- Email: [email protected]
The effect of effluent high in chromium on marine sediment aerobic heterotrophic bacteria
Marine Environmental Research 13 (1984) 69-79
The Effect of Effluent High in Chromium on Marine Sediment Aerobic Heterotrophic Bacteria J. Aislabie & M. W. Loutit Microbiology Department, University of Otago, PO Box 56, Dunedin, New Zealand (Received: 13 March, 1984)
ABSTRACT ,4 survey of the aerobic heterotrophic bacteria present in sediments at Sawyers Bay, New Zealand, receiving tannery effluent high in Cr, and a control site, indicates that the populations present are different and show seasonal variation. The bacterial population present at the polluted site appears more able to tolerate C r l l I at concentrations less than 0.21zmolm1-1
INTRODUCTION Chromium (Cr) is one of the most ubiquitous metals used in industry (Dean et al., 1972) and its use is increasing (UNEP, 1978). Because the effluents from many of these industries are discharged into bodies of water, point sources of pollution are increasing. The fate of Cr in the effluents depends on the form of the Cr, the composition of the effluents and the conditions of the receiving waters (Pfeiffer et aL, 1980; Smillie et al., 1981). In marine systems much of this Cr is deposited into sediments (Smillie et al., 1981) and little information exists on its subsequent availability to biological systems. That Cr does enter the food chain is evident from the work of Mearns et al. (1976) and Elwood et al. (1980). Although bacteria have been shown to be involved in metal transformations (Silverman & Ehrlich, 1964; Jernelov & Martin, 1975; 69 Marine Environ. Res. 0141-1136/84/$03-00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
70
J. Aislabie, M. W. Loutit
Summers & Silver, 1978), little work has implicated them in the passage of Cr through food chains in the marine environment (Lee et al., 1975; Johnson et al., 1981) although some work shows their involvement in freshwater systems (Loutit et al., 1973; Patrick & Loutit, 1976, 1977). To understand the interaction between bacteria and Cr in the marine system, information is required on the types of bacteria found in sediments polluted with Cr. This paper reports such an investigation into sediments in Otago Harbour where a tannery deposits effluent, and is the first of a series on the inter-relationship between Cr and bacteria in sediments.
MATERIALS AND METHODS The study area
Two sites in the Otago Harbour (Fig. 1) were sampled at intervals over a period. Sawyers Bay has received tannery effluent for 100 years and, although the volume and strength of this effluent has now decreased, the sediment in the Bay is high in Cr. The effluent is released into a small stream which then empties into the Harbour. The control site, the Leith
9
(k,, Lowyers Heocl
Fig. 1. Sedimentsampling sites in Otago Harbour, Dunedin, New Zealand.
Chromium and marine sediment bacteria
71
Mouth, is the site at which the Leith stream enters Otago Harbour. Sediment samples (1 cm layer) were collected at low tide into sterile acidcleaned screw-capped jars and the samples were processed within an hour of collection. Enumeration and isolation of bacteria
Difco Plate Count Agar (PCA) prepared in an estuarine salts solution (ESS) (Austin et al., 1977), alone or supplemented with Cr, was used for estimating viable bacteria and Cr tolerant bacteria and for the TABLE 1 Determinative Scheme for the Presumptive Identification of Marine Sediment Bacteria 1. Gram positive Gram negative 2. Rods, filaments Cocci 3. Branching Non-branching 4. Oxidative Fermentative 5. Large rods, spores Pleomorphic forms 6. Catalase positive Catalase negative Fermentative 7. Oxidative Fermentative 8. Rods, coccobacilli, occurring in filaments, singly or in pairs: Pigmented Non-pigmented 9. Yellow Purple 10. Oxidase positive Oxidase negative 11. Oxidative Fermentative 12. Motile Non-motile 13. Motile Non-motile
2 8 3 6 4 5 Nocardia Streptomyces Bacillus Coryneform 7 Streptococcus Micrococcus Staphylococcus
9 10 Flavobacterium Chromobaeterium 11 Enterobacteriaceae 12 13 Pseudomonas A lcaligenes Aeromonas/Vibrio Aeromonas
72
J. Aislabie, M. W. Loutit
propagation of isolates. Tenfold dilutions of sediment were prepared in ESS, the first dilution being 10 g of sediment in 90 ml ESS. Pour plates with I ml volumes of each dilution were prepared in triplicate and incubated at 25 °C for 1 week. After counting the colonies, one hundred colonies were picked off on a non-selective basis (either all those on a plate or from a sector of a plate) and were streaked to obtain pure cultures. Presumptive identification of isolates A scheme adapted from Murchelano & Brown (1970) and Bergey's Manual of Determinative Bacteriology (1974) was used to group isolates. Tests used were morphology, Gram reaction, marine oxidation fermentation reaction (Leifson, 1963) catalase and oxidase tests. The grouping was based on the scheme given in Table 1. Tolerance to chromium Sediment samples diluted in ESS were incorporated into PCA supplemented with Cr(III) (as CrC13.6H~O) or Cr(VI)(as Na2CrO4) to give a final concentration from 0-1/~mol ml - 1 to 5 pmol ml - 1. The Cr was added from sterile stock solutions to the cooled molten agar immediately prior to pouring the plates. Determination of chromium concentration and organic matter in sediments The methods used have been described previously (Smillie et al., 1981).
