Hydrobiologia 159: 119-132 (1988)T. Berman (ed) The Role of Microorganisms in Aquatic Ecosystems 119© Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands

Biochemical markers for measurement of predation effects on the biomass,community structure, nutritional status, and metabolic activity ofmicrobial biofilms

David C. White1 & Robert H. Findlay2

'Institute for Applied Microbiology, University of Tennessee, 10515 Research Drive, Suite # 300,Knoxville, TN37932-2567, USA2 School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami,FL 33149

Key words: Predation on biofilms, microbial biofilms, benthic grazer effects, biofilm response to predation,biomass shifts with predation, signature biomarkers.

Abstract

Chemical measures for the biomass, community structure, nutritional status, and metabolic activities of mi-crobes in biofilms attached to detrital or sediment surfaces based on analysis of components of cells and ex-tracellular polymers represent a quantitative and sensitive method for the analysis of predation. These methodsrequire neither the quantitative removal of the organisms from the surfaces nor the efficient culture of eachgroup of microbes for analysis of predation effects on the biofilm. The biomass of microbes can be determinedby measuring the content of cellular components found universally in relatively constant amounts. If thesecomponents have a high natural turnover or are rapidly lost from viable cells, they can be utilized to measurethe viable cell mass. The membrane phospholipids have a naturally high turnover, are found in all cellular mem-branes, are rapidly hydrolyzed on cell death, and are found in reasonably constant amounts in bacterial cellsas they occur in nature. Estimates of the viable biomass by phospholipid content correspond to estimates fromthe content of muramic acid, ATP, several enzyme activities, direct cell counts, and in some cases viable countsof subsurface sediments. The analysis of the ester-linked fatty acids of the phospholipids (PLFA) using capillarygas chromatography/mass spectrometry (GC/MS) provides sufficient information for the detection of specificsubsets of the microbiota based on patterns of PLFA. With this technique shifts in community structure canbe quantitatively assayed. Some of the microbiota form specific components such as poly beta-hydroxyalkanoate (PHA) under conditions of unbalanced growth. Others form polysaccharide glycocalyxwhen subjected to mechanical or chemical stress. The combination of analysis of phospholipids, PLFA, PHA,and glycocalyx provides a definition of the biomass, community structure, and metabolic status of complexmicrobial communities. These methods involve chromatographic separation and analysis so rates of incorpora-tion or turnover into specific components can be utilized as measures of metabolic activities. With thesemethods it has proved possible to show that amphipod grazing can induce shifts in biofilm community struc-ture, nutritional status, and metabolic activities. With this technology it proved possible to show resource parti-tioning amongst sympatric detrital feeding amphipods, prey specificity of feeding of benthic microvores, effectsof sedimentary microtopology on predation, and shifts in the microbiota by exclusion of top epibenthic preda-tors.

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Introduction

The microbes that form biofilms on detrital surfacesor sediment granules present a complex problem forassay. Classical microbiological methods that in-volve the quantitative detachment and subsequentculturing of organisms on Petri plates can lead togross underestimations of the numbers of organisms(White, 1983).

Our laboratory has been involved in the develop-ment of assays to define microbial consortia inwhich the bias of cultural selection of the classicalplate count is eliminated. Since the total communityis examined in these procedures without the necessi-ty of removing the microbes from surfaces, themicrostructure of multi-species consortia ispreserved. The method involves the measurement ofbiochemical properties of the cells and their extracel-lular products. Those components generally dis-tributed in cells are utilized as measures of biomass.Components restricted to subsets of the microbialcommunities can be utilized to define the communi-ty structure. The concept of 'signatures' for subsetsof the community based on the limited distributionof specific components has been shown for manymonocultures (Lechevalier, 1977; White, 1983). Thispaper will review a series of experiments from ourlaboratory to elucidate the role of predation in theecology of microbial biofilms.

Description of sites studied

Detrital microbiota developed on the surface of oakleaves (Quercus virginiana Mill) or Teflon sheets ex-posed in Apalachicola Bay, Florida (29°43.2'N;84°57.3 'W), sediments at that site or at one near theFlorida State marine laboratory (29°54'N;84°27.5'W) were utilized in these experiments.Complete descriptions of the study sites can befound in: (Morrison & White, 1980; Findlay &White, 1983a, b; Federle et al., 1983a, b).

Methods

Biomass estimation

Phospholipids, intercellular adenosine nucleotides,

and cell wall amino-sugars are biochemical compo-nents of cells that have been utilized to estimatemicrobial biomass (White, 1983). Of these, phos-pholipids have proven the most useful when examin-ing predation effects on microbial biofilms. Phos-pholipids are found in the membranes of all cells.Under the conditions expected in natural communi-ties the bacteria contain a relatively constant propor-tion of their biomass as phospholipids (White et al.,1979c). Phospholipids are not found in storage lipidsand have a relatively rapid turnover in some sedi-ments so the assay of these lipids gives a measure ofthe 'viable' cellular biomass when compared to en-zyme activities, total intra-cellular adenosinenucleotides; cell wall muramic acid (White et al.,1979b). Our laboratory has found it useful to meas-ure the phosphate, the various polar head groups,and the ester-linked fatty acids that form the phos-pholipids. The phosphate of the phospholipids orthe glycerol-phosphate and acid-labile glycerol fromphosphatidyl glycerol-like lipids that are indicatorsof bacterial lipids can be asayed to increase the speci-ficity and sensitivity of the phospholipid assay(Gehron & White, 1983). As shown by Smith et al.(1986a) there appears to be a unique microbialcommunity in uncontaminated subsurface sedi-ments from below the root zone. The microbiota aresparse and have a small cocco-bacillary morpholo-gy. In these subsurface sediments the biomass andcell numbers estimated from direct cell count afteracridine orange staining agree with the numbers andbiomass estimated from the extractible phospholipidphosphate and total fatty acids, the total adenosinetriphosphate, the fatty acids from the lipopolysac-charide lipid A, and the cell wall muramic acid con-tent (Balkwill et al., 1987).

