Speed bumps and barricades in the carbon cycle: substrate structural effects on carbon cycling

The observation that a fraction of organic matter produced in marine systems evades the concerted efforts of microbial communities and is buried in sediments suggests that there are ‘speed bumps’ in carbon degradation pathways that impede microbially driven remineralization processes. The initial step in degradation of macromolecules, extracellular enzymatic hydrolysis, is often stated to be ‘the’ rate-limiting step in carbon remineralization. Experimental investigations described here, however, demonstrate that at least in certain cases, microbes produce extracellular enzymes on time scales of hours to tens of hours in response to substrate addition, and hydrolysis is extremely rapid. If enzymatic hydrolysis can be rapid, what factors slow or stop organic matter degradation? A lack of the correct inducer to initiate enzyme production, and/or a lack of the correct organism to produce the required enzyme, may result in a complete lack of hydrolysis in certain environments—a barricade, rather than a speed bump. Preliminary evidence supporting this hypothesis includes a comparison of polysaccharide hydrolysis in seawater and sediments, which demonstrates that the spectrum of enzymes active in seawater and sediments are fundamentally different. Furthermore, a survey of enzyme activities in surface waters from a range of locations suggests that pelagic microbial communities also differ widely in their abilities to express specific extracellular enzymes. Trans-membrane transport through porins is yet another potential location of structure-related selectivity.Our efforts to identify speed bumps and barricades are hampered by our inability to structurally characterize in sufficient detail the macromolecular structures present in marine systems. Furthermore, assessments of organic matter ‘quality’ from a chemical perspective do not necessarily accurately reflect the availability of organic carbon to microbial communities. For these communities, in fact, ‘quality’ may be a variable, which depends on the enzymatic and uptake capabilities of community members. To begin to assess substrate structure and quality from a microbial perspective, we will have to combine specific knowledge of macromolecular structures with detailed investigations of the enzymatic and transport capabilities of heterotrophic marine microbes.     

Anoxic carbon degradation in Arctic sediments: Microbial transformations of complex substrates

Complex substrates are degraded in anoxic sediments by the concerted activities of diverse microbial communities. To explore the effects of substrate complexity on carbon transformations in permanently cold anoxic sediments, four substrates—Spirulina cells, Isochrysis cells, and soluble high molecular weight carbohydrate-rich extracts of these cells (Spir-Ex and Iso-Ex)—were added to sediments collected from Svalbard. The sediments were homogenized, incubated anaerobically in gas-tight bags at 0°C, and enzyme activities, fermentation, and terminal respiration were monitored over a 1134 h time course. All substrate additions yielded a fraction (8%-13%) of carbon that was metabolized to CO₂ over the first 384 h of incubation. The timecourse of VFA (volatile fatty acid) production and consumption, as well as the suite of VFAs produced, was similar for all substrates. After this phase, pathways of carbon degradation diverged, with an additional 43%, 32%, 33%, and 8% of Isochrysis, Iso-Ex, Spirulina, and Spir-Ex carbon respired to CO₂ over the next 750 h of incubation. Somewhat surprisingly, the soluble, carbohydrate-rich extracts did not prove to be more labile substrates than the whole cells from which they were derived. Although Spirulina and Iso-Ex differed in physical and chemical characteristics (solid/soluble, C/N ratio, lipid and carbohydrate content), nearly identical quantities of carbon were respired to CO₂. In contrast, only 15% of Spir-Ex carbon was respired, despite the initial burst of activity that it fueled, its soluble nature, and its relatively high (50%) carbohydrate content. The microbial community in these cold anoxic sediments clearly has the capacity to react rapidly to carbon input; extent and timecourse of remineralization of added carbon is similar to observations made at much higher temperatures in temperate sediments. The extent of carbon remineralization from these specific substrates, however, would not likely have been predicted on the basis of general substrate characteristics.     

Long lifetimes of β-glucosidase,leucine aminopeptidase,and phosphatase in Arctic seawater

The active lifetime of extracellular enzymes is a critical determinant of the effectiveness of enzyme production as a means for heterotrophic marine microbes to obtain organic substrates. Here, we report lifetimes of three classes of extracellular enzyme in Arctic seawater. We also investigated the relative importance of photochemical processes and particle-associated processes in inactivating extracellular enzymes. Enzyme inactivation in filtered seawater was slow, with apparent half-lives of enzyme activities on the order of hundreds of hours. The presence of particles (including cells) did not significantly change inactivation rates, suggesting that the long half-lives observed in filtered seawater were realistic for enzymes in unfiltered seawater. Phosphatase and leucine aminopeptidase were susceptible to photoinactivation, but only under high intensity UV-B and UV-C illumination; there was no evidence for increased inactivation rates under natural illumination at our study site in Ny Ålesund, Svalbard. Comparison of inactivation rates of commercially-obtained enzymes from non-marine sources with the extracellular enzymes naturally present in Arctic seawater suggests that the natural enzymes contain structural features that confer longer lifetimes, consistent with observations reported by others from a range of field sites that cell-free enzymes can contribute a substantial fraction of total hydrolytic activity in the water column.     

