Dataset Information

Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND).

ABSTRACT: A protocol was developed to extract, fractionate, and quantitatively analyze periphyton extracellular polymeric substances (EPS), which obtains both information on the molecular weight (M r) distribution and protein and polysaccharide content. The EPS were extracted from freshwater periphyton between July and December 2011. Organic carbon (OC) compounds from different EPS extracts were analyzed using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND), and total protein and polysaccharide content were quantified. Four distinct OC fractions, on the basis of M r, were identified in all extracts, corresponding to high M r biopolymers (?80-4 kDa), degradation products of humic substances (M r not available), low M r acids (10-0.7 kDa), and small amphiphilic/neutral compounds (3-0.5 kDa). Low C/N ratios (4.3 ± 0.8) were calculated for the biopolymer fractions, which represented 16-38 % of the measured dissolved organic carbon (DOC), indicating a significant presence of high M r proteins in the EPS. Protein and polysaccharide represented the two major components of EPS and, when combined, accounted for the measured DOC in extracts. Differences in specific OC fractions of EPS extracts over the course of the study could be quantified using this method. This study suggests that LC-OCD-OND is a new valuable tool in EPS characterization of periphyton.

SUBMITTER: Stewart TJ

PROVIDER: S-EPMC3633784 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND).

Stewart Theodora J TJ Traber Jacqueline J Kroll Alexandra A Behra Renata R Sigg Laura L

Environmental science and pollution research international 20121012 5

A protocol was developed to extract, fractionate, and quantitatively analyze periphyton extracellular polymeric substances (EPS), which obtains both information on the molecular weight (M r) distribution and protein and polysaccharide content. The EPS were extracted from freshwater periphyton between July and December 2011. Organic carbon (OC) compounds from different EPS extracts were analyzed using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND), and tota ...[more]

PMID: 23065603

Similar Datasets

Project description:While soil microbial ecology, soil organic carbon (SOC) and soil physical quality are widely understood to be interrelated - the underlying drivers of emergent properties, from land management to biochemistry, are hotly debated. Biological binding agents, microbial exudates, or 'extracellular polymeric substances' (EPS) in soil are now receiving increased attention due to several of the existing methodological challenges having been overcome. We applied a recently developed approach to quantify soil EPS, as extracellular protein and extracellular polysaccharide, on the well-characterised soils of the Highfield Experiment, Rothamsted Research, UK. Our aim was to investigate the links between agricultural land use, SOC, transient binding agents known as EPS, and their impacts on soil physical quality (given by mean weight diameter of water stable aggregates; MWD). We compared the legacy effects from long-term previous land-uses (unfertilised grassland, fertilised arable, and fallow) which were established > 50 years prior to investigation, crossed with the same current land-uses established for a duration of only 2.5 years prior to sampling. Continuously fallow and grassland soils represented the poorest and greatest states of structural integrity, respectively. Total SOC and N were found to be affected by both previous and current land-uses, while extractable EPS and MWD were driven primarily by the current land-use. Land-use change between these two extremes (fallow → grass; grass → fallow) resulted in smaller SOC differences (64% increase or 37% loss) compared to MWD (125% increase or 78% loss). SOC concentration correlated well to MWD (adjusted R 2 = 0.72) but the greater SOC content from previous grassland was not found to contribute directly to the current stability (p < 0.05). Our work thus supports the view that certain distinct components of SOC, rather than the total pool, have disproportionately important effects on a soil's structural stability. EPS-protein was more closely related to aggregate stability than EPS-polysaccharide (p values of 0.002 and 0.027, respectively), and ranking soils with the 5 greatest concentrations of EPS-protein to their corresponding orders of stability (MWD) resulted in a perfect match. We confirmed that both EPS-protein and EPS-polysaccharide were transient fractions: supporting the founding models for aggregate formation. We suggest that management of transient binding agents such as EPS -as opposed to simply increasing the total SOC content- may be a more feasible strategy to improve soil structural integrity and help achieve environmental objectives.

Project description:Sinking marine oil snow was found to be a major mechanism in the transport of spilled oil from the surface to the deep sea following the Deepwater Horizon (DwH) oil spill. Marine snow formation is primarily facilitated by extracellular polymeric substances (EPS), which are mainly composed of proteins and carbohydrates secreted by microorganisms. While numerous bacteria have been identified to degrade oil, there is a paucity of knowledge on bacteria that produce EPS in response to oil and Corexit exposure in the northern Gulf of Mexico (nGoM). In this study, we isolated bacteria from surface water of the nGoM that grow on oil or Corexit dispersant. Among the 100 strains isolated, nine were identified to produce remarkable amounts of EPS. 16S rRNA gene analysis revealed that six isolates (strains C1, C5, W10, W11, W14, W20) belong to the genus Alteromonas; the others were related to Thalassospira (C8), Aestuariibacter (C12), and Escherichia (W13a). The isolates preferably degraded alkanes (17-77%), over polycyclic aromatic hydrocarbons (0.90-23%). The EPS production was determined in the presence of a water accommodated fraction (WAF) of oil, a chemical enhanced WAF (CEWAF), Corexit, and control. The highest production of visible aggregates was found in Corexit followed by CEWAF, WAF, and control; indicating that Corexit generally enhanced EPS production. The addition of WAF and Corexit did not affect the carbohydrate content, but significantly increased the protein content of the EPS. On the average, WAF and CEWAF treatments had nine to ten times more proteins, and Corexit had five times higher than the control. Our results reveal that Alteromonas and Thalassospira, among the commonly reported bacteria following the DwH spill, produce protein rich EPS that could have crucial roles in oil degradation and marine snow formation. This study highlights the link between EPS production and bacterial oil-degrading capacity that should not be overlooked during spilled oil clearance.

Project description:Purpose: To obtain an overview of the metabolic changes associated to the absence of KpsM in Synechocystis, in particular on carbon-related metabolic pathways Methods: The trancriptomes of Synechocystis wild type and kpsM mutant were analyzed by RNA sequencing, using three biological replicates. Cultures were grown until reaching an OD730nm of 1.5. Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina®. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina). Raw data (raw reads) of FASTQ format were firstly processed through in-house scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. Paired-end clean reads were mapped to the reference genome using HISAT2 software.HTSeq was used to count the read numbers mapped of each gene, including known and novel genes. And then RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. RPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels. Differential expression analysis between two conditions/groups (three biological replicates per condition) was performed using DESeq2 R package. DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted P value < 0.05 found by DESeq2 were assigned as differentially expressed. Results: Approximately 700 genes (out of the 3636 identified and 215 quantified, representing a coverage of 85,12%) were significantly differentially expressed (P 216 value < 0.05) in the kpsM mutant compared to the wild type. Of those, 406 were down217 regulated, while 297 were up-regulated. Conclusions: Overall, our transcriptomic data indicates alterations in the mechanisms of energy production and conversion in the kpsM mutant compared to the wild type. The approach resulted in the identification of altered levels of transcripts belonging to a variety of functional categories and highlighting a number of key metabolic processes affected by the disruption of kpsM, namely photosynthesis, oxidative phosphorylation and carbon metabolism.

Project description:Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977 Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest.IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.

Dataset Information

Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND).

Publications

Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets