Microbial degradation of sorbed and dissolved protein in seawater

Limnol. Oceanogr., 40(5), 1995, 875-885 0 1995, by the American Society of Limnology and Oceanography, Inc.

Microbial degradation of sorbed and dissolved protein in seawater

Gordon T. Taylor Marine Sciences Research Center, State University of New York, Stony Brook 11794-5000

Abstract The effects of adsorption and concentration on rates of protein degradation by marine bacteria were

examined by measuring hydrolysis and remineralization of the radiolabeled protein, ribulose- 1 +bisphosphate carboxylase-oxygenase ([meth$3H]Rubisco). Protein adsorbed at low surface concentrations on glass beads had hydrolysis and remineralization rate constants 247% and 282% higher than protein bound at high surface concentrations. Moreover, thin films of bound protein were hydrolyzed and remineralized 522% and 1,033% faster than comparable pool sizes of dissolved protein. At high concentrations, however, rates of’ hydrolysis and remineralization were not significantly different between proteins in dissolved or sorbed pools. Results demonstrate that proteins adsorbed to surfaces can be as bioavailable as those in solution. In fact, the sorption process may actually aid bacterial degradation by concentrating the protein and hydrolysates at an interface and by unfolding proteins to relax steric hindrance and expose a higher proportion of bonds to bacterial endo- and exohydrolases.

In natural waters, high-molecular-weight (HMW) materials, including protein, represent important growth substrates for bacteria and may account for 20-35% of the total dissolved organic matter (DOM) pool (Benner et al. 1992). HMW materials can support significantly higher bacterial growth than can comparable amounts of low-molecular-weight (LMW) DOM (Amon and Benner 1994). Pertinent to proteins, dissolved combined amino acids (DCAA = adsorbed free amino acids + peptides + proteins) can be 20-100 more abundant than dissolved free amino acids (DFAA) in surface waters but utilized 2-10 times slower than monomeric counterparts (Azam et al. 1992; Keil and Kirchman 1993). Disparities in reported pool sizes and utilization rates may reflect dissim- ilar rates of supply and consumption as well as differences in lability among DCAA, DFAA, and other DOM con- stituents. The relative labilities of LMW and HMW DOM in seawater remain poorly understood and probably depend upon the composition, particle reactivity, physical state, and age of these materials as well as the chemistry and biology of the receiving waters (e.g. Keil and Kirch- man 1993, 1994; Mayer 1994; Keil et al. 1994).

After release to seawater and before significant degradation, macromolecules such as proteins have several possible physical states in seawater, including native state, denatured state, solute, sorbate, or complexed with other dissolved organic matter (Keil and Kirchman 1993, 1994; Taylor et al. 1994a). Proteins, like most macromolecules,

Acknowledgments I am indebted to D. Zheng, G. Gyananath, and P. J. Troy for

technical assistance and to D. M. Karl for use of laboratory instruments. I am grateful to J. Gulnick, C. Lee, S. Pantoja, and L. Palmer for comments and assistance in manuscript preparation. I am also grateful to D. L. Kirchman, R. G. Keil, and L. M. Mayer for constructive skepticism and comments which helped immensely in improving this communication.

Research was supported in part by Office of Naval Research contract NO00 14-88-K-0044.

Marine Sciences Research Center Contribution 990.

tend to be very particle-reactive in seawater and partition out of the aqueous phase as functions of protein concentration, protein composition, and substratum properties (Kirchman et al. 1989; Taylor et al. 1994a,b). Conse- quently, enrichments of these materials are expected at interfaces such as the surfaces of seston and sediment particles (e.g. Tanoue 1992; Mayer et al. 1986).

The process of adsorption can induce denaturation. Molecular unfolding occurs when the attractive forces between substratum and internal hydrophobic moieties of protein molecules exceed the molecule’s stabilizing forces, especially on hydrophobic surfaces (Soderquist and Walton 1980; Lundstrom 1985; Norde and Lyklema 199 1). Denaturation results in exclusion of water and reductions of interfacial free energies. Arnebrant et al. (1985) proposed that under in vitro conditions, adsorbed proteins organize as submono- to monolayers of molecules mostly in denatured state or as composite multilayers with native state molecules bound on top of the base monolayer (Fig. 1). Organization depends on solute concentration which controls sorbate interactions (i.e. competition for binding sites and steric limitations to molecular unfolding). Kinetic, thermodynamic, and spec- troscopic studies of the autotrophic enzyme, Rubisco, in seawater have yielded results consistent with this model (Taylor et al. 1994a,b).

The effects of surface adsorption on organic matter (OM) degradation and bacterial activity have been under dis- pute (see van Loosdrecht et al. 1990). Enhanced as well as diminished rates of hydrolysis, remineralization, and bacterial growth have been reported for OM bound to seston, sediments, sediment surrogates, and soils (e.g. Dashman and Stotsky 1986; Griffith and Fletcher 199 1; Smith et al. 1992; Mayer 1994). Whether sorbed OM is degraded or preserved will depend on the age, location, and structure of the material. Preservation in sediments will be favored if OM is covalently bound to surface sites, is adsorbed into smaller mesopores (2-50 nm), is geo- polymerized, or is buried in anoxic sediments (Mayer 1994; Keil and Kirchman 1993; Keil et al. 1994). For

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876 Taylor

Native state Denatured monolayer Composite bilayer Fig. 1. Schematic representation of the Rubisco molecule in native state (A) and presumed

organization of proteins on the surface of experimental glass beads in thin ‘(I$ and thick (C) films as modeled for other systems by Arnebrant et al. (1985). Secondary and quaternary structures are those presented by Andersson et al. (1989) and Miziorko and Lorimer (1983), respectively.

example, surfaces of coastal sediments exceeding several years in age seem to be coated with monomolecular layers of OM that remain relatively constant downcore, indicating preservation of this material. This residual layer may result from the process called sorptive protection or sorptive preservation (Mayer 1994; Keil et al. 1994).

