Anoxic and sulfidic sediments formed during intensive milkfish farming in Bolinao Bay (Lingayen Gulf, Luzon, Philippines) showed concentrations of total proteinaceous matter roughly three times higher (up to16.9 mg cm−3) than those of less reduced sediments. Ectoprotease activities in these coastal sediments suggested an inhibition of enzymatic recycling of proteins by sulfide. Sodium sulfide proved capable of inhibiting protease activity produced by a bacterial isolate from a sulfidic sediment site (Ki = 20 mM Na2S · 9H2O). Reoxidation and removal of H2S from sulfidic sediments, however, proved inadequate to recover proteolytic activity. Hence, H2S formed during organic matter mineralization via bacterial sulfate respiration may have initiated an irreversible inhibition of the enzymatic degradation of proteinaceous matter from fish farming. Ectoprotease activity was positively correlated (r = .93) with redox potential values. Lowest proteolytic activities were found in strongly reduced sediments, that were predominantly fine grained (very fine sand and silt) and sulfidic at the same time. Low ectoprotease activity in these sulfide-rich sediments can be interpreted as a kind of negative feedback mechanism that would mitigate the immediate recycling of excessive amounts of feed borne protein-rich deposits. Proteins may be further protected from degradation by selective particle adsorption in the finest grained deposits of fish farming waste.

Introduction

In coastal fish-farming areas of the Philippines (Lingayen Gulf) proteinaceous compounds accumulate particularly in the anoxic and sulfidic silty marine sediments (Reichardt et al., 2007). A mass fish kill in 2002 caused a particularly high deposition of organic matter in the Bolinao Bay part of the gulf. Protein-rich fish feed and waste particles deposited on the sediment can be expected to undergo depolymerization in the presence of extracellular proteolytic enzymes such as ectoproteases. Extracellular enzymatic processes are considered as rate limiting steps in processes of organic particle remineralization (Hoppe, 1991; Patel et al., 2001).

Sulfide-rich sediments of Bolinao Bay indicate a predominance of anaerobic organic matter recycling via sulfate-respiration in the fish farming zone (Holmer et al., 2003; Reichardt et al., 2007). In situ inhibition of extracellular enzymes by H2S has so far been reported for the water column, but not for marine sediments (Hoppe et al., 1998). In the latter, selective adsorption processes are known to interfere with protein degradation (Ding and Henrichs, 2002). Inhibition of the primary enzymatic steps of protein degradation in the fine grained, sulfidic surface deposits typically accumulating at fish farming sites of Bolinao Bay (Reichardt et al., 2007) would dampen eutrophying effects resulting from the deposited fish farming waste and favor their burial.

Protease assays based on the solubilization of scleroprotein particles seemed an adequate target to compare the proteolytic degradation potential in sediment samples with important environmental variables. Analyses of sediment samples covered a period of about two years after the extraordinary input of organic matter by a mass fish kill in 2002. As both sulfidic conditions and protective adsorption could play a role as inhibitors of protein degradation, redox potential and sediment texture were determined in addition to concentrations of proteinaceous compounds in situ. Further environmental variables such as viable counts of proteolytic bacteria were also considered. Pairwise measures of protein concentrations and ectoprotease activities were to mirror in situ substrate pools with their pertinent extracellular enzymes. Samples from fish cages and mariculture-free areas were chosen to quantify and compare the concentration of proteinaceous compounds with environmental variables of potential significance. Short term laboratory assays with manipulated sediment samples were to elucidate potential mechanistic aspects arising.

Materials and Methods

Sampling sites

Coastal marine sediments from subtidal (at water depths between 5 and 20 m) and intertidal zones were investigated in the NW part of Lingayen Gulf (South China Sea) which is here referred to as “Bolinao Bay.” A channel formed by the Bolinao peninsula to the West and by several islands to the East has become a major resource base for intensive finfish and shellfish farming since the mid 1990s. A lead-enforced (6 cm diameter × 25 cm) plexiglass gravity sampler with a plastic membrane as a valve, served as core sampler for a total of 21 subtidal and intertidal surface sediments from September to December 2003. A subsequent collection of core samples from 7 intertidal and 2 subtidal sediments in November 2004 was also used for texture analysis. Vertical sediment profiles were obtained by divers using plexiglass cores of more than 1 m length at four subtidal sites (1 = 16° 22.60’N, 119°54.35’E, 20 m; 2 = 16° 23.06’N, 119°54.88’ E, 20 m; 3 = 16° 20.5’N, 120°11.5’E, 6 m; 4 = 16° 23.96’N, 119°54.95’E, 14 m) from September to November 2004.

