Observations of Bacterial Microcolonies on the Surface of Ferromanganese Nodules from Blake Plateau by

Scanning Electron Microscopy

PAUL A. LAROCK AND HENRY L. EHRLICH

Department of Oceanography, Florida State University, Tallahassee, Florida 32306, and Department of Biology,Rensselaer Polytechnic Institute, Troy, New York ]2181

Abstract

Examination of the surface of freshly collected ferromanganese nodules by scanning electron microscopy revealed the presence of microcolonies of rod- and coccus-shaped bac- teria which appeared to be anchored to the nodule surface by slime. The attachment of microcolonies by slime to the surface of freshly collected nodules argues against their being contaminants introduced during nodule collection or processing. These results corroborate cultural and biochemical detection of bacteria on ferromanganese nodules.

Introduction

Unti l now, the presence of bacter ia on and in f e r romanganese nodules has only been demons t ra ted by cul tural and b iochemica l tests. Such studies have revea led the presence of Mn(II ) ox id iz ing bacter ia , Mn( IV) reducing bac ter ia , and bac ter ia which nei ther ox id ize nor reduce manganese [ 7 ] . The Mn(II ) ox id iz ing bac ter ia are able to promote manganese accre t ion to nodules by ca ta lyz ing ox ida t ion o f Mn(I I ) adso rbed to nodules . The Mn( IV) reducing bacter ia have the abi l i ty to reduce Mn( IV) oxides in the nodule matr ix. A role for these o rgan isms in nodule genesis has been d iscussed by Ehrl ich [6 ] . Since s igni f icant numbers of these bac te r ia have been shown to res ide on freshly co l lec ted nodules , visual cor robora t ion of their presence in nodules has become important . In the fo l lowing s tudy, we have examined the surfaces of f reshly col lec ted nodules from Blake Plateau in the At lant ic Ocean for the presence of bacter ia by scanning e lect ron

mic roscopy (SEM).

Materials and Methods

Nodule samples were collected on cruise E-9-73 of the RV Eastward at 32 ~ 00.0'N, 78 ~ 48.0'W and 31 ~ 54.7'N, 77 ~ 21.5'W. The approximate water depth at these stations was

84

MICROBIAL ECOLOGY, Vol. 2, 84-96 (1975) �9 1975 by Springer-Verlag New York Inc.

Microcolonies of Ferromanganese Modules 85

380 and 800 m, respectively. Small nodules between 3 to 10 mm diameter were collected using a Shippex dredge which closed on impact with the bottom. Nodules 5 cm and greater were collected in a chain dredge. Retrieval time of the dredges ranged between 5 to 10 rain depending on water depth. As soon as the sampling devices were secured on deck the appropriate samples were withdrawn and fixation for SEM started within 5 min. Small nodules of approximately 4-6 mm were used intact whereas fragments were chipped from larger specimens. Large nodules were cut by placing them in a Whirl-Pak and then cutting through the envelope with a sterile chisel. The nodules from the Blake Plateau are very brittle, fracturing readily, and thus the cutting procedure described is not unduly harsh. The reason for using fragments of large nodules is that corals and sea fans are frequently found growing on their surfaces and thus the top and bottom sides can be differentiated after collection.

The SEM fixation technique used was that of Arnold et al . [1] as later modified by Arnold et al . [2] concerning ethanol dehydration and critical point drying. The critical point dryer used was built according to our specifications and used liquid carbon dioxide. There are, however, commercially available critical point drying units. The theory of critical point drying for electron microscopy has been discussed by Cohen et al . [3]. After shipboard fixation, the samples were mounted on pedestals and stored in a dessicator until they were gold-palladium (60:40 w/w) coated on shore. All observations were made using a Kent- Cambridge Mark 2A Steroscan electron microscope.

Resu l t s

Examinat ion o f nodule surfaces at low magni f ica t ion under the scan-

ning electron microscope revealed smooth areas (Fig. 1) as well as o ther

areas with adherent debris , including sand grains, coccol i ths; and other

biogenic structures (Fig. 2). In some instances, it was possible to distin-

guish between upper surfaces, i .e . , those directly in contact with the water phase, and lower surfaces, i .e . , those resting on the sediment. In Fig. 2 the

lower surface of a nodule is represented. In Fig. 3 one sees an example of an upper surface re la t ively free o f debris but with upright structures. Al-

though these upright structures are of bacterial d imensions , some of them

may be hol low tubes, resembl ing part or all of a coccol i thophore skeleton.

Figures 4 - 6 show success ive enlargements of a cluster of typical

rod-shaped bacteria lying lengthwise on a nodule surface. Figure 4 shows

the posi t ion and size relation of the bacterial cluster (microcolony) relative

to other debris. Some o f the debris may be cover ing up other bacterial cells

on the nodule surface. Enlargements o f the microcolony (Figs. 5 and 6)

reveal a network of strands by which the bacterial cells appear to be

anchored to the nodule surface and to each other. In at least one area (Fig. 6) a band of ret iculate material seems to be connected with the strands. We

interpret this material to be bacterial sl ime which anchors the cells to the

nodule surface.

