Substrate specificity in polysaccharide hydrolysis: Contrasts between bottom water and sediments

Six structurally distinct polysaccharides were fluorescently labeled and used to compare potential hydrolysis rates and substrate specificities of extracellular enzymes in bottom water and surface sediments. Potential hydrolysis rates differed by factors of 10–100 among the six polysaccharides. The relative order of potential hydrolysis rates in sediments was distinctly different from that of the bottom water samples. In surface sediments, pullulan hydrolysis was extremely rapid, and relative hydrolysis rates decreased in the order pullulan ≫ laminarin > chondroitin sulfate > xylan > arabinogalactan ≫ fucoidan. In bottom water, in contrast, pullulan, arabinogalactan, and fucoidan were barely hydrolyzed, whereas chondroitin sulfate, xylan, and laminarin were hydrolyzed relatively rapidly. Hydrolysis rates decreased in the order xylan > chondroitin sulfate > laminarin >>> arabinogalactan ≅ pullulan ≅ fucoidan. The differences among the relative hydrolysis rates might reflect fundamental differences in seawater and sedimentary microbial communities with disparate extracellular enzymatic capabilities. Carbohydrates are significant constituents of dissolved organic carbon in seawater and are detectable as molecularly distinct structures in sediments of significant geologic age. Slow hydrolysis may provide the time required for geochemical transformations that further increase resistance to remineralization

Particulate organic matter in the sea: The composition conundrum

As organic matter produced in the euphotic zone of the ocean sinks through the mesopelagic zone, its composition changes from one that is easily characterized by standard chromatographic techniques to one that is not. The material not identified at the molecular level is called "uncharacterized". Several processes account for this transformation of organic matter: aggregation/disaggregation of particles resulting in incorporation of older and more degraded material; recombination of organic compounds into geomacromolecules; and selective preservation of specific biomacromolecules. Furthermore, microbial activities may introduce new cell wall or other biomass material that is not easily characterized, or they may produce such material as a metabolic product. In addition, black carbon produced by combustion processes may compose a fraction of the uncharacterized organic matter, as it is not analyzed in standard biochemical techniques. Despite these poorly-defined compositional changes that hinder chemical identification, the vast majority of organic matter in sinking particles remains accessible to and is ultimately remineralized by marine microbes

Extensive Microbial Processing of Polysaccharides in the South Pacific Gyre via Selfish Uptake and Extracellular Hydrolysis

Primary productivity occurs throughout the deep euphotic zone of the oligotrophic South Pacific Gyre (SPG), fueled largely by the regeneration of nutrients and thus recycling of organic matter. We investigated the heterotrophic capabilities of the SPG's bacterial communities by examining their ability to process polysaccharides, an important component of marine organic matter. We focused on the initial step of organic matter degradation by measuring the activities of extracellular enzymes that hydrolyze six different polysaccharides to smaller sizes. This process can occur by two distinct mechanisms: "selfish uptake," in which initial hydrolysis is coupled to transport of large polysaccharide fragments into the periplasmic space of bacteria, with little to no loss of hydrolysis products to the external environment, and "external hydrolysis," in which low molecular weight (LMW) hydrolysis products are produced in the external environment. Given the oligotrophic nature of the SPG, we did not expect high enzymatic activity; however, we found that all six polysaccharides were hydrolyzed externally and taken up selfishly in the central SPG, observations that may be linked to a comparatively high abundance of diatoms at the depth and location sampled (75 m). At the edge of the gyre and close to the center of the gyre, four of six polysaccharides were externally hydrolyzed, and a lower fraction of the bacterial community showed selfish uptake. One polysaccharide (fucoidan) was selfishly taken up without measurable external hydrolysis at two stations. Additional incubations of central gyre water from depths of 1,250 and 2,800 m with laminarin (an abundant polysaccharide in the ocean) led to extreme growth of opportunistic bacteria (Alteromonas), as tracked by cell counts and next generation sequencing of the bacterial communities. These Alteromonas appear to concurrently selfishly take up laminarin and release LMW hydrolysis products. Overall, extracellular enzyme activities in the SPG were similar to activities in non-oligotrophic regions, and a considerable fraction of the community was capable of selfish uptake at all three stations. A diverse set of bacteria responded to and are potentially important for the recycling of organic matter in the SPG

Extracellular enzyme activity in anaerobic bacterial cultures: Evidence of pullulanase activity among mesophilic marine bacteria