Sediment microcosm experiments

Batches of 35 cm3 of fine sandy intertidal sediment were mixed with equal volumes of 3.4% NaCl and transferred into 80 ml glass jars. Five replicates each were (a) continuously aerated, (b) kept air-tight or (c) sealed after supplementing Na2S .9 H2O at a final concentration of 3 mM. After 24 h of incubation in the dark, redox potential and ectoprotease activity were measured for each microcosm.

In re-oxidation trials equal pairs of anoxic and sulfidic sediment samples from four different sites at 14–15 m water depth were transferred into 100 ml glass jars. One half of each pair was aerated by intensive bubbling for five h and the other half kept as control to measure the effects of H2S removal by aeration on ectoprotease activities.

Analyses

Grain sizes were determined by sieving and weighing of dried sediment samples using a set of sieves according to the Wentworth scale (Buchanan, 1984). A Pt redox electrode (WTW) with an Ag/AgCl electrode at 194 mV serving as reference was used for (uncorrected) measurements of redox potentials either of the sediment surface (0–2 cm) or of 2 cm thick sediment slices obtained from long cores.

Concentrations of total proteinaceous compounds (“Lowry-protein”) in 0.5 cm3 aliquots of sediment samples from 0–2 cm surface layers or 2 cm slices obtained from long cores were determined using a modification of the Folin/Cu method Lowry et al. 1951 (Herbert et al., 1971; Reichardt, 1988). Aliquots of 0.5 cm3 sediment were extracted with 0.5 ml of 1 N NaOH in a boiling water bath for exactly 5 min. After immediate cooling in tap water, 2.5 ml of freshly prepared Cu reagent (mixture of 50 ml 5% Na2CO3 and 2 ml of 0.5% CuSO4 × 5 H2O in 1% Na/K tartrate) were allowed to react with the sample in the dark for 10 min, followed by the addition of 0.5 ml of 1 N Folin-Ciocalteu's phenol reagent (Merck, Darmstadt, Germany) and centrifugation, after the blue color complex had formed within 30 min. Absorbance of the supernatant was measured at 600 nm using a Milton Roy Spectronic 20 photometer. Calibration was done with lysozyme (Serva Biochemicals) which also served as an internal protein standard.

Extracellular protease (ectoprotease) activities were determined by incubating triton X 100 treated sediment extracts with 20 mg amounts of finely ground hide powder azure (Sigma-Aldrich, St. Louis, MO, USA) as substrate (Reichardt, 1988). Two ml of 20 mM tris-HCl buffer, pH 7.6, and 1 ml of 4% triton X-100 were added to 0.5 cm3 aliquots of sediment sample and incubated from 6 to 20 h at 30°C. After terminating the assay by adding 0.5 ml of formaldehyde followed by centrifugation, absorbance of the solubilized dye in the supernatant was measured at 595 nm using a Milton Roy Spectronic 20 photometer.

Viable counts of proteolytic (gelatinase-positive) bacteria were obtained as plate counts after spreading 0.1 ml aliquots of serial sediment dilutions on nutrient gelatine agar (Pitt and Dey, 1970) and incubating at 18°C, until colonies had formed pits in the semisolid medium. Plates were incubated both aerobically and anaerobically (using BBL Gas Pack anaerobic jars). The highest scores of CFUs obtained under either aerobic or anaerobic conditions were considered. Isolated colonies showing rapid growth in a liquid sea water based culture medium containing 1% gelatine were used as sources of extracellular protease (ectoprotease). After centrifugation, clear supernatants were used for inhibition assays with Na2S according to Dixon (1953).

Relationships between different sediment parameters were tested by Spearman's non-parametric rank correlation. Absence or presence of H2S in sediment samples vs. ectoprotease activity was based on Point biserial correlational analysis (Downie and Heath, 1983).

Results

Roughly two years after a devastating fish kill in Bolinao Bay (February 2002), concentrations of proteinaceous compounds in the 0–2 cm surface layer of 21 samples ranged from 0.3 to 16.9 mg cm−3. Protein concentrations below 5 mg cm−3 were confined to a relatively narrow range of positive redox potential values, whereas higher concentrations were found in sediments with a strongly negative redox potential (Figure 1). Approximately three years after the fish kill, peak protein values persisted in the areas designated for milkfish farming in net cages. Long sediment cores (>1 m) from different parts of the bay showed that the sites most affected by fish farming were characterized by several dm of thick surface layers of black sulfidic mud. Redox potential readings in the top 40 cm horizon of sediment cores from the margins of fish cages ranged between −200 and −400 mV (Figure 2a). Proteinaceous compounds had most intensively been accumulated in the strongly reduced sediment of a fish cage site (Figure 2b, site 4). Parallel assays for ectoprotease activities showed a steep (exponential) decline with sediment depth (Figure 2c). Mirror images obtained for profiles of protein and ectoprotease at the fish cage site 4 suggested an inverse relationship between protein concentrations and ectoprotease activity for this site (Figures 2b, c).