Coccoid cells were frequent ly encountered. Figure 7 is a view at low

magnif icat ion showing several microcolonies of cocci in a c revice and the

rough texture o f a nodule surface. Figures 8 and 9 are enlargements of the

86 Paul A. LaRock and Henry L. Ehrlich

central portion of Fig. 7 and show particularly well the contrast between cocci and larger inanimate structures, probably mineral gains. The cocci in this picture are about 0.8 /zm in diameter while the more typical inorganic structures are about 7 /zm in diameter. Figures 10 and 11, which are further enlargements of some of the microcolonies in Fig. 7, reveal that the cocci are anchored to the nodule surface by slime, as evidenced by the network of strands from the coccoid cells to the nodule surface. Figure 10 shows a long, branched strand from two cocci in one of the microcolonies that is attached to the upper surface of the nodule cavity (Fig. 12) which harbors the colony. This strand suggests a tether, as though one or several cells sticking together became dislodged from another site in the cavity at some previous time, but still remained attached to the nodule by a slime strand. The cells were thus afforded a limited degree of movement to their present site but were prevented from being washed away.

A somewhat, larger microcolony of cocci and its surroundings is shown in successive enlargements in Figs. 13-15. Anchoring slime is less apparent in these pictures, but attachments to the nodule can be seen in Fig. 15. Division furrows are very evident in some of the occi in Fig. 16 as are strands of slime connecting a number of the cells.

Discussion

The structures which we have identified as rod-shaped or coccoid bacteria are not likely to be deterital material because of the slime strands which emanate from them and anchor them to the nodule surface (Figs. 6, 10, and 11) and because of the cleavage furrows which are clearly evident among some of the cocci (Figs. 14-16). The aggregation of coccoid cells into microcolonies and the slime strands which anchor them to the nodule surfaces indicate that these bacteria must have resided at their present site on the nodule surface for some time prior to collection of the nodules. The fact that the nodules were processed for SEM immediately after recovery frorn the ocean floor further indicates that the microcolonies must have developed on the nodule surfaces when the nodules were in place on the sediment. The processing of nodules for electron microscopy immediately after recovery is very crucial since Ehrlich et al. [7] have previously shown that bacteria will grow extensively on and in nodules during storage at 4~ at atmospheric pressure.

The attachment of certain bacteria to solid surfaces by slime has been previously reported by Corpe [4] , Fletcher and Floodgate [8 ] , Marshall et

al, (9, 10], Meadows [11, 12], Meadows and Anderson [ 1 3 ] , and Paerl [14J. Some scanning electron photomicrographs of detritus in sediment from Lake Tahoe prepared by Paerl [14] show bacteria attached by slime

Microcolonies of Ferromanganese Modules 87

Fig. 1. Surface of a nodule relatively free of adherent debris.

Fig. 2. Lower surface of a nodule covered by adherent biogenic and inorganic debris. The ocean bottom at tills location consisted of sand and coccolith remains as is evidenced by the aHached material Io lhe nodule.

88 Paul A. LaRock and Henry L. Ehrlich

Fig. 3. Upright hollow structures on the upper surface of a nodule.

Fig. 4. Surface of a nodule harboring a rnicrocolony of rod-shaped bacteria (arrow) among the debris.

Microcolonies of Ferromanganese Modules 89

Fig. 5. Enlargement of the bacterial colony in Fig. 4 showing attachment by slime strands to the nodule.

Fig. 6. Enlargement of the bacterial microcolony in Fig. 4 showing attachment by slime strands.

90 Paul A. LaRock and Henry L. Ehrlich

Fig. 7. Crevice on the surface of a nodule harboring several microcolonies of cocci. The enclosed central area is enlarged in the next figure.

Fig. 8. Enlargement of the central portion of the previous figure. Seen are five distinct microcolonies and a number of single ceils indicated by arrows.

Microcolonies of Ferromanganese Modules 91

Fig. 9. Enlarged portion of Fig. 8 showing the attachment of the central colony to the nodule by filaments.

Fig. 10. Enlarged portion of the cental colony in Fig. 8 showing a long tether-like filament going up from the colony through the center of the figure. The termination of this strand is seen in Fig. 12. Also seen are two rod-shaped bacteria (arrows) that are attached to the nodule by strands of slime.

92 Paul A. LaRock and Henry L. Ehrlich

Fig. 11. An extreme enlargement of the enclosed area of Fig. 10 showing an extensive sl ime network and at lachmenl of the tether f i lament to the large cell in lhe foreground.

Fig. 12. The terminal a t tachment of the tether f i lament seen in Fig. l0 to the upper wall of the crevice. Also seen are a number of rod-like structures of an unknown nature. A single coccus is seen in a surface depression (arrow).

Microcolonies of Ferrornanganese Modules 93

Fig. 13. Microcolony of cocci and organic debris in a concavity of a nodule surface.

Fig. 14. Enlargement of the microcolony of Fig. 13.

