Craig J. Plante

Isolation of Surfactant-Resistant Bacteria from Natural, Surfactant-Rich Marine Habitats

ABSTRACT Environmental remediation efforts often utilize either biodegradative microbes or surfactants, but not in combination. Coupling both strategies holds the potential to dramatically increase the rate and extent of remediation because surfactants can enhance the bioavailability of contaminants to microbes. However, many surfactants permeabilize bacterial cell membranes and are effective disinfectants. An important goal then is to find or genetically modify microorganisms that possess both desirable degradative capabilities and the ability to thrive in the presence of surfactants. The guts of some marine invertebrates, particularly deposit feeders, have previously been shown to contain high levels of biosurfactants. Our primary aim was to mine these natural, surfactant-rich habitats for surfactant-resistant bacteria. Relative to sediment porewaters, the gut contents of two polychaete deposit feeders, Nereis succinea and Amphitrite ornata, exhibited a significantly higher ratio of bacteria resistant to both cationic and anionic surfactants. In contrast, bacteria in the gut fluids of a holothuroid, Leptosynapta tenuis, showed surfactant susceptibility similar to that of bacteria from sediments. Analyses of 16S rRNA gene sequences revealed that the majority of surfactant-resistant isolates were previously undescribed species of the genus Vibrio or were of a group most closely related to Spongiobacter spp. We also tested a subset of resistant bacteria for the production of biosurfactants. The majority did produce biosurfactants, as demonstrated via the oil-spreading method, but in all cases, production was relatively weak under the culture conditions employed. Novel surfactant-resistant, biosurfactant-producing bacteria, and the habitats from which they were isolated, provide a new source pool for potential microorganisms to be exploited in the in situ bioremediation of marine sediments.

Olive Ridley Sea Turtle Hatching Success as a Function of the Microbial Abundance in Nest Sand at Ostional, Costa Rica

Several studies have suggested that significant embryo mortality is caused by microbes, while high microbial loads are generated by the decomposition of eggs broken by later nesting turtles. This occurs commonly when nesting density is high, especially during mass nesting events (arribadas). However, no previous research has directly quantified microbial abundance and the associated effects on sea turtle hatching success at a nesting beach. The aim of this study was to test the hypothesis that the microbial abundance in olive ridley sea turtle nest sand affects the hatching success at Ostional, Costa Rica. We applied experimental treatments to alter the microbial abundance within the sand into which nests were relocated. We monitored temperature, oxygen, and organic matter content throughout the incubation period and quantified the microbial abundance within the nest sand using a quantitative polymerase chain reaction (qPCR) molecular analysis. The most successful treatment in increasing hatching success was the removal and replacement of nest sand. We found a negative correlation between hatching success and fungal abundance (fungal 18S rRNA gene copies g-1 nest sand). Of secondary importance in determining hatching success was the abundance of bacteria (bacterial 16S rRNA gene copies g-1 g-1 nest sand). Our data are consistent with the hypothesis that high microbial activity is responsible for the lower hatching success observed at Ostional beach. Furthermore, the underlying mechanism appears to be the deprivation of oxygen and exposure to higher temperatures resulting from microbial decomposition in the nest.

DISTURBANCE EFFECTS OF DEPOSIT FEEDING ON MICROALGAL COMMUNITY STRUCTURE AND MECHANISMS OF RECOLONIZATION1

Benthic microalgae (BMA) are important primary producers in intertidal and shallow subtidal sediments, serving as a vital food resource for heterotrophs. BMA also release extracellular polymeric secretions that inhibit resuspension of sediments. Key ecological parameters such as abundance, productivity, and species composition of BMA each contribute to the character of these roles. Our primary objectives were to (i) assess the importance of biotic disturbance to the structure of sedimentary microalgal communities and (ii) identify principal modes of recolonization. We employed field comparative studies to test whether deposit feeding by two invertebrates (Leptosynapta tenuis and Balanoglossus aurantiacus) caused removal of BMA, and manipulative experiments to assess rates and mechanisms of recolonization. Both deposit feeders were determined to significantly reduce BMA biomass via ingestion; however, little change in community composition was observed. Recovery of these disturbed patches was followed over the period of intertidal exposure. We distinguished between potential recolonization methods of migration and regrowth by monitoring fecal coils incubated naturally on underlying sediments (regrowth + migration treatment), hydrogen‐peroxide‐treated coils incubated on ambient sediment (migration only), and coils that were incubated on 0.2 μm filters and thereby isolated from underlying sediment (regrowth only). BMA biomass recovery was significant in <3 h, with migration from underlying sediments the dominant means of recolonization. Surprisingly, recovery appeared somewhat slower when natural egesta were exposed to underlying sediments (migration + regrowth treatments) as compared to migration into peroxide‐treated coils (migration only). This counterintuitive result was due to the dynamic, bidirectional vertical migrations of diatoms in surficial sediments.

