Production Of Exopolymeric Substances Research Articles

Cyclic di-peptide Cyclo (L-Phe-L-Pro) mitigates the quorum-sensing mediated virulence in Salmonella typhi and biofilm formation in poultry and plastic system

Salmonella typhi is a predominant foodborne pathogen, posing a significant threat to food quality and safety through typhoid outbreaks. Quorum sensing (QS) and its pathways are crucial for typhoid antigen regulation and S. typhi-mediated spoilage and pathogenesis. Therefore, this study intends to identify secondary metabolites from extracts of 15 bacterial isolates sourced from wild Mangifera indica (mango) brine pickles as novel anti-QS agents against S. typhi. Cell-free supernatants (CFS) of 15 bacterial isolates from fermented mango brine pickles were assessed and identified for anti-QS positive metabolites using GC-MS. Anti-QS metabolite was tested on early and mature biofilms, as well as violacein inhibition, EPS, cellular adherence, motility, antibiotics, and immune resistance of S. typhi. Gene expression and in-silico studies were also conducted to validate the compound's efficacy. The results depicted that among the 15 isolates, Bacillus subtilis P89 exhibited potent anti-QS properties. Gas chromatography-mass spectrometry (GC-MS) analysis identified twelve potential quorum-sensing inhibiting compounds, with Cyclo (L-Phenylalanine-L-Proline) (CPP) demonstrating the maximum anti-QS activity against S. typhi. CPP disrupted the pathogen's early and mature biofilms, reducing exo-polymeric substance production, cell hydrophobicity, and motility at sub-lethal concentrations. It also enhanced the immune response against S. typhi and improved traditional efficacy. Real-time qPCR analyses revealed CPP's modulation of essential quorum-sensing regulatory genes. Molecular docking studies indicated competitive binding of CPP with the SdiA receptor. CPP inhibited S. typhi biofilm formation on chicken skin, plastics, and eggshells. To our knowledge, this study is the first to highlight CPP's potential in food and clinical environments to combat S. typhi via quorum sensing-based mechanisms.

Properties of exopolymeric substances (EPSs) produced during cyanobacterial growth: potential role in whiting events

Abstract. Extracellular polymeric substances (EPSs) are an important organic carbon reservoir in many pelagic and benthic environments. The production of EPS is intimately associated with the growth of phyto- and picoplankton. EPS plays a critical role in carbonate precipitation through the binding of cations and by acting as a nucleation site for minerals. Large-scale episodes of fine-grained calcium carbonate precipitation in the water column (whiting events) have been linked to cyanobacterial blooms, including of Synechococcus spp. The mechanisms that trigger these precipitation events are still debated. We pose that the cyanobacterial EPS, produced during exponential and stationary growth phases, plays a critical role in the formation of whitings. The aim of this study was to investigate the production of EPS during a 2-month cyanobacterial growth, mimicking a bloom. The production and characteristics of EPS were examined in different growth stages of Synechococcus spp. using various techniques such as Fourier transform infrared (FT-IR) spectroscopy as well as colorimetric and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) assays. We further evaluated the potential role of EPS in carbonate precipitation through in vitro-forced precipitation experiments. EPS produced during the early and late stationary phase contained a larger amount of negatively charged groups than present in EPS produced during the exponential phase. Consequently, a higher Ca2+-binding affinity of the stationary-phase EPS led to the formation of a larger amount of smaller carbonate minerals (< 50 µm) compared to crystals formed in exponential-phase EPS, which were less abundant and larger (> 50 µm). These findings were used to establish a conceptual model for picoplankton-bloom-mediated CaCO3 precipitation that can explain the role of EPS in whitings.

