Microbial Attachment to Particles in Marine and F r e s h w a t e r Ecosystems HANS W,

PAERt.

Division o1" Environmental Studies,

Urn'versify ~f Cali/~)rnia, Davis, Cal(/'ornia 95616

Abstract Scanning electron microscopy observations of in situ suspended marine and fi'eshwater particles show diverse but similar modes of bacterial and funga/ attachment. A survey of Sierra Nevada mountain lakes and pelagic and near-shore waters in the Pacific Ocean indicates that attachment is most noticeable in the near-surface waters where fresh dissolved and particulate input of carbon from phytoplankton and elevated telnperatures favor microbial growth. The most common modes of attachment are: adhesive stalk formatibn, growth on adhesive webs, attachlnent by the use of pill-like appendages and slimy capsular secretions, and molecular or chemical sorption without the use of visualized structural appendages. Attached microbial growth is accelerated when particulate subslrates are supplied, even when they are not rich in organic nutrients. This is the case in the Lake Tahoe basin, where microflora attached to eroded silts can significantly modify tile organic carbon and nutrient content of such minerogenous particles.

Introduction Uncertainty exists as to the frequency and general occurrence of in situ microbial (bacterial and fungal) attachment in marine and freshwater

ecosystems. Although attachment to surfaces in enclosed vessels has been documented [18], current marine and freshwater in situ studies have yielded differing conclusions. Wiebe and Pomeroy [22] found very few attached bacteria in numerous samples from coral reefs, the central Sargasso Sea, and Antarctic and sub-Antarctic waters. In contrast, Jannasch and Pritchard [7] and Seki [17] observed bacteria attached to detritus (nonliving particulate organic matter) in the oceans, and Rodina [15] has obtained similar results in lakes. The conflict is based on the ability or inability to discern between detritus and microorganisms in freshly filtered and strained water samples. Scanning electron microscopy (SEM) has recently been used to examine detritus [10] and has allowed for a more definitive description of 73 MICROBIAL ECOLOGY, Vol. 2, 73-83 (1975) 9 1975 by Springer-Verlag New York Inc.

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Hans W. Paerl

detritat micro-environments. Scanning electron microscopy has revealed that microorganisms less than 0.5 /xm in diameter are tightly bound to suspended particles and are involved in increasing the production and size of particles by the secretion of organic matter while attached [4, l l ] . In this paper a SEM survey of freshly fixed suspended matter from freshwater and marine ecosystems reveals that microbial attachment to particles (both organic and inorganic) is common in both systems. Various modes of attachment due to morphological and physiological specialization of microbial cells are described. Certain modes have profound effects on the chemical composition and size of particles.