Neutral processes and species sorting in benthic microalgal community assembly: effects of tidal resuspension.

Benthic microalgae (BMA) provide vital food resources for heterotrophs and stabilize sediments with their extracellular secretions. A central goal in ecology is to understand how processes such as species interactions and dispersal, contribute to observed patterns of species abundance and distribution. Our objectives were to assess the effects of sediment resuspension on microalgal community structure. We tested whether taxa‐abundance distributions could be predicted using neutral community models (NCMs) and also specific hypotheses about passive migration: (i) As migration decreases in sediment patches, BMA α‐diversity will decrease, and (ii) As migration decreases, BMA community dissimilarity (β‐diversity) will increase. Co‐occurrence indices (checkerboard score and variance ratio) were also computed to test for deterministic factors, such as competition and niche differentiation, in shaping communities. Two intertidal sites (mudflat and sand bar) differing in resuspension regime were sampled throughout the tidal cycle. Fluorometry and denaturing gradient gel electrophoresis were utilized to investigate diatom community structure. Observed taxa‐abundances fit those predicted from NCMs reasonably well (R2 of 0.68–0.93), although comparisons of observed local communities to artificial randomly assembled communities rejected the null hypothesis that diatom communities were assembled solely by stochastic processes. No co‐occurrence tests indicated a significant role for competitive exclusion or niche partitioning in microalgal community assembly. In general, predictions about relationships between migration and species diversity were supported for local community dynamics. BMA at low tide (lowest migration) exhibited reduced α‐diversity as compared to periods of immersion at both mudflat and sand bar sites. β‐diversity was higher during low tide emersion on the mudflat, but did not differ temporally at the sand bar site. In between‐site metacommunity comparisons, low‐ and high‐resuspension sites exhibited distinct community compositions while the low‐energy mudflats contained higher microalgal biomass and greater α‐diversity. To our knowledge this is the first study to test the relevance of neutral processes in structuring marine microalgal communities. Our results demonstrate a prominent role for stochastic factors in structuring local BMA community assembly, although unidentified nonrandom processes also appear to play some role. High passive migration, in particular, appears to help maintain species diversity and structure communities in both sand and muddy habitats.

Olive Ridley Sea Turtle Hatching Success as a Function of Microbial Abundance and the Microenvironment of In Situ Nest Sand at Ostional, Costa Rica

Sea turtle hatching success at mass nesting beaches is typically lower than at solitary nesting beaches, presumably due in part to high rates of microbial metabolism resulting from the large input of organic matter from turtle eggs. Therefore, we tested the hypothesis that hatching success varies across areas of the beach in conjunction with differences in the physical nest environment and microbial abundance of in situ olive ridley sea turtle nests at Ostional, Costa Rica. We marked natural nests in high-density, low-density, and tidal-wash nesting areas of the beach and monitored clutch pO2 and temperature throughout the incubation period. We quantified hatching success and collected samples of nest sand during nest excavations. We quantified microbial abundance (bacteria and fungi) with a quantitative polymerase chain reaction (qPCR) analysis. Hatching success was lower in nests with lower pO2, higher temperatures, higher organic matter content, and higher microbial abundance. Our results suggest that the lower oxygen within the nest environment is likely a result of the high microbial abundance and rates of decomposition in the nest sand and that these factors, along with increased temperature of clutches in the high-density nesting area, are collectively responsible for the low hatching success at Ostional.

