Enzymes in the Environment: Activity, Ecology and Applications - Chapter 3 pdf

Dissolved enzymes 15 and large particles ⬎8 µm generally contribute only minor parts to the total enzyme activity.. Significant differences in the extracellular enzyme activity per cell b

3 Ecological Significance of Bacterial Enzymes in the Marine Environment

of enzymes in the sea specific marine environmental factors have to be considered, e.g.,

a) seawater is a highly diluted medium interspersed with hot spots of organic matter centration, aggregation, and decomposition (1) b) as a result of hydrographic conditions,the oceans are characterized by distinct horizontal and vertical zonations The deep sea,

con-in particular, with its enormous volume, depends entirely on substrate supply from scon-inkcon-ingmaterial produced in the surface layer (2) c) in the sea, low-molecular-weight organicmatter that persists as dissolved organic carbon (DOC) is less bioreactive and morestrongly diagenetically altered than the bulk of high-molecular-weight matter (3,4) d) thedeep seabed receives very little of the total surface-derived primary productivity, andmuch of this organic matter is strongly altered Finally, at chemical and hydrographicdiscontinuities in the sea (e.g., fronts, boundary layers, and oxygen, nutrient, and salinitygradients), drastic changes occur in microbial species diversity and enzymatic properties.Organic material, prone to bacterial degradation on nongeological time scales, isactively involved in biogeochemical cycles Such material originates from phytoplanktonprimary production and from ‘sloppy’ feeding of zooplankton as well as from the excre-tions of all kinds of organisms Currently, the spectrum of extracellular enzymes investi-gated in the sea is relatively limited, comprising principally hydrolytic enzymes such asproteases, glucosidases, chitinase, lipase, and phosphatase A larger variety has been inves-tigated in limnetic systems (5)

The principal focus of this chapter is on the ecological significance of extracellularenzymes in marine waters and sediments ranging from microscales to oceanwide scales

Investigations of extracellular enzymes from marine animals and enzymes isolated fromprokaryotes are considered only if a clear connection to marine ecology is established.

The term extracellular enzymes is used throughout this chapter, whereas Chro´st (5) guishes between ectoenzymes and extracellular enzymes Ectoenzymes are defined by

distin-Chro´st (5) and inChapter 2as enzymes located in the periplasmic space or attached tothe outer membrane of the bacterial cell Extracellular enzymes are enzymes freely dis-solved in the water or attached to particles other than the enzyme-synthesizing cell In

this chapter, however, the term extracellular enzymes refers to both ectoenzymes and

extracellular enzymes, unless otherwise stated

Early studies on the fate of organic aggregates and dissolved polymers in the seawere presented by Riley (6), Walsh (7), and Khailov and Finenko (8) Overbeck (9) re-viewed the early studies on extracellular enzyme activity in the aquatic environment

heterotro-as well heterotro-as cyanobacteria, form a food web of their own, loosely connected to the foodweb of the larger grazers In general, the nutritional basis of the microbial food web isprovided by the pool of dissolved organic matter (DOM) and particulate organic matter(POM) The DOM pool is a priori reserved for bacterial utilization, whereas competitionwith metazoans occurs for POM This competition is determined by the bacterial potentialfor enzymatic dissolution of POM on the one hand and the feeding activity of the metazo-ans on the other hand The bulk of both the dissolved and particulate resources, however,requires enzymatic hydrolysis prior to uptake by bacteria (Fig 1) Thus the enzymaticactivities of bacteria initiate organic carbon (C) remineralization and define the type andquantity of substrate available to the total microbial food web and, to certain extent, also

to the top predators in the system

B Free and Attached Enzyme Activity

Generally, extracellular enzymes may be bound to the cell (defined as ectoenzymes byChro´st [5]) or in the free and adsorbed state (11,12) Most of the total enzyme activity

in seawater has been found to be associated with the particle size class dominated bybacteria (⬎0.2 µm–3µm) (13,14) (Table 1) Dissolved enzymes (15) and large particles

⬎8 µm generally contribute only minor parts to the total enzyme activity In estuaries,however, which are characterized by strong gradients and fluctuations of turbidity andsalinity, total enzyme activity can be dominated by particle size classes⬎3 µm Enzymeactivity measured in such particles originated mainly from attached bacteria, leading tothe conclusion that particle-attached bacteria accounted for most of POM degradation inthese estuaries (16) Another example of the dominance of particle-associated enzymeactivity is marine snow Although bacterial production was not enhanced on snow parti-cles, enzyme activities (α- and β-glucosidases, leucine amino peptidase) of marine snow–

Figure 1 Extracellular enzyme activity (EEA), the initial step of the microbial loop The matic conversion of particulate organic matter and macromolecular exudates to dissolved organicmatter (DOM) triggers the microbial loop Arrows indicate the pathways of degradation, grazing,and predation.

enzy-attached bacteria were significantly higher than those of free-living bacteria, in terms ofboth absolute and per-cell rates (17) Similar observations, with respect to the relationshipbetween enzyme activities and amino acid incorporation of particle-associated bacteria inthe San Francisco Bay, were reported by Murrell, et al (18) The enzyme activities ofbacteria associated with the recently explored transparent exopolymer particles (TEPs)-which can harbor 2% to 25% of total bacteria in the sea (19,20)-have not yet been exam-ined

