetylene and grow at 3 gil and 4 gil (Table 3) which are the salinities of the sea-water in the Lune estuary and surface sediments, respectively. However, many of the bacteria isolated from MPN bottles at low salinities are able to grow at sea-water salinities. For example, in one MPN experiment using low salinity media, inocula from each positive bottle (18 in all) were grown aerobically on McConkey agar and retested for acetylene reduction in nitrogen-free glucose broth at 0 gil and 3.5 gil. All the cultures were positive for acetylene reduction at both salinities. This is not always the case especially when the species isolated is not K. pneumoniae. 6 strains of K. pneumoniae, 4 from the river and 2 from the sediments, were tested
516
K.Jones
Table 3. Effects of the salinity of media on the enumeration of nitrogen-fixing Enterobacteriaceae from sediments
Salinity (gIl)
MPN bacteria (g-l dry sediment)
o
14,779 3,142 5,269 3,675
1.5 3.0 4.0
6
Acetylene reduction
(pgC2 H4
7
culture-1 6 hour-1 )
5 Day 4
4
o
1
~
~
~
M
~
~
~
~
Salinity (g/Il
Fig. 1. The effects of salinity on acetylene reduction by Klebsiella pneumoniae isolated from marine sediments.
for their ability to reduce acetylene at varying levels of salinity. The results were similar in all cases and are typified by those in Fig. 1. Initially acetylene reduction at the higher salinities was less than at low salinities but after a period of adaptation substantial activity occurred at the higher levels. Discussion Nitrogen-fixing (acetylene-reducing) Enterobacteriaceae are found throughout the Lune estuary in considerable numbers (Table 1). They occur both in the fresh
Salinity-tolerant Nitrogen-fixing Enterobacteriaceae
517
water reaches of the river Lune and in the sea-water at high tide. In the sediments they predominate in the surface layer (Table 2). Numbers in the sediments are generally higher in the spring and summer months but there is not an exact correlation with nitrogen fixation rates in the sediments (Jones, 1982). The bacteria enter the estuary from two main sources. The river Lune is contaminated with run-off from land in the fresh water reaches and the estuarine section is dosed directly with sewage. Lancaster sewage, apart from maceration for cosmetic reasons, is virtually untreated and is discharged into the river Lune 15 min after high tide. MPN experiments carried out at the sewage outfall give the number of nitrogen-fixing Enterobacteriaceae as 11,000 bacteria per ml water which is 100 times that for the river above the outfall. These bacteria are mainly lost into the Irish Sea due to the scouring action of the tide but some may reach the sediments. Nitrogen-fixing Enterobacteriaceae could only be isolated using the following sequence of media and conditions: anaerobic growth in nitrogen-free glucose broth under nitrogen followed by aerobic growth on McConkey agar plates in air. Nitrogen-fixing strains could not be isolated if the medium first used was McConkey broth. This is broadly in agreement with results obtained by Wright and Weaver (1981) working with forage grass roots. They obtained many fewer nitrogen-fixing colonies when McConkey medium rather than nitrogen-free medium was first used to isolate Enterobacteriaceae. The predominant nitrogen-fixing species of the Enterobacteriaceae in the waters and the sediments is Klebsiella pneumoniae. Herbert (1975), who carried out a study on marine sediments off the coast of Scotland, also isolated nitrogen-fixing strains of K. pneumoniae. However, he found that they were of fresh water origin and were unable to grow at the salinity of sea-water. Nitrogen fixation has not been detected in the water column of the Lune estuary (Jones, 1982) but it has been quantified for the sediments where the salinity of the surface layers can be as high as 4.1 gil. Therefore if K. pneumoniae is a contributor of fixed nitrogen it should be able to grow at this salinity. Most isolates from MPN experiments on the sediments, whether from low or high salinity media, are able to reduce acetylene at seawater salinities (3.5 gil) and 6 strains of K. pneumoniae tested for growth in a range of salinities could reduce acetylene at 5 gil after a period of adaptation (Fig. 1). It seems likely, therefore, that salinity is not a major factor in limiting nitrogen fixation by K. pneumoniae in estuarine sediments although it may cause a slow down in the rate of growth. Werner et al. (1974) have isolated nitrogen-fixing Enterobacteriaceae from marine habitats in North America which reduced acetylene at salinities 66% that of sea-water. Acknowledgements. I wish to thank Mr.]. V.Davies for help with the preparation of
media.
References El-Abagy, M. M., El-Zanialy, H. T., EI-Hawaary, S.: Direct MPN for faecal coliform. Zbl.
Bakt., II. Abt. 135, 396-401 (1980)
Collins, C.H.: Microbial methods, 2nd Ed. London, Butterworth 1967 Fogg, G.E.: Nitrogen fixation in the oceans. Ecol. Bull. (Stockh.) 26, 11-29 (1978) Herbert, R. A.: Heterotrophic nitrogen fixation in shallow estuarine sediments. J. Exp. Mar.
BioI. 18, 215-225 (1975)
518
K.Jones
Jones, K.: Nitrogen fixation in the temperate estuarine intertidal sediments of the river Lune. Limno!' Oceanogr. 27, 455-460 (1982) Prouasoli, L., McLaughlin, j., Droop, D. R.: The development of artificial media for marine algae. Arch. Microbiol. 25, 392-428 (1957) Stewart, W.D.P., Fitzgerald, G.P., Burris, R.H.: In situ studies of nitrogen fixation using the acetylene reduction technique. Proc. nat. Acad. Sci. (Wash.) 58,2071-2078 (1967) Werner, D., Evans, H.j., Seidler, R.J.: Facultatively anaerobic nitrogen-fixing bacteria from the marine environment. Canad. J. Microbiol. 20, 59-64 (1974) Wright, S.F., Weaver, R. W.: Enumeration and identification of nitrogen-fixing bacteria from forage grass roots. Appl, Environ. Microbiol. 22, 97-101 (1981) Dr. Keith Jones, Dept. of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, U.K.