The fate of freshly adsorbed protein in the presence of active aerobic bacteria in surficial sediments or on seston may be quite different than that of aged material. For example, Mayer et al. (1986) observed that proteins bound to coastal sediment, accounting for lo-20% of total hy- drolyzable amino acids, decreased downcore much more rapidly than total organic nitrogen. Preferential degradation of macromolecules over sorbed peptides, amino acids, and other nitrogenous compounds is implicit in these observations.

The following study explores ways in which physical state and concentration influence bacterial rates of protein degradation. Specifically, I tested the hypothesis that adsorption and surface denaturation increase bioavailability of protein to marine bacteria by examining hydrolysis and remineralization of protein in adsorbed and dissolved states. Effects of adsorption and surface denaturation were separated by adsorbing protein films, approximating the molecular organizations depicted in Fig. 1B and C, onto smooth glass beads. Rates of degradation (i.e. hydrolysis and remineralization) of adsorbed protein are compared with those observed for a wide range of dissolved protein concentrations.

Materials and methods

Protein preparation - Rubisco, key enzyme in the Cal- vin-Benson cycle, was chosen because it is the most ubiq- uitous protein (Miziorko and Lorimer 1983) and is therefore a logical model for behavior of large proteins in natural waters. Furthermore, rates of hydrolysis and bacterial uptake of Rubisco appear to be similar to a variety of proteins (Keil and Kirchman 1993).

Radiolabeled protein was prepared by reductive meth- ylation of commercially available spinach Rubisco (Sig- ma Chemical Co.) with [3H]borohydride (50-75 Ci mmol-l) as previously described by Kirchman et al. (1989) and Taylor et al. (1994b). Specific activity of radiolabeled

protein preparations was 47.6 nCi pg- l. Proteins prepared in this manner had adsorption-desorption and bacterial degradation (hydrolysis) characteristics indistinguishable from unlabeled Rubisco (Kirchman et al. 1989; Samuels- son and Kirchman 1990). However, apparent assimilation and respiration of [methyZ-3H]Rubisco by marine bacteria are less than for uniformly labeled [3H]Rubisco, suggesting discrimination against uptake of 3H-methylated amino acids (Keil and Kirchman 1992), a fact un- published at the time of these experiments. Therefore, assimilation is not reported here, and respiration rates are considered minimum estimates of bacterial remineralization.

Degradation of adsorbed protein-The effects of adsorption and surface concentration on degradation rates were evaluated by incubating bacterial suspensions with glass beads coated with relatively thin and thick [methyl- 3H]Rubisco films. Before incubations with bacterial suspensions, an adsorption isotherm was run with Pyrex glass plates to determine the solute concentration dependence of adsorption (see Taylor et al. 19946). Results from the adsorption isotherm were used to select two dissolved protein concentrations that would yield glass surfaces coated with either submonolayers or mono- to multilayers of adsorbed protein, designated henceforth as thin and thick films. Smooth borosilicate glass beads, 6 mm in diameter, were placed in eight 250-ml Pyrex incubation flasks (170 beads per flask) and thoroughly washed in detergent, then 10% HCl, and rinsed exhaustively in distilled water before drying and combusting at 475°C for 14 h to remove organic contamination.

Relatively thin and thick films were obtained by equil- ibrating beads with 25 ml of either 0.5 or 50 pg ml-’ solutions of [methyZ-3H]Rubisco in sterile UV-oxidized seawater for 2 h at 24°C on an orbital shaker at 150 rpm. Gravimetric rather than molar quantities are presented because surface adsorption of macromolecules varies more as a function of molecular mass than number. For molar conversions, a molecular weight of 560 kDa may be applied (Miziorko and Lorimer 1983). The radioactive bulk solution was discarded, and the flasks and beads were rinsed for 2 min with 50 ml of distilled water, which was also discarded. Beads and flask were drained of excess

Adsorption and protein degradation 877

rinse water over a nylon mesh. This procedure resulted in 80 and 95% of the total label being adsorbed to the beads and flask walls in the low and high protein treatments; the balance resided in interstitial water. The surface area of flask exposed to protein was 72 cm2, and the beads provided an additional 192 cm2 of exposed surface area.

Continuous cultures of mixed bacterial populations from the south shore of O’ahu, Hawaii, were maintained at a specific growth rate of 0.5 d- 1 in unamended filtered seawater (0.22 pm). Half of this dilute cell suspension was equilibrated in 0.2% sodium azide (NaN,) for 30 min to serve as a poisoned control. The experiments were ini- tiated by adding 130 ml of uninhibited cell suspension to two flasks with beads coated with thin films and to two with beads coated with thick films. The same volume of the azide-control cell suspension was added to four additional flasks with thin and thick films. Flasks were incubated 12 h at 24°C on an orbital shaker at 150 rpm.

Desorption, hydrolysis, and remineralization of adsorbed protein were examined by monitoring the partitioning of radiolabel into total dissolved ([3H]DIS = GF/F filtrate), TCA-soluble (LMW 5 2 kDa), and nonvolatile residue ([3H]RES) fractions through time. Immediately after addition of bacterial suspensions (To), one of the azide control flasks from both surface concentrations was sacrificed for detailed analysis of label partitioning. At each subsequent time point (2, 4, 8.5, and 11.5 h), triplicate lo-ml aliquots of the overlying suspension from one live treatment at both surface concentrations were passed through Whatman GF/F filters to remove bacteria and the filtrates captured for subsequent processing. In separate control experiments under comparable culture conditions, bacterioplankton labeled with [3H]leucine were retained equally well on GF/F, Millipore GS (0.22 pm), and Nuclepore 0.2-pm filters, so only a negligible portion of labeled bacteria or particles was likely to have passed into this GF/F filtrate. The unsampled live and poisoned flasks were sacrificed at 12 h for detailed analysis.