Intertwined synergistic abiotic and biotic degradation of polypropylene pellets in marine mesocosms

The accumulation of plastic waste in the oceans has caused growing concern for its effects on marine life. The interactions of plastics with environmental factors have been linked to fragmentation to micro- and nanoparticles with different properties and consequences, but the mechanism of fragmentation has not been fully understood yet. In this work, we investigate the combined effect of marine communities and ultraviolet (UV) radiation towards the degradation of virgin and artificially weathered polypropylene (PP) pellets after a long-term incubation period in marine mesocosms. The surface chemical alterations and deterioration of the polymer, in conjunction with the attachment and evolution of marine bacterial communities, the development of biofilm and exopolymeric substances (EPS), as well as the colloidal properties (zeta-potential and hydrodynamic diameter) of the mesocosms were studied. The surface area of both types of pellets decreased over time, despite no concrete weight change being observed. Cell growth, EPS production and colloid particle size were correlated to the loss of area. Therefore, we propose that surface area could be effectively monitored, instead of weight loss, as an alternative indicator of polymer degradation in biodegradation experiments. Changes in the chemical structure of the polymer, in addition to the evolution of the biological factors, implied that a complex degradation process alternated between two phases: an abiotic phase, when UV irradiation contributes to the deterioration of the polymer surface layers and a biotic phase, when marine communities degrade the weathered polymer surface to reveal the underlying layer of virgin polymer. Finally, microscopic particles, produced as a result of the decrease in pellet area, promoted the aggregation of colloidal particles. The role and impacts of these colloidal particles in marine ecosystems are yet as unidentified as that of micro- and nano-sized plastic particles and call for further investigation.

Exopolymeric substances production by Bacillus cereus KMS3-1 enhanced its biosorption efficiency in removing Cd2+ and Pb2+ in single and binary metal mixtures

The present study investigated the growth, exopolymeric substance (EPS) production, and biosorption efficiency of strain Bacillus cereus KMS3-1 in the Cd2+ and Pb2+ ions containing single and binary metal-treated broth (50 mg/L). In addition, the interaction of the KMS3-1 strain with Cd2+ and Pb2+ ions in single and binary metal-treated broths was investigated using SEM–EDS, FTIR, and XRD analyses. The results showed that the biosorption efficiency (%) and EPS production of KMS3-1 biomass in both single and binary metal-treated broths had increased with increasing incubation time and were higher for Pb2+ ions than for Cd2+ ions. In the single and binary metal-treated broths, the maximum biosorption efficiency of KMS3-1 for Pb2+ ions were 70.8% and 46.3%, respectively, while for Cd2+ ions, they were 29.3% and 16.8%, respectively, after 72 h. Moreover, the biosorption efficiency of strain KMS3-1 for both metal ions was dependent on its EPS production and peaked at the maximum EPS production. The copious EPS production by KMS3-1 was observed in metal-treated media (50 mg/L), in the following order: Pb2+ ions (1925.7 μg/mL) > binary metal mixtures (1286.8 μg/mL) > Cd2+ ions (1185.5 μg/mL), > control (1099 μg/mL) after 72 h of incubation. This result indicates that the metal biosorption efficiency of the KMS3-1 strain was enhanced by the increased EPS production in the surrounding metal-treated broth. SEM–EDS and FTIR characterization studies revealed that the KMS3-1 biomass effectively adsorbed Cd2+ and Pb2+ ions from the medium by interacting with their surface functional groups (hydroxyl, carbonyl, carboxyl, amide, and phosphate). Moreover, the biosorbed Cd2+ and Pb2+ ions were transformed into CdS and PbS, respectively, by the KMS3-1 biomass. This study suggests that the Bacillus cereus KMS3-1 strain may be a promising candidate for the treatment of metal contamination.

Culture Supernatant of Enterococcus faecalis Promotes the Hyphal Morphogenesis and Biofilm Formation of Candida albicans.