Importance of the Sedimentary Matrix for Anaerobic Oil Degradation by Marine Bacterial Assemblages

ABSTRACT The microbial conversion of petroleum hydrocarbons is increasingly employed in bioremediation efforts. In marine sediments, oxygen levels are characteristically depleted, so anaerobic degradation coupled to sulfate reduction dominates. Prior studies have noted that anaerobic degradation is much reduced in the absence of sediments. In this study, a simple centrifugation protocol was used to extract sediment porewaters to obtain a sediment-free bacterial assemblage capable of anaerobic hydrocarbon degradation. In addition, the factors in sediment that were important to degradation rates were determined. Experiments were designed to differentiate among the effects of increased surface area associated with individual grains in sediments, differing levels of organic constituents in sediments and water, and disparate microbiota within the sedimentary matrix and those free living. Anaerobic alkane degradation, sulfate levels and bacterial community structure were monitored over 90 days in five treatments consisting of Bonny Light crude oil added to (1) intact sediment, (2) sediment-free supernate from centrifuged sediment, (3) supernate plus autoclaved sediment, (4) supernate plus organic-free (combusted) sediment, and (5) a control, with autoclaved supernate plus autoclaved sediment. Lack of surface area associated with sediment grains had little effect on degradation. Separation of porewaters from the sedimentary matrix resulted in loss of bacterial biomass, although this had only a temporary negative effect on degradation rates. Reduction of organic matter due to sediment removal had the largest effect, resulting initially in lower degradation rates. However, sulfate depletion in low organic treatments was also reduced so that long-term loss of alkanes was enhanced.

Tidal Stage Changes in Structure and Diversity of Intertidal Benthic Diatom Assemblages: a Case Study from Two Contrasting Charleston Harbor Flats

Benthic microalgae are key contributors to near-shore food webs and sediment stabilization. Temporal variability in microalgal biomass and production throughout the tidal cycle has been well documented; however, due to limitations of traditional methods of analysis patterns of community composition and diversity over such time scales have not been revealed. To explore the latter and better understand how short-term changes throughout the tidal cycle may affect community functioning, we compared benthic diatom composition and diversity over tidal stage shifts. We employed two disparate molecular techniques (denaturing gel gradient electrophoresis with Sanger DNA sequencing of excised bands and high-throughput community metagenome sequencing) to characterize diatom assemblages in representative muddy and sandy intertidal sites in Charleston Harbor, SC, USA. In support of prior studies, we found higher diatom diversity in sandbar as compared to mudflat sediments. Spatial differences were stronger relative to tidal temporal differences, although diversity metrics generally were highest after prolonged tidal immersion as compared to low-tide emersion or just after immersion at flood tide. Composition of the diatom assemblage differed markedly between sites, with species in genera Halamphora, Amphora, and Navicula dominating the sandbar, whereas Cyclotella, Skeletonema, and Thalassiosira were the most prevalent genera on the mudflat. Diatom composition differed by tidal stage, with assemblages during low-tide exposure distinct from samples taken after immersion. Both sandbar and mudflat sediments exhibited increases in relative proportion of epipelic diatoms and decreases in planktonic taxa during low-tide exposure. Our findings of short-term changes in species composition and dominance could inform primary productivity models to better estimate understudied diatom contributions in heterogeneous and highly variable tidal systems.

Propidium monoazide-denaturing gradient gel electrophoresis (PMA-DGGE) assay for the characterization of viable diatoms in marine sediments

Molecular-based community profiles often overestimate species richness and diversity as they are incapable of distinguishing DNA signals derived from extracellular or moribund sources from that of viable organisms. Propidium monoazide (PMA), a double stranded DNA-binding dye, was used to selectively isolate the genetic signature of viable diatoms from marine cultures and environmental samples. Upon optimization of photoactivation time using a 600-W halogen lamp, PMA concentrations, and number of treatments, the compromised genetic signals from heat-killed samples, visualized by denaturing gradient gel electrophoresis, were either greatly reduced or removed entirely while those derived from intact, viable samples remained relatively unaffected. Subsequent tests illustrated that bands associated with extracellular and moribund DNA were removed from combinations of viable and nonviable cells initially derived from two sedimentary diatom isolates. In addition, viable and nonviable samples of Skeletonema marinoi that were spiked into fresh deposit-feeder (Balanoglossus aurantiacus) faecal-coil sediments demonstrated the effectiveness of the assay within an environmental matrix. Thus, we propose the use of PMA as a quick and necessary addition to any protocol relying on genetic profiles to discern changes in viable benthic diatom community structure.