Undesirable adsorption of soluble [methyZ-3H]Rubisco was minimized during sample processing by rinsing the filters and filtration equipment before each sampling with 10 ml of chilled, filter-sterilized seawater containing 2 yg ml-’ of unlabeled Rubisco in an attempt to saturate adsorption sites. Despite this precaution, control experiments with sterile, particle-free seawater revealed that 15- 24% of the LMW material was adsorbed to filters and filtration apparatus. Values presented are not corrected for adsorptive losses, so production of LMW material is systematically underestimated.

Total dissolved label ([3H]DIS) was determined by directly radioassaying 2 ml of filtrate. The TCA-soluble fraction was partitioned by mixing 5 ml of filtrate, 5 ml of chilled 10% TCA (trichloroacetic acid), and 0.1 ml of a 100 mg ml-l solution of bovine serum albumin (BSA) in phosphate-buffered saline and centrifuging at 1,100 x g for 20 min as described by Hollibaugh and Azam ( 198 3). The BSA functioned as a carrier to precipitate radiolabeled HMW material. Radiolabeled TCA-soluble mate-

rials were determined by radioassaying 2 ml of the su- pernatant. Nonvolatile label ([3H]DIS-3H20) was as- sayed by evaporating 2-ml aliquots of the filtrate directly in their respective scintillation vials to dryness with a Buchler vortex evaporator at 45”C, rehydrating the resi- dues ([3H]RES) in 2 ml of distilled water, and vigorously vortexing before adding scintillant and radioassaying.

Amounts of [methyZ-3H]Rubisco bound to the beads and flask wails at the beginning and end of incubations were determined by radioassaying aliquots of separate acid digests of rinsed beads and empty, rinsed flasks (TO and T12), obtained from extractions with 70% formic acid at 55°C for 3 h in a sonicating bath. Total available radiolabel at the beginning of the experiment was calculated from surface coverage (nCi cm-2 or ng cm-2) and total immersed surface area (beads + flask wall). All radioas- says were conducted in Aquasol 2 (New England Nuclear) with a Packard Tri-Carb 4640 scintillation counter. Dif- ferential quenching resulting from the solvent systems was normalized on the basis of control experiments. Pro- duction of 3H20 was calculated by the difference between [3H]DIS and [3H]RES. Flux rates (dpm ml- 1 h-l) were derived from end-point differences between the flasks sampled only at TO and T12, and rate constants (h-l) were calculated by dividing rates by the total available pool. All reported rates are calculated from means of triplicate subsamples or least-squares regression analysis.

Degradation of dissolved protein -Dependence of protein degradation rates on dissolved protein concentration was evaluated in three end-point experiments 10, 10, and 24 h in duration. In the first experiment, 10 ml of nine dilutions of sterile [ methyZ-3H]Rubisco in UV-oxidized seawater were added to 40 ml of the picoplankton suspension used in the bead experiments (final concn = 1.4- 13,240 ng Rubisco ml-l). Azide-poisoned controls were prepared as described above for three protein concentrations.

Variability in activity among different bacterial populations was assessed by performing two more kinetics experiments. The first experiment used picoplankton in seawater freshly collected from Kewalo Basin, south shore of O’ahu, on 9 October 1990. The suspension passing through a 2.0-pm Nuclepore membrane was added to seven dilutions of protein, yielding concentrations of 2.2- 9,366 ng [methyZ-3H]Rubisco ml-l. Azide controls were prepared as before at four protein concentrations span- ning most of this concentration range. Azide controls were subsequently found to vary in effectiveness. Biotic and abiotic processes were better distinguished by using 0.008% HgC12 controls. The last kinetics experiment was performed with the picoplankton fraction in freshly collected seawater from the same field site on 23 October 1990 and with a more effective negative control (0.008% HgCl2).

Incubation conditions, glassware preparation, sample processing, radioassay procedures, and pool calculations were the same as described for the bead experiment. Total available protein in each flask was determined from 1 -ml

878 Taylor

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[methyl-3HlRubisco (pg ml-9 Fig. 2. Adsorption isotherm of [methyl-3H]Rubisco irre-

versibly bound to Pyrex surfaces (0) and surface concentrations measured on borosilicate glass beads \. Least-squares regression line described by rirr = 0.03 C00.585, r2 = 0.960. Dotted lines represent calculated range for establishment of a monomolecular layer of native state protein (see Taylor et al. 1994b).

aliquots of unfiltered suspension taken at the beginning of the experiments.

Bacterial abundances and growth-Subsamples of seawater from each treatment were preserved with borate- buffered 2% formaldehyde at the beginning and end of the experiments. Enumeration of bacteria attached to beads and flask walls was not attempted. Bacterial abundances, N (cells ml-l), were determined by epifluores- cence microscopy with acridine orange as the fluoro- chrome (Hobbie et al. 1977). Growth rate constants (h-l) were calculated assuming exponential growth; p = (In Nt - In No)/& where No and Nt equal bacterial concentrations at time 0 and t. Bacterial abundances at the midpoint of the experiment were calculated as NJ2 = N,exp(@/2), which is equivalent to the geometric mean.

Results

Protein adsorption and molecular organization - Sur- face concentrations of [methyZ-3H]Rubisco irreversibly bound to Pyrex surfaces (I’,,) varied as a Freundlich function (Ii, = kC01/n) of dissolved protein concentration, Co, where k and n were 0.03 and 1.7 1, respectively (Fig. 2, diamonds). Partitioning of protein onto borosilicate glass

Table 1. Release rate constants and surface concentrations of [methyl-3H]Rubisco irreversibly bound to glass beads after a brief rinse in distilled water (initial) and after 12-h exposures (final) to seawater suspensions of bacterioplankton and controls.

