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

H A N S 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 indi- cates 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.

In t roduct ion

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 (nonliv- ing 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.

74 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 micro- bial cells are described. Certain modes have profound effects on the chemi- cal 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 phyto- plankton 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 traas- ferred 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 gEM 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 [ i6] , 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 con- verted 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 bo th m a r i n e and 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 was 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 ex t en t s . Nea r s u r f a c e , o p e n o c e a n w a t e r s

c o n t a i n e d smal l pa r t i c l e s , c o m p o s e d o f d i a t o m f rus tu le s and r e m a i n s o f

algal ce l l s coa t ed wi th m u c o i d - l i k e ma te r i a l and bac te r i a . A g g r e g a t e s var ied

in s ize f rorn 5 to 30 pan. In d e e p e r w a t e r ( > 100 m) pa r t i c l e s w e r e m o r e

d i f f i cu l t to i den t i fy , w e r e g e n e r a l l y s m a l l e r , and 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 m a r k e d d e c r e a s e . D i v e r s e t ypes o f a t t a ch ed bac te r ia w e r e ob-

s e rved , h a v i n g var ious m e c h a n i s m s o f a t t a c h m e n t . A t t a c h e d f o r m s w e r e

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 ime mater ia l (Fig. 1A). B o t h

rod and coccal forms were present and s ingle cel ls could be seen on part icles

wi th s t r ings o f s e c r e t e d mater ia l t ra i l ing b e h i n d them. The d ive r s i t y o f cel l

t y p e s a t t a ched on a s ing le pa r t i c le was s u r p r i s i n g l y h igh . C o m m o n l y ,

s ta lked bacteria were a t tached to part icles also suppor t ing small rods.

S a m p l e s taken neat" the Pt. Loxna s e w a g e out fa l l s h o w e d d r ama t i c

d i f f e r e n c e s in t e rms o f a t t a c h m e n t as wel l as cel l s ize . Large rods ( 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 micro- autoradiographic 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 embed- ded 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 prep- arations viewed with light optics. These cells were also found in a free- floating 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 charac- teristics were shown for large (4 -5 /zm 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 amorph- ous 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). En- richment 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 sedi- ments. 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) embed- ded 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 ob- served 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 --

24(

20( -

Q}

Q.

E 1 6 0 -

O t'N

1 2 0 -

8 0 -

40

Partic

Partic

CHO

AT P ]

T O

Partic

CHO

T 1 Steri le T 1 Live

Fig. 3. Comparative accumulation of ATP-biomass carbon (ATP), carbohydrate- carbon (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 par- ticulate 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 obser- vations 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 parti- cles not much larger than cellular size. One possible explanation for at- tachment in oligotrophic waters is the ability for bacteria to remain near

f 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 parti- cle 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 "micro layers" of concentrated nutrients in dilute aquatic systems may offer an attractive growth site for organisms capable of attach- ing to or being adsorbed on a particle.

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 re- quires 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 enrich- ment 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 reac- tion, 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 cytop- lasmic components of the attached cells, are also noticed, but such increases are not as pronounced as increases in carbohydrates.

82 Hans W. Paerl

T h e a t t achmen t and g rowth p roces se s desc r ibed here add fu r the r c o m -

p l ex i ty to the a l ready c o m p l i c a t e d p r o c e s s e s o f mic rob ia l d e c o m p o s i t i o n o f

pa r t i cu la te mat ter . Even though sotne o f the a t t a chmen t s i tes may be

m i n e r a l i z e d th rough e n z y m a t i c p roces se s not d i scussed he re , there is a lso a

net f low of d i s so lved o rgan ic ca rbon ( D O C ) into the pa r t i cu la te state. As a

resu l t , m ine ra l i za t ion and o rgan i c ca rbon loss f rom par t i c l e s may be masked

by up take o f D O C at depths w h e r e a t t a c h m e n t and g rowth are f avo rab l e .

T h e s e dep ths appea r to be near the su r face o f lakes and o c e a n s , for e l e v a t e d

t empera tu res in these r e g i o n s wil l e n h a n c e net g r o w t h and the re fo re D O C

up take . In add i t ion , n e w l y g e n e r a t e d sources o f D O C are in g rea te r abun-

dance in the upper wa te r masses s ince algal p h o t o s y n t h e t i c ca rbon r educ t ion

is l imi t ed to these zones . In o rde r to ob ta in a rnore c o m p l e t e p ic tu re of

bacterial convers ion and re lease o f growth l imi t ing nutrients such as phos-

phate, nitrate, and ammonia , o ther facets of minera l iza t ion must be invest i-

gated. A m o n g these are bac te r ia l au to lys i s , d iges t ion and re l ease by pro to-

zoans and c rus t acean 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 of

s ink ing a g g r e g a t e s .

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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