The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan [α(1,6)-linked maltotriose units] was directly assessed with a combination of gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydrolysis products of pullulan were separated by GPC into three fractions with molecular weights of ≥10,000, -5,000, and ≤1,200. NMR spectra of these fractions demonstrated that pullulan was rapidly and specifically hydrolyzed at α(1,6) linkages by pullulanase enzymes, most likely type II pullulanase. Although isolated pullulanase enzymes have been shown to hydrolyze pullulan completely to maltotriose (S. H. Brown, H. R. Costantino, and R. M. Kelly. Appl. Environ. Microbiol. 56:1985-1991, 1990; M. Klingeberg, H. Hippe, and G. Antranikian, FEMS Microbiol. Lett. 69:145-152, 1990; R. Koch, P. Zablowski, A. Spreinat, and G. Antranikian, FEMS Microbiol. Lett. 71:21-26, 1990), the smallest carbohydrate detected in the bacterial cultures consisted of two maltotriose units linked through one α(1,6) linkage. Either the final hydrolysis step was closely linked to substrate uptake, or specialized porins similar to maltoporin might permit direct transport of large oligosaccharides into the bacterial cell. This is the first report of pullulanase activity among mesophilic marine bacteria. The combination of GPC and NMR could easily be used to assess other types of extracellular enzyme activity in bacterial cultures

Substantial Carbohydrate Hydrolase Activities in the Water Column of the Guaymas Basin (Gulf of California)

The Guaymas Basin spreading center situated in the Gulf of California is characterized by a thick layer of organic-rich sediments that are thermally altered by hydrothermal fluids, thereby providing a bottom water source of dissolved organic carbon (DOC) to the water column. The potential for heterotrophic microbial communities in the water column to metabolize this organic matter source has not yet been investigated, however. In order to assess heterotrophic potential in the water column of the Guaymas Basin, we measured the activities of carbohydrate-hydrolyzing extracellular enzymes at the chlorophyll maximum, the oxygen minimum, the deep-water turbidity plume, and bottom waters. These measurements were carried out using water obtained from repeat CTD casts over the course of a week, and from bottom water collected by HOV Alvin at hydrothermally active areas with extensive chemosynthetic microbial mats. Repeat measurements at subsurface depths were very comparable across sampling dates and CTD casts. Exo-acting (terminal-unit-cleaving) monosaccharide hydrolase activities were typically higher in deeper waters than in surface waters, despite colder temperatures. In bottom water, the spectrum of endo-acting (mid-chain-cleaving) polysaccharide hydrolase activities was broader than at shallower depths. The high enzyme activities in Guaymas Basin bottom waters indicate an unusually active heterotrophic community that is responding to influx of DOC and nutrients into bottom waters from the hydrothermally affected sediments, or to the availability of chemosynthetically produced biomass

Heterotrophic extracellular enzymatic activities in the atlantic ocean follow patterns across spatial and depth regimes

Heterotrophic microbial communities use extracellular enzymes to initialize degradation of high molecular weight organic matter in the ocean. The potential of microbial communities to access organic matter, and the resultant rates of hydrolysis, affect the efficiency of the biological pump as well as the rate and location of organic carbon cycling in surface and deep waters. In order to investigate spatial- and depth-related patterns in microbial enzymatic capacities in the ocean, we measured hydrolysis rates of six high-molecular-weight polysaccharides and two low-molecular-weight substrate proxies at sites spanning 38°S to 10°N in the Atlantic Ocean, and at six depths ranging from surface to bottom water. In surface to upper mesopelagic waters, the spectrum of substrates hydrolyzed followed distinct patterns, with hydrolytic assemblages more similar vertically within a single station than at similar depths across multiple stations. Additionally, the proportion of total hydrolysis occurring above the pycnocline, and the spectrum of substrates hydrolyzed in mesopelagic and deep waters, was positively related to the strength of stratification at a site, while other physichochemical parameters were generally poor predictors of the measured hydrolysis rates. Spatial as well as depth-driven constraints on heterotrophic hydrolytic capacities result in broad variations in potential carbon-degrading activity in the ocean. The spectrum of enzymatic capabilities and rates of hydrolysis in the ocean, and the proportion of organic carbon hydrolyzed above the permanent thermocline, may influence the efficiency of the biological pump and net carbon export across distinct latitudinal and depth regions

Enhanced protein and carbohydrate hydrolyses in plume-associated deepwaters initially sampled during the early stages of the Deepwater Horizon oil spill

Oil spilled in the ocean can be biodegraded through a cascade of microbial processes, including direct degradation of petroleum-derived hydrocarbons, as well as subsequent degradation of transformation byproducts and exopolymeric substances (EPS) that are produced by microbes to emulsify hydrocarbons and facilitate access to oil. In the aftermath of the Deepwater Horizon oil spill, we measured enzymatic hydrolysis of carbohydrates and peptides in waters initially collected from within and outside of the deep hydrocarbon plume. The rationale is that the presence of EPS and other transformation byproducts in the deepwater plume may have enhanced heterotrophic bacterial metabolism in the cold deepwater environment. Our investigation targets carbohydrate and peptide hydrolase activities as indicators of the degradation of high molecular weight organic matter, including EPS substrates. Deepwater associated with the hydrocarbon plume revealed higher peptidase activity compared to non-plume deepwater samples. Enzymatic hydrolysis of carbohydrates, measured by the means of exo-acting enzyme activity (β-glucosidase), was also more rapid inside compared to outside the deepwater plume. Hydrolysis rates and patterns of endo-acting polysaccharide hydrolases, measured by means of distinct polysaccharide substrates in longer-term incubations, demonstrated more rapid plume-associated hydrolysis of two (laminarin and xylan) of the three substrates hydrolyzed in deepwaters. Our results indicate that microbial communities associated with the deepwater plume exhibited 'primed' responses to addition of specific substrates, which may structurally resemble components found in bacterial EPS and oil degradation byproducts. Bacterial transformation of oil-degradation byproducts thus likely contributed to microbial growth and respiration measured inside the deepwater plume