Significant differences in the extracellular enzyme activity per cell between attached and free-living bacteria frequently have been reported although these differencesare not always observed and most likely depend on the quality and composition of theparticles as well as on the nature of the colonizing bacteria (17,21–26) (Table 2) Metaboli-cally active particle-attached bacteria commonly have a larger polysaccharidic capsulethan do free-living bacteria (27) Because the majority of extracellular enzymes are embed-ded in the capsular envelope of metabolically active bacteria, a larger capsule potentiallycould harbor a greater quantity of enzymes It also has been observed that the capsular

particle-Table 1 Particle-Attached and Free (⬍0.2-µm) Extracellular Enzyme Activity of Different Enzymes in Different Habitats

Percentage Percentage PercentageEnzyme, Size of total Size of total Size of totalEnvironment Conditions substrate class (µm) activity class (µm) activity class (µm) activity Reference

turbidity β-glucosidase ⬍3 ⬃5 to ⬃50 ⬎3 ⬃50 to ⬃95gradient

Adriatic enzyme re- α-glucosidase 4–11

Sea lease β-glucosidase 0.6–10

California Surface sea leu-AMP ⬍0.2 0.2 ⬍1 40–80 ⬎1 ⬃20–60 (37)Bight water

Santa Monica Above 100 m leu-AMP ⬍0.2 ⬍30 0.2–0.8 70–75 (14)Basin

San Francisco Spring and leu-AMP ⬎1 47–76 av 65 (18)Bay summer β-glucosidase ⬎1 15–87 av 56

Kiel Fjord, Mesotrophic phosphatase ⬍0.2 33 0.2–3 14 3–150 53 (219)

Northern Red Oligotrophic phosphatase 0.2–2 50–71 2–20 12–27 ⬎20 3–37 (77)Sea

Table 2 Specific Extracellular Enzyme Activity per Bacterial Cell (amol cell h ) of Enzymes in Different Habitats

Environment Conditions leu-AMP Lipase P-ase α-Glucosidase β-Glucosidase Chitobiase ReferenceTrophic gradient Eutrophic 31.6 10.3 1.69 0.18 (113)

Basin

Seawater av 52.5⫾ 15

California Bight 44 Isolates from 4–3810 0.2–584 0.7–410 0–8 0–35 0–559 (148)

marine sources

Baltic Sea Summer 0.3–5 0.1–3.3 0.2–3.7 0.7–3.3 (115)

Arabian Sea Euphotic zone 6.6–23.2 0.4–3.6 0.16–0.22 (61)

Deep water 33–118 5.6–23.4 0.27–1.18Oman coast, upwel- Euphotic zone 12.6–46.9 1.2–8.3 0.02–1.2 (61)ling Deep water 455–1817 10.8–86.2 7.7–52.5

Coastal lagoon Hypertrophic LT 218–478 6.9–25.0 (221)

Japan Bottom water 21.1–270

LT, low tide; HT, high tide.

layer is continuously renewed by the bacteria (28) Metabolically inactive bacteria, incontrast, are usually devoid of a capsule (27), and a larger fraction of active bacteria hasbeen found in marine snow than in free-living bacteria (27) Consequently, a higher pro-portion of active, particle-associated bacteria might result in an overall higher extracellularenzyme activity per cell.

The pool of free dissolved enzymes (i.e., enzymes that pass through size filters) is fueled by various sources In addition to enzymes released by bacteria, theycan be derived from protozoa such as flagellates and ciliates and from mesozooplankton(e.g chitinase) Obviously, during periods of high zooplankton grazing activity, selectedenzymes can contribute the majority of the bulk activity (29) (Table 1), but this is not acommon feature Special patterns of distribution were recorded for phosphatases(13,30,31), which are generated not only by bacteria but also by phytoplankton, cyanobac-teria (32,33), and macroalgae (34)

0.2-µm-pore-C The Particle Decomposition Paradox and the Biological C Pump

Organic particles represent the nutritional basis for bacteria, and life in general, in theaphotic zone of the marine environment However, microscopic analysis has revealed thatparticles are frequently less heavily colonized by bacteria than expected Nevertheless,below the euphotic zone, particle decomposition has to supply the entire microbial commu-nity including the free-living bacteria A key to understanding this paradox lies in theenhanced individual enzyme activity of the attached bacteria (18,21,35,36) and probablyalso in the extracellular release of endo-enzymes by these bacteria (37,38) By hydrolyzingmacromolecular linkages in an endo- fashion (i.e., hydrolyzing the nonterminal linkages

