Passage of metals in effluents, through bacteria to higher organisms

T,t a:¢r Re~eurch VoL It). pp. 333 to 335 Pergamen Press I9"b Printed in Great Britain.

PASSAGE OF METALS IN EFFLUENTS, THROUGH BACTERIA TO HIGHER ORGANISMS F. M. PATRICK and MARGARET LOUTIT Microbiolog.~ Department, University of Otago, Dunedin, New Zealand

IReceit'ed 18 February 1975) A~tract--Bacteria were greater in numbers in sediment in that part of a river receiving effluents from various sources. Isolates from the sediment were able to concentrate Cr, Cu, Mn, Fe, Pb and Zn. When tubificids were fed bacterial cultures containing these metals the tubificids showed increased concentration of metals. It is thought that small amounts of metals in effluents are concentrated by bacteria and are then passed to other organisms in a food chain.

Isolation of bacteria

INTRODUCTION

In an investigation of a river receiving effluent from treated sewage and from wool scouring and dyeing factories, large marts of the bacterium Sphaerotilus were found below the effluent entry points. The effluents contained small a m o u n t s of various metals (Patrick and Loutit, 1972). As soil bacteria have been shown to concentrate metals (Loutit, Loutit and Brooks, 1967) an investigation was begun to see if cultures of Sphaerotihts could also concentrate metals. It was found that both laboratory cultures and Sphaerotih~s from the river contained high levels of metals (Patrick a n d Loutit. 1972: Loutit, Patrick and Malthus, 1973). During the investigation a large kill of eels occurred a n d the eels were found to have high levels of Z n a n d P b in their flesh and gut (Loutit, Patrick and Malthus, 1973). Investigations also showed that levels of various metals were higher in river sediments, water plants and tubificids below the effluent entry points than above. It was postulated that bacteria below the effluent entry were concentrating the metals from the effluent a n d the metals were being passed up the food chain to higher animals (Loutit, Patrick and Malthus, 1973). The paper reports the results of investigations of passage of metals through part of a food chain.

MATERIALS AND METHODS

Media Both Bacto-Sphaerotilus agar (Difco) supplemented with 10'/o soil extract (L~ihnis. 1920) (SASE) and the same medium without agar (SMSE) was used. Doubly-distilled water was used in media and solutions in experiments measuring metal uptake. All sterilisation was at 121:C for 15 mill.

Estimation of bacterial numbers

Sediment samples collected as described were diluted 1:2 in 0"85°;~ saline and shaken vigorously. Loopsful were streaked on to the surface of SASE plates. After incubation at 28~C for 5 days representatives of the eleven colony types on the plates were picked off. streaked on to SASE and pure cultures obtained. The isolates were identified to generic level by following Skerman's Key (Skerman 1967).

Concentration of metals by bacteria Each of the eleven bacterial isolates obtained was grown in SMSE for 48 h at 28°C. One millilitre of each culture was inoculated into 150 ml of(l) SMSE, (2) SMSE containing six metals each at a concentration of l ltg ml-t and (3) SMSE with the same metals at 2/.,g ml-t. The metals were from BDH standard solutions for Atomic Absorption Spectrometry at l mg m l - ' . They were added to the medium before sterilisation and the pH of the medium adjusted. After incubation for 7 days at 28°C in a shaking water bath, the cells were harvested by centrifugation, washed twice in doubly-distilled water and prepared for atomic absorption (Loutit, Patrick and Malthus, 1973).

Worm feeding experiments (1) Sediment (200 g) and 500 ml of water from a stream supporting large numbers of tubificids were placed in 2 l glass tanks. To each tank was added 150 worms of a species of Tubifex and 150 of a species of Limnodrilus. The mud and water was stirred every day for l0 days and samples removed and numbers of bacteria in the slurry estimated by the dilution pour plate procedure. 12) Ballotini spheres (150 g, diameter 0"053 ram) after washing in acid followed by rinsing until achieving a neutral pH were placed in tanks together with 500 ml of autoclaved stream water, and 200 worms (60~0 Tubifex and 40~'~ Limnodrilus). After two days during which time faecal material from the worms was removed by suction, washed bacteria grown either with or without metals for 10 days were added. The worms were harvested after a week, washed twice in doubly-distilled water, and left in doublydistilled water overnight to allow the evacuation of their gut contents. The worms were then prepared for atomic absorption. A portion of the sample of worms as collected from the stream was also washed and readied for analysis of metals.

River sediment to the 15 crn level was collected aseptically, pooled, mixed and sub-sampled. Samples (10 g) were diluted in 0-85~o saline and dilution pour plates prepared using SASE. Plates were incubated aerobically and anaerobically for 5 days and colonies counted.

