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 bacteria 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
Until now, the p r e s e n c e of bacteria on and in f e r r o m a n g a n e s e nodules has only been d e m o n s t r a t e d by cultural and b i o c h e m i c a l tests. Such studies have r e v e a l e d the p r e s e n c e o f Mn(II) o x i d i z i n g bacteria, M n ( I V ) reducing b a c t e r i a , and b a c t e r i a w h i c h neither o x i d i z e nor r e d u c e m a n g a n e s e [ 7 ] . T h e Mn(II) o x i d i z i n g b a c t e r i a are able to p r o m o t e m a n g a n e s e a c c r e t i o n to n o d u l e s by c a t a l y z i n g o x i d a t i o n o f M n ( I I ) a d s o r b e d to n o d u l e s . T h e M n ( I V ) reducing bacteria have the a b i l i t y to reduce M n ( I V ) o x i d e s in the nodule matrix. A role for these o r g a n i s m s in nodule genesis has been d i s c u s s e d by Ehrlich [ 6 ] . Since significant numbers o f these b a c t e r i a have been shown to reside on freshly c o l l e c t e d n o d u l e s , visual c o r r o b o r a t i o n of their presence in nodules has b e c o m e important. In the f o l l o w i n g study, we have e x a m i n e d the surfaces of freshly c o l l e c t e d nodules from B l a k e Plateau in the Atlantic O c e a n for the presence o f bacteria by scanning electron m i c r o s c o p y (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.

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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 KentCambridge Mark 2A Steroscan electron microscope.

Results
E x a m i n a t i o n o f nodule surfaces at low m a g n i f i c a t i o n under the scanning e l e c t r o n m i c r o s c o p e r e v e a l e d s m o o t h areas (Fig. 1) as well as o t h e r areas with adherent debris, including sand grains, co cco l i t h s; and other b i o g e n i c structures (Fig. 2). In s o m e instances, it was possible to distinguish b e t w e e n upper surfaces, i . e . , those directly in contact with the water phase, and l o w e r surfaces, i . e . , those resting on the sediment. In Fig. 2 the l o w e r surface o f a nodule is represented. In Fig. 3 one sees an e x a m p l e o f an upper surface r e l a t i v e l y free o f debris but with upright structures. Although these upright structures are o f bacterial d i m e n s i o n s , s o m e o f them may be h o l l o w tubes, r e s e m b l i n g part or all of a c o c c o l i t h o p h o r e skeleton. Figures 4 - 6 show s u c c e s s i v e e n l a r g e m e n t s o f a cluster o f typical rod-shaped bacteria lying l e n g t h w i s e on a nodule surface. F i g u r e 4 shows the position and size relation of the bacterial cluster ( m i c r o c o l o n y ) relative to other debris. S o m e o f the debris may be c o v e r i n g up other bacterial cells on the nodule surface. E n l a r g e m e n t s o f the m i c r o c o l o n y (Figs. 5 and 6) r e v e a l a network o f strands by w h i c h the bacterial cells appear to be anchored to the nodule surface and to each other. In at least one area (Fig. 6) a band o f reticulate material seems to be c o n n e c t e d with the strands. W e interpret this material to be bacterial slime which anchors the cells to the nodule surface. C o c c o i d cells were frequently e n c o u n t e r e d . Figure 7 is a v i e w at low m a g n i f i c a t i o n s h o w i n g several m i c r o c o l o n i e s o f cocci in a c r e v i c e and the rough texture o f a nodule surface. F i g u r e s 8 and 9 are e n l a r g e m e n t s of the

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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

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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.

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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.

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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.

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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.

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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.

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Fig. 11. An extreme e n l a r g e m e n t of the enclosed area of Fig. 10 showing an extensive slime network and atlachmenl of the tether filament to the large cell in lhe foreground.

Fig. 12. The terminal attachment of the tether filament 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).

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

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

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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).

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webs r e s e m b l i n g those which a n c h o r bacteria to f e r r o m a n g a n e s e nodule surfaces as shown in this paper. Marshall et a l . [ 9 ] have d e s c r i b e d a bacterial succession in the process o f c e l l u l a r attachment. Excess slime or slime left behind after d i s l o d g e m e n t of bacteria from nodule surfaces may be m e t a b o l i z a b l e by M n ( I V ) reducing b a c t e r i a as their source of carbon and e n e r g y . If the slime is p o l y s a c c h a r i d e in c o m p o s i t i o n , as found by C o r p e [ 4 ] and by F l e t c h e r and F l o o d g a t e [ 8 ] , it may be a source o f reducing p o w e r with which these bacteria convert M n ( I V ) to Mn(II). The finding o f cocci on nodules from Blake Plateau was p r e v i o u s l y reported by Ehrlich [ 5 ] . The cocci may represent m i c r o c o c c i or the c o c c o i d phase o f A r t h r o b a c t e r , which was also found on nodules from B l a k e Plateau [5]. The frequency o f bacterial o c c u r r e n c e on nodules c a l a c u l a t e d on the basis o f m i c r o c o l o n y d i s t r i b u t i o n in Fig. 11 is about 6 x l 0 s p e r c m 2. Ehrlich et a l . [7] p r e v i o u s l y reported on the o r d e r o f 103 o r g a n i s m s per gram o f surface scrapings o f nodules from Blake Plateau. T h e f o r e g o i n g e l e c t r o n rnicrographic o b s e r v a t i o n s p r o v i d e visual confirmation o f the previous detection by cultural means o f bacteria in association with f e r r o m a n g a n e s e nodules. The bacteria are o f m o r p h o l o g i c a l l y distinct types that a p p e a r well suited for growth on solid surfaces by the formation o f a slime a n c h o r i n g 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. Arnold, J.D., Barnes, W.G., and Berger, A.E. 1974. Clinical experience with Cefzolin: Results with fifty patients. Infection 2: 97-101. Cohen, A.L., Marlow, D.P., and Garner, G.E. 1968. A rapid critical point method using fluorocarbons ("Freons") as intermediate transitional fluids. J. Microscopie7: 331-342.

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Paul A. LaRock and Henry L. Ehrlich Corpe, W.A. 1970. An acid polysaccharide produced by a primary filnl-forming marine bacterium. Devel. Industr. Microbiol. 1 1 : 4 0 2 - 4 1 2 . Ehrlich, H.L. 1963. Bacteriology of manganese nodules. I. Bacterial action on manganese in nodule enrichn/ents.. Appl. Microbiol. 11: 15-19. Ehrlich, H.I,. 1972. The role of microbes in manganese nodule genesis and degradalion. 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. 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. 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. Marshall, K.C., Stout, R., anti Mitchell, R. 1971. Selective sorption of bacteria from seawater. Can. J. Microhiol. 17: 1413-1416. 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. Meadows, P.S. 1965. Attachment of marine and fresh water bacteria to solid surfaces. Nature (London) 20'7: 1108. Meadows, P.S. 1971. The attachment of bacteria to solid surfaces. A r c h . f . Mikrobiol. 75: 374-381. Meadows, P.S. and Anderson, J.G. 1966. Microorganisms attached to marine and freshwater sand grains. Nature (LomlolO 212: 1059-1060. Paerl, H.W. 1973. Detritus in Lake Tahoe: Structural modification by attached microfIota. Science 180: 496-498.

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