in a polymer), these enzymes are able to break up the complex polymers inside the cles Both processes potentially create a surplus of dissolved monomeric or oligomerichydrolysis products from the particles that are not entirely taken up by the attached bacteria(loose hydrolysis-uptake coupling) Escaping into the surrounding water, these substratessupport the nutrition of free-living bacteria (39,40) A loose hydrolysis-uptake couplingfrequently has been reported for particle-attached bacteria, whereas tight coupling has beenreported between hydrolysis of DOM and uptake of the resulting monomers (17,22,40).Other studies, however, have not revealed a difference in the hydrolysis-uptake cou-pling between attached and free-living bacteria (24,36) In two recently published investi-gations on the extracellular enzyme activity of marine snow–associated bacteria, no evi-dence was found that glucosidase and aminopeptidase activity in marine snow–associatedbacteria were less tightly coupled to the uptake of the respective monomers than in free-living bacteria (23,26) Furthermore, in a number of studies using thymidine and leucineincorporation into bacterial deoxyribonucleic acid (DNA) and protein (41,42), respec-tively, as an estimate of bacterial C production, growth and the hydrolytic activities ofattached and free-living bacteria were compared (17,24,36,40) On the basis of such bacte-rial production estimates and concurrently measured hydrolytic activity, it was concludedthat the C demand of attached bacteria was lower than the amount of C cleaved by enzy-matic activity, hence indicating a loose hydrolysis-uptake coupling (43) However, it iswell known that leucine and especially thymidine are efficiently adsorbed to polysaccha-rides This adsorbed (radiolabeled) thymidine and leucine is taken up at significantly lowerrates than their nonadsorbed, truly dissolved counterparts (44) Since determinations ofthe saturating substrate concentrations are rarely made in such investigations (for logisticreasons), the amount of radiolabeled thymidine and leucine actually available for bacteria

parti-in the free form, and consequently available for rapid uptake, remaparti-ins unknown Thisadsorption and the concurrent lower availability for bacterial uptake might cause an under-estimation of the actual bacterial production on and in polysaccharide-rich material such

as marine snow (44), relative to bacterial enzyme activity

The coupling between hydrolysis and uptake of DOM in particle-associated and freebacteria is still not fully understood The reasons why the attached bacteria benefit so littlefrom their strong hydrolytic activities, if there are no limiting factors interfering with theuptake of enzymatic hydrolysis products, are unknown This fundamental discrepancyshould be more thoroughly investigated in order to improve understanding of the biogeo-chemical flux of organic matter and the role of bacteria in the cycling of DOM in the ocean

In any case, it is well accepted that particle decomposition (45) contributes significantly tothe loss of organic material from settling particles during sinking and thus determines theefficiency of the biological C pump (organic matter transport from the sea surface to theseabed)

D Environmental Factors Influencing Enzymatic Activity

The magnitude of the main extracellular enzyme activities in marine water is frequently

in the order aminopeptidase⬎ phosphatase ⬎ β-glucosidase ⬎ chitobiase ⬎ esterase ⬎α-glucosidase However, exceptions may occur, as observed by Christian and Karl (46)

in the equatorial Pacific, whereβ-glucosidase was about four times higher than tidase This suggests that there may be factors regulating activities on a large scale How-ever, knowledge of global regulating factors is scarce Christian and Karl (47) found thathistidine and phenylalanine inhibited aminopeptidase expression in Antarctic waters Like-wise, Kim and Lipscomb (48) suggested that metals may be regulating factors for proteases(leucine amino peptidase seems to be principally a Zn2 ⫹-dependent enzyme) This wasespecially due to Zn2 ⫹(which is rare in marine waters), but Mn2 ⫹, Co2 ⫹, Fe2 ⫹, and Mg2 ⫹

aminopep-might also play a role (47–50) In the surface layer of the ocean, ultraviolet-B radiationcan be important, mainly through photochemical degradation of the extracellular enzymes(51,52) With respect to phosphatase activity, the abundance of inorganic P is regarded as

a regulating factor, particularly for the P-limited regions in the oceans (53–55) However,dissolved organic phosphorus (DOP) and particulate organic P also should be considered(56) Furthermore, mechanisms of phosphatase regulation are different for bacteria andphytoplankton While the phosphatases of phytoplankton seem to be regulated strictly

by inorganic P concentrations (49,57–59), this mechanism is not so clear for bacterialphosphatases The latter may target C and N rather than P supply, as pointed out for thelimnetic environment by Siuda and Gu¨de (60) and for the deep and C-limited, but phos-phate-replete, ocean by Hoppe and Ullrich (61) In any case, regardless of environmentalfactors, variation of species composition within the bacterial community can significantlyinfluence the distribution of enzyme activities in the sea (62,63)

The effects of environmental factors on enzyme regulation are reflected by the

diver-sity of extracellular enzymes, as expressed in the possible ranges of K mand the patterns

of individual cell-specific enzyme potentials (Table 2,Table 3) Information on the Km

values of marine bacteria, however, is scarce Proteinase affinities seem to be higher in

oligotrophic than in eutrophic regions K mvalues observed in Antarctic regions at in situtemperature were similar to those in warmer regions; the relationship does not seem tohold for Arctic environments (Table 3) Cell-specific enzyme activities vary over a widerange They are low in eutrophic waters, but relatively high in oligotrophic waters and

Table 3 K mValues (Apparent) of Extracellular Enzymes in Different Marine Habitats

K m

Environment Conditions Enzyme, substrate (µmol L⫺1) Reference

After storm induced β-d-glucosidase 21.9–57.6

Lena River plume Arctic, eutrophic leu-AMP 28.6–83.3 (222)