Treatment

Surface concn (ng Rubisco cm-2)

Initial Final

Release rate (h-7

Low [P] -live 8 6 0.023 Low [P] -azide 8 7 0.012 High [P] -live 1,053 507 0.043 High [P] -azide 1,053 708 0.027

beads exposed to 0.5 and 50 hg [methyZ-3H]Rubisco ml- 1 was very similar to that of Pyrex, yielding I’i, values of 8 and 1,053 ng [methyZ-3H]Rubisco cm-2 (Fig. 2, squares). Sorbate is considered irreversibly bound in an operational sense because these molecules remain on surfaces after a brief (2 min) distilled-water rinse (Taylor et al. 19943). However, some desorption is evident over extended exposure to seawater (presented below). Adsorption to Py- rex slides is indicative of the partitioning of dissolved [methyZ-3H]Rubisco to flask walls in all experiments. In the bead experiments, flask walls are an additional source of adsorbed protein, contributing 27 and 9% to the total inventory in the thin and thick film treatments, respectively. In experiments with dissolved proteins, adsorption to flask walls represents a negligible sink for dissolved pools, varying from 1.43% down to 0.03% of the total inventory for the lowest to highest protein concentrations.

The monolayer domain (Fig. 2, dotted lines) was derived from a range of literature values for molecular size and weight (see Taylor et al. 1994b) and represents the range of surface concentrations (60-930 ng cm-2) that could potentially form a monolayer of molecules in native state (Fig. 1A). Beads and flasks equilibrated with 0.5 pg [methyZ-3H]Rubisco ml-’ formed thin protein films an order of magnitude below the monolayer threshold. In this case, the most probable molecular organization for adsorbate is in a submono- to monolayer of highly unfolded molecules, where maximum contact with binding sites is possible (Fig. 1B). In contrast, beads and flasks equilibrated with 50 pg [methyZ-3H]Rubisco ml- 1 formed relatively thick protein films that coincided with the upper boundary of the monolayer domain. Given the heterogeneous nature of protein adsorption (Taylor et al. 1994a,b), thicker films probably varied spatially from mono- to multilayers comprised of molecules in native as well as denatured states (Fig. 1C).

Release of adsorbed protein -Mass transfer of bound protein from glass surfaces to the aqueous phase resulted from both passive desorption and bacterial degradation. These fluxes varied as functions of both initial surface concentration and biological activity (Table 1). Release rate constants for thin films exposed to live bacterioplankton (0.023 h-l) were about half of those derived from thick films (0.043 h-l). These results illustrate that protein molecules bound to beads with higher surface


concentrations are either more easily desorbed or hydrolyzed. Evidence presented below suggests that desorption of more loosely bound outer molecular layers from thick films accounts for much of the difference in release rates. Differences in release rates are consistent with the model for interfacial organization of protein presented in Fig. 1B and C and with previous observations (Taylor et al. 1994b). In the azide controls, release of bound protein was evident, but rate constants were half those observed in comparable live treatments (Table 1). These results indicate that both passive desorption and bacterial activity play significant roles in altering surface concentrations of bound protein.

Hydrolysis of adsorbedprotein -The TCA-soluble fraction includes LMW products hydrolyzed from macromolecules, 3H20 produced by biological respiration, and organic metabolites released by bacteria but excludes material incorporated into biomass. During the time-course with protein-coated beads, dissolved concentrations of TCA-soluble products increased linearly, resulting in LMW production rat,es from thick films 70 times greater than from thin films: 11,480 vs. 163 dpm ml-l h-l (Fig. 3A). However, relative to total protein pool size, which varied by a factor of 105, the pool of molecules in thin films was turned over 160% faster by hydrolysis than that in thick films (H, = 0.076 h-l vs. 0.049 h-l). TCA-soluble products were also released in the azide controls, but at rates 39 and 32% of the live counterparts for thin and thick films (Fig. 3A; solid symbols). Release of TCA- soluble products in poisoned controls may result from several processes, including chemical hydrolysis, residual active exoenzymes, and incomplete biological inhibition. None of these alternatives can be adequately evaluated with existing data.

Remineralization of adsorbed protein -As with the TCA-soluble pool, production of 3H20 in live treatments increased linearly over 12-h incubations (Fig. 3B). When exposed to thick films, comparable bacterial populations produced 3Hz0 78 times faster than those exposed to thin films: 95 vs. 7,378 dpm ml-l h-l. However, the entire pool was respired somewhat faster in thin films than thick films: R, = 0.044 vs. 0.032 h-l. Production of 3H,0 is clearly dependent on biological activity, as indicated by low yields of 3H20 in the azide controls (Fig. 3B; solid symbols). Low but measurable yields were obtained for the controls at both protein surface concentrations, amounting to 23 and 29% of the live treatments. Incom- plete inhibition of bacterial activity in the controls is apparent.

Mean concentrations and growth rates of suspended bacteria were comparable in both treatments: N = 2.20 and 2.25 x lo6 cells ml-l and p = 0.036 and 0.040 h-l for thin and thick films, respectively. Therefore, normalizing rates by bacterial abundances does not alter trends significantly. Results from both the 3H,0 and TCA- soluble assays suggest that protein molecules at low surface concentrations are more readily hydrolyzed and respired by bacteria than those present at high surface con-

Adsorbed

- ,

Time ( h ) Fig. 3. Release of TCA-soluble label (A) and tritiated water

(B) into solution from [methyl-3H]Rubisco adsorbed to beads at low (8 ng cm-*, triangles) and high (1,053 ng cm-*, circles) surface concentrations. Solid symbols are 0.2% NaN, controls. Least-squares regression lines described by C,,, = 138 + 163 t, r* = 0.945, Chi = 4,491 + 11,480 t, r2 = 0.995 for TCA- soluble label and by C,,, = - 101 + 95 t, Y* = 0.975, Chi = 1,139 + 7,378 t, r* = 0.985 for 3H20. Dotted lines represent 95% C.I. of regressions.

centrations. The alternate explanation, that hydrolytic and uptake systems saturated (i.e. approached V,,,) during these experiments, is evaluated by kinetics experiments presented below.