(1) Background: Enterococcus faecalis and Candida albicans are often isolated from infected root canals. The interaction between these two species is not clear enough. Therefore, the objective of this study was to investigate the effect of E. faecalis on the biofilm formation, hyphal morphogenesis and virulence gene expression of C. albicans. (2) Methods: We used the culture supernatant of E. faecalis (CSE) to treat the biofilms of C. albicans. Then, crystal violet staining and colony-forming unit (CFU) counting were performed to evaluate biofilm biomass. Scanning electron microscopy (SEM) and confocal laser scanning microscope (CLSM) were applied to observe fungal morphology. Subsequently, exopolymeric substances (EPS) production, cellular surface hydrophobicity (CSH) and adhesion force of biofilms were investigated by CLSM, water–hydrocarbon two-phase assay and atomic force microscopy (AFM), respectively. Finally, the expression of C. albicans virulence genes (ALS1, ALS3, HWP1 and EFG1) were measured by RT-qPCR assay. (3) Results: The exposure of CSE promoted the biofilm formation and hyphal morphogenesis of C. albicans, increased the EPS production, CSH and adhesion force of C. albicans biofilms, and increased the expression level of EFG1. (4) Conclusions: Our data indicated that CSE promoted the hyphal morphogenesis and biofilm formation of C. albicans.

Effect of experimentally increased nutrient availability on the structure, metabolic activities, and potential microbial functions of a maritime Antarctic microbial mat.

The role of competitive interactions based on resource utilisation was explored in a phototrophic microbial mat from Byers Peninsula (Maritime Antarctica). Shotgun metagenomic profiling of the mat showed a taxonomic and functionally diverse microbial community. The heterotrophic bacterial community was dominated by Proteobacteria, where genera typically found in polar habitats, such as Janthinobacterium, Pseudomonas, and Polaromonas, were highly prevalent. Cyanobacteria played the main role as primary producers, accompanied by diatoms and chlorophytes. To test the potential effects of the inorganic nutrient (N and P) availability on this community, a fully factorial nitrate and phosphorus addition experiment was conducted in situ. The mat exhibited a functional and structural response to the nutrient amendments. Compared to the undisturbed mat, phosphorus fertilisation favoured the growth of (non-heterocystous) cyanobacteria relative to that of diatoms, as indicated by changes in the carotenoid pigment biomarkers. Although no mat accretion was visible, fertilisation improved the phototrophic activity, and, mainly, when P was amended, the production of exopolymeric substances was favoured, whereas further changes in the vertical distribution of primary production activity were observed as well. Illumina amplicon sequencing of the 16S rRNA gene also demonstrated changes in the relative abundance of heterotrophic prokaryotes, which were detectable from the phylum to the genus level and mainly related to the amendment of nitrogen. Predictions made on the functional skills of these shifted prokaryotic communities indicated changes in abundance selecting taxa with a metabolic adaptation to the new nutrient scenarios. They mainly consisted of the enhancement of ecological strategies and metabolic regulatory mechanisms related to the uptake and metabolising of either nitrogen or phosphorus, regulated by its availability whether in a balanced way or not. This study is a pioneer in demonstrating how shifts in the regional dynamic of nutrients might alter the metabolic equilibrium of these initially considered homeostatic benthic communities. They can be accordingly considered as taxonomically diverse microbiomes with a functional repertoire still inclined to respond to the biogeochemical alteration of nutrient cycles, although occurring in a cold extreme environment where biological activity is partially restricted by environmental harshness.

Effect of Culture pH on Properties of Exopolymeric Substances from Synechococcus PCC7942: Implications for Carbonate Precipitation

The role of culture conditions on the production of exopolymeric substances (EPS) by Synechococcus strain PCC7942 was investigated. Carbonate mineral precipitation in these EPS was assessed in forced precipitation experiments. Cultures were grown in HEPES-buffered medium and non-buffered medium. The pH of buffered medium remained constant at 7.5, but in non-buffered medium it increased to 9.5 within a day and leveled off at 10.5. The cell yield at harvest was twice as high in non-buffered medium than in buffered medium. High molecular weight (>10 kDa) and low molecular weight (3–10 kDa) fractions of EPS were obtained from both cultures. The cell-specific EPS production in buffered medium was twice as high as in non-buffered medium. EPS from non-buffered cultures contained more negatively charged macromolecules and more proteins than EPS from buffered cultures. The higher protein content at elevated pH may be due to the induction of carbon-concentrating mechanisms, necessary to perform photosynthetic carbon fixation in these conditions. Forced precipitation showed smaller calcite carbonate crystals in EPS from non-buffered medium and larger minerals in polymers from buffered medium. Vaterite formed only at low EPS concentrations. Experimental results are used to conceptually model the impact of pH on the potential of cyanobacterial blooms to produce minerals. We hypothesize that in freshwater systems, small crystal production may benefit the picoplankton by minimizing the mineral ballast, and thus prolonging the residence time in the photic zone, which might result in slow sinking rates.