β-d-glucosidase 14.3–40

particularly high on organic particles and in deep water (Table 2) In general, the istics of these variables indicate (in some cases clearly) a dependency on the prevailingenvironmental conditions

IN MARINE ENVIRONMENTS

A The Size Continuum of Organic Matter from DOM to POM

Dissolved organic matter (DOM) in the ocean is recognized as one of the three mainreservoirs of organic matter on the planet, equal to the organic matter stored in terrestrialplants or soil humus (64) DOM of natural waters is chemically complex: less than 40%

of the oceanic DOM pool is chemically characterized The concentration of DOM is,therefore, frequently measured as dissolved organic carbon (DOC) DOC in the oceantypically decreases from the euphotic zone with concentrations ranging from 100 to 150

µM C to around 40 µM C in the ocean’s interior (65) Despite the lower concentrations

in the deep ocean, the major fraction of the DOM is found in the aphotic zone of theocean, comprising⬃90% of the total oceanic DOC (66)

Chemical characterization of oceanic DOM is hampered by both the low tions of DOM and the high salt content of ocean waters, which interferes with chemicalanalysis Typically, 20–30% of oceanic DOC is recovered via 1000-Da ultrafiltration (67)

concentra-In estuarine environments, recoveries of DOM are usually higher (up to 70%), as a result

of the higher average molecular weight of the DOM in fresh water (68) On the basis ofsize fractionation studies performed over the past two decades on oceanic DOM, it appearsthat most of the DOM retained by ultrafiltration through 1000-Da filter cartridges is com-posed of compounds in the size range of 1000 to 30,000 Da (67–71) This high-molecular-weight DOM has been shown to be of contemporary origin (67,69) and derived fromrelease processes taking place during photosynthesis of phytoplankton, grazing, and lysis

of organisms (72–75) Phytoplankton extracellular materials have a similar carbohydrate

signature to that of the oceanic DOM in surface layers (76–78) Phytoplankton activityand mortality (grazing and viral lysis) have been suggested to be the major source ofoceanic DOM (70,74,75,78–82).

The molecular weight fraction of the DOM larger than 1000 Da but smaller than0.2µm is also frequently termed colloidal organic matter (COC), in contrast to the truly

dissolved DOM of⬍1000 Da Freshly produced high-molecular-weight DOM consistsmostly of carbohydrates, as indicated also by overall C : N ratios ranging from 15 to 25(67,69,70) In addition to polysaccharides, proteins and lipids are present as chemicallycharacterizable DOM components Polysaccharides, however, are by far the most abundantmacromolecular class of oceanic DOM

DOM is likely present as a size continuum in seawater; molecular weight and dynamic volume of DOM may vary with specific environmental conditions For example,the fibrillar structure of polysaccharides allows them to form bundles of molecules boundtogether via cationic bridges mediated by Mg and Ca (83) Thus, coagulation processes

hydro-of DOM, and particularly hydro-of the polysaccharides, are likely to be more important in oceanicseawater than in freshwater systems, as a result of the higher ionic concentrations in seawa-ter These coagulation processes lead to the formation of colloidal and ultimately micropar-ticulate organic material; thus DOM may be transformed to POM In this coagulationprocess, polysaccharides play a major role as a result of their relatively high concentrationand the physicochemical characteristics of the fibrillar structure (84–86) In 1998 Chin,Orellana, and Verdugo showed that even low-molecular-weight DOM has the potential

to coagulate spontaneously to form polymeric gels (87) These gels represent condensedorganic matter at a higher concentration relative to that of the surrounding water and mighttherefore be of considerable importance for bacterioplankton (1) and enzymatic hydrolysis.Furthermore, these microgels might interact with other colloidal matter, forming distinctsubmicrometer particles that are ubiquitously present in seawater at concentrations of up

to⬃109ml⫺1(88–93) Whereas these submicrometer particles are not colonized by ria, the larger transparent exopolymer particles (TEPs) are frequently densely colonized

bacte-by bacteria (94,95) TEPs have been shown to originate mainly from phytoplanktonblooms and their decay (96) In addition to these polysaccharidic TEPs, protein particleshave been reported to be abundant in the surface layers of the ocean (97)

At the upper end of the size continuum of condensed colloidal organic matter, marinesnow is commonly present, although at highly varying concentrations, in the surface aswell as in the deep waters The structural frame of this marine snow is also provided bypolysaccharides: they are highly hydrated structures larger than 0.5 mm and range up tometers in diameter as observed in the subpycnocline layers of the Adriatic and Mediterra-nean Seas and in the deep ocean (24,98–100)

Whether there are close links between different size categories of condensed organicmatter and whether smaller aggregates are really the precursors for the next larger group

of particles remain unclear at the moment There are indications, however, based on thecommon chemical signatures of the polysaccharide pool (which dominates the macromo-lecular fraction of all these particles), that there is a link between them and that they arederived mainly from auto- and heterotrophic microorganisms

Irrespective of the exact relationships between submicrometer particles, TEP, andmarine snow, all of this polysaccharide-based condensed matter interacts with the sur-rounding chemical environment by adsorbing inorganic and organic nutrients This results

in a higher nutrient concentration on these particles than in the ambient water (99,24)

