Abstract
The degradation of particulate organic matter in the ocean is a central process in the global carbon cycle, the ‘mode and tempo’ of which is determined by the bacterial communities that assemble on particle surfaces. Although recent studies have shed light on the dynamics of community assembly on particles –which serve as hotspots of microbial activity in the ocean, the mapping from community composition to function, i.e. particle degradation, remains completely unexplored. Using a collection of marine bacteria cultured from different stages of succession on chitin micro-particles we found that the hydrolytic power of communities is highly dependent on community composition. Different particle degrading taxa –all of which were early successional species during colonization– displayed characteristic particle half-lives that differed by ~170 hours, comparable to the residence time of particles in the ocean’s mixed layer1. These half-lives were in general longer in multispecies communities, where the growth of obligate cross-feeders limited the ability of degraders to colonize and consume particles. Remarkably, above a certain critical initial ratio of cross-feeder to degrader cells, particle degradation was completely blocked along with the growth of all members of the community. We showed that this interaction occurred between a variety of strains of different taxonomic origins and that it only appears when bacteria interact with particles, suggesting a mechanism by which non-degrading secondary consumers occlude access to the particle resource. Overall, our results show that micro-scale community ecology on particle surfaces can have significant impact on carbon turnover in the ocean.
Introduction
Learning how the composition of ecological community impacts their function is arguably one of the central challenges in ecology2–4. In the case of microbes, this problem is particularly complex, not only because of the extreme diversity of taxa and genes that make up microbial communities, but also because community function depends on micro-scale processes that are hard to measure such as aggregation, dispersal and cell-cell interactions5. A prime example of the link between micro-scale community ecology and large-scale ecosystem function is found in the biological turnover of particulate organic matter. In the marine environment, biopolymer particles formed by aggregation of fragments of decaying organisms, fecal pellets, and extracellular polysaccharides are degraded and consumed by heterotrophic bacteria that attach to particle surfaces and form dense microbial communities of large taxonomic and metabolic diversity6–9. Because particulate matter tends to sink in the water column, its degradation in the upper layers of the ocean where oxygen abounds is crucial to sustain the marine food web and prevent the sequestration of carbon and nitrogen into the deep sea9–11. Therefore, particle-attached microbial communities play a fundamental role by closing the loop of the global carbon cycle and maintaining the balance of nutrients in marine ecosystems. Although many physical aspects of the bacteria-particle interaction such as attachment or the effects of flow12,13 have been well characterized, the possible role that ecological interactions between microbes may play in controlling the dynamics of particle colonization and degradation –and thus the ‘mode and tempo’ of the global carbon cycle– is much less clear.
Previous studies have shown that ecological interactions between microbes can play a significant role in controlling the dynamics of community assembly on particles. Competition for particle surface and thus primary resource access is likely to be strong among particle-attached bacteria and interference competition mediated by secondary metabolites can be a powerful strategy to deter competitors14,15. Moreover, over the time scales of particle turnover, trophic interactions mediated by byproducts of degradation and primary metabolism can strongly influence the overall dynamics of bacterial growth16: To release the carbon trapped in particulate matter, bacteria secrete hydrolytic enzymes that deconstruct complex biopolymers and release soluble sugars into the environment. The bioavailable sugars can in turn be taken up by nearby cells, thus unlocking a niche for ‘cheaters’ that consume resources but do not contribute to degradation16,17. Likewise, byproducts of primary metabolism such as organic acids or amino acids that are released to the local environment can be consumed by crossfeeding bacteria that co-assemble on the particle. On chitin particles, these types of trophic interaction have been shown to lead to successional waves and invasion of secondary consumers, which eventually become the numerically dominant members of the community16. These findings led us to hypothesize that interactions across trophic levels at the micro-scale might alter the catabolism of chitin and consumption of byproducts, possibly affecting the rate of particle turnover and the conversion from particle to bacterial biomass.