Co-cultivation, metal stress and molasses: strategies to improving exopolymeric yield and metal removal efficacy

This study investigated and compared several improvement strategies to increase the yield and quality of exopolymeric substances (EPS) from Bacillus cereus. This includes co-culturing of B. cereus with Trichoderma asperellum, cultivation in media with metal (Zn) stress and supplementation with molasses. EPS is subsequently extracted from these different cultures and subjected to characterization and metal removal tests in single-metal systems (Cu, Pb, Zn, Cd, Cr). Results indicate that co-cultivation of B. cereus and T. asperellum produced EPS which have attributes differing from single cultivation. These changes were detected via functional group changes using Fourier-Transform Infrared Spectroscopy, as well as the increase in carbohydrate and protein content. However, the interaction of these two microbes were merely additive and did not result in improved EPS yield nor the subsequent metal removal efficacy in comparison to single cultivation (control). By contrast, supplementation of Zn (metal stress with 50 mg L− 1 Zn) improved EPS quality and metal removal, but decreased EPS yield. The application of 1% molasses was the only strategy demonstrating high yield and efficient metal removal. EPS quality and yield (0.45 mg mL− 1) and metal removal efficacy (Cu: 58%, Pb: 98%, Zn: 83%, Cd: 73%, Cr: 96%) were improved significantly. This study showed that among the three improvement strategies (co-cultivation, metal stress, molasses), supplementation with molasses was the most effective as it improved both yield and quality of EPS significantly, suggesting that this approach may be adopted for future production of bulk EPS for up-scaling of wastewater treatment.

Glucose-Induced Biofilm Formation in Bacillus thuringiensis KPWP1 is Associated with Increased Cell Surface Hydrophobicity and Increased Production of Exopolymeric Substances.

Bacillus thuringiensis is an agriculturally and medically important bacteria as it produces insecticidal Cry proteins and can form biofilm on different plant surfaces. Previous studies reported that the ubiquitous carbon source glucose could induce restricted motility and fractal pattern formation in the growing colonies of pH, salt and arsenate tolerant Bacillus thuringiensis KPWP1. As bacteria are evolved with the ability to exhibit multicellular behavior and biofilm formation under limiting conditions for survival, the present study was focused on exploring the effect of glucose in biofilm formation by Bacillus thuringiensis KPWP1. A significant rise in biofilm loads was observed with increased glucose concentrations in growth media. Compared to control, six times more biofilm load was marked in presence of 2% of glucose. Interestingly, it was observed that the effect was glucose specific and also not due to any change in the sugar-induced physicochemical property of the growth media as the addition of galactose or arabinose could not induce any significant increase in KPWP1 biofilm load. Scanning electron-, confocal laser scanning-microscopic studies and biochemical tests revealed that increased concentrations of glucose could induce increased production of exopolymeric substances, increased number of densely-packed micro-colonies in KPWP1 biofilm and increased hydrophobicity and adherence properties in KPWP1cells.

Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers (ADDOMEx): Toward a Synthesis of Processes and Pathways of Marine Oil Snow Formation in Determining the Fate of Hydrocarbons