Figure 1.

Concentration of proteinaceous compounds vs. redox potential in 0–2 cm surface sediment layers at 14 sites of Bolinao Bay (intertidal to subtidal with maximum depth at 19 m).

Figure 1.

Concentration of proteinaceous compounds vs. redox potential in 0–2 cm surface sediment layers at 14 sites of Bolinao Bay (intertidal to subtidal with maximum depth at 19 m).

Figure 2.

(A) Depth profiles of redox potential values of long sediment cores from subtidal Bolinao Bay sites characterized by most extensive sulfidic mud surface layers at (site 2) and near fish cages (sites 1,4), and bioturbated sediment without impact of fish farming (site 3); (B) depth profiles of protein concentrations (mg cm−3); (C) depth profiles of ectoprotease (relative units).

Figure 2.

(A) Depth profiles of redox potential values of long sediment cores from subtidal Bolinao Bay sites characterized by most extensive sulfidic mud surface layers at (site 2) and near fish cages (sites 1,4), and bioturbated sediment without impact of fish farming (site 3); (B) depth profiles of protein concentrations (mg cm−3); (C) depth profiles of ectoprotease (relative units).

Possible linkages of protein turnover with both sediment texture and redox potential were investigated with a further set of surface sediment (0–2 cm horizon) samples collected from 7 intertidal and 2 subtidal sites (Table 1). Ectoprotease activities showed a strong positive correlation with the redox potential (0.93, Spearman's non-parametric rank correlation). Larger grain size fractions (medium sand) were positively correlated with ectoprotease activity (0.82), whereas the fine grained fractions of silt to very fine sand that are most predominant in the fish farming zone showed a negative correlation (0.66) with ectoprotease activities. In contrast to these extracellular enzyme activities, correlations between viable counts of ectoprotease producing (proteolytic) bacteria and sediment texture lacked a clear trend.

Table 1.

Predominant texture, redox potential, protein content, scleroprotease activity, and proteolytic bacterial CFU's in predominantly intertidal (*) sediment samples (** = at fish cage; *** = off fish farming zone) obtained from 4–17 November 2004.

Sample depth (m) coordinatesPredominant grain size,%Redox potential (mV)Protein content (mg·cm−3)Ectoprotease activity (μg cm−3 h−1)Proteolytic bacteria (CFUs·cm−3 10−3)
0.4 * N 16°20.34’ E119°48.28’ Coarse sand 32.8% +90 0.45 (±0.08) 989 (±120) 120 (±355) 
0.6 * N 16°24.99’ E119°54.54’ Coarse sand 30.4% +222 0.62 (±0.09) 949 (±91) 267 (±62) 
11 ** N 16°22.82’ E119°55.74’ Fine sand 45.8% −201 1.07 (±0.19) 69 (±41) 38 (±10) 
0.4 * N 16°23.03’ E119°53.38’ Fine sand 63.4% +64 1.27 (±0.49) 719 (±87) 15 (±12) 
0.4 * N 16°20.34’ E119°48.28’ Very fine sand 38.3% +7 0.42 (±0.10) 295 (±51) 75 (±23) 
0.4 * N 16°20.34’ E119°48.28’ Very fine sand 58.7% +79 0.34 (±0.05) 481 (±58) 117 (±39) 
0.4 * N 16°20.34’ E119°48.28’ Very fine sand 60.5% −158 1.33 (±0.21) 249 (±51) 35 (±19) 
12 *** N 16°23.66’ E119°54.51’ Very fine sand 66.79% −124 0.87 (±0.25) 143 (±41) 93 (±10) 
0.1 * N 16°20.34’ E119°48.28’ Very fine sand 70.4% −210 1.11 (±0.14) 134 (±28) 120 (±27) 
Sample depth (m) coordinatesPredominant grain size,%Redox potential (mV)Protein content (mg·cm−3)Ectoprotease activity (μg cm−3 h−1)Proteolytic bacteria (CFUs·cm−3 10−3)
0.4 * N 16°20.34’ E119°48.28’ Coarse sand 32.8% +90 0.45 (±0.08) 989 (±120) 120 (±355) 
0.6 * N 16°24.99’ E119°54.54’ Coarse sand 30.4% +222 0.62 (±0.09) 949 (±91) 267 (±62) 
11 ** N 16°22.82’ E119°55.74’ Fine sand 45.8% −201 1.07 (±0.19) 69 (±41) 38 (±10) 
0.4 * N 16°23.03’ E119°53.38’ Fine sand 63.4% +64 1.27 (±0.49) 719 (±87) 15 (±12) 
0.4 * N 16°20.34’ E119°48.28’ Very fine sand 38.3% +7 0.42 (±0.10) 295 (±51) 75 (±23) 
0.4 * N 16°20.34’ E119°48.28’ Very fine sand 58.7% +79 0.34 (±0.05) 481 (±58) 117 (±39) 
0.4 * N 16°20.34’ E119°48.28’ Very fine sand 60.5% −158 1.33 (±0.21) 249 (±51) 35 (±19) 
12 *** N 16°23.66’ E119°54.51’ Very fine sand 66.79% −124 0.87 (±0.25) 143 (±41) 93 (±10) 
0.1 * N 16°20.34’ E119°48.28’ Very fine sand 70.4% −210 1.11 (±0.14) 134 (±28) 120 (±27) 