Methods and Materials Seven freshwater lakes were sampled, ranging in altitude from 800 to 2800 m and in surface area from 6 km 2 (Convict Lake) to 500 km ~ (Lake Tahoe). Tile lakes are situated in the central and southern Sierra Nevada mountain range. This range is located along the eastern border of California. All lakes were sampled March 23, 1973. The aerial distance from the northernmost to southernmost lake is approximately 200 miles. Water samples for SEM were taken with a alcohol-rinsed (95%) Van Dorn PVC sampler at a depth of 2 m in each lake except Lake Tahoe. At Lake Tahoe, samples were taken at 20 m because phytoplankton were highly light inhibited at 2 m in this ultra-oligotrophie lake. June and Convict Lakes were ice-covered during the sampling period. Marine sampling was done during two cruises of the R. V. Alexander Agassiz (June 1973, October 1973) off tile coast of southern California. Scanning electron microscopy samples were taken with Van Dorn samplers from a 0-500 m profile 150 km west of San Diego (32 ~ 41' 2 sec N, 117 ~ 51' 0 sec W) and a 0-50 m profile taken near a sewage outfall 6 km west of Pt. Loma, San Diego, Samples laken from the Sierra Nevada lakes were transported in polyethylene gallon jugs, placed in coolers, and processed as rapidly as possible. Maximum elapsed time between sampling and filtration was 5 hr at lake temperatures. Marine samples were filtered and fixed immediately after sampling. Scanning electron microscopy preparations were made according to steps described by Paerl and Shimp [13] briefly outlined here. For both marine and freshwater, 50-100 ml subsamples were filtered at 800 torr through 25 mm diameter, 0.2 ,gin porosity Nuclepore (General Electric Corp.) filters. After filtration, all filters were folded in half and inserted into similarly sized squares of aluminmn foil used to clamp and retain the filters while fixation and drying steps were performed. Filters and clamps were fixed in 2% glutaraldehyde solutions buffered with 0.1 M sodium cacodylate in freshwater or filtered seawater (marine) for 1 hr at 4~ After fixation, marine filters were desalinized by immersing them for 10 Mill in decreasing concen~,rations of seawater (90%, 75%. 50%, 25%, 0%). Filters were then traasferred to distilled water and taken through stepwise ethyl alcohol dehydratmn (10 rain in 25%, 50%, 75%, 100% ethyl alcohol solutions). Filters were immersed in amyl acetate for at least 15 min prior to carbon dioxide critical point drying as outlined by Anderson [ 1]. Small squares of dried filters were illounted oil g E M stubs. Scanning electron microscopy stubs were coated with gold-palladiuln (60/40) or silver-gold (50/50) to a thickness of 150 ,~ and viewed with a Cambridge MK II stereoscan scanning electron microscope at an accelerating voltage of 20 kV.

Microbial Attachment to Panicles

75

In addition to SEM sampling, stream silt slurries eroded by an October 22, 1973 rainstorm in the Ward Creek watershed (Ward Creek is a tributary in the Tahoe basin) were collected in polyefllylene gallon jugs and filtered through precombusted, acid cleaned, 25 mm Whatman GFC filters for analyses of the following total particulates: organic carbon [2], carbohydrate [ i 6 ] , protein [8], and phosphorus [5]. Fillers were rinsed with 20 ,n[ of 0.1 N HCl and 20 ml of distilled water prior to analyses to avoid adsorbed sources of carbon and phosphorus. Turbidity blanks were run on clean filters. The silt slurry sampled was equally dispensed into duplicate (for each analysis) 1 liter dialysis bags made from cellulose dialysis tubing. Slurries were incubated with natural lake microorganisms sampled at 20 m in Lake Tahoe. The dialysis bags were closed at each end with monofilament fishing line. The ends were then sealed by dipping them in paraffin wax. The final concentrations of silt in dialysis bags were similar to those at the mouth of Ward Creek in Lake Tahoe. After sealing the ends the bags were transferred to a darkened double-walled container with 1 cm nonoverlapping holes in the sides and incubated in situ in Lake Tahoe at 20 m depth. Dialysis bags allowed lor diffusion of soluble mitrients and metabolic waste products. To check on chemical adsorption to particulate matter under investigation, sterile bags were made by dipping sealed bags in 2,4-dinitrophenol (DNP) (10 -~M) 24 hi- prior to incubation along with live samples. Repeated 2 week incubations with 2,4-DNP treated bags, followed by microscopic and plate counts as well as ATP analyses for live organisms, indicated that wax-sealed bags remained sterile. After a 2 week incubation period bolh sets of bags were analyzed for the particulate fractions as outlined above. In addition to particulate analyses, adenosine triphospbate (ATP) deterlninations, using the hlciferin-luciferase reaction, were made. The contents of dialysis bags were filtered at 800 torr through sterile 25 mm GFC filters. Extractions and ATP analyses were done according to Holm-Hansel~ and Booth [6], and ATP values were converted to live cellular carbon by multiplying ATP content by 250. Earlier work [6] has shown ATP content of live cells to be a constant percentage of total living microbial biomass expressed as dry weight. The concentrations of silt in the bags were low enough to avoid measurable adsorption of ATP to particles.