Temperature induced decoupling of enzymatic hydrolysis and carbon remineralization in long-term incubations of Arctic and temperate sediments

Extracellular enzymatic hydrolysis of high-molecular weight organic matter is the initial step in sedimentary organic carbon degradation and is often regarded as the rate-limiting step. Temperature effects on enzyme activities may therefore exert an indirect control on carbon mineralization. We explored the temperature sensitivity of enzymatic hydrolysis and its connection to subsequent steps in anoxic organic carbon degradation in long-term incubations of sediments from the Arctic and the North Sea. These sediments were incubated under anaerobic conditions for 24 months at temperatures of 0, 10, and 20 °C. The short-term temperature response of the active microbial community was tested in temperature gradient block incubations. The temperature optimum of extracellular enzymatic hydrolysis, as measured with a polysaccharide (chondroitin sulfate), differed between Arctic and temperate habitats by about 8-13 °C in fresh sediments and in sediments incubated for 24 months. In both Arctic and temperate sediments, the temperature response of chondroitin sulfate hydrolysis was initially similar to that of sulfate reduction. After 24 months, however, hydrolysis outpaced sulfate reduction rates, as demonstrated by increased concentrations of dissolved organic carbon (DOC) and total dissolved carbohydrates. This effect was stronger at higher incubation temperatures, particularly in the Arctic sediments. In all experiments, concentrations of volatile fatty acids (VFA) were low, indicating tight coupling between VFA production and consumption. Together, these data indicate that long-term incubation at elevated temperatures led to increased decoupling of hydrolytic DOC production relative to fermentation. Temperature increases in marine sedimentary environments may thus significantly affect the downstream carbon mineralization and lead to the increased formation of refractory DOC.     

Pyrolysis-GC characterization of whole rock and kerogen-concentrate samples of immature Jurassic source rocks from NW-Germany

Nine rock samples from three Jurassic stratigraphic units of a shallow core from NW Germany were analyzed by pyrolysis-gas chromatography. The units contain a mixed Type-II/III kerogen (Dogger-α), a hydrogen-rich Type-II kerogen (Lias-), and a hydrogen-poor Type-III kerogen (Lias-δ). All of the kerogen was immature (R_o = 0.5%). Two sets of kerogen concentrates (“AD”: HCl/HF followed by a density separation, and “A”: only acid treatment) prepared from the rock samples were also analyzed to make a detailed comparison of the pyrolysates of rock and corresponding kerogen-concentrates.Hydrogen-index (HI) values of the kerogen concentrates prepared from organic-carbon poor rock were nearly 200% higher than HI values of the rock samples. Changes in HI were minimal for the samples containing Type-II kerogen. The A and AD samples from the C_org-poor rock yielded pyrolysates with n-alkane series of very different molecular lengths. Pyrograms of the rock samples had n-alkane series extending to n-C₁₄; the chromatograms of the A samples reached the n-C₁₄-nC₂₀ range. The AD samples from C_org-poor rock and all three sample types from the C_org-rich rock had n-alkane series up to n-C₂₉. The benzene/hexane and toluene/heptane ratios for the C_org-poor rock and A samples were far higher than for the AD samples, which had ratios similar to those of all three sample types from the C_org-rich rocks. These results indicate that choice of kerogen preparation method is critical when C_org-poor samples are analyzed.     

Surface associations of enzymes and of organic matter: Consequences for hydrolytic activity and organic matter remineralization in marine systems

The extent to which marine organic matter is associated with surfaces and the consequences of such associations for organic matter remineralization are the focus of considerable attention. Since extracellular enzymes operating outside microbial cells are required to hydrolyze organic macromolecules to sizes sufficiently small for substrate uptake, the effects of surface interactions–on enzymes as well as on substrates–for hydrolytic activity also require investigation. We used a simplified laboratory system consisting of a free (dissolved) polysaccharide (pullulan) and the same polysaccharide tethered to agarose beads to restrict mobility, plus the corresponding free enzyme (pullulanase) and the same enzyme sorbed to montmorillonite (Mte), to investigate systematically the consequences of surface associations of enzymes and of substrates on hydrolytic activity. Changes in substrate molecular weight were monitored with time to measure the course of enzymatic hydrolysis. Although hydrolysis of free substrate was nearly complete after 2 min incubation with the free enzymes, the sorbed enzymes also effectively hydrolyzed free substrate, and the data suggest that they retained activity longer in solution compared to the free enzymes. Sorbed enzymes progressively hydrolyzed the free substrate from > 50 kD to lower molecular weights during a 24 h incubation, with a final product distribution on average showing only 1.4% and 10.3% of substrate still in the > 50 kD and 10 kD size classes, while 46.6%, 29.3%, and 12.5% of substrate was in the 4 kD, monomer, and free tag size classes, respectively. This product distribution was very similar to that of the free substrate/free enzyme experiment. Tethering the substrate to agarose beads led to lower substrate release (2–3% of total substrate after 98 h incubation) into solution compared to the free substrate case. For tethered substrates, the state of the enzyme (free or sorbed) measurably affected the molecular weight distribution of the hydrolysis products, with free enzymes producing a higher fraction of high molecular weight hydrolysis products (28.7 ± 5.4% of substrate > 50 kD at the end of the incubation) compared to sorbed enzymes (11.6 ± 2.8% of substrate > 50 kD at the end of the incubation.) Tethered substrates were also hydrolyzed in a sediment slurry from surface sediments from Cape Lookout Bight, North Carolina; 0.1% of total substrate was released by enzymes naturally present in 1 cm³ of sediment after 144 h incubation, demonstrating that the enzymes naturally present in marine sediments are also capable of accessing tethered substrates. These investigations suggest that surface associations of enzymes in marine systems may extend the active lifetime of such enzymes, providing an opportunity for hydrolysis over longer periods of time and producing a different size spectrum of hydrolysis products relative to free enzymes. Furthermore, in well-mixed systems, surface-associated enzymes can hydrolyze substrates whose mobility is restricted, highlighting the importance of processes such as resuspension and bioturbation on organic matter remineralization.     