In a set of 21 samples, from predominantly subtidal sediments collected in 2003, existed two groups: either releasing or lacking H2S. Ectoprotease activities in the group releasing H2S (10 samples) were confined to a lower and threefold narrower range (36–215 μg h−1 cm−3) than activities in the group (of 11 samples) without noticeable release of H2S that ranged from 125 to 1957 μg h−1 cm−3. Correlation analysis of H2S presence and ectoprotease activity showed a moderately negative correlation (rpb = −0.45, Point Biserial).

Furthermore, in vitro assays of extracellular protease produced in batch cultures of a bacterial isolate from the investigated sediment indicated a direct inhibition by Na2S · 9 H2O. This was characterized by an inhibition constant of ki = 20 mM using a Dixon plot (Dixon, 1953). Significant direct effects of H2S were, however, not observed in sediments that had been artificially adjusted to oxic (+81 mV), suboxic (−89 mV), and anoxic-sulfidic (−292 mV) conditions and incubated for 24 h. Differences among the resulting ectoprotease activities proved significant only between the oxic vs. the two other treatments, but not between suboxic and anoxic-sulfidic conditions (P < 0.05, ANOVA-TUKEY). Aeration to remove H2S from anoxic-sulfidic sediment triggered no increase of the endogenous ectoprotease activity in the sediment (Table 2).

Table 2.

Impact of aerating individual anoxic-sulfidic sediment batches on ectoprotease activity {μg h−1 cm−3).

Batch of SedimentRedox potential of non-aerated control (mV)Ectoprotease activity in non-aerated controlRedox potential of aerated batch (mV)Ectoprotease activity in aerated batch
−357 197 − 113  81 
− 344 107 − 141  90 
− 170 143  − 82 152 
− 355 260  − 28 170 
Batch of SedimentRedox potential of non-aerated control (mV)Ectoprotease activity in non-aerated controlRedox potential of aerated batch (mV)Ectoprotease activity in aerated batch
−357 197 − 113  81 
− 344 107 − 141  90 
− 170 143  − 82 152 
− 355 260  − 28 170 

Discussion

N-organic matter pools in marine sediments are governed by diverse and incompletely understood processes (Hedges and Keil, 1995). Despite increasing inputs of proteinaceous nitrogen in fish feed and fish farming waste (typically ranging between 20–35%) by intensified mariculture in tropical Asia, mechanisms underlying the enzymatic turnover of proteins in marine sediments still remain to be investigated (Kader et al., 2005). In oxic marine environments proteinaceous biopolymers seem to be rapidly depolymerized and remineralized, even in sediments near fish farming sites (Pantoja and Lee, 1999; Vezzulli et al., 2002). On the other hand, accumulation of proteinaceous compounds increases with biogeochemical and early diagenetic changes (Keil et al., 1994; Mayer et al., 1989; Dauwe et al., 1999).

Protein assays based on the Folin-Ciocalteu reagent according to Lowry et al. (1951) have been widely applied to a variety of samples (Herbert et al., 1971). In soils and sediments, phenolic hydroxyl groups as the main targets of this assay gain particular importance during the transformational processes that ultimately lead to humification. Thus proteins at various stages of their transformation into humic substances are also measured as proteinaceous constituents of partly humified organic matter depending on the efficiency of the preceding hydrolytic step. Consequently the term “proteinaceous organic matter” for the analysis of protein concentrations used in this investigation more aptly describes the real target of the assay.