Microbial community structure

The ester-linked fatty acids in the phospholipids(PLFA) are presently both the most sensitive and themost useful chemical measures of microbial biomassand community structure thus far developed (Bob-bie & White, 1980; Guckert et al., 1985; White et al.,1984). The specification of fatty acids that are ester-linked in the phospholipid fraction of the total lipidextract greatly increases the selectivity of this assay

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as many of the anthropogenic contaminants as wellas the endogenous storage lipids are found in theneutral or glycolipid fractions of the lipids. Byisolating the phospholipid fraction for fatty acidanalysis it proved possible to show bacteria in thesludge of crude oil tanks. The specificity and sensi-tivity of this assay has been greatly increased by thedetermination of the configuration and position ofdouble bonds in monoenoic fatty acids (Nichols etal., 1985; 1986a; Edlund et al., 1985) and by the for-mation of electron capturing derivatives which afterseparation by capillary GLC can be detected afterchemical ionization mass spectrometry as negativeions at femtomolar sensitivities (Odham et al., 1985).This makes possible the detection of specific bacter-ia in the range of 10 to 100 organisms. Since manyenvironments such as marine sediments often yield150 ester-linked fatty acids derived from the phos-pholipids, a single assay provides a large amount ofinformation. Combining a second derivatization ofthe fatty acid methyl esters to provide informationon the configuration and localization of the doublebonds in monounsaturated components provideseven deeper insight. By utilizing fatty acid patternsof bacterial monocultures, Myron Sasser of theUniversity of Delaware in collaboration withHewlett Packard has been able to distinguish be-tween over 8000 strains of bacteria (Sasser et al.,1984; Hewlett-Packard, 1985). Thus analysis of thefatty acids can provide insight into the communitystructure of microbial consortia as well as an esti-mate of the biomass from the total PLFA.

Despite the fact that the analysis of PLFA cannotprovide an exact description of each species or phys-iological type of microbe in a given environment, theanalysis provides a quantitative description of themicrobiota in the particular environment sampled.With the techniques of statistical pattern recogni-tion analysis it is possible to provide a quantitativeestimate of the differences between samples withPLFA analysis.

Potential problems with defining communitystructure by analysis of PLFA come with the shiftsin fatty acid composition of some monocultureswith changes in media composition or temperature(Lechevalier, 1977) some of which were defined inthis laboratory (Joyce et al., 1970; Frerman & White,

1967; Ray et al., 1971). There is as yet little publishedevidence for such shifts in PLFA in nature where thegrowth conditions that allow survival in highly com-petitive microbial consortia would be expected to se-verely restrict the survival of specific microbialstrains to much narrower conditions of growth. Theshifts in microbial PLFA patterns with changes inphysiological conditions can be utilized to gain in-sight into the nutritional status of the organisms ina particular biofilm so long as the behavior of thespecific groups of microbes under consideration iscarefully.validated by studies of cultures under de-fined conditions. A potentially very useful findingis the detection of increased proportions of transmonoenoic fatty acids in the minicells that resultfrom starvation of some marine bacteria (Guckert etal., 1986). This biomarker appears to indicate starva-tion with attachment to biofilms in the initialmicrofouling community.

From the residue of the lipid-extracted biofilm,muramic acid, a unique component of the bacterialcell wall can be recovered (Findlay et al., 1983a).Muramic acid in the bacterial cell wall exists in a 1: 1molar ratio with glucosamine. Since the analysisgives both glucosamine and muramic acid, and thechitin walls of many microeukaryotes yield glucosa-mine, the glucosamine to muramic acid ratio givesinsight into the prokaryote to eukaryote ratio. Thiscomplements the information developed from theester-linked PLFA.

Nutritional status

The nutritional status of biofilms or microbial con-sortia can be estimated by monitoring the propor-tions of specific endogenous storage compoundsrelative to the cellular biomass. Thus, the nutritionalstatus of microeukaryotes (algae, fungi, or protozoa)in biofilms can be monitored by measuring the ratioof triglyceride glycerol to the cellular biomass(Gehron & White, 1982).

Certain bacteria form endogenous lipid PHA un-der conditions when the organisms can accumulatecarbon but have insufficient total nutrients to allowgrowth with cell division (Nickels et al., 1979). (Amore sensitive assay based on GLC of the compo-

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nents of the PHA polymer showed the presence ofa 3-OH acid longer than 4-carbons in these polymers(Findlay & White, 1983b) which accounts for thechanging of the name from poly beta-hydroxybutyrate (PHB) to PHA).

Assays for extracellular polysaccharide glycoca-lyx based on the specific content of uronic acids havebeen developed (Fazio et al, 1982). These have beenutilized to show that poor growth conditions stimu-late the formation of uronic acid containing ex-opolymers by a marine Pseudomonas (Uhlinger &White, 1983).