RESULTS Sediments were sampled at intervals from Sawyers Bay and the control site at the mouth of the Leith and the numbers of aerobic heterotrophic bacteria were estimated. These results, together with the organic matter content and Cr concentration of the sediments, are given in Table 2. Isolates from each sample were grouped according to the system described and the results are given in Fig. 2. Statistical analysis of the populations by the Chi-square test showed that there is a significant difference in the percentages of the different groups of bacteria present at the two sites (P < 0.001) and there is, in addition, a seasonal variation. To
73
Chromium and marine sediment bacteria
TABLE 2 Numbers of Bacteria, Chromium Concentration and Organic Content of Sediment Samples from Sawyers Bay and the Leith Mouth Sampling date
CFU* (Per gram dried sediment)
Sawyers Bay Cr (#mol g- 1 dried sediment)
July, 1980 2.745 x 106 28.84 + 0.00"* Oct. 1980 7.94x 106 20.38_+2.21 April, 1981 4.835 × 1 0 6 47"49+0"81 May, 1982 1.96x l0 T 71.15_+6.92 Jan. 1983 9.05 × 1 0 7 26"92 +_2-69
Organic content (~)
CFU* (Per gram dried sediment)
Leith Mouth Cr (pmol g- 1 dried sediment)
NDt 9.76_+0.62 13'89+2'03 ll.79_0.28 25"99_+ 1'02
7.65 x l0 s 5.29 x l0 s 4'09 x l0 s 1.41 x l 0 s 4"70x i07
0.36J; 6.99_ 0-26** 0-23_+0.09 6-65_+0.30 0-29_+0"02 11"52_+0"96 0.27-+0.01 8.60_+0.95 0"41_+0'24 22"53 -+ 1"29
"
Organic content (%)
* Numbers of bacteria measured as colony-forming units (CFU). t Not determined. ** Mean of three subsamples _ the standard error of the mean. :~ One subsample measured.
zo
mmmmsowors ~ 1 D L t ~ Mouth
15
JULY'OO
~i -
F-1
OCT'80
-
0
,
.
presumptiveidentificotion Fig. 2, The frequency of occurrence of isolates of aerobic heterotrophic bacteria in sediments from the Leith Mouth and Sawyers Bay sampled on 5 occasions.
74
J. Aislabie, M. W. Loutit
make sure that the difference was not due to sample variation, three sediment samples were collected 1 m apart at the same time, and analysed as described. The results (Fig. 3), when analysed statistically, indicated that there was no significant difference between the bacterial populations of the three samples, collected on the same day. The observed difference in composition of the biota between the sites and at different times therefore holds. It was postulated that the high Cr in the sediment at Sawyers Bay may have exerted a selective pressure on the bacterial populations, accounting not only for the different composition of the population but resulting in isolates from Sawyers Bay being better able to tolerate Cr. Certainly, when sediment dilutions were, incorporated in PCA plates containing various concentrations of Cr, there appeared to be more bacteria from Sawyers Bay sediment growing on the plates containing 0.1/~mol m l - z of Cr(III) than on plates without Cr (Fig. 4). At concentrations above 0.2 pmol m l - 1, however, there were fewer colony-forming
40
5owyers Boy
30
10 C QJ
3°S 0
LeithHouth
2O
,0 0
Fig. 3.
presumptiveidentification The variation in types of bacteria in three sedimentsamplesA, B and C collected 1 m apart at the same time from the Leith Mouth and Sawyers Bay.
75
Chromium and marine sediment bacteria
i
J,,
s, n B,y
10
J.ae
~ -
1
2
3
1
2
3
[Cr] jumoI rn[Iin isolationplates Fig. 4. The effect of increasing Cr(III) and Cr(VI) concentration on the numbers (colony forming units, CFU, per gram of oven-dried sediment) of aerobic heterotrophic sediment bacteria isolated from the Leith Mouth and Sawyers Bay in January and June 1983.