Strong seasonal differences of bacterial polysaccharide utilization in the North Sea over an annual cycle

Marine heterotrophic bacteria contribute considerably to global carbon cycling, in part by utilizing phytoplankton-derived polysaccharides. The patterns and rates of two different polysaccharide utilization modes - extracellular hydrolysis and selfish uptake - have previously been found to change during spring phytoplankton bloom events. Here we investigated seasonal changes in bacterial utilization of three polysaccharides, laminarin, xylan and chondroitin sulfate. Strong seasonal differences were apparent in mode and speed of polysaccharide utilization, as well as in bacterial community compositions. Compared to the winter month of February, during the spring bloom in May, polysaccharide utilization was detected earlier in the incubations and a higher portion of all bacteria took up laminarin selfishly. Highest polysaccharide utilization was measured in June and September, mediated by bacterial communities that were significantly different from spring assemblages. Extensive selfish laminarin uptake, for example, was detectible within a few hours in June, while extracellular hydrolysis of chondroitin was dominant in September. In addition to the well-known Bacteroidota and Gammaproteobacteria clades, the numerically minor verrucomicrobial clade Pedosphaeraceae could be identified as a rapid laminarin utilizer. In summary, polysaccharide utilization proved highly variable over the seasons, both in mode and speed, and also by the bacterial clades involved

Microbial enzymatic activity and secondary production in sediments affected by the sedimentation pulse following the Deepwater Horizon oil spill

A large fraction of the spilled oil from the Deepwater Horizon (DwH) blowout in April 2010 reached the seafloor via sinking oil aggregates (oil snow) in a massive sedimentation that continued until late summer 2010 ("Dirty blizzard"). We measured heterotrophic microbial metabolic rates as well as porewater and sedimentary geochemical parameters at sites proximate to and distant from the wellhead to investigate microbial responses to the "Dirty Blizzard". Lipase activity and rates of bacterial protein production were highest and leucine-aminopeptidase activity was lowest in 0-2 cm sediment layers at the sites proximate to the wellhead. These results suggest that the presence of the oil snow stimulated benthic microbial enzymatic hydrolysis of oil-derived organic matter that was depleted in peptide substrates at the time of our sampling. The strong gradients in porewater DOC, NH4+, and HPO43- concentrations in the upper 6 cm of the sediments near the wellhead likewise indicate elevated heterotrophic responses to recently-sedimented organic matter. In addition to enhanced microbial activities in the 0-2 cm sediment layers, we found peaks of total organic carbon and elevated microbial metabolic rates down to 10 cm at the sites closest to the wellhead. Our results indicate distinct benthic metabolic responses of heterotrophic microbial communities, even three months after the ending of the "Dirty Blizzard". Compared to other deep-sea environments, however, metabolic rates associated with the recently deposited particulate matter around the wellhead were only moderately enhanced. Oil contaminants at the seafloor may therefore have prolonged residence times, enhancing the potential for longer-term ecological consequences in deep-sea environments

A mechanistic microbial underpinning for the size-reactivity continuum of dissolved organic carbon degradation

The reservoir of dissolved organic carbon (DOC) in the ocean is modified by multiple input and removal processes. Incubation experiments as well as measurements of oceanic DOC have demonstrated that the high molecular weight (HMW) fraction of DOC typically has a younger radiocarbon age and is more reactive biologically than the low molecular weight (LMW) fraction of DOC. These observations have been summarized as a ‘size-reactivity continuum’ of DOC reactivity, but mechanistic explanations for these observations have been lacking. Here we describe how our recent discovery of ‘selfish’ HMW organic matter uptake among bacteria in surface ocean waters may help explain the rapid removal of HMW DOC. ‘Selfish’ substrate uptake by bacteria encompasses rapid binding and partial hydrolysis of intact polysaccharides on the outer membrane of bacteria, seamlessly followed by the transport of large oligosaccharide fragments into the periplasm with little to no loss of LMW hydrolysis products. ‘Selfish’ bacteria therefore process HMW substrates in a manner distinct from bacteria that carry out extracellular hydrolysis that yields LMW hydrolysis products in the environment. Recognition of the presence and prevalence of selfish bacteria in the ocean has profound implications for carbon flow – the source and quantity of LMW substrates made available to non-extracellular-enzyme producing bacteria – as well as for efforts to model and measure bacterial interactions during organic matter degradation. This discovery also highlights the importance of targeted substrate binding and uptake as key (often understudied) factors in geochemical investigations of microbially driven carbon cycling in the ocean. We conclude with some speculative thoughts about the factors that may determine the prevalence of selfish substrate uptake in the environment