Atomic absorption spectrometry Media, water and other solutions were sprayed directly into the flame. Other samples were treated as described previously (Loutit et al., 1973). 333

334

F . M . P~,TglCK and M. LOUT~T R E S U L T S AND D I S C C S S I O N

The postulate that bacteria concentrate metals in effluents which are then passed up aquatic food chains was based on observations that levels of metals were higher in sediments, tubificids, plants and eels collected below effluent points than in those collected above. Bacteria in the sediments or the copious quantities of Sphaerotihls found could form the basis of the chain. Sphaerotih,s collected from the river a n d cultured in the laboratory showed high levels of certain metals. As tubificids are reputed to feed on bacteria (Coler, G u n n e r and Zuckerman. 1967: W a v r e a n d Brinkhurst, 1971) and m u d (Marples, 1962). the next step in the investigation was to see if bacteria in m u d were ingested by tubificids, if bacteria isolated from m u d could concentrate metals and whether the bacteria containing the metals would in turn be ingested by tubificids. Estimates of n u m b e r s of bacteria in sediments were higher below the effluent entry points than above (Table 1), suggesting that the effluents provide nutrients for bacterial growth. Sediment and water samples from the c o n t a m i n a t e d area were placed in glass tanks and tubificid worms of two types found in the river added. Every day for 10 days the m u d and water was well stirred, samples removed a n d the n u m b e r s of aerobic bacteria estimated. A similar procedure was adopted for mud and water samples to which no worms had been added. The results of such an experiment are given in Fig. 1. The bacterial population is obviously kept in check by the worms. In further experiments the eleven most prevalent types were isolated from the sediment and identified. Single strains of Aloinomonas, Cellribrio, Brecibacteriron, Sarcina, Cory~lebacterium, Caulobacter a n d Zooolea a n d two strains of each of Bacilhls a n d Achromohatter were found. The 11 isolates were cultured and inoculated into media with and without metals. After incubation for seven days at 28°C, the cells were har-

Table I. Bacteria g-t oven dr) mud taken from a river above and below effluerit entry points. Estimations were made by the dilution plate count technique using Sphaerotilus agar-soil extract medium. Results are the average of 3 plates'dilution, incubated for 4 days at 28:C Station

Effluent

Aerobes

Anaerobes

1

7.9 x 10 "~

5'3 x I0-"

2

5.0 x 10"*

1.0 x 10J

8.7 5-3 1.7 1.6

5'8 1.1 8'2 2'2

Wool dyeing 2b 2c 3 4

x x x x

10~' l0 n 10~' 10n

x x x x

103 10"~ 10J 10"~

Wool scouring 5 5a 6

9.2 x 105 9-9 x 105 1.3 x 10"

2'0 × 10"t 2"0 x 10'* l-6 x 10't

1.3 x l0 n

4-6 x 104

Treated sewage 7

vested by centrifugation, washed and prepared for atomic absorption. The results of these experiments are given in Table 2. The bacterial cells have concentrated the metals in the growth medium and even in those cultures to which no metals were added, the cells have concentrated the small a m o u n t s of metals present. 0 , COr~ Ionkwith noworms

10g~

o

~

I

2

3

O. r n o ~ tut~tizian

t.

5

6

7

8

9

10

Fig. l. Effect of tubificids on numbers of bacteria in river sediment.

Table 2. Concentration of Cr. Cu. Mn. Fe. Pb and Zn by a mixed culture of It bacterial isolates from sediment

Mixed cells--no metal added Mixed cells +1 ttg ml - t of all 6 metals Mixed cells + 2 / a g ml -~ of all 6 metals

Cr

Cu

37"19 1415.42

376-23 395.62

2973-41

1512.84

Mn Fe (,ug g - t dry ~eight)

Pb

Zn

9.00 126.45

212.79 1276.80

173.28 1572.18

527-92 1153.40

456.34

2664.70

1881.31

1127-24

Itg ml- ' *SMSE medium--no metal added SMSE medium + I l~g ml- t of all 6 metals SMSE medium + 2 ,ug ml-1 of all 6 metals

"t'ND

0-24

0-03

0-06

0"08

0"14

1-00

1.20

0-78

0"85

1"05

1"28

2-05

2-40

1.66

1.82

1-90

2"00

* SMSE sphaerotilus agar-soil extract medium. t N D = Not detectable at a level of 0.10/~g ml -t.