Metabolic activity

substrate concentrations in the biofilms that are justabove the natural levels. This is not possible withradioactive precursors. Improvements in analyticaltechniques have increased the sensitivity of this anal-ysis. Utilizing a chiral derivative and fused silicacapillary GLC with chemical ionization and nega-tive ion detection of selected ions, it proved possibleto detect 8 pg (90 femtomoles) of D-alanine from thebacterial cell wall (the equivalent of 1000 bacteriathe size of E. coli (Tunlid et al., 1985). In this analysisit proved possible to reproducibly detect a 1% en-richment of 15-N in the 14-N-D-alanine.

Reproducibility

The analyses described above all involve the isola-tion of components of microbial consortia. Sinceeach of the components is isolated, the incorpora-tion of labeled isotopes from precursors can be uti-lized to provide rates of synthesis or turnover inproperly designed experiments. Measurements ofthe rates of synthesis of DNA with 3-H-thymidineprovide an estimate of the rates of heterotrophic bac-terial growth if short incubation times are utilized,isotope dilution is utilized to estimate precursor con-centration, and DNA is purified (Moriarty & Pol-lard, 1982). Incorporation of 35-S-sulfate into sul-folipid can be utilized to measure activity in themicroeukaryotes (White et al., 1980; Moriarty et al.,1985). Incorporation of 32-P phosphoric acid intophospholipids can be utilized as a measure of the ac-tivity of the total microbiota. The inhibition ofphospholipid synthesis in the presence of cyclohexi-mide represents the microeukaryote portion of thelipid synthesis (White et al, 1980; Moriarty et al.,1985). Measurement of rates of synthesis and turno-ver of both carbon and phosphate in individualphospholipids showed different turnover for the var-ious lipids (King et al., 1977).

Analysis of signatures by GC/MS makes possiblethe utilization of mass labeled precursors that arenon-radioactive, have specific activities approaching100%, include isotopic marker for nitrogen, and canbe efficiently detected using the selective ion modein mass spectroscopy. The high specific activitymakes possible the assay of critical reactions using

The changes in biofilm or sedimentary microbiotainduced shifts in the environment of the micronichethat are reproducible. The shifts in the terminal elec-tron acceptors from high potential (oxygen or nitrate)to lower potential (sulfate, or carbon dioxide) induceschanges in the microbial community structure. In ex-periments utilizing an inoculum from marine sedi-ments it has proved possible to manipulate the com-munity structure of the benthic microbiota byshifting from aerobic to anaerobic conditions(Guckert et al., 1985). The PLFA of independentflasks showed reproducible shifts when manipulatedidentically and significant differences when manipu-lated with different treatments. The absence of longchain polyenoic fatty acids indicated that the com-munities were predominantly prokaryotic and thatthe differences in the PLFA were primarily in theproportions of cyclopropane fatty acids and theproportions and geometry of the monounsaturatedfatty acids.

In similar experiments a subsurface sediment in-oculum was grown through two cycles of aerobicgrowth and compared to organisms from the sameinoculum grown through two cycles of anaerobicgrowth with no supplement, or with sulfate or nitrate(Hedrick & White, 1986). Again there were repro-ducible shifts in the microbial community structureas reflected in the profiles of PLFA. Not only werethe PLFA patterns reproducible but the ratio ofrespiratory quinones reflected the redox environ-ment in the bacterial biofilms. Benzoquinone

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isoprenologues are formed by microbes grown withhigh potential terminal electron acceptors such asoxygen or nitrate (Hollander et al., 1977).Naphthoquinones are formed by bacteria utilizinglow-potential electron acceptors such as sulfate ororganic substrates. Aerobic consortia formed themost benzoquinone relative to naphthoquinone, thenitrate supplemented anaerobic culture formed lessbenzoquinone, the sulfate supplemented cultureformed still less benzoquinone, and the anaerobicfermentation formed the least.

These methods (summarized in Fig. 1) can be ap-plied to biofilms attached to substrata (detrital sur-faces, sediments, biofouled surfaces) or from sus-pended particulate matter in the water column after

Biomass

Cell Wall - Muramic AcidLipopolysaccharide Lipid A fatty acids

Membrane - Phospholipids (analysis of lipid phosphate, lipidglycerol phosphate, polar lipid total fatty acids)

Cellular Adenosine Triphosphate

Community Structure

'Signature Biomarkers'Phospholipid Ester - Linked Fatty Acids (PLFA)Lipopolysaccharide Lipid A hydroxy fatty acidsPhytopigments and CarotenoidsSteroids (microeukaryotes)

Nutritional Status

Ratio of PLFA to poly beta-hydroxy alkanoate (PHA)Proportion of trans monoenoic PLFARespiratory Quinone ratio

Metabolic ActivityRatio of rates of incorporation into PHA/PLFA. Excellent for

determination of the 'disturbance artifact' in analysis of sedi-ments

Incorporation of 13-C or 15-N labeled precursors into 'signaturebiomarkers' with gas chromatographic/mass spectral analysis

Rates of incorporation of 32-P or 35-S into lipidsRates of 3-H thymidine incorporation into DNARates of enzyme activity (FDA)Cellular Adenosine Energy Charge

collection through methanol-washed Nucleoporefilters with an extraction technique using hexane-isopropanol (Guckert, 1986). This solvent systemdoes not affect the Nucleopore membrane.