The nutrient-enriched zones might be in the micrometer range, similar to the microzonesproposed by Azam (1) and elegantly visualized by Blackburn et al (101), or marine snow

of meters in diameter In any case, they are attractive to indigenous bacteria because ofnutrient concentrations up to three orders of magnitude higher than in the ambient water(102,103) Similarly, bacteria also have been reported to be enriched by up to three orders

of magnitude on these particles (24)

B Enzyme Activity and DOM/POM Reactivity

Contemporary DOM of high molecular weight has been shown to be efficiently utilized bybacterioplankton (3,4), while the majority of oceanic low-molecular-weight DOM, whichpersists long enough to be measured, can be considered as refractory This finding led tothe formulation of the size-reactivity model (3,4) proposing that the majority of the low-molecular-weight DOM pool is the consequence of chemical and biological degradation(4) Since bacterioplankton can take up molecules only smaller than 600 Da without priorcleavage by extracellular enzymes, the efficient utilization of this high-molecular-weightDOM indicates the importance of bacterial extracellular enzymes This enzyme pool in-cludes both endo- and exohydrolases (104) Endohydrolases cleave polymers into oligo-meric compounds, and, subsequently, exohydrolases generate monomers, which are taken

up by bacteria In order to cleave complex molecules, several endohydrolases act in cert, as has been demonstrated for the cellulase complex (105) With commonly usedfluorogenic substrate analogs, such as methylumbelliferyl derivatives (13,21), only thefinal step of the cleavage of the monomer (i.e., the exohydrolase activity) can be measured.The majority of this cleavage activity by hydrolases is bound to the cell wall or occurs

con-in the periplasmic space of Gram-negative bacteria (106), and only a small percentage offreely dissolved enzymatic activity can be detected (see Sec III A.) Fluorescently labeledhigh-molecular-weight substrates (see Section IV F.) can be used to measure endohydro-lase activities

C Significance of Enzyme Activity for Substrate Supply

Bacteria have different possibilities to respond to nutrient limitation (107) because theycan use inorganic as well as combined and monomeric organic molecules to supply theircellular demands for energy, growth, and maintenance The relative contribution of differ-ent sources to bacterial nutrition depends essentially on the availability of inorganic nutri-

ents (108) and on the C : N : P ratios (e.g., 106 : 16 : 1; the Redfield ratio) of organic matter

(109), which determine its nutritional value after hydrolysis Examples of the utilization

of the N pools are presented inTable 4 Using15NH⫹4 techniques, Tupas and Koike (110)demonstrated that natural bacterial assemblages in nutrient-enriched seawater cultures fu-eled 50–88% of their N demands for growth by NH⫹4 even in the presence of large amounts

of DON This DON consisted mostly of combined amino acids and contributed, togetherwith NH⫹4 uptake, 70–260% of bacterial N production However, an average of 80% ofthe DON used was subsequently remineralized to NH4⫹by the bacteria (110) In general,information is too scarce to derive principles about the preferences of bacteria for specific

N sources in the sea, however, hydrolysis of dissolved combined amino acids is always

a prominent feature

Table 4 Contribution of Different N Sources to Bacterial N Demand

DFAA DCAA NH⫹4

Environment Conditions (% of N demand) (% of N demand) (% of N demand) Experiment ReferenceDelaware Bay estuary Exponential growth ⬍34 (C% ⬍ 14) ⬍24 (C% ⬍ 10) n.d Batch cultures enriched (224)

with C and NC-limited 37–62 4–10 27–59

Sargasso Sea Surface water, depth ⬍20 20–65 n.d Radiotracer incubations (118)

profilesAburatsubo Bay Enriched seawater cul- n.d 70–260 50–88 15

NH⫹4 techniques (110)and Ohtsuchi Bay tures

Santa Rosa Sound Seawater incubations DFAA dominant DCAA secondary NH4⫹tertiary 14C-AA, enzyme (111)Gulf of Mexico N source N source N source essay

Uptake(nM h⫺1) Uptake(nM h⫺1) Uptake(nM h⫺1)Delaware Estuary Salinity gradient 114 117 n.d 14C-AA, algal protein (171)

LMW-DOC HMW-DOC

% uptake d⫺1 % uptake d⫺1Gulf of Mexico, tropical Seawater incubations 3–6.6 4.5–22.5 Tangential flow DOM (4)

Generally, the combined substrates DCAA, dissolved combined amino acids, require enzymatic hydrolysis, and their utilization therefore reflects indirectly the contribution of enzyme activity to bacterial growth DFAA, dissolved free amino acids; LMW-DOC, low-molecular-weight dissolved organic carbon; HMW-DOC, high-molecular-weight dissolved organic carbon.