Hydrolysis of dissolvedprotein -Three separate kinetics experiments were performed to examine the variability of protein degradation among bacterial communities. Production rates of TCA-soluble material appeared to behave as a first-order function of dissolved protein concentration and did not approach saturation levels over the concentration range examined in any of the three

880 Taylor

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Fig. 4. Dependence of hydrolysis (A) and remineralization (B) rates on protein concentration in seawater suspensions of picoplankton amended with dissolved [methyZ-3H]Rubisco, as measured by TCA-soluble radioactivity and by 3H20. O-Ex- periment 1, using picoplankton from continuous culture (see materials and methods); A-experiment 2, using freshly collected (9 October 1990) picoplankton from Kewalo Basin, O’ahu, Hawaii; 0 -experiment 3, using picoplankton collected on 23 October 1990 from the same source as experiment 2; O-adsorbed protein, calculated from end-point (lower) and time- course (upper) experiments. Results from least-squares regressions for experiment 1: V,,, = 0.361 C01.323, Y* = 0.952 and V H*O = 0.111 co1.305, r* = 0.967. Values of zero for respiration were arbitrarily assigned a value of 1 for presentation purposes only. Dotted lines represent 95% C.I.

kinetics experiments (Fig. 4A). Normalizing I$,, by the geometric means of bacterial abundance for each protein concentration did not appreciably change the trend apparent in Fig. 4A (not presented). Cell-specific hydrolysis rates varied from 0 to 9 5 fg Rubisco cell- l h- l, with only a slight suggestion of curvilinearity, i.e. rate saturation. However, the data were subjected to Lineweaver-Burk,

1 2 3 4 5

Bacteria (xl O6 ml-‘) Fig. 5. Production rates of TCA-soluble material in relation

to the geometric mean of bacterial abundances. Symbols same as Fig. 4.

Eadie-Hofstee, and Woolf plot transformations, and results never conformed to the Michaelis-Menten kinetic model. Consequently, V,,, and K, could not be derived. Rate constants, H,, for production of TCA-soluble material calculated per sample were relatively slow (0.002- 0.074 h-l) and varied most at the lowest Rubisco concentrations.

Remineralization of dissolved protein -Production of 3H20 was evident at all protein concentrations in the first experiment and only at select higher concentrations in subsequent experiments (Fig. 4B). In fact, 3H20 production was only detectable in 50% of the observations. How- ever, if only nonoutliers from the first experiment (n = 8) are included in a regression analysis, 3H,0 production rates appear to vary as a first-order function of Co, with no indication of rate saturation. Rate constants for production of 3H20 from dissolved [methyZ-3H]Rubisco were low, varying from 0.000 to 0.026 h-l and averaging 0.007 h-l for all observations.

Dissolved protein and bacterial populations -The geometric means of bacterial concentrations varied from 1.63 to 4.24 x lo6 cells ml-’ among all live treatments and from 0.25 to 2.86 x lo6 cells ml-l among all poisoned controls. Hydrolysis of dissolved protein among treatments, but not experiments, varied as nonlinear functions of bacterial abundance (Fig. 5). However, most of the variance in all observations of V,,, (n = 45) is explained by protein concentration (r2 = 0.823, P < 0.0 1). Inclusion of bacterial concentrations in a multiple linear regression does not significantly improve prediction of V,,, (r2 = 0.856, P < 0.01). Even though bacteria were exposed to essentially the same range of protein concentrations in all three experiments, significant release of TCA-soluble material was evident only when unique thresholds in bac-


terial populations were exceeded (Fig. 5). The maximum cell-specific hydrolysis rate for cultured bacteria was clearly greater than those of field populations (95 vs. 48 and 45 fg Rubisco cell-l h- ‘).

Bacteria were actively growing in all three experiments, but growth rates only increased with Rubisco concentrations in the first two (Fig. 6A,B). Ambient protein concentrations were not measured, so contributions of [meth- yZ-3H]Rubisco to total pools are unknown. However, bacteria in the first kinetics experiment were obtained from a low nutrient chemostat (as in bead experiments), so usable substrates are expected to be near depletion. Ad- dition of protein was stimulatory to these populations, which is consistent with higher cell-specific hydrolysis and remineralization rates observed in this experiment. Protein concentration-dependent growth rates suggest that bacterioplankton in the second experiment were nutrient limited and that additional protein provided them with usable growth substrate (Fig. 6B). In contrast, the con- stancy of p in the third experiment (Fig. 6C) suggests that these bacterioplankton were either nutrient-replete or un- able to efficiently utilize Rubisco as a growth substrate. Both explanations are possible because uniformly high growth rates support the former hypothesis and the low rate constants for protein-related fluxes (V,,, and VH20) support the latter.

In the azide controls, growth rates were positive but slower than in live treatments, and rates were negative or very low in the HgC12 controls (closed symbols; Fig. 6). These results demonstrate that even in the presence of inhibitors at previously recommended concentrations, actively metabolizing bacteria were present. Consequent- ly, differentiating between biological and strictly chemical hydrolytic processes is not possible with existing data, and rates derived from poisoned controls are not presented.