Results In b o t h m a r i n e a n d f r e s h w a t e r , m i c r o b i a l a t t a c h m e n t w a s c o m m o n l y o b s e r v e d , a l t h o u g h to v a r y i n g e x t e n t s . N e a r s u r f a c e , o p e n o c e a n w a t e r s contained small particles, composed of diatom frustules and remains of algal c e l l s c o a t e d w i t h m u c o i d - l i k e m a t e r i a l a n d b a c t e r i a . A g g r e g a t e s v a r i e d in s i z e f r o r n 5 to 30 p a n . In d e e p e r w a t e r ( > 100 m) p a r t i c l e s w e r e m o r e d i f f i c u l t to i d e n t i f y , w e r e g e n e r a l l y s m a l l e r , a n d m i c r o b i a l a t t a c h m e n t s h o w e d a marked decrease. Diverse types of attached bacteria were observed, having various mechanisms of attachment. Attached forms were o b s e r v e d on e x t e n s i v e w e b - l i k e n e t w o r k s o f s l i m e m a t e r i a l ( F i g . 1A). B o t h rod and coccal f o r m s w e r e p r e s e n t and single cells c o u l d be s e e n on particles w i t h s t r i n g s o f s e c r e t e d m a t e r i a l t r a i l i n g b e h i n d t h e m . T h e d i v e r s i t y o f cell types

attached

on

a single particle

was

surprisingly

high.

Commonly,

stalked bacteria w e r e a t t a c h e d to particles also s u p p o r t i n g small rods. S a m p l e s t a k e n neat" the Pt. Loxna s e w a g e o u t f a l l

showed dramatic

d i f f e r e n c e s in t e r m s o f a t t a c h m e n t as w e l l as cell s i z e . L a r g e r o d s ( I - 3 ,u.m

76

Hans W. Paerl

Fig. 1. SEM observations on marine particulate matter. (A) Surface morphology of a large (50 p.m diameter) detrital aggregate sampled from 100 m at the pelagic station 150 km west of San Diego. The length of attached rod-shaped bacteria is 1 /~m. (B) Detrital aggregate sampled from 20 m near the Point Loma sewage outfall. Large flocs supporting from 50 to 100 bacteria were routinely observed from all depths at this station.

Microbial Attachment to Particles

77

in length) were present in abundance on particles and aggregates (Fig. 1B). Aggregates were larger in size, 10-50 ~ m , than in open ocean waters. Few cellular appendages were seen except flagella, which in the case of the predominant rods were polar. These rods were also seen as free floating cells but their adhesive nature was evident in extensive clumping of cells and dense populations on particles. It appeared that many attached bacteria at this location were directly discharged from the sewage plant because these bacteria were morphologically similar to cells sampled near the mouths of outlet pipes. Bacteria were viable in seawater since microautoradiographic examinations of subsamples from the same location [11] revealed bacteria actively assimilating tritiated organic substrates. Freshwater attachment was also diverse. At Lake Tahoe, surfaces of large particles, 10-30 ~ m , supported small colonies of rods firmly embedded in secreted networks resembling capsular material (Fig. 2A). Small cocci were seen on complex webbing which was laid down on diatom frustule aggregates. Rods having adhesive stalks were also observed on particles. Cells which appeared adsorbed to suspended matter were seen in virtually all samples. Similar observations were noted by Marshall et al. [9]. Adsorbed cells from Lake Tahoe showed no clear mechanism for attachment and were predominantly nonmotile when present in fresh preparations viewed with light optics. These cells were also found in a freefloating state. Pyramid Lake, which is an alkaline lake located in western Nevada, contained cocci having fibrillar appendages which served to keep the cells tightly anchored to suspended matter (Fig. 2B). Similar characteristics were shown for large ( 4 - 5 / z m in length) nonmotile rods attached to periphytic algae flowing into Lake Tahoe from Ward Creek (Fig. 2C). Ice-covered June and Convict Lakes contained large aggregates of amorphous organic material interlaced with filamentous bacteria and fungal hyphae (Fig. 2D). Filaments revealed extensive amounts of extracellular organic materials. Mono Lake, an extremely alkaline lake (pH 10.8) with a low diversity of phytoplankton and zooplankton, also contained particles rich in attached filamentous microorganisms. Freshly eroded silt incubated in Lake Tahoe showed showed a marked enrichment by particulate carbon, expecially carbohydrate (Fig. 3). Enrichment by protein and total phosphorus was noticed as well. Effects of microbial growth on mineral particles in terms of shifts in C and P to total sediment weight ratios were observed. The ratio of sediment weight to total carbon and phosphorus weights changed from 100:20:0.2 for fresh silts to 100:50:0.5 for colonized silts, indicating substantial enrichment of sediments. Scanning electron microscopy observations on dialysis bag contents indicated extensive colonization of particles by bacteria. A 2 to 1 ratio of attached to free-floating cells was observed. Colonization of fresh silts and concurrent deposition of cellular structural and secreted materials was evi-