units (CFU). By contrast, isolates from the Leith Mouth sediment were inhibited by all the concentrations of Cr(III) tested, compared with control plates containing no Cr. Similarly, Cr(VI) inhibited the numbers of CFU, at all concentrations tested, compared with the controls containing no Cr. It was of interest that fungi were frequently isolated on the plates containing higher concentrations of Cr(III). DISCUSSION Analysis of the types and frequency of occurrence of the aerobic heterotrophic bacteria from a sediment receiving tannery effluent (Sawyers Bay) indicated that the populations differed significantly (Fig. 2) from that of a sediment which did not receive the effluent. Further, there was a seasonal variation in the populations (Fig. 2). It was of interest to find that Gram positive isolates made up from 32 to
76
J. Aislabie, M. W. Loutit
92 ~ of the population, depending on the sampling time. Few studies have considered Gram positive isolates in sediment (Boeye et al., 1975; Austin et al., 1977; Timoney et al., 1978). Sieburth (1979) has suggested that an intensive study of their occurrence and in situ activity is long overdue. Perhaps the lack of study of these Gram positive sediment bacteria is related to the difficulty experienced in identifying them. They are, on the whole, metabolically inert in the biochemical tests used to identify isolates to generic level (Bergey's Manual o f Determinative Bacteriology, 1974). Many of them also appear to be morphologically atypical (Sieburth, 1979) and cause problems parallel with those experienced by Johnson et al. (1978) who were attempting to identify soil isolates from the Antarctic. Moriarty & Hayward (1982) reported the predominance of Gram negative organisms in sediments using the electron microscope technique as opposed to cultural conditions in which the organisms isolated are influenced by the media used. The Gram negative organisms noted by Moriarty & Hayward (1982) could, for example, be photosynthetic bacteria which would not grow on the media and under the conditions we used. Our attempts to follow the procedures described by Moriarty & Hayward (1982) were unsuccessful because of the size of particles in the sediment. The attempt to assess whether the presence of Cr in the polluted sediment had selected isolates tolerant to Cr gave some interesting results. If Cr(III) (0.1 pmol ml- 1 Cr) was incorporated into the isolating media a larger number of organisms could be isolated from Sawyers Bay sediment than if no Cr was used. This effect was not seen with organisms isolated from the control site, the Leith Mouth. This increased efficiency of isolation in the presence of a metal has been reported by others (Goyne & Jones, 1973; Traxler & Wood, 1981). Why this stimulation should occur is not known. It is postulated that some Sawyers Bay isolates may require Cr for growth or that, in the presence of chromium, some bacterial colonies develop such that they are large enough to be seen and counted. Hines & Jones (1982) have suggested that stimulation of bacterial populations by micromolar metal supplements to agar media may more properly be attributed to the displacement of ions which were lower on the avidity series than to direct stimulation by the toxic supplement. When Cr(VI) was added, the number of colonies on the isolating medium was less than on the plates containing no Cr. The tolerance of certain organisms to quite high concentrations of Cr(VI) is, however, evident from the results in Fig. 4. Some isolates can tolerate Cr(VI) to at
Chromium and marine sediment bacteria
77
least 3 #mol ml- x. This held for isolates from both Sawyers Bay and the Leith Mouth. It is postulated that these organisms may possess a plasmid that confers resistance to the chromate ion. Plasmid mediated resistance to the chromate ion has been reported in Pseudomonas aeruginosa by Summers & Jacoby (1978) and in Streptococcus lactis by Efstathiou & McKay (1977). In view of the very high concentration of Cr in the Sawyers Bay sediment, 20.38-71.15/~mol g - 1 (Table 2), one might have expected that bacteria surviving in that environment would have a tolerance beyond 0.1 #mol ml- 1 Cr(III) in PCA prepared in ESS. The detected ability of some of the organisms from both the polluted and control sites to tolerate Cr(VI) is probably not due to Cr(VI) contamination in the sediment since Cr(VI) is not readily detected in the polluted sediment (Smillie et al., 1981), most being in the Cr(III) form. The selective agent may be some other substance and the information mediating chromate resistance is carried on the same plasmid. The results support the contention that most of the Cr in the tannery effluent is deposited on entry in an unavailable form: certainly, little is detectable in the overlying water (Smillie et al., 1981). As Cr does not form sulphides it must, therefore, be deposited either as oxides and hydroxides or bound to organic matter. The latter appears to be the most likely in view of recent work (C. J. Pillidge, pers. comm.). Cr tolerance studies on bacteria carried out on solid media could be unsatisfactory since the Cr can be bound to the agar or components in the agar (Ramamoorthy & Kushner, 1975; Washington et al., 1978). It is necessary, therefore, to attempt to study Cr tolerance in a synthetic liquid medium. Work on this, and the interaction between Cr and other groups of organisms, is now in progress. The results so far are of interest environmentally since they indicate that very little of the Cr deposited in Sawyers Bay sediment is biologically available. Since the concentration of Cr in the overlying water is low (Smillie et aL, 1981) and the concentration of biologically available Cr in the sediment is low, the means by which Cr enters food chains requires further investigation. REFERENCES Austin, B., Allen, D. A., Mills, A. L. & Colwell, R. R. (1977). Numerical taxonomy of heavy-metal tolerant bacteria isolated from an estuary. Canadian Journal of Microbiology, 23, 1433-47.