Adsorbed vs. dissolved protein -An adsorption effect was verified by comparing degradation rates derived from the end-point bead experiment with those derived from regressions of the first kinetic experiment (Fig. 4). This comparison is the most valid because both experiments shared a common bacterial source and culture history, were lo- 12-h end-point determinations of samples main- taining constant volumes throughout the incubations, and had comparably sized Rubisco pools. On the basis of this comparison, sorption to glass beads presented no clear impediment to degradation of Rubisco. In fact, the highest rate constants for hydrolysis and remineralization were observed for proteins adsorbed at low surface concentrations (Table 2 and Fig. 4). Proteins sorbed in thin films were hydrolyzed 247% and remineralized 282% faster than those in thick films, whereas low concentrations in solution were hydrolyzed at 2 1% and remineralized at 27% of the high concentration rates (Table 2). Proteins sorbed in thin films were hydrolyzed 522% and remineralized 1,033% faster than comparable dissolved pools (22 ng Rubisco ml-l) (Table 2). Rates of hydrolysis and remineralization of high surface and dissolved concentra-

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[methyL3H]Rubisco concentrations for isotherm 1 (A), isotherm 2 (B), and isotherm 3 (C). Open symbols same as for Fig. 4 and closed symbols are azide controls in panels A and B and HgCl, controls in panel C. Standard error bars presented when larger than symbols.

tions (2,260 ng Rubisco ml-l), however, were indistinguishable (Fig. 4 and Table 2).

The ratio of bacterial turnover to hydrolysis rates (T, : H,) has been exploited to examine the degree of coupling between hydrolysis and subsequent bacterial utilization of hydrolysate (Hoppe et al. 1988). Because bacterial assimilation cannot be adequately assessed with [methyl-

882 Taylor

Table 2. Rate constants for hydrolysis and remineralization (3H20 production) of [methyZ-3H]Rubisco and their ratios. Low and high [P] = 22 and 2,260 ng [methyl-3H]Rubisco ml-l.

Protein Hydrolysis* (h-l) 3H20 (h-l)

state Mean (SE) Mean (SE) Rr : K-t

Adsorbed* Low [P] 0.047 (0.004) 0.03 1 (0.001) 0.660 High [P] 0.019 (0.001) 0.011 (0.000) 0.579

Dissolved# Low [P] 0.009 (0.002) 0.003 (0.001) 0.333 High [P] 0.042 (0.005) 0.011 (0.002) 0.262

* Minimum hydrolysis rate, H,, equivalent to net LMW production + hydrolysate remineralized and excreted by bacteria. Estimates do not include radioactivity incorporated into bacteria nor adsorbed onto surfaces.

t Ratio of remineralization to hydrolysis rates (an index of coupling between protein degradation and bacterial utilization).

$ Mean and standard error calculated from triplicate analyses of end points.

0 Least-squares regressions in Fig. 4 were used to obtain mean values and standard errors of the estimates (n = 24) from experiment using populations subjected to same culture conditions as adsorbed protein study.

3H]Rubisco as a tracer, the conservative estimates of remineralization and hydrolysis reported here are used analogously to T, : H,. Coupling between the two processes for bacteria degrading adsorbed proteins seems to be greater than for those using proteins in solution, particularly at low surface concentrations (Table 2). Like- wise, the fraction of the total pool remineralized is greater for adsorbed proteins; R, : Co = l . l-3.1% h-l for adsorbed protein and 0.3-l. 1% h-l for dissolved protein.

Observed differences in the degree of coupling and rates of remineralization could be attributed to a number of factors relating to the physical state of the protein, unique features of the boundary layer, species composition as it affects enzymatic potential, and bacterial concentration. The first two factors are discussed below and the third is unexamined. The last factor, bacterial concentration, did vary between experiments but not significantly within an experiment. Normalized hydrolysis rates (fg Rubisco cell- l h-l) were essentially the same for high protein concentrations in dissolved and adsorbed state (Table 3). How- ever, cell-specific hydrolysis rates for thin protein films were 9 times faster than for the same pool size of dissolved protein (Table 3).

Discussion

Proteins in solution - Extracellular proteins have three common fates after release to seawater: adsorption to surfaces, complexation with other solutes, and degradation by bacterioplankton. In the kinetics experiments, most Rubisco remained in solution during the incubations, with only a negligible portion adsorbed to flasks or seston. Therefore, observed hydrolysis rates represent

Table 3. Estimates of cell-specific hydrolysis rates based on observed fluxes of TCA-soluble radioactivity (including 3H,0) and geometric means (+ SE, n = 10) of cell concentration. Rates for adsorbed and dissolved proteins calculated from end-point experiments and regression presented in Fig. 4, respectively. Low and high [P] = 22 and 2,260 ng [methyZ-3H]Rubisco ml- l.

Bacterial concn Hydrolysis rates

Protein state

(X lo6 ml-l)

Mean SE (ng ml-’ (fg cell-’

h-‘) h-l)

Adsorbed Low [P] High [P]

Dissolved Low [P] High [P]

2.20 (0.11) 1.0 0.45 2.25 (0.10) 42.3 18.80

3.80 (0.5 1) 0.2 0.05 4.24 (0.33) 94.4 22.26

bacterial interactions with molecules in solution. In the aqueous phase, polyelectrolytic molecules as large as Rub- isco can be considered colloidal particles, which may be of little chemical consequence per se, but being a large and structurally complex molecule may have biological significance (e.g. steric hindrance of bacterial proteolytic enzymes). Furthermore, complexation of dissolved proteins with other solutes by processes such as glucosylation may be very common and may render these molecules much more resistant to bacterial degradation (Keil and Kirchman 1993, 1994). The extent of complexation with other solutes cannot be evaluated in these experiments, but it may have influenced observed rates.

Hydrolysis rates per milliliter and per cell behaved as a first-order kinetics reaction varying in proportion to the amount of protein in solution, with little evidence of rate saturation. Explanation of total variance in hydrolysis rates was only slightly improved by a multilinear function including both bacterial abundance and Co. These observations suggest that excess enzymatic capacity existed in these microcosms and that individual populations do vary in their proteolytic abilities. Bacterial growth rates were stimulated by increasing Co in two experiments, indicating that enzymatic capacity within microcosms varied in response to available substrate.