To test this hypothesis, in this study we used an isolate collection obtained directly from particle-attached communities previously shown to colonize in micro-scale successions16. In brief, these communities were enriched on ~50 μm paramagnetic chitin hydrogel particles incubated in seawater from the coastal ocean (Nahant, MA, USA). Bacteria were isolated directly from the particles, resulting in a collection that includes taxa such as Alteromonadales, Flavobacteriaceae, Rhodobacteriales, Vibrionaceae, and Oceanospiriliae. Notably, the composition of our collection coincides well with the taxonomic profiles of natural chitinous marine particles collected at 200–500 meters depth in the North Pacific gyre18. This overlap between our isolate collection and the taxonomic composition of natural particle-attached communities suggests that isolates obtained from model particles represent a relevant set of strains with which to study the effect of ecological interactions on particle turnover.
Bacterial isolates in our collection fall into two coarse-grained functional groups, defined on the basis of shared physiological characteristics and colonization dynamics16. The first group comprises primary degraders, which secrete chitinolytic enzymes, are motile, can grow rapidly on degradation byproducts and belong to species that tend to appear early during particle colonization. The second group corresponds to secondary consumers, which in general do not secrete enzymes, cannot grow on chitin, grow poorly if at all on monomers, are not motile and tend to belong to late successional species (Fig 1A, Fig S1). Although secondary consumers cannot grow on chitin particles alone, they can reach 100–1000 fold higher abundance in the presence of primary degraders16 due to their ability to utilize metabolic byproducts released by primary degraders during colonization.
Our goal in this study is to provide a quantitative description how particle degradation kinetics depend on the assembly of primary degraders and secondary consumers during particle colonization. To this end, we first studied how mono-cultures of primary degraders consumed particles by tracking changes in particle volume over time using high-throughput, high-resolution time-lapse microscopy (Fig 1B) and guiding our analysis with simple mathematical models of colonization and resource consumption. Subsequently, we assembled two-strain communities of primary degraders and secondary consumers and developed a quantitative phenomenological characterization of the impact of secondary consumers on degradation. Our results reveal that early colonizing taxa can differ significantly in their hydrolytic power to break down chitin, that particle degradation is limited by the number of enzyme-secreting bacteria that colonize the particle surface, and that secondary consumers effectively become parasites that increase in abundance at the cost of the primary degraders when co-colonizing on particle surfaces. Furthermore, the presence of parasitic secondary consumers can delay or even obstruct particle degradation. All these effects suggest that micro-scale community ecology on particle surfaces plays a major role in controlling community function by primarily slowing down resource turnover rates.
Results
Variability in hydrolytic power: the effect of primary degrader identity and abundance
We tracked the dynamics of particle consumption by measuring changes in particle volume over time, V (t), using high-throughput time-lapse microscopy of individual chitin microbeads. We chose an initial concentration of degrader cells of 5×105 cells/ml –an upper-bound estimate of the concentration of degrading bacteria in coastal waters19- and quantified V (t) over a period of 240 h, for four primary degraders and four secondary consumers incubated in media with no carbon source other than the particle. As expected, secondary consumers did not grow on particles in monoculture and therefore did not affect V(t) over the course of the ten-day time-lapse. For primary degraders, instead, V(t) was characterized by a long period of no detectable change, followed by a swelling of the particle and an abrupt collapse (Fig 1C, Sup. movie 2). Measurements of bacterial growth during degradation showed that bacteria grew steadily from the beginning of the incubation, despite no apparent change in particle volume, indicating that depolymerization was a continuous process and that swelling and collapse occurred only after a critical amount of polymer was consumed (Fig 1C). Particle swelling indicates that the degradation of cross-linked chitin in the hydrogel allows water molecules to expand the matrix20, while the transition from swelling to collapse indicates the point at which depolymerization ‘outcompetes’ swelling. The type of degradation curves observed for primary degraders (Fig. S2), with most of the dynamics concentrated on long transients, allowed us to quantify the ability of bacteria to consume particles with a single quantity, the particle half-life, τ1/2, i.e. the time it took for the particle to decrease to half its volume (see methods).
We found a remarkable variation in τ1/2 among the four different primary degraders, despite the fact that all of these isolates appeared early on in the ecological succession on chitin particles (Fig 1A). At an initial cell concentration of 5×105 cells/ml for all primary degraders, particle half-lives varied from ~30 h for the fastest degrader (a strain of the genus Psychromonas, named psych6C06) to ~200 h for the slow degraders (a strain of Vibrio nigripulchritudo named vnigri6D03) (Fig 1D). The large number of chitinase copies in psych6C06 (19 copies) suggested that gene dosage played a role in controlling the hydrolytic power of the strains. However, overall the differences between τ1/2 among primary degraders could not be clearly correlated to variation in gene content, suggesting instead that expression levels and the ‘quality’ of extracellular enzymes played a more significant role. Gene content did however distinguished primary and secondary consumers: degraders tended to encode the genomic potential to transport chitin monomers (N-acetylglucosamine specific PTS transporters), use monomers as chemotaxis signals and attach to chitin surfaces, features which tended to be absent in secondary consumers (Fig 1E, Table S1).
Chitin degradation is intrinsically linked to the production of public goods such as chitinases and as such can be subject to cooperative growth dynamics21, i.e. a positive dependency between cell densities and growth or depolymerization rates. If cooperativity does play a role, half degradation times would be highly sensitive to cell numbers, increasing disproportionally in cases where cell load is low. To test the relevance of this phenomenon and, in general, to study how τ1/2 depended on initial conditions, we measured degradation kinetics as a function of the initial concentration of primary degrader [Bp]0, which until now was arbitrarily set to 5×105 cells/ml. In addition, we guided our analysis with simple models of particle degradation and bacterial growth. To construct these models we assumed that particle depolymerization was proportional to the density of bacteria. We studied two possibilities, i) that bacteria grew cooperatively, i.e. with growth rate proportional to Bn and n > 1, and ii) that cooperativity played no significant role and growth and occurred at fixed, density independent per capita rates. Assuming that τ1/2 depends linearly on the speed of depolymerization, model i) predicts that τ1/2 should scale as − 1/[Bp]0, whereas model ii) predicts that τ1/2 should scale as −log ([Bp]0) (Methods and Supplementary Text, Fig. 2F).
In agreement with the simplest model with no cooperativity (ii), we find a linear relation between τ1/2 and log ([Bp]0) (Fig 2, Table S2). This behavior implies that the particle half-life is controlled by simple mass action kinetics22 that—at least in the conditions of our experiment—are not influenced by cooperativity. More precisely, we find that τ1/2 is well described by the following expression, where t0 is the intercept of the lines in Fig 2E and represents a timescale to degradation that is intrinsic to each strain, β is the slope and represents the per-capita contribution to the degradation process and –log captures the effect of the primary degrader concentration in the local environment, akin to a chemical potential for the cell-particle reaction.
The relationship found in (1) shows that the turnover of particulate organic matter depends on the load of primary degraders in the milieu in a simple, predictable manner. The lack of a cooperativity observed suggests that the possible benefits that bacteria may derive from ‘teaming up’ are effectively offset by local competition for resources between neighbors. Overall, our results indicate that variation in the composition and abundance of primary degraders can have a significant impact on the rate of particulate organic matter turnover.
Secondary consumers behave as parasites during particle degradation
To understand how ecological interactions between primary degraders and secondary consumers influence particle degradation, we focused our analysis on two primary degraders and one secondary consumer. We chose the relatively ‘slow’ degrader, vsple1A01 (Fig 1CD) a member of the Vibrio splendidus clade, the most abundant group of marine vibrios in coastal seawaters23, and the relatively ‘fast’ degrader, Pseudoalteromonas sp. palte3D05 (Fig S2), a common member of heterotrophic bacterioplankton communities24,25. Secondary consumers, or strains unable to degrade chitin, have previously been found to invade particle-attached communities and to become numerically dominant during community assembly16 (Fig. 1A). We focused our efforts on a secondary consumer cultivated from seawater-incubated chitin particles, a strain of the genus Maribacter (a type of marine Flavobacteria), that we here call marib6B07. As with other secondary consumers, marib6B07 is able to crossfeed when grown in co-culture with degraders16. Interestingly, genome sequences marib6B07 and other secondary consumers show that, despite their inability to degrade chitin under laboratory conditions, these organisms can contain chitinases (marib6B07 has two), but in general lack genes for N-acetylglucosamine specific chemotaxis, N-acetylglucosamine specific phosphotransferase (PTS) transport and chitin-binding, all of which tend to be present in multiple copies in the genomes of primary degraders (Table S1). These differences in the genomes of primary degraders and secondary consumers suggest that their functional roles in the community may be determined by the interaction between multiple traits, such as the ability to chemotax towards breakdown products of chitin and to transport them into the periplasm.