78
J. Aislabie, M. W. Loutit
Bergey's Manual of Determinative Bacteriology (1974) (8th edn). (Buchanan, R. E. & Gibbons, N. E. (Eds)), Baltimore, Williams and Wilkins Company. Boeye, A., Wayenbergh, M. & Aerts, M. (1975). Density and composition of heterotrophic bacterial populations in North Sea sediment. Marine Biology, 32, 263-70. Dean, J. G., Bosqui, F. L. & Lanouette, K. L. (1972). Removing heavy metals from wastewater. Environmental Science and Technology, 6, 518-22. Efstathiou, J. D. & McKay, L. L. (1977). Inorganic salts resistance associated with a lactose fermenting plasmid in Streptococcus lactis. Journal of Bacteriology, 130, 257-65. Elwood, J. W., Beauchamp, J. J. & Allen, C. P. (1980). Chromium levels in fish from a lake chronically contaminated with chromates from cooling towers. International Journal of Environmental Studies, 14, 289-98. Goyne, E. R. & Jones, G. E. (1973). An ecological survey of the open ocean and estuarine microbial populations. II. The oligodynamic effect of Ni on marine bacteria. In: Marine microbial ecology. (Stevenson, H. L. (Ed.)) Columbia, SC, University of South Carolina Press. Hines, M. E. & Jones, G. E. (1982). Microbial metal tolerance in Bermuda carbonate sediments. Applied and Environmental Microbiology, 44, 502-5. Jernelov, A. & Martin, A. L. (1975). Ecological implications of metal metabolism by micro-organisms. Annual Review of Microbiology, 29, 61-77. Johnson, R. M., Madden, J. M. & Swafford, J. R. (1978). Taxonomy of Antarctic bacteria from soils and air primarily of the McMurdo Station and Victoria Land Dry Valleys Region. In: Terrestrial biology. III. Antarctic Research Series Volume 30 (Paper 3). (Parker, B. C. (Ed.)), American Geophysical Union. Johnson, I., Flower, N. & Loutit, M. W. (1981). Contribution of periphytic bacteria to the concentration of chromium in the crab Helice crassa. Microbial Ecology, 7, 245-52. Lee, Y., Patrick, F. M. & Loutit, M. W. (1975). Concentration of metals by a marine bacterium Leucothrix and a periwinkle Melarapha. Proceedings oj The University of Otago Medical School, 53, 17-18. Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. Journal of Bacteriology, 85, 1183-4. Loutit, M. W., Patrick, F. M. & Malthus, R. S. (1973). The role of metalconcentrating bacteria in a food chain in a river receiving effluent. Proceedings of The University of Otago Medical School, 51, 37-8. Mearns, A. J., Oshida, P. S., Sherwood, M. J., Young, D. R. & Reish, D. J. (1976). Chromium effects on coastal organisms. Journal of the Water Pollution Control Federation, 48, 1929-39. Moriarty, D. J. W. & Hayward, A. C. (1982). Ultrastructure of bacteria and the proportion of Gram negative bacteria in marine sediments. Microbial Ecology, 8, 1-14. Murchelano, R. A. & Brown, C. (1970). Heterotrophic bacteria in Long Island Sound. Marine Biology, 7, 1-6.
Chromium and marine sediment bacteria
79
Patrick, F. M. & Loutit, M. W. (1976). Passage of metals in effluents through bacteria to higher organisms. Water Research, 10, 333-5. Patrick, F. M. & Loutit, M. W. (1977). The uptake of heavy metals by epiphytic bacteria on Alisma plantago-aquatica. Water Research, l l, 699-703. Pfeiffer, W. C., Fiszman, M. & Carbonell, N. (1980). Fate of chromium in a tributary of the Iraja River, Rio de Janeiro. Environmental Pollution (Series B), 1, 117-26. Ramamoorthy, S. & Kushner, D. J. (1975). Binding of mercuric and other metal ions by microbial growth media. Microbial Ecology, 2, 162-76. Sieburth, J. McN. (1979). Sea microbes. New York, Oxford University Press. Silverman, M. P. & Ehrlich, H. L. (1964). Microbial formation and degradation of minerals. Advances in Applied Microbiology, 6, 153-205. Smillie, R. H., Hunter, K. & Loutit, M. (1981). Reduction of chromium(VI) by bacterially produced hydrogen sulphide in a marine environment. Water Research, 15, 1351-4. Summers, A. O. & Jacoby, G. A. (1978). Plasmid determined resistance to boron and chromium compounds in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 13, 637-40. Summers, A. O. & Silver, S. (1978). Microbial transformations of metals. Annual Review of Microbiology, 32, 637-72. Timoney, J. F., Port, J., Giles, J. & Spanier, J. (1978). Heavy metal and antibiotic resistance in the bacterial flora of sediments of the New York Bight. Applied and Environmental Microbiology, 36, 455-72. Traxler, R. W. & Wood, E. M. (1981). Multiple metal tolerance of bacterial isolates. Developments in Industrial Microbiology, 22, 521-8. UNEP (1978). Data profiles for the evaluation of their hazards to the environment of the Mediterranean Sea. In: International Register of Potentially Toxic Chemicals, 487-549. Washington II., J. A., Snyder, R. J., Kohner, P. C., Wiltse, C. G., Ilstrup, D. M. & McCall, J. T. (1978). Effect of cation content of agar on the activity of Gentamicin, Tobramycin and Amikacin against Pseudomonas aeruginosa. Journal of Infectious Diseases, 137, 103-11.