The contribution of fish farm waste to the sinking flux of particles approaching peak values of 1 kg (dry weight) m−2 d−1, net sedimentation rates of organic particles in Bolinao Bay were several times higher at fish cages than at nearby subtidal sites not used for fish farming. Up to 3–4 cm thick layers of watery sulfide-rich sediment may accumulate annually and are usually covered with sulfide-oxidizing Beggiatoa mats (Holmer et al., 2003; Reichardt et al., 2007). Redox potential profiles of subtidal sediment cores from sites exposed to fish farming were roughly 200 mV lower than at mariculture-free sites (Figure 2a). Mineralization of fish farming residues under these anoxic conditions is governed by sulfate respiration turning sediments sulfidic (Holmer and Kristensen, 1995; Holmer et al., 2003).

In general, anoxic and sulfidic conditions tend to reduce the functional microbial diversity in marine sediments (Freitag et al., 2003). Moreover, benthic macrofauna that is often crucial in terms of conditioning organic particles for enzymatic attacks (Gripsholt et al., 2003) is widely lacking in the H2S-rich sediments of Bolinao Bay. Most importantly, however, ectoenzymatic activities such as ectoproprotease can be inhibited by H2S (Hoppe et al., 1988). From this point of view, limitation of remineralization rates for proteinaceous particles in marine environments by the initial ecto-enzymatic steps (Patel et al., 2001; Guldberg et al., 2002) would explain the observed relative increase of protein content in anoxic and sulfidic sediments (Figure 1).

Coastal marine sediments harbor common types of sediment proteases sharing similar functional properties and responses to pollution (Nakamura, 2006). Cores from Bolinao Bay suggest an exponential decrease of proteolytic recycling capacities with sediment depth (Figures 2b, c), as described for deposition-dependent microbial activity profiles in stratified sediments (Reichardt, 1987). Negative correlations between ectoprotease activities from sediments with different texture and oxidation status (Table 1) with both redox potential and grain sizes, suggest a negative feedback response of proteolytic activities to increasing waste particle deposition and anoxia.

Mechanistic explanations for negative feedback effects of small particles on enzymatic protein decomposition are provided by selective protein adsorption to minerals or by protective coating by submicron particles (Pantoja and Lee, 1999; Ding and Henrichs, 2002; Borch and Kirchman, 1999). Whereas little is known about the supply and regulation of proteolytic enzyme activities in marine sediments (Mayer, 1989; Tholosan et al., 1999), inhibition of ectoprotease by hydrogen sulfide would explain why the lowest ectoprotease activities were measured in the most reduced and at the same time strongly sulfidic sediments of Bolinao Bay (Table 1). Inhibition experiments with the culture fluid of a proteolytic bacterial isolate from the sediments studied confirmed the assumed inhibition by H2S.

Simple re-oxidation experiments using aerated sediment microcosms designed to examine the reversibility of this inhibition failed to recover most of the ectoprotease activity (Table 2). Although final sulfide concentrations after raising the redox potential remained undetermined, the lack of any positive response at redox potential increases of 88–327 mV favors the assumption of an irreversible ectoprotease inhibition under the sulfidic conditions in the sediments studied. Overall, proteolysis in anoxic and sulfidic sediments can only continue, when active proteolytic enzymes accompany their deposited substrates. Inclusion of viable counts of proteolytic bacteria in correlation analyses did not provide any further mechanistic clues.

Conclusions

Pollution of tropical marine sediments with fish farming waste can produce thick layers of sulfidic sediment, since excess hydrogen sulfide accumulates as a byproduct of organic matter remineralization via sulfate respiration. Watery fine-grained sulfide-rich top sediment layers that are characteristic of sediments affected by intensive milkfish farming reveal drastically lower enzymatic decomposition capacities for proteins. Formation of these sulfidic sediments apparently confers a negative feedback inhibition on rate limiting steps in the enzymatic breakdown of organic matter, viz., at least those steps that are catalyzed by ectoprotease. Further linkage of low ectoprotease activities with fine grained sediment texture may suggest an inhibitory effect of selective particle adsorption, too. Deposition and burial of protein-rich fish farming waste in sulfidic sediments can not only retard biogeochemically important steps of organic matter recycling in “external” sediment environments. Wide spread build up of oxygen consuming layers of fish farming deposits turning sulfidic can also be harmful to fish farming itself by contributing to reduced dissolved O2 levels in non-aerated fish cages (Reichardt et al., 2007; San-Diego-McGlone et al., 2008).

Acknowledgements

This investigation was supported by an equipment grant from German Academic Exchange Service (DAAD) to Wolfgang Reichardt.

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