Predation effects on microbial biofilms

Biofilm succession

In the absence of predation a natural succession ofmicrobes during the maturation of marine biofilmshas been detected. Shifts in morphology seen usingscanning electron microscopy (SEM) can be meas-ured quantitatively with PLFA analysis (Morrison etal., 1977; Nickels et al., 1981a). The morphology bySEM shows an initial colonization by coccobacillarybacteria followed by bacteria with more complexmorphology and microeukaryotes (predominatelydiatoms) that are then followed by other algae andmicroeukaryote larvae. The analysis of the initialmicrofouling film shows PLFA typical of gram-negative marine bacteria (Odham et al., 1985). Thisis followed by signature patterns typical of thefilamentous bacteria, the diatoms, the algae andmicroeukaryote larvae (Smith et al., 1982).Thesestudies form controls for analysis of predation ef-fects.

Effects of predation by amphipods on the detritalmicrobiota

Brown fallen oak leaves from a live oak (Quercus vir-giniana Mill) provide a surface on which a succes-sion of microbiota attach (Morrison et al., 1977).When these leaves are exposed to seawater in a sub-tropical esturary such as Apalachicola Bay, Floridaa biofilm forms on the detritus. The biofilm is ini-tially comprised of bacterial microcolonies that arequickly overlain by a more diverse communitycharacterized by filamentous bacteria, fungalhyphae, cyanobacteria, diatoms and microalgae asdetected by scanning electron microscopy (SEM).The initial microfouling community is characterizedby a high ratio of extractible adenosine triphosphate(ATP) to muramic acid. This is followed by a com-

Fig. 1. Biochemical characterization of microbes in biofilms onDetritus or sediment granules. The same analysis has been appliedto particulates recovered from the water column on membranes(Guckert, 1986).

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munity with a lowered ratio of ATP to muramic acid.Muramic acid is a unique component of the bacterialcell wall.

After a two week exposure in the bay in litterbaskets, the leaves were frozen and thawed threetimes to decrease the macrofaunal populations andexposed to filtered estuarine seawater for an addi-tional day. The dominant amphipod Gammarusmucronatus Say 1818 is a surface biofilm feeder.These animals were recovered, counted, and starvedfor 24 hours by incubation in the absence of leaveswith a biofilm. These starved amphipods rapidlygrazed the surface of the leaves when exposed to themature biofilm. They appear to graze the biofilm ex-clusively as delicate stellate trichome structures onthe ventral sides of the leaves were never damaged inthese experiments. The experiments consisted of ex-posing the mature detrital biofilm generated by atwo week exposure in the bay to grazing amphipodsat a natural density (one amphipod/8.5 cm2 leafsurface area and recovering leaves over a period of20 days. The exposures were maintained at ambientconditions and there was no amphipod mortality.Leaves were sampled by removing 6.5 mm diameterdisks with a cork borer and utilizing the disks for thedeterminations described below (Morrison & White,1980).

Amphipod grazing resulted in a progressive de-crease in the total colonizing microbiota with an in-crease in the exposed leaf surface. In the first daythere was a rapid loss of biomass followed by a rapidrecovery of the biofilm. There was essentially nochange in the biofilms not exposed to the grazingamphipods throughout the experiment. In thegrazed biofilms high resolution SEM showed a shiftin morphology with grazing. The mature biofilmwith the fungal hyphae, complex bacterial coloniescontaining filaments, diatoms, cyanobacteria, andmicroalgae were replaced by a bacterial biofilm con-taining much more extracellular polymer glycoca-lyx. The shift in biofilm community structure de-fined by SEM was confirmed by the changes in thepatterns of PLFA. There was a shift in the propor-tions of monoenoic PLFA from those characteristicof both the bacteria and microeukaryotes to thoseformed by bacterial anaerobic pathways, a markeddecrease in the polyenoic PLFA characteristic of the

microeukaryotes, and an increase in the short andbranched saturated PLFA characteristic of gram-negative bacteria (Smith et al., 1982). The biofilm bi-omass estimated by the extractible ATP and the lipidphosphate both showed increases by a factor of twoover the ungrazed control that paralleled the forma-tion of secondary bacterial biofilm. The unexposedleaves contained about 10% of the phospholipid and0.04% of the ATP found in the bacterial biofilm.This indicated that the biomass indicators wereprimarily related to the biofilm microbes. These ex-periments were repeated with teflon coupons so thatchanges observed with SEM could be confirmed byanalysis of PLFA without interference from oak leaflipids.

The biofilm microbial activity in the grazedmicrobiota showed increases. The oxygen utilizationexpressed per gram dry weight of leaf increased4-fold over the ungrazed detritus. The rate of the lossof 14-C carbon dioxide from pre-labeled detrital bi-ofilms was more than twice that of the ungrazed con-trol. The synthesis of PHA and total microbial lipidwas likewise higher in the grazed biota. The alkalinephosphatase however was roughly equal in the firstfew days of the experiment but then was consistentlylower in the grazed bacterial biofilms. The rate ofloss of 14-C labeled PHA was significantly greaterin the grazed microbiota (Fig. 2).