Metals in effluents

335

Table 3. The passage of six hea~? metals from bacteria to tubificid worms, the bacteria being used as the sole food source for the worms Sample

Cr

Cu

Mn Fe ,ug g-, dry weight

Pb

Zn

Worms as collected Worms fed cells grown in medium, no metals added Worms fed cells grown in medium +1 /ag m l - ' metals Worms fed ceils grown in medium +2 ,ug ml-t metals Cells--no metals added Cells + 1 lLg ml- t of each metal Cells +2 ,ug ml -t of each metal

19-10 3.92

80-70 236.17

113.04 13.49

1285.96 737.71

151.14 178.95

529.80 261.86

14-08

397.05

17.92

1242.06

559.18

685"08

29.86

621.19

24.97

1944.15

568.31

868.35

109-09 983-00

213.22 765.87

16"53 292.83

289-26 2252-56

119-01 409-56

206-61 1648-46

2850.00

1068.00

558.00

3450.00

720.80

2010.00

fLg mlSMSE~--no metals added SMSE + ltg ml- z of each metal SMSE + 2 ,ug ml-t of each metal 3N HCI (to dissolved ash)

NDI" 1-00 2-05 ND

0.24 1"20 2.40 ND

0.03 0.78 1.66 ND

t

0.06 0"85 1"82 0"22

0-08 1.05 1.90 0.14

0"14 1-28 2-00 ND

* Mean of 5 replicates. "t ND not detectable at levels: Cr 0"10. Cu 0.04. Mn 0-02, Fe 0.05, Pb 0"10. Zn 0.01 ,ttg ml -~. ++SMSE = sphaerotilus agar-soil extract medium. Having shown that bacteria from mud concentrate metals, the next step was to see whether tubificids by ingesting bacteria containing metals would show an increased level of metals. Worms were placed in glass tanks containing a layer of Ballotini spheres in place of sediment. Autoclaved stream water was added after an initial settling in period, equal wet weights of the bacterial cell mixtures which had been grown with and without metals were added. After one week the worms were harvested, washed and left overnight in doubly-distilled water, to allow discharge of their gut contents. The worms were then analysed for metal content and the results are given in Table 3. Worms collected at the same time as those in these experiments were analysed for metal content on the day of collection following washing. The stream water was also analysed for metal content. From the results it is apparent that bacteria from river sediments can concentrate metals which can then be passed to tubificids. The results show that following ingestion of bacteria high in metal concentration the worms showed increased metal level. The reason for the generally lower levels in the worms kept under experimental conditions compared with those fresh from the stream is not known. It is possible that the worms were not ingesting as much food as usual. The differing levels of metal concentration in the cells used in these experiments compared with those recorded in Table 2 probably arises from using mixed cultures and from incubating the cells for 10 rather than 7 days. Different bacterial strains are able to concentrate metals to differing levels and metal concentration tends to increase late in the growth cycle (Loutit, unpublished results). There are likely to be interactions between the different bacterial strains when pooled and in

spite of using inocula prepared under similar conditions in each experiment, some variation in numbers of each strain is likely to occur. These experiments support the hypothesis that bacteria can concentrate metals from water and these metals are then passed to higher organisms following ingestion of the bacteria. Small amounts of metals in effluents can be concentrated by bacteria. Provided material is present in the effluent to cause increased numbers of bacteria, the metal will be held and not dispersed. In this way heavy metals can be passed up food chains. Acknowledyements---This work was supported by a grant from the Scientific Research Distribution Committee. REFERENCES

Coler R. A., Gunner H. B. & Zuckerman B. M. {1967} Selective feeding to tubificids of bacteria, Nature, Lond. 216, 1143-1144. L/Ahnis F. (1920) Laml Wirtscht~lich-Bakteriologisches Praktikum. 2 Auft. Gebriider Born traeger. Bcrlin. Loutit M. W., Loutit J. S. & Brooks R. R. (1967) Differences in molybdenum uptake by microorganisms from the rhizosphere of Raphanus saticus L. grown in two soils of similar origin. Plant. Soil 27, 335-346. Loutit M. W., Patrick F. M. & Malthus R. S. (1973) The role of metal-concentrating bacteria in a food chain in a river receiving effluent, Proc. Unic. Otaffo Med. School 51, 37-38. Patrick F. M. & Loutit M. W. (1972) Concentration of metals by a water bacterium of the genus Sphaerotilus, Proc. Univ. Otayo Med. School 50. 30-3 I. Skerman V. B. D. (1967) A yuide to the Identification of the Genera of Bacteria. 2nd edition. Williams and Wilkins, Baltimore. Wavre M. & Brinkhurst R. O. (197l) Interactions between some tubificid oligochaetes and bacteria found in the sediments of Toronto Harbour, Ontario. J. Fish. Res. Brd Can. 28, 335-341.