78

Hans W. Paerl

Fig. 2. SEM observations on freshwater particulate matter. (A) High-magnification view of a attached colony of rod-shaped bacteria (bacterial width is 1 /xm) embedded in a web of organic fibers. Particles revealing attached microcolonies were present in the upper 100 m of Lake Tahoe. (B) Small coccal bacterium (diameter 0.75 p,m) attached to a diatom frustule with the aid of fibrillar appendages. Sample taken at Pyramid Lake, Nevada. (C) Bacterial attachment in Lake Tahoe with the

Microbial Attachment to Particles

79

aid of fibrillar appendages. Dense populations of rods (4-5 p.m in length) were observed on stream-borne periphytic algae which were deposited in pelagic waters of Lake Tahoe. (D) Extensive aggregation of particulate organic material as observed in ice-covered Convict Lake. Interlaced fungal and bacterial growth formed the framework of these large aggregates (150 /zm diameter).

80

Hans W. Paerl 280

--

Partic 24(

20( Q} Q.

E

160Partic

O t'N

Partic

120-

80CHO

40 CHO AT P]

TO

T 1 Sterile

T 1 Live

Fig. 3. Comparative accumulation of ATP-biomass carbon (ATP), carbohydratecarbon (CHO), and total particulate carbon (partic) in duplicate sterile vs. nonsterile dialysis bags. Twenty milliliters were removed from each 1 liter bag for individual analysis. The incubation period lasted 2 weeks in situ in Lake Tahoe.T, represents carbon levels at the start of incubation and TI represents carbon levels after a 2 week incubation period.

dent when dialysis bag ATP-biomass values were compared to increases in total particulate carbon and carbohydrate (Fig. 3). These results indicated that during the colonization period dead cellular and excreted material was deposited on particles while the living carbon fraction remained in a near steady state. Sterile dialysis bags showed virtually no accumulation of particulate carbon or ATP (Fig. 3). Scanning electron microscopy observations of particles in dialysis bags revealed fibrillar and capsular deposition by bacteria on particles. Discussion

Microbial attachment to particles in aquatic ecosystems appears to be common rather than exceptional. Scanning electron microscopy observations on a wide variety of in situ suspended particles, beyond examples shown in this paper, substantiate earlier conclusions that particles serve as growth-conducive surfaces in both freshwater and marine systems [7, 18,

Microbial Attachment to Particles

81

21], as well as soils [19, 20]. Even in oligotrophic waters, extensive attachment was observed and in some cases bacteria were present on particles not much larger than cellular size. One possible explanation for attachment in oligotrophic waters is the ability for bacteria to remain near articulate nutrient sources in ultra-low dissolved nutrient environments 18]. Jannasch and Pritchard [7] have shown, however, that particles may stimulate bacterial growth even when they contain virtually no detectable nutrients. Similarly, studies with low organic silt additions presented here as well as hydrolyzed (nutrient stripped) silt additions made to Lake Tahoe water in earlier nutrient-independent experiments [12] point out that particle surface area is a variable of considerable importance in promoting in situ 9 microbial growth. As in soil-clay systems, adsorption of nutrients leading to the formation of " m i c r o l a y e r s " of concentrated nutrients in dilute aquatic systems may offer an attractive growth site for organisms capable of attaching to or being adsorbed on a particle.