Microbes (bacteria, phytoplankton) in the ocean are responsible for the copious production of exopolymeric substances (EPS) that include transparent exopolymeric particles. These materials act as a matrix to form marine snow. After the Deepwater Horizon oil spill, marine oil snow (MOS) formed in massive quantities and influenced the fate and transport of oil in the ocean. The processes and pathways of MOS formation require further elucidation to be better understood, in particular we need to better understand how dispersants affect aggregation and degradation of oil. Toward that end, recent work has characterized EPS as a function of microbial community and environmental conditions. We present a conceptual model that incorporates recent findings in our understanding of the driving forces of MOS sedimentation and flocculent accumulation (MOSSFA) including factors that influence the scavenging of oil into MOS and the routes that promote decomposition of the oil post MOS formation. In particular, the model incorporates advances in our understanding of processes that control interactions between oil, dispersant, and EPS in producing either MOS that can sink or dispersed gels promoting microbial degradation of oil compounds. A critical element is the role of protein to carbohydrate ratios (P/C ratios) of EPS in the aggregation process of colloid and particle formation. The P/C ratio of EPS provides a chemical basis for the “stickiness” factor that is used in analytical or numerical simulations of the aggregation process. This factor also provides a relative measure for the strength of attachment of EPS to particle surfaces. Results from recent laboratory experiments demonstrate (i) the rapid formation of microbial assemblages, including their EPS, on oil droplets that is enhanced in the presence of Corexit-dispersed oil, and (ii) the subsequent rapid oil oxidation and microbial degradation in water. These findings, combined with the conceptual model, further improve our understanding of the fate of the sinking MOS (e.g., subsequent sedimentation and preservation/degradation) and expand our ability to predict the behavior and transport of spilled oil in the ocean, and the potential effects of Corexit application, specifically with respect to MOS processes (i.e., formation, fate, and half-lives) and Marine Oil Snow Sedimentation and Flocculent Accumulation.

Colonisation dynamics of Listeria monocytogenes strains isolated from food production environments

Listeria monocytogenes is a ubiquitous bacterium capable of colonising and persisting within food production environments (FPEs) for many years, even decades. This ability to colonise, survive and persist within the FPEs can result in food product cross-contamination, including vulnerable products such as ready to eat food items. Various environmental and genetic elements are purported to be involved, with the ability to form biofilms being an important factor. In this study we examined various mechanisms which can influence colonisation in FPEs. The ability of isolates (n = 52) to attach and grow in biofilm was assessed, distinguishing slower biofilm formers from isolates forming biofilm more rapidly. These isolates were further assessed to determine if growth rate, exopolymeric substance production and/or the agr signalling propeptide influenced these dynamics and could promote persistence in conditions reflective of FPE. Despite no strong association with the above factors to a rapid colonisation phenotype, the global transcriptome suggested transport, energy production and metabolism genes were widely upregulated during the initial colonisation stages under nutrient limited conditions. However, the upregulation of the metabolism systems varied between isolates supporting the idea that L. monocytogenes ability to colonise the FPEs is strain-specific.

Transcriptome-wide responses of aggregates of the diatom Odontella aurita to oil

Diatom aggregates can play an important role in the formation of marine oil snow and the transport of oil from the sea surface to the benthos, yet their molecular response to oil has not been characterized. Here we use RNA-seq to analyze the transcriptome-wide responses of aggregates of the common Gulf of Mexico diatom Odontella aurita exposed to the water accommodated fraction of 2 different types of oil, Macondo surrogate and Refugio Beach oil. We identify a common set of 353 genes that are differentially expressed in response to both Macondo and Refugio oil exposure, relative to controls. Genes related to photosynthesis, and nuclear and ribosomal processes were all down-regulated in oil treatments, while genes related to repairing membrane damage, cellular stress, and the production of exopolymeric substances were up-regulated. Differential expression of genes was often greater in magnitude in the Refugio than the Macondo oil treatment, which may be due to differences in oil concentration in the treatments or to the physiochemical characteristics of the oils. Exposure to Refugio oil induced more severe nucleolar stress and more damage to chloroplasts than the lighter Macondo oil, which triggered an up-regulation of a more diverse suite of stress-response genes.

Developing Liquid Rhizobium Inoculants with Enhanced Long-Term Survival, Storage Stability, and Plant Growth Promotion Using Ectoine Additive.