The Effect of Effluent High in Chromium on Marine Sediment Aerobic Heterotrophic Bacteria J. Aislabie & M. W. Loutit Microbiology Department, University of Otago, PO Box 56, Dunedin, New Zealand (Received: 13 March, 1984)
ABSTRACT ,4 survey of the aerobic heterotrophic bacteria present in sediments at Sawyers Bay, New Zealand, receiving tannery effluent high in Cr, and a control site, indicates that the populations present are different and show seasonal variation. The bacterial population present at the polluted site appears more able to tolerate C r l l I at concentrations less than 0.21zmolm1-1
INTRODUCTION Chromium (Cr) is one of the most ubiquitous metals used in industry (Dean et al., 1972) and its use is increasing (UNEP, 1978). Because the effluents from many of these industries are discharged into bodies of water, point sources of pollution are increasing. The fate of Cr in the effluents depends on the form of the Cr, the composition of the effluents and the conditions of the receiving waters (Pfeiffer et aL, 1980; Smillie et al., 1981). In marine systems much of this Cr is deposited into sediments (Smillie et al., 1981) and little information exists on its subsequent availability to biological systems. That Cr does enter the food chain is evident from the work of Mearns et al. (1976) and Elwood et al. (1980). Although bacteria have been shown to be involved in metal transformations (Silverman & Ehrlich, 1964; Jernelov & Martin, 1975; 69 Marine Environ. Res. 0141-1136/84/$03-00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
70
J. Aislabie, M. W. Loutit
Summers & Silver, 1978), little work has implicated them in the passage of Cr through food chains in the marine environment (Lee et al., 1975; Johnson et al., 1981) although some work shows their involvement in freshwater systems (Loutit et al., 1973; Patrick & Loutit, 1976, 1977). To understand the interaction between bacteria and Cr in the marine system, information is required on the types of bacteria found in sediments polluted with Cr. This paper reports such an investigation into sediments in Otago Harbour where a tannery deposits effluent, and is the first of a series on the inter-relationship between Cr and bacteria in sediments.
MATERIALS AND METHODS The study area
Two sites in the Otago Harbour (Fig. 1) were sampled at intervals over a period. Sawyers Bay has received tannery effluent for 100 years and, although the volume and strength of this effluent has now decreased, the sediment in the Bay is high in Cr. The effluent is released into a small stream which then empties into the Harbour. The control site, the Leith
9
(k,, Lowyers Heocl
Fig. 1. Sedimentsampling sites in Otago Harbour, Dunedin, New Zealand.
Chromium and marine sediment bacteria
71
Mouth, is the site at which the Leith stream enters Otago Harbour. Sediment samples (1 cm layer) were collected at low tide into sterile acidcleaned screw-capped jars and the samples were processed within an hour of collection. Enumeration and isolation of bacteria
Difco Plate Count Agar (PCA) prepared in an estuarine salts solution (ESS) (Austin et al., 1977), alone or supplemented with Cr, was used for estimating viable bacteria and Cr tolerant bacteria and for the TABLE 1 Determinative Scheme for the Presumptive Identification of Marine Sediment Bacteria 1. Gram positive Gram negative 2. Rods, filaments Cocci 3. Branching Non-branching 4. Oxidative Fermentative 5. Large rods, spores Pleomorphic forms 6. Catalase positive Catalase negative Fermentative 7. Oxidative Fermentative 8. Rods, coccobacilli, occurring in filaments, singly or in pairs: Pigmented Non-pigmented 9. Yellow Purple 10. Oxidase positive Oxidase negative 11. Oxidative Fermentative 12. Motile Non-motile 13. Motile Non-motile
2 8 3 6 4 5 Nocardia Streptomyces Bacillus Coryneform 7 Streptococcus Micrococcus Staphylococcus
9 10 Flavobacterium Chromobaeterium 11 Enterobacteriaceae 12 13 Pseudomonas A lcaligenes Aeromonas/Vibrio Aeromonas
72
J. Aislabie, M. W. Loutit
propagation of isolates. Tenfold dilutions of sediment were prepared in ESS, the first dilution being 10 g of sediment in 90 ml ESS. Pour plates with I ml volumes of each dilution were prepared in triplicate and incubated at 25 °C for 1 week. After counting the colonies, one hundred colonies were picked off on a non-selective basis (either all those on a plate or from a sector of a plate) and were streaked to obtain pure cultures. Presumptive identification of isolates A scheme adapted from Murchelano & Brown (1970) and Bergey's Manual of Determinative Bacteriology (1974) was used to group isolates. Tests used were morphology, Gram reaction, marine oxidation fermentation reaction (Leifson, 1963) catalase and oxidase tests. The grouping was based on the scheme given in Table 1. Tolerance to chromium Sediment samples diluted in ESS were incorporated into PCA supplemented with Cr(III) (as CrC13.6H~O) or Cr(VI)(as Na2CrO4) to give a final concentration from 0-1/~mol ml - 1 to 5 pmol ml - 1. The Cr was added from sterile stock solutions to the cooled molten agar immediately prior to pouring the plates. Determination of chromium concentration and organic matter in sediments The methods used have been described previously (Smillie et al., 1981).