Co-incubation of mari6B07 with vsple1A01 and palte3D05 showed that mari6B07 increased τ1/2 relative to primary degrader monocultures (Fig 3A), implying that the crossfeeder impaired the ability of degrader populations to depolymerize the particle. To study this phenomenon in a quantitative manner, we measured how τ1/2 responded to changes in the initial concentration of secondary consumer, [Bs]0, with the number of cells of the primary degrader fixed at a given concentration ([Bp]0 ≈ 1.25×105 cells/ml) (Fig 3A, Fig S5A). We found that over low [Bs]0, τ1/2 increased roughly linearly, such that a one-fold increase in the secondary consumer [Bs]0 had approximately the same effect as a ten-fold reduction of the primary degrader [Bp]0 in monoculture.
Surprisingly, at a threshold [Bs]0 we observed an abrupt increase in τ1/2, to the extent that particle degradation did not occur within the 240 h imaging period, suggesting that the population of primary producers might have been inhibited from colonization and/or growth. To investigate how this phenomenon depended on the composition of the two-strain community, we varied the abundance of the primary degraders, [Bp]0 and secondary consumer [Bs]0, in order to obtain degradation phase planes (Fig 3B). The degradation phase planes show that complete inhibition did not depend on the total concentration of the secondary consumer, [Bs]0, but on the ratio of secondary consumer to primary degrader, γ = [Bs]0/[Bp]0 (Fig 3B,C). For the slow degrader, vsple1A01, degradation was blocked at γ > ~ 1, whereas for the fast degrader, palte3D05, degradation was blocked above a ratio of γ > ~16, showing that the slow degrader was more sensitive to the inhibitory effects of secondary consumer marib6B07 than the fast degrader. This analysis indicates that the balance between the relative abundances of secondary consumers to primary degraders in the environment, in addition to the degradation kinetics of the primary consumer, may be an important parameter that dictates the turnover rates of carbon over short time-scales (see Discussion).
Quantification of the abundance of each strain in co-culture before and after particle degradation showed that the interaction between primary degrader and secondary consumer is parasitic i.e. positive for the consumer, negative for the degrader. CFU counts during the time course of degradation in co-cultures of vsple1A01 and marib6B07 showed that primary degrader growth rate and yield were lower than in monoculture, and that the “loss” of degrader cells was compensated by the growth of secondary consumers (Fig 3C). Secondary consumers doubled approximately 5 times by the time of particle collapse, in contrast to their zero doublings in monoculture (see Fig S5B). Notably, the total yield of the co-culture was always equal or lower to the yield of the mono-culture, highlighting the parasitic nature of the interaction. Thus, secondary consumers, whose growth is facilitated by primary degraders, exert a negative feedback on degraders, limiting their ability to consumer produced resources and potentially their own growth.
Given the higher ratio of secondary consumer to degrader (γ) required to inhibit palte3D05 compared to vsple1A01, we hypothesized that “slow degraders” might be more susceptible to the detrimental effect of secondary consumers. To test this hypothesis as well as whether the observed parasitic interactions can be generalized to other primary degrader – secondary consumer pairs, we measured the effect of co-culture at γ = 1 ratio on particle degradation for all primary degraders (Fig 1D) with four different secondary consumers (including marib6B07) of diverse taxonomic origins, all of which were co-isolated from the same chitin-attached communities. The results showed that while the fast degraders psych6C06 and palte3D05 were only mildly affected by co-culture with secondary consumers at γ = 1, the slow degraders vsple1A01 and vnigr6D03 were susceptible to the presence of secondary consumers (Fig 4), with the slowest degrader, vnigr6D03 being inhibited by all four secondary consumers, three of which caused total blockage of particle consumption. These data further indicate that parasitic interactions between degraders and consumers are not dependent on specific taxa, but rather on the hydrolytic power of the degrader.