The turnover of specific components labeled by ashort exposure to precursors and then incubatedwith a higher concentration of unlabeled precursorgives an indication of the minimal rate of metabo-lism of that component. If the 'pulse chase' experi-ment is done at the same time as the exposure to graz-ing then the loss of label from a specific componentbecomes the combination of the rates of metabolismby the bacteria plus the rate of removal from the bi-ofilm by the grazer. Examination of lipid classes inthis experiment showed that the deacylated phos-pholipid glycerol esters gave insight into the rate ofpredation. In the ungrazed biota the glycerol esterglyceryl-phosphorylcholine (GPC) derived fromphosphatidyl choline showed essentially no turno-ver. When the biofilm was exposed to the amphipodsthe rate of disappearance of GPC paralleled the rateof loss of muramic acid. Glyceroyl-phosphorylglycerol derived from phosphatidyl

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A40 OXYGEN GRAZED

UNGRAZED

20

o 4$o+

O 5 10 15 20

B1.0 14 C -TOTAL LIPID

0.5

o.o, *,, * l, * | | . | l,, * .... ,0 5 0 15

C 14C-PHA

2.0

+bo +

Xl.O .:

0 5 10 15 20DAYS

Fig. 2. Comparison of microbial activity in grazed and ungrazedmicrobial biofilms. Grazed biofilms are indicated with solid sym-bols, ungrazed with open symbols. Rates of oxygen utilization (A,top panel), 14-C released from prelabeled biofilms (B, middlepanel), and 14-C PHA biosynthesis (C, lower panel) (data fromMorrison & White, 1980).

glycerol has an active metabolism in bacteria. Its rateof turnover increased in the grazed microbiota in-dicating both the loss due to removal of the cells plusthe greatly stimulated metabolic activity induced inthe bacterial biofilm that is grazed.

In preliminary experiments it was possible to showthat there is an optimum density of grazing amphi-pods for stimulating the biofilm microbial activity.Densities too low or too high result in either a maturebiofilm with the complex morphology of the un-grazed control or at too high a density a grazing ratethat does not allow the secondary bacterial biofilmto develop. With exposures of detrital microbiota to

high enough grazing pressure the capacity for recov-ery by the biofilm is exceeded and the activity andbiomass of the detrital microbiota are depressed(Morrison, 1980).

These experiments clearly indicate that grazingshifts the detrital microflora from a metabolicallystable community with complex morphology to oneof metabolically active fast growing bacteria and di-atoms. The loss of alkaline phosphatase activity inthe grazed community suggests an increase in theavailability of phosphate released by the grazers.These are the conditions of 'perpetual youth' im-posed by grazing that were predicted by Johannes(1965).

Determination of the nutritional status of thegrazers

These methods also allow definition of the nutri-tional status of the grazing animal. The amphipodswhen allowed to feed freely on the detrital microbio-ta exhibit a ratio of triglyceride glycerol to mem-brane phospholipid of about 0.7. Starvation by iso-lation from the detritus for a week depressed theratio to 0.13 (Gehron & White, 1982). Similar experi-ments showed the same findings for fungi, yeast, ar-thropods and the protozoa of the detritus. With thistechnology it was possible to establish that the feed-ing of the amphipods on the estuarine detritalmicrobiota resulted in the same nutritional status asthe amphipods recovered from the field.

Resource partitioning between sympatricamphipod grazers

Using the PLFA analysis of the detrital biofilm it waspossible to show that two sympatric amphipodsGammarus mucronatus (Say) and Melita appen-diculata (Say) which have markedly different mouthpart morphology partition the biofilm microbiota(Smith et al., 1982). These experiments were per-formed much as those described above except thatthe substratum was teflon sheets rather than oakleaves. The morphology of the feeding apparatus oftwo sympatric estuarine detrital-feeding amphipods

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was reflected in partitioning of the microbial re-source as determined by biomarker analysis of themicrobial biofilm (Smith et al., 1982). The Melita ap-pendiculata with its less setosed and more complexlyarticulated mandibles and maxillae apparently fedon non-photosynthetic microeukaryote grazers andhad less effect in increasing the community microbi-al metabolic activity than the general feeding (bac-teria and diatoms) Gammarus mucronatus. Grazingby M. appendiculata left a detrital biofilm greatlyenriched in bacteria (increased muramic acid andhigher proportions of the bacterial PLFA such as cisvaccenic acid, and the short iso and anteisobranched saturated fatty acids), a larger photosyn-thetic biomass (increased glycolipid galactose, in-creased rate of sulfolipid biosynthesis), and a highertotal microbial biomass (greater extractible lipidphosphate and rate of phospholipid biosynthesis)than the biofilm remaining after grazing by Gamma-rus mucronatus. The biofilm exposed to Melitashowed an enrichment in bacteria and diatoms bySEM. This possibly reflected the removal of themicrofaunal grazers by the selective feeding of theMelita. The enrichment of the residual biofilm by di-atoms was reflected in the increase in the twenty car-bon pentaunsaturated polyenoic PLFA with alphalinolenic acid type of unsaturation characteristic ofdiatoms.

The surface grazed by the Gammarus with itshighly setosed and less articulated mandibles andmaxilla showed removal of the bacteria and cleanerdetrital surface by SEM. More of the microeukary-otes remained, as reflected in the higher total lipidglycerol (primarily found in the triglyceride). Thegrazed surface showed intense metabolic activity asreflected in the higher total extractible adenosinenucleotide relative to the microbial biomass estimat-ed by the extractible phospholipid. This was alsoreflected in the higher ATP, and the higher adenylateenergy charge. The residual microbiota after grazingby Gammarus showed a ratio of phospholipid syn-thesis to phospholipid (essentially a measure of bac-terial activity) and of sulfolipid synthesis toglycolipid galactose (a measure of sulfolipid synthe-sis) that is about half that of the Melita grazed detri-tus. The more generalized grazing of the Gammarusproduced a smaller, more active residual microbiota

that had a smaller specific synthetic activity. Thesetwo sympatric amphipods clearly partition in thedetrital microbiota as evidenced by the chemical bi-omarker technique.

In these experiments the nutritional status of theamphipods in the laboratory exposures and in thefield were essentially identical as measured by theadenylate energy charge and the ratio of triglycerideglycerol to phospholipid (Smith et al., 1982).

Effects of predation in sediments

Another experiment shows that the effects of preda-tion from the top of the food chain can affect thebenthic microbiota. It has been postulated for a longtime that changes in rates of predation at the top ofestuarine food chains would reverberate throughthe various trophic stages and finally affect themicrobiota at its base. After developing methods forpreserving sediment samples (Federle & White, 1982)and sampling strategies for mud flats (Federle et al.,1983a) it was possible to show statistically signifi-cant differences in the community structure of thesedimentary microbiota by eliminating predation bythe crabs and fish at the top of the food chain withproperly designed caging experiments (Federle et al.,1983b). These experiments also showed significantdifferences in the benthic microbiota between con-tinuous predation (crabs and fish caged inside) andthe random predation of control areas. A stepwisediscriminant analysis showed distinct differences be-tween the PLFA of the benthic microbiota betweenthe control, the sediments subjected to continuouspredation and those with random predation. Thepredator exclusion cages were rapidly overgrown bya dominant polychaete Mediomastus ambiseta andshowed markedly decreased proportions of the poly-enoic fatty acids characteristic of nematodes and al-gae and a markedly increased proportion of PLFAcharacteristic of bacteria. There followed a rapid in-crease in linoleic acid which is characteristic ofmicroeukaryotes and could represent an increase inprotozoa. The PLFA from the sediments suggesteda shift in the bacterial community to a more anaero-bic sulfate-reducing consortium.

With this technology it was possible to validate

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microcosms set up to model estuarine sediments(Federle et al., 1986). The laboratory microcosmsshowed microbial biomass and community struc-tures that were detectably different but the degree ofdifference was not large and did not increase withtime when compared to the field in the system takenfrom a shallow, turbid, highly disturbed bay that isenriched by riverine runoff and is characterized bylow macroscopic species diversity and high biomass.Microcosms prepared from a more stable, higher sa-linity, system with a much more diverse macroscopiccommunity that is controlled by epibenthic preda-tors showed a great difference from the field site. Thedifferences between the microcosms in the laborato-ry and the field site increased drastically with timein this system.

Effects of bioturbation

In sediments in which a redox gradient exists theanalysis of the effects of predation is complicated bythe effects of disturbance. The addition of oxygen toreduced sediments can markedly affect the benthicmicrobiota. Findlay developed a sensitive measureof disturbance based on the ratio of the rate of 14-Cacetate incorporation into PHA and PLFA (Findlayet al., 1985). PHA is formed by bacteria under condi-tions when cell division is compromised (un-balanced growth, Nickels et al., 1979). PLFA synthe-sis accompanies cell growth and cell division,conditions during which PHA formation stops andutilization accelerates. Findlay (1986) described theconsequences of disturbing sediment. The distur-bance of sieving through 998 tm sieve resulted in arapid initial increase in the ratio of 14-C incorpora-tion into PLFA/PHA. This burst of growth indicat-ed bacterial growth on the reduced carbon compo-nents in the anaerobic sediment induced in thepresence of the added oxygen. This response wasmaximal in 2 hours after sieving a mud flat sedi-ment. Subsequently there was a drop in the ratio tobelow predisturbance levels by 8 hours. The decreasecontinued for 3 days after which the ratio increasedagain to predisturbance levels by the fifth day. Totalgrowth rates measured as phospholipid synthesis orincorporation of thymidine into DNA relative to theextractible phospholipid decreased initially in the

first hour, then increased for a short time with amaximum in 2-4 hours and then decreased to amaintenance level at about half the predisturbancerate throughout the 5 day experiment. The microbialbiomass showed an initial fall in the first 2- 4 hoursthen a slow recovery to predisturbance levels meas-ured as muramic acid or total extractible phos-pholipid phosphate. The PLFA patterns showedshifts during the initial fall and rebound of growthrates. The long-chain polyunsaturated PLFAdecreased. These PLFA are characteristic of themicroeukaryotes. PLFA characteristic of anaerobicbacteria initially decreased and then showed a dis-proportionate increase that paralleled the increasedrate of PLFA synthesis. PHA initially disappearedthen did not accumulate for the 2-4 hour period ofrecovery. Increased proportions of trans monoenoicPLFA shown to be characteristic of starved bacteriaby Guckert et al. (1986) were detected in the2- 4 hour period. The microbiota apparently under-went an initial shock from which a few microbesrecovered and exploited the increased nutrients andhigh potential electron donor. This secondarygrowth spurt continued until growth depleted the en-vironment of an essential micronutrient and un-balanced growth ensued (PHA and trans monoenoicPLFA increased). This community was eventuallyreplaced by a microbially dominated assembly great-ly enriched in anaerobes and facultative hetero-trophs.