f

Scanning electron microscopy observations have shown that some bacteria possess varied means of assuring attachment. Among these are adhesive stalk formation, capsular secretions, fibrillar appendages which serve as anchors, attached webbing on which cells are located, and sorption of bacterial cells to particles without the aid of cellular appendages or secretions. The formation of appendages and secretions undoubtedly requires energy in the form of reduced organic structural compounds. It is likely that energy expenditure is rewarded by the ability to increase growth and replication on favorable physical and chemical substrates. Microbial colonization of particles alters the chemical properties of such particles in dialysis bags. Dialysis bags cannot perfectly duplicate natural conditions because they alter turbulence and are enclosed, though being diffusive vessels. However, results from these studies confirm in situ evidence off stream mouths [10] and in the oceans [14], citing enrichment of particulate matter by the dissolved organic pool. Of the particulate carbon added to particles, carbohydrate appears to be a significant fraction. Carbohydrate analyses, using the anthrone reaction, provide underestimates of total carbohydrate present, since the method only detects carbohydrates composed of six-carbon sugars. Other numbered carbon sugars and carbohydrates not detected by the anthrone method may be included in the webbing and slime-like materials laid down on particles. Corpe [3] and Floodgate [4] have identified acidic polysaccharides as being important components of capsular material used as adhesives by aquatic bacteria. Increases in protein, probably due to structural and cytoplasmic components of the attached cells, are also noticed, but such increases are not as pronounced as increases in carbohydrates.

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T h e a t t a c h m e n t and g r o w t h p r o c e s s e s d e s c r i b e d h e r e add f u r t h e r c o m p l e x i t y to the a l r e a d y c o m p l i c a t e d p r o c e s s e s o f m i c r o b i a l d e c o m p o s i t i o n o f p a r t i c u l a t e matter. E v e n t h o u g h sotne o f the a t t a c h m e n t sites m a y b e m i n e r a l i z e d t h r o u g h e n z y m a t i c p r o c e s s e s not d i s c u s s e d h e r e , there is also a net f l o w of d i s s o l v e d o r g a n i c c a r b o n ( D O C ) into the p a r t i c u l a t e state. As a r e s u l t , m i n e r a l i z a t i o n and o r g a n i c c a r b o n loss f r o m p a r t i c l e s m a y b e m a s k e d by u p t a k e o f D O C at d e p t h s w h e r e a t t a c h m e n t and g r o w t h are f a v o r a b l e . T h e s e d e p t h s a p p e a r to be n e a r the s u r f a c e o f l a k e s and o c e a n s , for e l e v a t e d t e m p e r a t u r e s in these r e g i o n s will e n h a n c e net g r o w t h and t h e r e f o r e D O C u p t a k e . In a d d i t i o n , n e w l y g e n e r a t e d s o u r c e s o f D O C are in g r e a t e r abund a n c e in the u p p e r w a t e r m a s s e s s i n c e algal p h o t o s y n t h e t i c c a r b o n r e d u c t i o n is l i m i t e d to t h e s e z o n e s . In o r d e r to o b t a i n a rnore c o m p l e t e p i c t u r e of bacterial c o n v e r s i o n and release o f growth limiting nutrients such as phosphate, nitrate, and a m m o n i a , other facets of mineralization must be investigated. A m o n g t h e s e are b a c t e r i a l a u t o l y s i s , d i g e s t i o n and r e l e a s e by p r o t o z o a n s and c r u s t a c e a n z o o p l a n k t o n , and o x i d a t i o n - r e d u c t i o n b r e a k d o w n o f sinking aggregates.

Acknowledgments Marine sampling was through the facilities of Scripps Institution of Oceanography. Particular thanks to Drs. C. R. Goldman, A. F. Carlucci, O. Hohn-Hansen, and S. L. Shimp for their help and advice during these studies. Scanning electron micrographs were taken at the Facility for Advanced Instrumentation, U.C. Davis. The author appreciates the helpful comments of Drs. M. P. Starr and A. F. Carlucci in their review of this manuscript. This work was funded by NSF-RANN Grant GI-22 to the Tahoe Rcsearch Group.