94 Pau~ A. LaRock and Henry L. Ehrlich

Fig. 1~. Enlargement of the microcolony of Fig. 13. Notice the attachment of the cells to the nodule by slime strands and the smooth appearance of the ceils relative to those in Fig. 15.

Fig. 16. Enlargement of the microcolony of cocci in Fig. 13 showil~g cleavage furrows and strands connecting clumps of cells (arrows).

Microcolonies of Ferromanganese Modules 95

webs resembl ing those which anchor bacter ia to f e r romanganese nodule surfaces as shown in this paper . Marshal l et a l . [9] have descr ibed a bacterial success ion in the process of ce l lu la r a t tachment .

Excess s l ime or s l ime left behind after d i s lodgemen t of bacter ia f rom nodule surfaces may be metabo l i zab le by Mn( IV) reducing bac ter ia as their source of carbon and energy. If the s l ime is po lysacchar ide in compos i t ion , as found by Corpe [4] and by F le tcher and F loodga te [ 8 ] , it may be a source of reducing power with which these bacter ia conver t Mn( IV) to Mn(II) .

The f inding o f cocci on nodules from Blake Plateau was prev ious ly reported by Ehrl ich [5 ] . The cocci may represent micrococc i or the coccoid phase o f A r t h r o b a c t e r , which was also found on nodules from Blake Plateau [5 ] .

The f requency of bacter ia l occurrence on nodules ca lacula ted on the basis of mic roco lony d is t r ibut ion in Fig . 11 is about 6 x l 0 s per cm 2. Ehrl ich et a l . [7] p rev ious ly repor ted on the order o f 103 organisms per gram of surface scrapings of nodules from Blake Pla teau.

The foregoing e lec t ron rnicrographic observa t ions provide visual con- f i rmat ion of the previous detec t ion by cultural means o f bacter ia in associa- tion with fe r romanganese nodules . The bacter ia are o f morpho log ica l ly dis t inct types that appear well sui ted for growth on solid surfaces by the format ion of a s l ime anchor ing network.

Acknowledgment

We thank Roland Walker for helpful discussion of the interpretation of the tubular. structures in Fig. 3. Thanks are due Carol D. Litchfield and the Duke University marine lab for providing space aboard cruises E-9-73 and E-4-74 of the RV Eastward. We also express our gratitude to the electron microscope facilities at the Florida State University and to William Miller for his expert skill on the SEM.

References

I. Arnold, J.D., Berger, A.E., and Allison, O.L. 1971. Some problems of fixation of selected biological samples for SEM examination. Proceedings of the Fourth Annual Scanning Electron Microscope Symposium, liT Research Institute, Chicago.

2. Arnold, J.D., Barnes, W.G., and Berger, A.E. 1974. Clinical experience with Cefzo- lin: Results with fifty patients. Infection 2: 97-101.

3. Cohen, A.L., Marlow, D.P., and Garner, G.E. 1968. A rapid critical point method using fluorocarbons ("Freons") as intermediate transitional fluids. J. Micros- copie7: 331-342.

96 Paul A. LaRock and Henry L. Ehrlich

4. Corpe, W.A. 1970. An acid polysaccharide produced by a primary filnl-forming marine bacterium. Devel. Industr. Microbiol. 11:402-412.

5. Ehrlich, H.L. 1963. Bacteriology of manganese nodules. I. Bacterial action on man- ganese in nodule enrichn/ents.. Appl. Microbiol. 11: 15-19.

6. Ehrlich, H.I,. 1972. The role of microbes in manganese nodule genesis and degrada- lion. 117.' Ferromanganese Deposits on the Ocean Floor. D.R. Horn, editor, pp. 63-70. The Office of the International Decade of Ocean Exploration. National Science Foundation, Washington, D.C.

7. Ehrlich, H.L., Ghiorse, W.C., and Johnson II, G.L. 1972. Distribution of microbes in manganese nodules from the Atlantic and Pacific Oceans. Devel, lndustr. Microbiol. 13: 57-65.

8. Fletcher, M. and Floodgate, G.D. 1973. An electron-microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J. Gen. Microbiol. 74 325-334.

9. Marshall, K.C., Stout, R., anti Mitchell, R. 1971. Selective sorption of bacteria from seawater. Can. J. Microhiol. 17: 1413-1416.

If). Marshall, K.C., Stout, R., and Mitchell, R. 1971. Mechanisms of the initial events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68: 337-348.

1 I. Meadows, P.S. 1965. Attachment of marine and fresh water bacteria to solid surfaces. Nature (London) 20'7: 1108.

12. Meadows, P.S. 1971. The attachment of bacteria to solid surfaces. Arch. f . Mikrobiol. 75: 374-381.

13. Meadows, P.S. and Anderson, J.G. 1966. Microorganisms attached to marine and freshwater sand grains. Nature (LomlolO 212: 1059-1060.

14. Paerl, H.W. 1973. Detritus in Lake Tahoe: Structural modification by attached microf- Iota. Science 180: 496-498.