RESULTS Sediments were sampled at intervals from Sawyers Bay and the control site at the mouth of the Leith and the numbers of aerobic heterotrophic bacteria were estimated. These results, together with the organic matter content and Cr concentration of the sediments, are given in Table 2. Isolates from each sample were grouped according to the system described and the results are given in Fig. 2. Statistical analysis of the populations by the Chi-square test showed that there is a significant difference in the percentages of the different groups of bacteria present at the two sites (P < 0.001) and there is, in addition, a seasonal variation. To
73
Chromium and marine sediment bacteria
TABLE 2 Numbers of Bacteria, Chromium Concentration and Organic Content of Sediment Samples from Sawyers Bay and the Leith Mouth Sampling date
CFU* (Per gram dried sediment)
Sawyers Bay Cr (#mol g- 1 dried sediment)
July, 1980 2.745 x 106 28.84 + 0.00"* Oct. 1980 7.94x 106 20.38_+2.21 April, 1981 4.835 × 1 0 6 47"49+0"81 May, 1982 1.96x l0 T 71.15_+6.92 Jan. 1983 9.05 × 1 0 7 26"92 +_2-69
Organic content (~)
CFU* (Per gram dried sediment)
Leith Mouth Cr (pmol g- 1 dried sediment)
NDt 9.76_+0.62 13'89+2'03 ll.79_0.28 25"99_+ 1'02
7.65 x l0 s 5.29 x l0 s 4'09 x l0 s 1.41 x l 0 s 4"70x i07
0.36J; 6.99_ 0-26** 0-23_+0.09 6-65_+0.30 0-29_+0"02 11"52_+0"96 0.27-+0.01 8.60_+0.95 0"41_+0'24 22"53 -+ 1"29
"
Organic content (%)
* Numbers of bacteria measured as colony-forming units (CFU). t Not determined. ** Mean of three subsamples _ the standard error of the mean. :~ One subsample measured.
zo
mmmmsowors ~ 1 D L t ~ Mouth
15
JULY'OO
~i -
F-1
OCT'80
-
0
,
.
presumptiveidentificotion Fig. 2, The frequency of occurrence of isolates of aerobic heterotrophic bacteria in sediments from the Leith Mouth and Sawyers Bay sampled on 5 occasions.
74
J. Aislabie, M. W. Loutit
make sure that the difference was not due to sample variation, three sediment samples were collected 1 m apart at the same time, and analysed as described. The results (Fig. 3), when analysed statistically, indicated that there was no significant difference between the bacterial populations of the three samples, collected on the same day. The observed difference in composition of the biota between the sites and at different times therefore holds. It was postulated that the high Cr in the sediment at Sawyers Bay may have exerted a selective pressure on the bacterial populations, accounting not only for the different composition of the population but resulting in isolates from Sawyers Bay being better able to tolerate Cr. Certainly, when sediment dilutions were, incorporated in PCA plates containing various concentrations of Cr, there appeared to be more bacteria from Sawyers Bay sediment growing on the plates containing 0.1/~mol m l - z of Cr(III) than on plates without Cr (Fig. 4). At concentrations above 0.2 pmol m l - 1, however, there were fewer colony-forming
40
5owyers Boy
30
10 C QJ
3°S 0
LeithHouth
2O
,0 0
Fig. 3.
presumptiveidentification The variation in types of bacteria in three sedimentsamplesA, B and C collected 1 m apart at the same time from the Leith Mouth and Sawyers Bay.
75
Chromium and marine sediment bacteria
i
J,,
s, n B,y
10
J.ae
~ -
1
2
3
1
2
3
[Cr] jumoI rn[Iin isolationplates Fig. 4. The effect of increasing Cr(III) and Cr(VI) concentration on the numbers (colony forming units, CFU, per gram of oven-dried sediment) of aerobic heterotrophic sediment bacteria isolated from the Leith Mouth and Sawyers Bay in January and June 1983.