Liquid microbial inoculants have recently received great attention due to their vital roles for sustainable agricultural practices. However, long-term conservation under ambient temperature conditions and deleterious environmental factors might negatively impact microbial cell survival and limit their efficacy in the field. Thus, developing efficient liquid formulation providing prolonged survival of rhizobia in the final product and after an application is crucial. Therefore, this study investigates the effect of various additives on the long-term survival of rhizobia stored in liquid cultures at room temperature (25°C) for 12months. Various yeast sucrose media amended with polyvinylpyrrolidone (PVP) or gum arabic as colloidal agents in combination with ectoine (as a compatible solute) and/or glycerol were evaluated. A dramatic decline in viable cell count was obtained in formulas amended only with PVP from Log 8.5 to Log 5 in the first six months and then to Log 1.5 after 12months. In contrast, rhizobia stored at PVP-based formulas amended with 10 mg L‒1ectoine exhibited almost constant survival level till the end of the storage period. The same trend was obtained using formulas based on gum arabic as a colloidal dispersing agent; however, less decline in cell count using a formula containing gum arabic alone as compared to using PVP. On the other hand, PVP based formulas exhibited higher viscosity compared with another formula. Increased viscosity till the 8th month of storage was achieved in the presence of ectoine indicating the increase of exopolymeric substances production. Electrophoretic protein pattern of rhizobial cells (stored for 12months) exhibited several low molecular weight protein bands in cells stored in PVP based formula with ectoine as compared to the other treatments. Thus, the amendment of the liquid formulation of rhizobia bioinoculant with PVP plus ectoine not only improved cell survival but also enhanced the culture viscosity and consequently ameliorate the colonization and performance of rhizobial inoculants.

Physical properties of epilithic river biofilm as a new lead to perform pollution bioassessments in overseas territories

Chlordecone (CLD) levels measured in the rivers of the French West Indies were among the highest values detected worldwide in freshwater ecosystems, and its contamination is recognised as a severe health, environmental, agricultural, economic, and social issue. In these tropical volcanic islands, rivers show strong originalities as simplified food webs, or numerous amphidromous migrating species, making the bioindication of contaminations a difficult issue. The objective of this study was to search for biological responses to CLD pollution in a spatially fixed and long-lasting component of the rivers in the West Indies: the epilithic biofilm. Physical properties were investigated through complementary analyses: friction, viscosity as well as surface adhesion were analyzed and coupled with measures of biofilm carbon content and exopolymeric substance (EPS) production. Our results have pointed out a mesoscale chemical and physical reactivity of the biofilm that can be correlated with CLD contamination. We were able to demonstrate that epilithic biofilm physical properties can effectively be used to infer freshwater environmental quality of French Antilles rivers. The friction coefficient is reactive to contamination and well correlated to carbon content and EPS production. Monitoring biofilm physical properties could offer many advantages to potential users in terms of effectiveness and ease of use, rather than more complex or time-consuming analyses.

Biometallurgical recovery of metals from waste printed circuit boards using pure and mixed strains of Acidithiobacillus ferrooxidans and Acidiphilium acidophilum

Bioleaching ability of two acidophiles, viz. Acidithiobacillus ferrooxidans, a gammaproteobacteria, and Acidiphilium acidophilum, an alphaproteobacteria in pure and mixed strains was comparatively evaluated for the recovery of metals from waste printed circuit boards (WPCBs) of computer at pulp density in the range of 7.5−15 g/L. Results indicated a variation in metal leachability, showing improved bioleaching by the mixed strain. Mixed strain demonstrated the highest bioleaching of 96% Cu, 94.5% Zn, 75% Ni, and 74.5% Pb from the pulverized particles ranging from 0.075–1 mm size with a pulp density of 7.5 g/L at 18 days with asymptoticity from 12 days onward. Further, the corresponding maximum bioleaching efficiency by the mixed culture for precious and rare earth metals achieved was 46% Ag, 38% Au, and 31% Sc, 27% each of Ce and La, and 24% Nd. Improved bioleaching by the mixed culture may be due to the synergistic growth of the bacterial strains producing a collective effect leading to efficient metal solubilization. Higher production of exopolymeric substances (EPS) by the mixed culture demonstrated their remarkable role in metal bioleaching. A yield of greater than 99% of base metals by fractional chemical precipitation of bioleachate served as a concluding step in metal recovery.