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Anderson, T. F. 1951. Techniques for the preservation of three dimensional structure in preparing specirnens for the electron microscope. Trans. N.Y. Acad. Sci., Sec. 2, 13: 130-134.

2.

Armstrong, R., Goldman, C. R., and Fujita, D. K. 1971. A rapid method for the estimation of carbon content of seston and periphyton. Limm~l. Oceanogr. 16: 137-139.

3.

Corpe, W. A. 1970. Attachment of marine bacteria to solid surfaces. In: Adhesion in Biological Systems. R. Manly, editor, pp. 73-85. Academic Press, New York.

4.

Floodgate, G. M. 1972. The mechanism of" bacterial attachment to detritus in aquatic systems. Mere. 1st. Ital. ldrobiol.; Suppl. 29: 309-323.

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Henriksen, A., 1970. Determination of total nitrogen, phosphorus and iron in freshwater by photo-oxidation with ultra-violet irradiation. Analyst 95: 601-608.

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Hohn-Hansen, O. and Booth, C. R. 1966. The measurement of adenosine triphosphate in the oceans and its ecological significance. Limnol. Oceanogr. 11: 510-519.

7.

Jannasch, H. W. and Pritchard, P. H. 1972. The role of inert particulate matter in the activity of aquatic microorganisms. Mere. 1st. Ital. ldrobiol.. Suppl. 29" 289308.

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Lowry, O. H., Roseborough, N. J., Farr, A. L., and Ranstall, R. J, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

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Marshall, K. C., Stout, R., and Mitchell, R. 1971. Selective sorplion of bacteria from seawater. Can. J. Microbiol. 17: 1413-1416.

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

l 1.

Paerl, H. W . , 1974. Bacterial uptake of dissolved organic matter in relation to detrital aggregation in marine and fresh-water systems. Limnol. Oceanogr. 19" 966972.

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Paerl, H. W. and Goldman, C. R. 1972. Stimulation ofheterotrophic and autotrophic activities of a planktonic microbial community by siltation at Lake Tahoe, California. Mere. 1st. ltal. ldrobiol., Suppl. 29: 129-147.

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Paerl, H. W. and Shimp, S. L. 1973. Preparation of filtered plankton and detritus for study with scanning electron microscopy. Limnol. Oceanogr. 18: 802-805.

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Riley, G. A., 1963. Organic aggregates in sea water and the dynamics of their formation and utilization. Limnol. Oceanogr. 8: 372-381.

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Rodina, A. G., 1963. Microbiology of detritus of lakes. Limnol. Oceanogr. 8: 388393.

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Scott, T. A. and Melvin, E. H. 1953. Determination of dextran with anthrone. Anal. Chem. 25: 1656-1666.

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Seki, H., 1972. The role of microorganisms in the marine food chain with reference to organic aggregates. Mere. 1st. ltal. ldrobiol., Suppl. 29: 245:259.

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Stark, W. H., Stadler, J., and McCoy, E. 1938. Some factors affecting the bacterial populations of freshwater lakes. J. Bacteriol. 36: 653-654.

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Stotzky, G., 1967. Clay minerals and microbial ecology. Trans. N.Y. Acad. Sci. 30: 11-21.

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Stotzky, 1973. Techniques to study interactions between microorganisms and clay minerals in vivo and in vitro. Bull. Ecol. Res. Comm. (Stockholm) 17: 17-28.

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W a k s m a n , S. A. and Carey, C. L. 1935. Decomposition of organic matter in seawater by bacteria. J. Bacteriol. 29: 531-543.

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Wiebe, W. J. and Pomeroy, L. R. 1972. Microorganisms and their association with aggregates and detritus in the sea: A microscopic study. Mem. 1st. ltal. Idrobiol., Suppl. 29: 325-352.

Microbial attachment to particles in marine and freshwater ecosystems.

Scanning electron microscopy observations ofin situ suspended marine and freshwater particles show diverse but similar modes of bacterial and fungal a...
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