units (CFU). By contrast, isolates from the Leith Mouth sediment were inhibited by all the concentrations of Cr(III) tested, compared with control plates containing no Cr. Similarly, Cr(VI) inhibited the numbers of CFU, at all concentrations tested, compared with the controls containing no Cr. It was of interest that fungi were frequently isolated on the plates containing higher concentrations of Cr(III). DISCUSSION Analysis of the types and frequency of occurrence of the aerobic heterotrophic bacteria from a sediment receiving tannery effluent (Sawyers Bay) indicated that the populations differed significantly (Fig. 2) from that of a sediment which did not receive the effluent. Further, there was a seasonal variation in the populations (Fig. 2). It was of interest to find that Gram positive isolates made up from 32 to
76
J. Aislabie, M. W. Loutit
92 ~ of the population, depending on the sampling time. Few studies have considered Gram positive isolates in sediment (Boeye et al., 1975; Austin et al., 1977; Timoney et al., 1978). Sieburth (1979) has suggested that an intensive study of their occurrence and in situ activity is long overdue. Perhaps the lack of study of these Gram positive sediment bacteria is related to the difficulty experienced in identifying them. They are, on the whole, metabolically inert in the biochemical tests used to identify isolates to generic level (Bergey's Manual o f Determinative Bacteriology, 1974). Many of them also appear to be morphologically atypical (Sieburth, 1979) and cause problems parallel with those experienced by Johnson et al. (1978) who were attempting to identify soil isolates from the Antarctic. Moriarty & Hayward (1982) reported the predominance of Gram negative organisms in sediments using the electron microscope technique as opposed to cultural conditions in which the organisms isolated are influenced by the media used. The Gram negative organisms noted by Moriarty & Hayward (1982) could, for example, be photosynthetic bacteria which would not grow on the media and under the conditions we used. Our attempts to follow the procedures described by Moriarty & Hayward (1982) were unsuccessful because of the size of particles in the sediment. The attempt to assess whether the presence of Cr in the polluted sediment had selected isolates tolerant to Cr gave some interesting results. If Cr(III) (0.1 pmol ml- 1 Cr) was incorporated into the isolating media a larger number of organisms could be isolated from Sawyers Bay sediment than if no Cr was used. This effect was not seen with organisms isolated from the control site, the Leith Mouth. This increased efficiency of isolation in the presence of a metal has been reported by others (Goyne & Jones, 1973; Traxler & Wood, 1981). Why this stimulation should occur is not known. It is postulated that some Sawyers Bay isolates may require Cr for growth or that, in the presence of chromium, some bacterial colonies develop such that they are large enough to be seen and counted. Hines & Jones (1982) have suggested that stimulation of bacterial populations by micromolar metal supplements to agar media may more properly be attributed to the displacement of ions which were lower on the avidity series than to direct stimulation by the toxic supplement. When Cr(VI) was added, the number of colonies on the isolating medium was less than on the plates containing no Cr. The tolerance of certain organisms to quite high concentrations of Cr(VI) is, however, evident from the results in Fig. 4. Some isolates can tolerate Cr(VI) to at
Chromium and marine sediment bacteria
77
least 3 #mol ml- x. This held for isolates from both Sawyers Bay and the Leith Mouth. It is postulated that these organisms may possess a plasmid that confers resistance to the chromate ion. Plasmid mediated resistance to the chromate ion has been reported in Pseudomonas aeruginosa by Summers & Jacoby (1978) and in Streptococcus lactis by Efstathiou & McKay (1977). In view of the very high concentration of Cr in the Sawyers Bay sediment, 20.38-71.15/~mol g - 1 (Table 2), one might have expected that bacteria surviving in that environment would have a tolerance beyond 0.1 #mol ml- 1 Cr(III) in PCA prepared in ESS. The detected ability of some of the organisms from both the polluted and control sites to tolerate Cr(VI) is probably not due to Cr(VI) contamination in the sediment since Cr(VI) is not readily detected in the polluted sediment (Smillie et al., 1981), most being in the Cr(III) form. The selective agent may be some other substance and the information mediating chromate resistance is carried on the same plasmid. The results support the contention that most of the Cr in the tannery effluent is deposited on entry in an unavailable form: certainly, little is detectable in the overlying water (Smillie et al., 1981). As Cr does not form sulphides it must, therefore, be deposited either as oxides and hydroxides or bound to organic matter. The latter appears to be the most likely in view of recent work (C. J. Pillidge, pers. comm.). Cr tolerance studies on bacteria carried out on solid media could be unsatisfactory since the Cr can be bound to the agar or components in the agar (Ramamoorthy & Kushner, 1975; Washington et al., 1978). It is necessary, therefore, to attempt to study Cr tolerance in a synthetic liquid medium. Work on this, and the interaction between Cr and other groups of organisms, is now in progress. The results so far are of interest environmentally since they indicate that very little of the Cr deposited in Sawyers Bay sediment is biologically available. Since the concentration of Cr in the overlying water is low (Smillie et aL, 1981) and the concentration of biologically available Cr in the sediment is low, the means by which Cr enters food chains requires further investigation. REFERENCES Austin, B., Allen, D. A., Mills, A. L. & Colwell, R. R. (1977). Numerical taxonomy of heavy-metal tolerant bacteria isolated from an estuary. Canadian Journal of Microbiology, 23, 1433-47.