Experimental formation of clay-coated sand grains using diatom biofilm exopolymers

Abstract In sedimentary environments, clay and sand are segregated by hydrodynamic processes. Yet, clay coats, thin clay envelopes lining sand grains, are abundant in modern and ancient coastal sedimentary deposits. Here, we present laboratory experiments in which we produced clay-coated quartz sands similar to those observed in modern and ancient estuarine sands. These coats were produced at ambient temperature by mixing exopolymeric substances (EPS) derived from intertidal diatom biofilms with clay minerals and quartz reference materials. The imaging of sediment-EPS mixes using cryo–scanning electron microscopy and atomic force microscopy demonstrated that EPS form organic bridges between clay and quartz. The physicochemical properties of the EPS were characterized independently through wet chemical assays and Fourier transform infrared spectroscopy. The results indicated that several EPS components (e.g., proteins, polysaccharides) had a potential to complex with quartz and clay. Our findings provide novel insights in the importance of biofilms in the aggregation of clay and sand. Detrital coats from ancient estuarine sandstones show textural similarities to the experimental clay-EPS complexes and could be considered as biosignatures of biofilm development and EPS production in past environments.

Removal of metronidazole from aqueous media by C. vulgaris

This current study investigated the removal of metronidazole from aqueous media by C. vulgaris. Two different initial sizes of inoculum (0.05 and 0.5 g L−1) were tested for a wide concentration range of metronidazole (1–50 μM). The effect of metronidazole concentrations on biomass production was studied for 20 days. The exopolymeric substances (EPS) were quantified and correlated with the removal of antibiotics from aqueous media. Specifically, MDZ stimulated the production of EPS in C. vulgaris, which played the major role in the adsorption of this antibiotic. Also, metronidazole significantly influenced the zeta potential of C. vulgaris in the test cultures, indicating a change in surface characteristics. This decrease in surface negative charge caused auto-flocculation phenomena at a stationary phase. Chronic and acute toxicity experiments showed that metronidazole was harmful to C. vulgaris at stationary phase. Results from this study would advance our knowledge on the treatment of metronidazole-contaminated waters with C. vulgaris as a green technology-oriented process.

The interplay of extracellular polymeric substances and oil/Corexit to affect the petroleum incorporation into sinking marine oil snow in four mesocosms

Large amounts of oil containing mucous-like marine snow formed in surface waters adjacent to the Deepwater Horizon spill that was implicated in oil delivery to the seafloor. However, whether chemical dispersants that were used increased or decreased the oil incorporation and sedimentation efficiency, and how exopolymeric substances (EPS) are involved in this process remains unresolved. To investigate the microbial responses to oil and dispersants in different oceanic settings, indicated by EPS production, petro- and non-petro carbon sedimentation, four mesocosm (M) experiments were conducted: 1) nearshore seawater with a natural microbial consortia (M2); 2) offshore seawater with f/20 nutrients (M3); 3) coastal seawater with f/20 nutrients (M4); 4) nearshore seawater with a natural microbial consortia for a longer duration (M5). Four treatments were conducted in M2, M3 and M4 whereas only three in M5: 1) a water accommodated fraction of oil (WAF), 2) a chemically-enhanced WAF prepared with Corexit (CEWAF, not in M5), 3) a 10-fold diluted CEWAF (DCEWAF); and 4) controls. Overall, oil and dispersants input, nutrient and microbial biomass addition enhanced EPS production. Dispersant addition tended to induce the production of EPS with higher protein/carbohydrate (P/C) ratios, irrespective of oceanic regions. EPS produced in M4 was generally more hydrophobic than that produced in M3. The P/C ratio of EPS in both the aggregate and the colloidal fraction was a key factor that regulated oil contribution to sinking aggregates, based on the close correlation with %petro-carbon in these fractions. In the short term (4–5 days), both the petro and non-petro carbon sedimentation efficiencies showed decreasing trends when oil/dispersants were present. In comparison, in the longer-term (16 days), petro-carbon sedimentation efficiency was less influenced by dispersants, possibly due to biological and physicochemical changes of the components of the oil-EPS-mineral phase system, which cooperatively controlled the sinking velocities of the aggregates.