IV DISTRIBUTION OF EXTRACELLULAR ENZYME ACTIVITIES

IN SEAWATER

A Enzymes in Coastal Regions, Lagoons, and Estuaries

Coastal regions represent the transition zone between land and the open sea Thus theyare frequently characterized by local morphological and hydrographical patterns and bygradients of salinity, eutrophication, pollution, and sediment resuspension These condi-tions are clearly reflected by specific patterns of extracellular organic matter degradation

In general, the enzymatic potential within the coastal regions affects the export of organicmatter to the adjacent open sea, which would otherwise only be supplied by autochthonousprimary production

Studies along trophic gradients have shown that extracellular enzyme activities react

in a specific manner, a response that differs from that of other microbial variables In acomparative study of a moderately eutrophic estuary and an open-water ecosystem,Jørgensen et al (111) observed a 2.5 times higher cell-specific leucine-aminopeptidaseactivity but a 2.4 to 18 times higher cell-specific free amino acid assimilation in the eutro-phic system Likewise, in a trophic gradient in the Adriatic Sea, Karner et al (112) foundpositive trends for leucine-aminopeptidase whereasα-glucosidase did not exhibit such aclear trend Trophic conditions also were clearly reflected by the patterns of a variety of

enzyme activities in a gradient at the Atlantic Barrier Reef off Belize Particularly, K m

values of leucine-aminopeptidase showed a much higher substrate affinity in oligotrophicwater than in the eutrophic region of the gradient (113) This corresponded to much higherper-cell activity in the oligotrophic environment In the salinity gradient between the Sac-ramento River and the central San Francisco Bay, increasing salinity was positively corre-lated with aminopeptidase activity and negatively correlated toβ-d-glucosidase activity(18)

In coastal regions, environmental factors, such as seasonal and diurnal variability intemperature and nutrient supply as well as stratification, are highly important for enzymaticactivity patterns In waters of the Uranouchi Inlet (Japan), phosphatase activity was similarthroughout the entire water column during the mixing period, whereas it was 20 timeshigher near the surface than in bottom waters during thermal stratification (114) Thisfinding suggests that phosphatase was limited by temperature and dissolved oxygen con-centration in the deep In the Pomeranian Bight (Baltic Sea), phosphatase activity was184–270 nmol L⫺1h⫺1in summer at about 18°C and 5.7 nmol L⫺1h⫺1in winter (0°C).Similar temperature effects were observed for α- and β-glucosidase and chitobiase Incontrast, peptidase activity was 9 to 72 times higher in autumn than in summer (115) In

a coastal station of the Ligurian Sea (Mediterranean Sea), Karner and Rassoulzadegan(116) measured the short-term variability of different extracellular enzymes, α- and β-glucosidase exhibited particularly strong diurnal variation, but such a variation was notobserved for aminopeptidase Sediment resuspension by storms in the coastal region alsocan have a strong impact, particularly on enzyme activity in bottom water This was shown

in mesocosm experiments in which aminopeptidase andβ-glucosidase were 24% and 43%higher, respectively, after a simulated storm event compared to the calm period (117)

Although the oceans generally have low concentrations of organic matter per volume ofwater, they play, because of their huge dimensions, a dominating role in organic matter

production and decomposition of the biosphere The production and transformations oforganic matter in the surface ocean and the burial of organic materials in the deep sedi-ments contribute significantly to the efficiency of the biogeochemical cycles and climatechange on Earth Nevertheless, enzyme investigations in truly offshore regions and ontransoceanic cruises rarely have been conducted.

In an extremely oligotrophic domain of Sargasso Sea, dissolved combined aminoacids dominated the N pools and also contributed the largest part (20–65%) of the bacterial

N demand in surface water A modified form of protein (glucosylated protein) accountedfor the highest portion of bacterial N demand in deeper waters (118) (Table 4) Comparingthe enzymatic properties of three oceanographic provinces of the Pacific (northern subtrop-ical, equatorial, and the Southern Ocean down to Antarctica), Christian and Karl (46)found significant variations in aminopeptidase andβ-glucosidase activities and their tem-perature characteristics The relative relationship between aminopeptidase andβ-glucosi-dase shifted from 0.3 at the equator to 593 in Antarctic waters This change was duemainly to an increase ofβ-glucosidase activity from 0.44 in Antarctica to 1519 nmol L⫺1

d⫺1at the equator The authors hypothesized that there was a longitudinal trend in bacterialutilization of polysaccharides relative to amino acids and proteins Investigating the north-ern Pacific from 45°N 165°E down to the south edge of the equatorial zone (8°S 160°E),Koike and Nagata (119) also found an increase ofβ-glucosidase activity in the surfacelayer from 0.11 to 3.1 nmol L⫺1h⫺1; this change, however, was far less dramatic as ob-served by Christian and Karl (46) In the northern Indian Ocean (Arabian Sea), enzyme(aminopeptidase,β-glucosidase, phosphatase) activities together with other microbial ac-tivity measurements reflected clearly the specific hydrographic conditions created by the

SW Monsoon (61).β-Glucosidase activity was not enhanced at the equator (⬃0.5 nmol

L⫺1h⫺1) but increased strongly in the upwelling regions off the coast of Oman (2 nmol L⫺1

h⫺1) In the same direction, the relationships between aminopeptidase andβ-glucosidaseactivities increased from⬃10 to ⬃50