In solution, H, for [rrtethyZ-3H]Rubisco varied from 0.002 to 0.074 h-l and averaged 0.025 h-l over the entire range, which equals a reaction half-time of - 1.2 d. These rate constants are conservative estimates of true hydrolysis because LMW materials incorporated by bacterioplankton or adsorbed are not included in the inventory owing to methodological limitations. The few previous marine studies examining hydrolysis of dissolved proteins report a range inclusive of the present observation: 0.007-0.07 1 h-l for Rubisco (Keil and Kirchman 1993), 0.005-0.02 1 h-l for bovine serum albumin (Hollibaugh and Azam 1983), and 0.015-0.038 h-’ for ambient proteins (calculated from Fontigny et al. 1987). Relevant to the present study, the upper limit of the concentration


range examined (0.002-13 mg Rubisco liter-l) exceeds that used in the bead experiment by a factor of almost six. In the larger context, this range is likely to encompass that found in most natural waters. In estuarine and coastal waters, the equivalent of 0.04-l .09 mg protein liter-l has been reported for dissolved protein concentrations (Fon- tigny et al. 1987; Azam et al. 1992; Keil and Kirchman 1993). Therefore, the relationships between concentration, hydrolysis rates, and bacterial populations can be applied more broadly to nature as well as to interpretation of the bead experiments.

Remineralization of [methyZ-3H]Rubisco in solution was generally slow and undetectable in half the samples. The average rate constant, R,, for all nonzero data was 0.007 h-l. It can be argued that low rates of remineralization of [ methyZ-3H]Rubisco are artifacts of discrimination against methylated amino acids by bacterial permeases (Keil and Kirchman 1992). Although discrimination undoubtedly occurs, bacterial permeases are known to transport oligopeptides as large as pentamers (Payne 1980). Relatively high values of R, were in fact observed in the bead and first kinetics experiments, indicating that 3H- methylated amino acids were clearly incorporated and metabolized by bacteria. Comparisons of R, among experiments reported above are valid because internally consistent incubation and analytical techniques were applied to samples similar in all respects except solute concentration and physical state of the labeled molecule- factors that have no bearing on uptake of 3H-methylated amino acids. Nonetheless, remineralization rates derived from [methyZ-3H]Rubisco must be viewed as conservative and relevant only to comparisons of experimental treatments within the present study.

Degradation of adsorbed protein -Differences in rate constants for protein adsorbed at low and high surface concentrations (Tables 1 and 2) could be argued to reflect kinetic saturation of enzymatic systems. For example, the protein pool represented by thick films may be large relative to maximal enzymatic rates and population growth potential. As a result, rate constants calculated for systems saturated by high surface concentrations will be less than for undersaturated systems. However, the exponential increase in V,,, observed over a wide range in dissolved protein and bacterial concentrations does not support such arguments. Alternatively, higher rate constants might be predicted for adsorbed protein than for the same pool size of dissolved protein because higher local concentrations of protein within the boundary layer of the beads might favor faster reaction kinetics. If this were the case, rate constants for protein adsorbed in thick films should exceed those for thin films, but this too was not observed. Consequently, unique features of the interface affecting bioavailability must be invoked to explain differences in rates of degradation and remineralization. I suggest that structural alterations of adsorbed proteins and conditions within the boundary layer rather than kinetic limitations of enzymatic systems and of cellular production account for observed trends.

Qualitative d$erences between films - Previous experimental results have demonstrated that exposure of adsorptive surfaces to a range of dissolved protein concentrations will yield films varying systematically in surface coverage and molecular organization, as presented in Fig. 1B and C (Arnebrant et al. 1985; Taylor et al. 1994a,b). In the present study, qualitative differences between thick and thin films were evident. Beads with thick films released radioactivity at twice the rate of beads with thin films, but thick films produced TCA-soluble material at less than half the rate of thin films. Release of radioactivity from glass beads is indicative of three processes: desorption, hydrolysis, and bacterial utilization. Desorp- tion is expected to be more significant for thick films because the outermost layer of molecules in native state (Fig. 1C) is more easily dissociated than the underlying layer. For example, Taylor et al. (19943) found that mul- tilayer films of Rubisco on titanium and copper surfaces Virr = 3.6 and 10 pg cm-2) desorbed into seawater at comparable rates (5.3 and 5.9 ng cm-2 h- ‘), but thinner films desorbed 1.8-4.0 times more slowly, indicative of stronger interactions between sorbent and sorbate. In the present study, the same trend was observed for poisoned controls, in which hydrolytic rates were reduced, suggesting that passive release from thick films plays a greater role in protein remobilization than it does from thin films.

Differences between binding energies (which control desorption rates) and the dominant physical state (which controls hydrolysis rates as discussed below) may explain the observed variance in bacterial degradation rates. In thick films, molecules desorbing from outer layers may escape the boundary layer, essentially in native state, to become part of a more dilute and less reactive dissolved pool. Consequently, rates of hydrolysis and remineralization derived from thick films can be indistinguishable from dissolved protein, as I observed. In thin films, however, unfolded molecules desorb slowly, and hydrolysis of proteins can account for most protein mobilization. In essence, these results suggest that materials released from thin, denatured protein films are qualitatively different - on average smaller and simpler and more bioavailable- than material released from thicker, multilayered films.

Surface-enhanced hydrolysis-It has become almost axiomatic to expect that degradation rates of surface- active monomers, particularly organic pollutants, will vary inversely with a molecule’s propensity for adsorption (e.g. polycyclic aromatic hydrocarbons, see Mihelcic and Lu- thy 1988). This relationship can be understood in terms of molecular activity in solution and the kinetics of transport from inert surfaces (e.g. sediments or detritus) through solution phase to cell-bound hydrolytic, oxidative, or transport systems. Many of the monomers examined, such as amino acids, are transported intracellularly with little or no modification (e.g. Dashman and Stotsky 1986). Because polymeric materials such as proteins need to be hydrolyzed prior to bacterial incorporation (Payne 1980; Hollibaugh and Azam 1983), being adsorbed to a surface does not necessarily present the same impediment to deg-

884 Taylor

radation as it does for monomers. Contrary to the oft- cited study of Dashman and Stotsky (1986) for amino acids and dipeptides bound to clays, I contend that the process of adsorption can facilitate degradation of proteins and other macromolecules in marine systems.