With the effects of mechanical disturbance care-fully documented, Findlay (1986) examined naturaldisturbances. The sting ray Dasyatis sabina digs ex-tensive pits in the sediment in search of infaunalprey. The disturbed sediments represent resuspendeddeep sediments brought to the surface in the feedingactivity. Disturbed sediments sampled after the noc-turnal feeding showed initially lower biomass andgrowth rates than control sediments with time de-pendent increases much like the sieving experiment.The PLFA patterns indicated shifts to more aerobicbacteria and microeukaryotes containing linoleicacid as these organisms colonized these newly aero-bic sediments. There was a greater decrease in theproportions of PLFA associated with facultative andanaerobic bacteria in disturbed than in the controlsediments.

Findlay (1986) then examined the effects of distur-

128

bance in the field combined with predation. The En-teropneust Ptchyodera bahaminasis feeds by ingest-ing sediment from near the surface and generatesmounds of fecal castings from the other end of a Ushaped feeding tube. The fecal castings initially con-tained a low bacterial biomass measured by muramic

acid and low rates of phospholipid synthesis andthymidine incorporation into DNA when comparedto control sediments. Initial high levels of phos-pholipid fatty acids and phosphate indicated non-bacterial lipids, possibly from the worm. These lipidsdisappeared rapidly and 2 hours after extrusion

Fig. 3. Comparison of the effects of mechanical disturbance (A, top panel), natural disturbance (B, middle panel), and predation-disturbance (C, lower panel) on microbial nutritional status, metabolic activity, and biomass in shallow marine sediments. Natural distur-bance was by the sting ray Dasyatis sabina creating pits in its search for infauna. Disturbance-predation was examined in the fecal castingsof the Enteropneust Ptchyodera bahaminasis. Nutritional status is assayed as the ratio of the relative rate of 14-C acetate incorporationinto PLFA versus PHA (solid squares). Microbial metabolic activity is given as the rate of 3-H-thymidine incorporation into DNA (opensquares) in units of pmoles thymidine/min/mnole lipid phosphate. Microbial biomas was assayed as PLFA (open diamonds) in nmolesPLFA/g dry weight of sediment. The arrow indicates the point at which the sediments were disturbed (* with the arrow is where the timeof disturbance is estimated). In the mechanical disturbance experiment (A, top panel) the t = - 2 hr samples were taken before the sedimentwas disturbed. Data for the natural disturbance (B, middle panel) and predation-disturbance (C, lower panel) is presented as average differ-ences (treatment - control). Values greater than zero indicate biomass or activity was greater in the disturbed sediments vs the controlsediments whereas values less than zero indicate biomass or activity was greater than the control sediments. Error bars represent plusand minus one standard deviation. Experimental details are presented in Findlay (1986).

A MECHANICAL DISTURBANCE14

C PLFA/14

C PHA

0.80 3 30 3 H THYMIDINE INTO DNATOTAL FAME

o.53 2 20. .I I . .

0.27 1 10

-2 0 2 4 6 8 10 12

IB NATURAL DISTURBANCE

0.5 10 i

-2 0 2 4 6 8 10 12

C PREDATION - DISTURBANCE2.0_ 3 80

.1--2.0 1 -3 l-80

-2 0 2 4 6 8 10 12

TIME IN HOURS

I

129

the muramic acid, phospholipid fatty acids andphosphate indicated a bacterial biomass less thanthe control sediments. The PLFA patterns suggestedthe loss of microeukaryotes, algae, aerobic andfacultative bacteria, and anaerobic bacteria. ThePLFA patterns indicated that the facultativeanaerobes appeared unchanged from the controlsediments. Six hours after extrusion, microbialgrowth rates were significantly greater in the fecalcastings than in control sediments. The fecal castsdegenerated after 8 hours, so long term effects couldnot be studied. The branched saturated fatty acidscharacteristic of the phospholipids of the sulfate-reducing bacteria Desulfovibrio and Desulfobacterseemed to accumulate preferentially in the maturefecal casts. The microbiota of the fecal casts indicatethe enteropneust fed rather nonselectively on the to-tal microbiota and although recolonization occurredsomewhat more slowly, the pattern of recovery wassimilar to that of sediments subjected to simplephysical disturbances. The effects of disturbanceand disturbance-predation are illustrated in Fig. 3.

Specificity of the feeding of benthic grazer

The sand dollar Mellita quinquiesperforata biotur-bates sandy sediments and grazes specific compo-nents of the benthic biota (Findlay & White, 1983b).Sediments through which the animal has passedshow an increase in the average depth of the oxidizedzone of the sediments from 0.2 cm in the control to0.8 cm. The total microbial biomass estimated as theextractible lipid phosphate or the phospholipid fattyacids was unchanged and the muramic acid andseveral PLFA characteristics of bacteria were slightlydecreased after passage of the sand dollar throughthe sediment. The microeukaryotic biomass estimat-ed as triglyceride glycerol, and total PLFA polyenoicfatty acids decreased, but the photosynthetic bi-omass measured as the chlorophyll was not signifi-cantly affected. Examination of the sediments for vi-tally staining harpacticoids, nemadodes, andforaminifera showed almost complete removal of theforaminifera without effect on the other two groupsof non-photosynthetic meiofauna. Confining thesand dollars to microcosms in the laboratory showed

that multiple feeding passes through the sedimentresulted in significant decreases in the bacterial bi-omass measured as their specific PLFA. PLFA anal-ysis of the grazing of sands by this organism wasshown to selectively remove the non-photosyntheticmicroeukaryotes from the sediment (Findlay &White, 1983a). This is in agreement with the studiesof the morphology of the organisms found in thefeeding apparatus. As described above the bioturba-tion associated with sand dollar predation increasedthe ratio of 14-C incorporation into PLFA anddecreased incorporation into PHA.