Aminopeptidase activity measured along a N-S Atlantic transect (54° N–62° S)(120) suggests a strong dependency of this variable on the global current system andclimate zones Lowest values were measured in the northern and southern subtropicalgyres (⬍10 nmol L⫺1d⫺1) Increased enzyme activity was detected near the southern edge

of the North Equatorial Current (7° N) and the northern edge of the South EquatorialCurrent (10° S), which are fueled by the Canary Current and the Benguela upwellingregions, respectively At the continental shelf edge (Patagonain Shelf) at 40° S (wherethe subtropical Brazil Current and the subantarctic Falkland Current meet and establish theSubtropical Convergence), aminopeptidase activity increased instantaneously by factors of

6 to 11 Surprisingly, aminopeptidase activities in the Antarctic Weddell Sea were nearly

as low as in the warm but nutrient-depleted subtropical regions (120)

Extreme environments in the marine biosphere are represented by the high-pressure waters

of the deep sea and the permanently cold polar regions, together with some zones enced by hot vent and anoxic (or even sulfidic) conditions

influ-Extracellular enzyme activities (aminopeptidase,α- and β-glucosidase, phosphatase)

in sea ice were similar to those reported from eutrophic, temperate marine environments.Though psychrophilic isolates showed temperature optima of their enzymes that weresimilar to those of mesophilic strains (⬃30°C), their activity at low temperature was rela-

tively much higher Activities of some enzymes in polar sediments, however, have ature optima much lower (ca 16°C) than those reported for temperate sites (121).Enzyme activity in the melted ice at 1°C was generally much higher than in thewater underneath the ice, 3.2–1702 times for aminopeptidase, and 2.4–42.2 times forphosphatase In poorly colonized ice cores, enzyme activity can be similar to or lower

temper-than in the water (122,123) K mvalues for aminopeptidase of bacterial communities fromAntarctic regions, measured at in situ temperature, were comparable to those observedfor a variety of aquatic environments (47) Studies on enzyme activities in waters of thedeep sea (mesopelagic and bathypelagic strata) are rare Koike and Nagata (119) measured

a very strong decrease of (particle-associated)α- and β-glucosidase activity down to 4000

m depth in the central Pacific Ocean, where activities were generally less than 1% of thesurface values In contrast, phosphatase activity at depth was up to 50% of that near thesurface This observation was confirmed by Hoppe and Ullrich (61), who found very lowglucosidase activity (in unfractionated samples) in the lower mesopelagic zone of theIndian Ocean At greater depths, aminopeptidase activity was equal to or much lower thanthe activity measured near the surface, but the phosphatase activities were up to seventimes higher than near the surface These phenomena were interpreted to be i) a result

of enzyme export by sinking particles and ii) a consequence of severe C limitation ofdeep sea bacteria meeting (partly) their C demands by the hydrolysis of organic P com-pounds (61) In deep coastal waters (Santa Monica Basin), proteolytic activities correlatedsignificantly with bacterial abundance and bacterial growth down to the sea floor at 900-

m depth (14) Effects of sulfidic conditions (H2S) on enzyme activities of bacterial nities from the Baltic Sea were investigated by Hoppe et al (124) In comparison tooxic control treatments, reduction of activity was particularly strong for peptidase and theglucosidases and to a lesser degree for chitinase and phosphatase (124)

commu-D Enzymatic Properties of Marine Bacterial Species and Other Organisms

The ability of marine bacterial isolates to support growth via freely released enzymes onparticulate organic substances (amylopectin, chitin, animal hide) was measured by Vetterand Deming (125) Under these conditions, the bacteria were able to grow, albeit at ratesgenerally lower than rates reported for growth on dissolved organic substances The inter-actions between corals and bacteria living in the mucus of corals were investigated by

Santavy et al (126) in the coral Colpophyllia natans If the corals were under stress by

infection (black band disease), they produced more mucus and the activity of the associatedbacteria was generally enhanced Phosphatase activity was especially enhanced, and themeasurement of phosphatase activity was recommended as a tool to quantify stress metab-

olism of corals Chitobiase (N-acetyl-β-d-glucosaminidase) activity, expressed during the premolt phase of crustaceans, was used as a measurement of copepod (Temora longi- cornis) secondary production in the sea (127) Other studies suggest that phagotrophic

nanoflagellates can contribute significantly to the pool of freeα-glucosidases and peptidases in marine environments (128), and the occurrence of phosphatase activity inred-tide dinoflagellates has been shown by Vargo and Shanley (129) From the appliedaspect, Sawyer et al (130) investigated enzymatic properties (including that of phospha-tase; it was not clear, however, whether extracellular or intracellular enzymes were stud-

amino-ied) of potentially pathogenic amoebae (Acanthamoeba sp.) from the sediments of marine

sewage dumping sites Morphologically similar species could be distinguished from each

other by the diversity of their enzymatic capabilities Dramatic effects of a geneticallyengineered bacterium with enhanced phosphatase activity on the growth of natural marinephytoplankton were recorded by Sobecky et al (131).