Proteins bound to surfaces represent enhanced local concentrations of labile substrate, potentially attractive to bacteria. Whether in native or denatured state, only a small proportion of sites on these large molecules can actually be bound to the surface. Therefore, peptidases can hydrolyze many peptide bonds away from surface binding sites to release polypeptides or individual amino acids. Although surface denaturation may arise from unfolding molecules binding to more surface sites than native state molecules, it also effectively relaxes steric hindrance, allowing hydrolytic enzymes greater access to tar- get bonds. As a result, bacteria may hydrolyze these unfolded molecules more efficiently. In fact, proteins in thin films, presumably dominated by slowly desorbing, denatured molecules, were turned over more rapidly than in any other state. Furthermore, cell-specific hydrolysis rates of thin films were 9 times higher than rates for comparable dissolved pools (Table 3). In contrast, the indistinguishable cell-specific hydrolysis rates of thick films and comparable dissolved pools suggest that sorption has no net effect on bacterial degradation of proteins at higher surface concentrations.

Surface-enhanced coupling of hydrolysis and remineralization -Despite the limitations imposed on respiration measurements, R, : H, ratios are useful for comparing internally consistent experiments. For adsorbed proteins, these ratios were a factor of -2 higher than those derived from dissolved protein at both concentrations (Table 2), indicating that coupling between hydrolysis and bacterial remineralization was tighter for bound than dissolved protein. These results suggest that although hydrolysis rates of adsorbed and dissolved protein can be comparable, adsorbed molecules are utilized more efficiently, presumably by bacteria on or near surfaces. In subsequent experiments, we observed that mere addition of organic- free siliceous sands to dilute marine broth (1 : 2 g g-l) can nearly double bacterial cell yield (Taylor and Gulnick unpubl. data). Higher efficiency and yield may be a consequence of hydrolysates from adsorbed proteins diffus- ing into a boundary layer comprised of concentrated solutes and enriched in bacteria equipped with efficient ec- toproteases and possessing rapid transport systems (Billen 199 1; Azam and Smith 199 1; Smith et al. 1992).

Within diffusive boundary layers, slower diffusion rates and the particle reactivity of large molecules as well as their breakdown products are expected to influence the kinetics of transport and degradation (B. Brownawell pers. comm.). For example, proteins and polypeptides at interfaces are slower to diffuse out of the boundary layer than are smaller solutes, thereby permitting bacteria more opportunity to degrade the larger molecules. A further consequence of adsorption is that diffusion of hydrolysate away from protein molecules is bounded by the solid surface and thereby directionally restricted to approxi-

mately a hemisphere. In contrast, diffusion of hydrolysate from dissolved protein is potentially isotropic (i.e. dif- fusing out in all directions). Consequently, the probability of bacterial permeases encountering released peptides is enhanced by a factor of -2 at interfaces, similar to the enhanced R, : H, observed for bound protein. Taken to- gether, trends in H,, R,, and R, : H, demonstrate that degradation rates of Rubisco are influenced as much by location and physical state of the molecules as by protein or bacterial concentrations. Results are consistent with the hypothesis that adsorption at low surface concentrations facilitates hydrolysis and bacterial turnover of glob- ular proteins.

Perspective-In nature, adsorption and its effects are undoubtedly more heterogeneous and complex than pres- ently observed for glass beads. Behavior of adsorbed proteins is likely to vary between proteins and substratum types and vary locally on the surfaces of individual particles (Samuelsson and Kirchman 1990; Taylor et al. 1994a,b; Mayer 1994). For example, adsorption of proteins and peptides into smaller mesopores of mineral phases may actually exclude bacteria and exoenzymes and thereby promote preservation in sediments (discussed by Mayer 1994). Sequestering of proteins into smaller mesopores may also impede degradation by preventing significant molecular unfolding, thereby protecting bonds susceptible to hydrolysis. Furthermore, residual amino acids and small peptides irreversibly bound to the surface after the bulk of the polymer is degraded may become more or less refractory in aging material, as reported by Dashman and Stotsky (1986), and lead to the sorptive protection or preservation of OM monolayers discussed by Mayer (1994) and Keil et al. (1994).

More than 50 yr ago with benefit of few data, ZoBell (1943) perceptively envisioned mineral-water interfaces as foci for bacterial activity. He proposed that interfaces promote enhanced rates of production because higher local concentrations of sorbed substrate, bacteria, and enzymes favor faster reaction kinetics. My model for fresh polymeric OM builds on ZoBell’s concept by incorpo- rating another physical-chemical term, sorption-medi- ated denaturation. Macromolecules unfold upon sorption, exposing bonds susceptible to enzymatic or chemical hydrolysis, and thereby facilitate the functioning of endo- and exohydrolases. In contrast to molecules in solution, diffusion of hydrolysate away from adsorbed molecules is anisotropic, bounded by the interface. Therefore, the probability of further encounter with bacterial hydrolytic and transport systems within the diffusive boundary layer surrounding solid phases is enhanced. As a consequence, proteolytic bacteria associated with surfaces potentially derive higher yields of hydrolysate per unit of enzyme and are capable of a higher degree of coupling between hydrolysis and remineralization. In essence, mineral surfaces and their boundary layer can serve a catalytic function. They concentrate reactants (colloids, polymers, bacterial hydrolases, and hydrolysates) in close proximity to one another. They induce conformational changes that relax steric limitations, thereby lowering the activation


energy of the reaction and increasing reaction rates and efficiencies. Whether all proteins and those associated with nonsiliceous mineral phases as well as biogenic de- bris behave similarly warrants closer examination.

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Submitted: 18 November 1993 Accepted: 11 July 1994

Amended: 11 April 1995

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Microbial degradation of sorbed and dissolved protein in seawater