78
J. Aislabie, M. W. Loutit
Bergey's Manual of Determinative Bacteriology (1974) (8th edn). (Buchanan, R. E. & Gibbons, N. E. (Eds)), Baltimore, Williams and Wilkins Company. Boeye, A., Wayenbergh, M. & Aerts, M. (1975). Density and composition of heterotrophic bacterial populations in North Sea sediment. Marine Biology, 32, 263-70. Dean, J. G., Bosqui, F. L. & Lanouette, K. L. (1972). Removing heavy metals from wastewater. Environmental Science and Technology, 6, 518-22. Efstathiou, J. D. & McKay, L. L. (1977). Inorganic salts resistance associated with a lactose fermenting plasmid in Streptococcus lactis. Journal of Bacteriology, 130, 257-65. Elwood, J. W., Beauchamp, J. J. & Allen, C. P. (1980). Chromium levels in fish from a lake chronically contaminated with chromates from cooling towers. International Journal of Environmental Studies, 14, 289-98. Goyne, E. R. & Jones, G. E. (1973). An ecological survey of the open ocean and estuarine microbial populations. II. The oligodynamic effect of Ni on marine bacteria. In: Marine microbial ecology. (Stevenson, H. L. (Ed.)) Columbia, SC, University of South Carolina Press. Hines, M. E. & Jones, G. E. (1982). Microbial metal tolerance in Bermuda carbonate sediments. Applied and Environmental Microbiology, 44, 502-5. Jernelov, A. & Martin, A. L. (1975). Ecological implications of metal metabolism by micro-organisms. Annual Review of Microbiology, 29, 61-77. Johnson, R. M., Madden, J. M. & Swafford, J. R. (1978). Taxonomy of Antarctic bacteria from soils and air primarily of the McMurdo Station and Victoria Land Dry Valleys Region. In: Terrestrial biology. III. Antarctic Research Series Volume 30 (Paper 3). (Parker, B. C. (Ed.)), American Geophysical Union. Johnson, I., Flower, N. & Loutit, M. W. (1981). Contribution of periphytic bacteria to the concentration of chromium in the crab Helice crassa. Microbial Ecology, 7, 245-52. Lee, Y., Patrick, F. M. & Loutit, M. W. (1975). Concentration of metals by a marine bacterium Leucothrix and a periwinkle Melarapha. Proceedings oj The University of Otago Medical School, 53, 17-18. Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. Journal of Bacteriology, 85, 1183-4. Loutit, M. W., Patrick, F. M. & Malthus, R. S. (1973). The role of metalconcentrating bacteria in a food chain in a river receiving effluent. Proceedings of The University of Otago Medical School, 51, 37-8. Mearns, A. J., Oshida, P. S., Sherwood, M. J., Young, D. R. & Reish, D. J. (1976). Chromium effects on coastal organisms. Journal of the Water Pollution Control Federation, 48, 1929-39. Moriarty, D. J. W. & Hayward, A. C. (1982). Ultrastructure of bacteria and the proportion of Gram negative bacteria in marine sediments. Microbial Ecology, 8, 1-14. Murchelano, R. A. & Brown, C. (1970). Heterotrophic bacteria in Long Island Sound. Marine Biology, 7, 1-6.
Chromium and marine sediment bacteria
79
Patrick, F. M. & Loutit, M. W. (1976). Passage of metals in effluents through bacteria to higher organisms. Water Research, 10, 333-5. Patrick, F. M. & Loutit, M. W. (1977). The uptake of heavy metals by epiphytic bacteria on Alisma plantago-aquatica. Water Research, l l, 699-703. Pfeiffer, W. C., Fiszman, M. & Carbonell, N. (1980). Fate of chromium in a tributary of the Iraja River, Rio de Janeiro. Environmental Pollution (Series B), 1, 117-26. Ramamoorthy, S. & Kushner, D. J. (1975). Binding of mercuric and other metal ions by microbial growth media. Microbial Ecology, 2, 162-76. Sieburth, J. McN. (1979). Sea microbes. New York, Oxford University Press. Silverman, M. P. & Ehrlich, H. L. (1964). Microbial formation and degradation of minerals. Advances in Applied Microbiology, 6, 153-205. Smillie, R. H., Hunter, K. & Loutit, M. (1981). Reduction of chromium(VI) by bacterially produced hydrogen sulphide in a marine environment. Water Research, 15, 1351-4. Summers, A. O. & Jacoby, G. A. (1978). Plasmid determined resistance to boron and chromium compounds in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 13, 637-40. Summers, A. O. & Silver, S. (1978). Microbial transformations of metals. Annual Review of Microbiology, 32, 637-72. Timoney, J. F., Port, J., Giles, J. & Spanier, J. (1978). Heavy metal and antibiotic resistance in the bacterial flora of sediments of the New York Bight. Applied and Environmental Microbiology, 36, 455-72. Traxler, R. W. & Wood, E. M. (1981). Multiple metal tolerance of bacterial isolates. Developments in Industrial Microbiology, 22, 521-8. UNEP (1978). Data profiles for the evaluation of their hazards to the environment of the Mediterranean Sea. In: International Register of Potentially Toxic Chemicals, 487-549. Washington II., J. A., Snyder, R. J., Kohner, P. C., Wiltse, C. G., Ilstrup, D. M. & McCall, J. T. (1978). Effect of cation content of agar on the activity of Gentamicin, Tobramycin and Amikacin against Pseudomonas aeruginosa. Journal of Infectious Diseases, 137, 103-11.