Application of fluorescence spectroscopic techniques and probes to the detection of biopolymer degradation in natural environments

The activities and substrate specificities of extracellular enzymes in natural systems are not well understood, despite their critical role in microbial remineralization of organic carbon. These enzymes initiate organic carbon degradation by selectively hydrolyzing high molecular weight substrates to lower molecular weight products which can be transported into cells. A set of single- and dual-labeled fluorescent polysaccharides was synthesized and characterized to explore a variety of approaches for measuring enzymatic hydrolysis of biopolymers via photophysical techniques, focusing particularly on rapid and robust optical techniques which are amenable to field measurements in remote locales. A shotgun-labeling approach yielded dual-labeled probes that exhibited substantial donor fluorophore quenching. The photophysical response of these probes to hydrolysis via purified enzymes was investigated in the lab, and fluorescence polarization proved to be a rapid and reliable technique for monitoring probe hydrolysis. Initial field results were also obtained from hydrolysis experiments in sediment porewaters. Because polarization measurements are rapid and simple, this approach could be used to follow the extracellular enzymatic hydrolysis of a wide range of biopolymers which fuel microbial metabolism.     

Enzyme activities in the water column and in shallow permeable sediments from the northeastern Gulf of Mexico

The activities of extracellular enzymes that initiate the microbial remineralization of high molecular weight organic matter were investigated in the water column and sandy surface sediments at two sites in the northeastern Gulf of Mexico. Six fluorescently labeled polysaccharides were hydrolyzed rapidly in the water column as well as in permeable sediments. This result contrasts with previous studies carried out in environments dominated by fine-grained muds, in which the spectrum of enzymes active in the water column is quite limited compared to that of the underlying sediments. Extracts of Spirulina, Isochrysis, and Thalassiosira were also used to measure hydrolysis rates in water from one of the sites. Rates of hydrolysis of the three plankton extracts were comparable to those of the purified polysaccharides. The broad spectrum and rapid rates of hydrolysis observed in the water column at both sites in the northeastern Gulf of Mexico may be due to the permeable nature of the sediments. Fluid flux through the sediments is sufficiently high that the entire 1.5 m deep water column could filter though the sediments on timescales of a few days to two weeks. Movement of water through sediments may also transport dissolved enzymes from the sediment into the water column, enhancing the spectrum as well as the rate of water column enzymatic activities. Such interaction between the sediments and water column would permit water column microbial communities to access high molecular weight substrates that might otherwise remain unavailable as substrates.     

Electron paramagnetic resonance spectroscopy as a novel approach to measure macromolecule–surface interactions and activities of extracellular enzymes

The dynamics of high molecular weight organic matter in marine systems are influenced by molecular conformation, interactions with surfaces and susceptibility to enzymatic hydrolysis, parameters that are difficult to observe experimentally. Here we use electron paramagnetic resonance spectroscopy (EPR) and spin-labeled (SL-) polysaccharides to monitor the sorption of SL-polysaccharides to natural sediment surfaces and to montmorillonite and to observe decreases in polysaccharide size due to enzymatic hydrolysis. SL-pullulan, SL-xylan and SL-maltoheptaose all sorbed rapidly to muddy sediments but not to sandy sediments. SL-pullulan and SL-maltoheptaose also both sorbed to montmorillonite; however, SL-pullulan reached substantially greater final surface loadings than did SL-maltoheptaose. Using EPR has the advantages of being rapid (spectra can be acquired in 100 seconds), non-destructive and functional in complex media, including sediment slurries, muddy water or other optically opaque samples, permitting investigation of the interactions between biomacromolecules, extracellular enzymes and mineral surfaces in aquatic environments.