Consistent with the observation that interactions are not specific to strains or species but to functional roles (i.e. secondary consumer, primary degrader), we did not find evidence of chemical antagonism from secondary consumers to degraders. Agar plate assays designed to detect secreted inhibitory factors showed no interaction between the secondary consumer and primary degraders. Moreover, co-cultures of vsple1A01 and palte3D05 with marib6B07 in liquid media supplemented with #-acetylglucosamine (the monomer of chitin), as sole carbon source showed no decrease in growth rates (Fig S8). This suggests that either an antagonistic factor is only secreted in the particle environment, or what is more likely, that the observed inhibition of primary degrader growth is based on interference with physical processes that only take place when resources are concentrated on particles (e.g. colonization, attachment, etc.).
Discussion
Despite the significant efforts put into understanding the factors that drive the turnover of organic matter in the ocean26,27, the potential role that microbial interactions may play in this process has remained relatively unexplored. Our study leveraged a simplified model based on wild isolates that naturally colonize chitin particles to dissect this question. We provided evidence that both differences in primary degrader type and the ratio of primary degrader to secondary consumer can significantly alter particle degradation kinetics. Remarkably, we show that even in the ideal conditions of our experiments (no N limitation, high number of cells pre-grown in rich media) particle turnover times can be as high as 200 hours or more, that is, in the same range as the residence time of particles in the ocean’s mixed layer. Moreover, we showed interactions between primary degraders and secondary consumers lead to a significant increase in particle turnover times. This result is in agreement with our previous observation of colonization dynamics in natural seawater, which showed that secondary consumers “displace” primary degraders from particles, becoming the dominant members of the particle attached community after a brief initial period of colonization by degraders16. Taken together these results suggest that the micro-scale community ecology of particle-attached bacteria plays an important role in controlling rates of carbon turnover in the ocean.
Although in this study we do not identify a direct mechanism for the inhibitory effect of secondary consumers on primary degraders, our results suggest that the effect is not dependent on chemical interactions, which tend to be strain specific. Instead, the fact that we were able to observe degradation inhibition with different secondary consumers in a dose-specific manner suggests that a role of physical processes such as occlusion of the particle surface or an alteration of resource gradients around the particle, which are likely to occur regardless of species identity. Furthermore, this notion is consistent with the fact that degradation inhibition was only observed when bacteria grow on particles, and that the consequences of adding secondary consumers to the environment are similar to those of reducing the primary degrader load (and hence their particle colonization rate). Finally, the fact that the secondary consumer load required to induce degradation inhibition is anticorrelated with the hydrolytic power of the degrader reinforces the notion that particle depolymerization and secondary consumer growth are competing processes. Further work should aim at identifying the precise mechanisms that mediate the negative feedback from secondary consumers to degraders, tracking single cell behavior on and around particles as well as the interplay between spatial structure enzymatic activity.
Materials and Methods
Bacterial culturing conditions
Bacterial strains used in this study were previously isolated from model chitin particles16. Strains were streaked from glycerol stocks onto Marine Broth 2216 (Difco #279110) 1.5 % agar (BD #214010) plates. After 48 h, single colonies were transferred to 2 ml liquid Marine Broth 2216 and incubated at room temperature, shaking at 200 rpm. Saturated liquid cultures were harvested after 48 h by centrifugation for 8 minutes at 3000 rpm (Eppendorf 5415D, Rotor F45–24–11) and washed two times with Tibbles-Rawling minimal media (see supplemental material of ref 16 for a detailed recipe). Optical density (OD) 600 nm was determined in 200 μl (50 μl culture, 150 μl minimal media) in a clear 96-well plate (VWR 10062–900) with a spectrophotometer (Tecan Infinite F500). Cell numbers were normalized to the desired initial concentrations using a three-point linear calibration between OD 600 nm and direct cell counts determined with a Guava easyCyte Benchtop Flow Cytometer for each strain.