Role of Polysaccharides in Diatom Thalassiosira pseudonana and its Associated Bacteria in Hydrocarbon Presence.

Diatoms secrete a significant amount of polysaccharides, which can serve as a critical organic carbon source for bacteria. The 2010 Deepwater Horizon oil spill exposed the Gulf of Mexico to substantial amounts of oil that also impacted the phytoplankton community. Increased production of exopolymeric substances was observed after this oil spill. Polysaccharides make up a major fraction of exopolymeric substances; however, their physiological role during an oil spill remains poorly understood. Here, we analyzed the role of polysaccharides in the growth and physiology of the oil-sensitive diatom Thalassiosira pseudonana and how they shape the surrounding bacterial community and its activity in the presence of oil. We found that inhibition of chrysolaminarin synthesis had a negative effect on the growth of T pseudonana and intracellular monosaccharide accumulation, which in turn suppressed photosynthesis by feedback inhibition. In addition, by acting as a carbon reserve, chrysolaminarin helped in the recovery of T pseudonana in the presence of oil. Inhibition of chrysolaminarin synthesis also influenced the bacterial community in the free-living fraction but not in the phycosphere. Exposure to oil alone led to increased abundance of oil-degrading bacterial genera and the activity of exoenzyme lipase. Our data show that chrysolaminarin synthesis plays an important role in the growth and survival of T pseudonana in the presence of oil, and its inhibition can influence the composition and activity of the surrounding bacterial community.

The role of microbially-mediated exopolymeric substances (EPS) in regulating Macondo oil transport in a mesocosm experiment

Large-scale exopolymeric substances (EPS) formation was observed in contaminated surface waters of the Gulf of Mexico, after the Deepwater Horizon oil spill. The conditions under which EPS are produced can cause variations in their physico-chemical properties, affecting the fate of the oil through either emulsification or coagulation with particles, followed by aggregation and marine oil snow (MOS) sedimentation. Here, we report results from a four-day mesocosm experiment that examined EPS production, the change in the bulk chemical composition (mainly polysaccharides and proteins), as well as the partitioning into different size fractions (suspended particulate matter (SPM), colloidal fraction, and sinking MOS). The treatments included 1) natural coastal seawater (Control), 2) a water accommodated fraction of oil (WAF), 3) a chemically-enhanced WAF with addition of oil dispersant Corexit (CEWAF), and 4) a diluted CEWAF (DCEWAF). Generally, the presence of WAF stimulated more extracellular polysaccharide production, whereas the addition of Corexit promoted more protein production. Moreover, marine aggregates changed from a terrestrial signature (higher aromaticity, lower EPS content and more uniform monosaccharide distribution) in the early stages of the experiment, to a fresher autochthonous signature (less aromatic, higher EPS content and glucose-dominated monosaccharide distribution) in the later stage of the mesocosm experiment. Corexit promoted the association between oil and proteins, assisting to emulsify the oil in colloids and SPM and delay the settling of EPS and oil (MOS) for the duration of the mesocosm. Furthermore, when oil, especially Corexit-dispersed oil, was present, extracellular polysaccharides were preferentially incorporated into sinking MOS relative to proteins, as evidenced from a lower protein-C/carbohydrate-C ratio of the sinking MOS than those of SPM and colloidal fraction in WAF, CEWAF and DCEWAF, respectively. This is in contrast to a higher protein-C/carbohydrate-C ratio in the sinking marine snow than those of SPM and colloidal fraction in the control. Likely, microbially-mediated extracellular polysaccharides are the key component that anchors the mineral ballast until the aggregates become dense enough and overcome the buoyancy added to the aggregates as a result of their association with oil/Corexit. We conclude that interactions between Corexit and EPS components regulate petroleum hydrocarbon distribution between the water column and sinking MOS.