On a global scale, marine sediments fulfill a critical function as a sink for reduced C: asmall fraction of the CO2fixed by phytoplankton in the surface ocean sinks through thewater column as fast-settling particles An even smaller fraction of these particles escapesthe efficient remineralization processes in sediments and is ultimately buried Burial ofthis reduced organic C represents long-term removal of CO2from the ocean (64) Marinesediments are also diverse and variable environments and serve as habitats for a widerange of benthic organisms The nutritional basis for many of these organisms is the rain

of particles from the surface ocean, so successful existence in the benthos is directly related

to the ability to gain C and energy from POM flux

Marine sediments are also dynamic systems, subject to physical as well as chemicalchanges on a wide range of time scales The flux of particles from the surface ocean isvariable in time and space; surface sediments may be resuspended as a result of slumping

or turbidity currents Activities of infaunal and epifaunal organisms lead to formation oftubes and burrows and extensive bioturbation Frequently, the net result is a ‘‘patchy’’environment characterized by physical and chemical discontinuities, redox gradients, andzonation of microbial activities, where biological and chemical parameters can vary greatly

on small spatial and temporal scales Effectively assessing these variations and discerningthe patterns that may underlie this variability are major challenges in studying sedimentaryenvironments

A Seasonal and Spatial Patterns in Coastal and Temperate Sediments

Coastal temperate regions are subject to significant seasonal changes in a range of physicaland chemical parameters, including light, temperature, and nutrient availability Cycles

of productivity respond to these seasonal changes; variations in extracellular enzyme ities in shallow and temperate sediments have in turn been linked to seasonal variations

activ-in activ-input of organic matter Reichardt (132) observed that an approximately twofold activ-crease in extracellular enzyme activity in surface sediments followed the sedimentation

in-of a phytoplankton bloom Protease activity, measured by using hide powder azure, wasparticularly high At the same site, Meyer-Reil (133) observed annual maxima in leucineamino peptidase activity in surface sediments, which occurred simultaneously with themain sedimentation events in Kiel Bight Pantoja and Lee (38) likewise observed an annualmaximum in peptide hydrolysis rate constants in Flax Pond sediments, which also corre-lated with sedimentary input of fresh organic matter Changes in enzyme activities havealso been correlated with annual temperature cycles in intertidal sediments in Maine (134).Pantoja and Lee (38), however, found that there was no direct relationship between temper-ature and peptide hydrolysis rate constant over an annual cycle at another coastal site,although rate constants in the upper 10 cm of the sediments were higher in the spring/summer than in the winter

Because C influx to the sediments occurs via settling particles, surface metabolism

is frequently stimulated above the levels observed in deeper layers of the sediments (135).Although organic matter in these deeper layers is often considered to be of lower nutri-tional quality, since it has already been reworked by the surface community, fresh organicmatter can be rapidly mixed to considerable depths by infaunal organisms (136), providingthe subsurface community with fresh organic C The depth trends observed in enzymeactivities generally correspond to these patterns Activities in surface layers of the sedi-ment are greater than those in lower depths (133,134,137) Shallow subsurface maxima,however, also have been observed; such maxima may correspond to ‘‘hot spots’’ of micro-bial activity and/or be related to infaunal organisms (38,134,138) Potential hydrolysisrates of carbohydrates and proteins measured at a wide range of coastal and temperatesediments vary over several orders of magnitude (Tables5,6, and7) Intertidal sediments

in Maine include the highest carbohydrate-hydrolysis rates reported to date (137) Thewide range of rates King (137) observed for a suite of monosaccharide substrates illustratesthe fact that extracellular enzymes in natural systems are sensitive to structural variationsamong closely related substrate analogs

In deep ocean sediments, enzyme activity likewise frequently is maximal in surface vals and decreases with depth in the sediments (139), although shallow subsurface maximaalso have been reported (140), perhaps also linked to biogenic structures and macrofaunaltubes (141,142) Surveys of the deep ocean consistently have shown patterns of decreasingoverall enzyme activities with increasing water column depth (139,143) No evidence has

inter-Table 5 Carbohydrate-Hydrolyzing Enzyme Activity in Coastal and ShallowMarine Sediments

Rate

Measurements made in sediment slurries, except as noted.

a Measured in intact cores, range of values.

b Recalculated from reference (Arnosti, in prep.)

Table 6 Carbohydrate-Hydrolyzing Enzyme Activity in Polar and Deep Sea Sediments

Rate

0.02–0.35 MUF- α-glucose 37–3427 Arctic continental slope (143) 0.02–3.02 MUF- β-glucose 37–3427 Arctic continental slope (143)

0.2–0.17 MUF- α-glucose 135–1680 Atlantic continental (139)

shelf

shelf

0.0049–0.0082 MUF- α-glucose 2879–4919 Northeast Atlantic (145) 0.002–0.016 MUF- β-glucose 2879–4919 Northeast Atlantic (145)

ca 0.005–0.006 MUF- α-glucose 4500 Northeast Atlantic (142)

Measurements made with sediment slurries, except as noted.

a Measured in intact cores; range of values.

b Recalculated from reference (Arnosti, in prep.).

c nmol g ⫺1 dry weight sediment h ⫺1

Table 7 Protein Hydrolysis in Coastal, Polar, and Deep Marine Sediments

Rate

Measured with l-leucine-4-methylcoumarinyl-7-amide (leu-MCA) Measurements made in sediment slurries, except as noted.

a Measurements made in intact cores, range of values.

b nmol g ⫺1 dry weight sediment h ⫺1