Particle degradation experiments
Particle degradation experiments were performed in clear 96 well plates (VWR 10062–900). Each well contained 180 μl Tibbles–Rawling minimal, bacterial cells at defined concentrations prepared as described above, and approximately 100 chitin magnetic beads (New England Biolabs #E8036L). Before being used in the experiments, the chitin magnetic beads storage buffer was removed using a neodymium magnet (McMaster-Carr #5862K38) to retain the beads. Beads were washed twice in Tibbles–Rawling minimal media and size selected using 100µm and 40μm strainers (VWR, #10199–658 and #10199–654, respectively).
For Fig 1B, the colonized particle was stained in the well after 24 by adding Syto9 (Thermo Fisher, S34854), 500 nM final concentration for 1h at room temperature in the dark. Microscopy was performed on an EVOS FL Auto Imaging System (Fisher #AMAFD1000) using a GFP lightcube (Thermo Fisher AMEP4651) and a 20x fluorite, long working distance objective (Fisher #AMEP4682, NA 0.40, WD 3.1 mm) and the softwares’ (revision 31201) Z-stack function. 3D-reconstruction was done using the ImageJ distribution Fiji (ImageJ 1.51N).
Time lapse imaging
Phase contrast time lapse images were acquired with an EVOS FL Auto Imaging System (Fisher #AMAFD1000) using the EVOS software (revision 31201) and a 20x fluorite, long working distance, phase-contrast objective (Fisher #AMEP4682, NA 0.40, WD 3.1 mm). Images were manually focused for each particle to capture the maximum cross section area (see Fig 1C, upper panel). Time lapses ran a maximum of 240 h, with images acquired every 2 h. To minimize evaporation effects, culturing plates were wrapped in para film during the time-lapse experiments and outer wells filled with 200 μl water.
Image processing and Volume quantification
Phase contrast images were analyzed using the ImageJ distribution Fiji (ImageJ 1.51N). A polygonal shape was manually drawn around the particle to determine the area of the particles’ cross-section. To convert from cross section area in square pixel (1 pixel = 0.4545 μm) to volume (in μm^3), we assumed a spherical shape of the particles. Volumes were normalized to initial volume at t=0 h to account for variation in particle sizes. In order to estimate the particle half-life, we fitted a sigmoidal function using MATLAB (Version R2016b) and the ‘fit’ function with initial values for k (0.5) and τ1/2 (initial estimates vary for each strain), constraining both variables to positive values (see also Fig S3 for examples of sigmoidal fits to the data).
Co-culture experiments
Cell counts were obtained by sampling 100 μl from 96 well culture plates (inoculated with 170 μl minimal media, 2x10 μl of the normalized bacterial culture, and 10 μl particles as described above). Imaging was performed as described above. For CFU counts, samples were vortexed thoroughly to detach cells from particles and 10 μl were plated in 10^-2 and 10^-3 dilutions in replicates on MB2216 agar plates using rattler beads (Zymo S1001). After 72 h, colonies were counted to obtain CFUs.
DNA quantification
To quantify DNA as a proxy for biomass from mono cultures in 96 well plates, wells were mixed thoroughly by pipetting and 100 μl of each well (including the particles) were sampled and frozen at −20 °C for subsequent analysis. Cells were lysed by thawing and boiling (95 °C, 10 min) 10 μl of each sample. Lysed samples were diluted 1:10 in TE buffer and quantified using Quant-it pico green (Fisher # P7589) standard protocols.
Strain genome annotation
The genomes are deposited at NCBI under Bioproject # PRJNA414740 and the respective accession numbers in Table S1. Assembled genomes were annotated using RAST and genome content was parsed using text parsing of the genome annotations for Chi, ChB, GTx.
Supplementary Figures
Supplemental Movie 1. 3D reconstruction of a chitin micro particle colonized by palte3D05 for 24 h, stained with SYTO9.
Supplemental Movie 1. Phase contrast time-lapse of a chitin particle cross section taken during degradation by vsple1A01, corresponding to frames shown in Figure 1C.
Acknowlegements
The authors wish to thank members of the Cordero lab for thoughtful discussions. This research was supported by NSF grant OCE-1658451, European Starting Grant no. 336938. OXC was also supported by the Simons Early Career Award 410104 and the Alfred P Sloan fellowship FG-20166236.