Conclusions

The methods described above provide quantitativeinsight into the biomass and community structure,nutritional status, and metabolic activities ofmicrobial consortia in biofilms that do not requirequantitative recovery of the organisms from the bi-ofilm or that they all be cultured successfully. Theexamples given in this review establish that the ef-fects of grazing can be measured easily in biofilmsand the complications in the analysis of grazing insediments by bioturbation can be resolved usingproperly designed experiments and these methods.These methods are not complicated by fossil compo-nents from non-viable cells remaining in themicrobial consortia. Phospholipids, adenosinenucleotides, muramic acid, and the lipopolysaccha-ride of dead bacteria are rapidly lost from marinesediments (Davis & White, 1980; White et al., 1979b;1979d; King et al., 1977; Moriarty, 1977; Saddler &Wardlaw, 1980). This indicates that these chemicalmarkers provide good estimates for the standingstock of viable microbiota. Rates of formation orloss of endogenous storage lipids or exocellularpolysacchride polymers or synthesis or turnover ofspecific membrane signature biomarkers provide in-sight into the nutritional status and actual metabolicactivities of these microbial consortia as they aregrazed. Selectivity in the grazing of biofilms andsediments by specific grazers can also be determinedbased on the analysis of signature biomarkers.

The effects of predation on the microbial commu-nity in biofilms on marine detritus or sediment sub-

130

Grazing of Biofilms

Increase in metabolic activity (oxygen uptake, carbon dioxiderelease, PHA formation, PHA turnover, lipid synthesis)

Increased biomass (ATP, total lipid, phospholipid)Increased bacteria (increase in muramic acid/ATP or phos-

pholipid, bacterial PLFA)Decreased alkaline phosphatase (increase in available phosphate)Shift in biofilm morphology (from complex climax in succession

to bacterial and diatom dominance)Specificity-sympatric amphipod grazers partition the biofilm

Sediments

Bioturbation of sediments with a redox gradientInitial anaerobic die-off (PLFA patterns, DNA and lipid syn-

thesis)Rapid bacterial proliferation (PHA disappearance, PLFA syn-

thesis)Recovery and unbalanced growth (PHA accumulation, trans

monoenoic PLFA accumulation)Shift in community structure (loss of microeukaryotes, in-

creased anaerobic bacteria - PLFA pattern shifts)

Predation of sedimentsDirect effects

Enteropneust fecal casts (loss of microbial biomass andactivity with rapid recolonization by bacteria - PLFApatterns)

Specific predation - Sand Dollar (minimal bioturbation +specific decrease in biomarkers for non-photosyntheticmicroeukaryotes)

Indirect effectsSting Ray feeding pits - bioturbation effects + recovery

with increased aerobic bacteria (PLFA patterns)Polychaete overgrowth - decreased mieofaunal PLFA, in-

crease in anaerobic bacterial PLFA followed by increasein possible protozoal PLFA (PLFA patterns)

[Refer to text for details]

Fig. 4. Summary of effects of grazing of detrital biofilms and bi-oturbation and/or predation in sediments. (refer to the text fordetails, PLFA and PHA defined in Fig. 1).

stratum are complex and far reaching (Fig. 4). Pre-dation by its very nature, initially decreasesmicrobial biomass. In most instances, unless grazingpressure is severe, the secondary microbial commu-nity that develops will have increased rates of meta-bolic activity and growth. Total microbial biomasswill be greater and the turnover rates of both the sub-

strates and microorganisms will also increase. Thecomplexity of the community will decrease with thecommunity structure shifted towards faster growingorganisms. Many predators appear selective andonly remove a subset of the microbial communityfurther altering community structure. In sediments,predation is usually accompanied by bioturbation.The effects of these two phenomena are similar ex-cept that predation makes detrital nutrients and car-bon available to higher trophic levels whereasnutrients liberated by death or injury of microorgan-isms during physical disturbance are likely to beavailable only to the microbial components of thebenthic food web.

Acknowledgements

This work would have been impossible without thededicated work of the colleagues who formed thislaboratory. Particularly we wish to acknowledge theessential functions of Norah Rogers who for13 years has managed this laboratory with exem-plary care and responsibility. This work has beensupported by grants N00014-82-C-0404 andN00014-83-K-0056 from the Office of NavalResearch, OCE-80-19757, DPP-82-13796, andINT-83-12117 from the National Science Founda-tion, and NAG2-149 from the Advanced Life Sup-port Office, National Aeronautics and Space Ad-ministration; and contracts CR-80-9994 andCR-81-3725 from the Robert S. Kerr EnvironmentalResearch Laboratory of the U.S. Environmental Pro-tection Agency, and AX-681901 from the E. I.DuPont de Nemours and Co., Atomic Energy Divi-sion, Savannah River Laboratory, Aiken, SouthCarolina; and the generous gift of the Hewlett Pack-ard HP-1000 RTE-6/VM data system for theHP5996A GC/MS system.

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