Effective methods for extracting extracellular polymeric substances from Shewanella oneidensis MR-1

2016 ◽  
Vol 74 (12) ◽  
pp. 2987-2996 ◽  
Author(s):  
You-Fen Dai ◽  
Yong Xiao ◽  
En-Hua Zhang ◽  
Li-Dan Liu ◽  
Ling Qiu ◽  
...  
Keyword(s):  
Extracellular Polymeric Substances ◽  
Ethylenediaminetetraacetic Acid ◽  
Heating Temperature ◽  
Three Dimensional ◽  
Shewanella Oneidensis ◽  
Cell Lysis ◽  
Extraction Methods ◽  
Bacterial Cells ◽  
Matrix Spectrum ◽  
Dry Cell

Extracellular polymeric substances (EPS) play crucial roles in bio-aggregate formation and survival of bacterial cells. To develop an effective but harmless method for EPS extraction from Shewanella oneidensis MR-1, five extraction methods, i.e. centrifugation (control), heating (40, 45, 50, and 60 °C), and treatments with H2SO4, ethylenediaminetetraacetic acid (EDTA) and NaOH, were examined, respectively. Results from scanning electron microscope and flow cytometric analyses indicate that MR-1 cells were severely broken by H2SO4, NaOH and heating temperature ≥45 °C. Proteins and polysaccharides in EPS extracted by heating at 40 °C were 7.12 and 1.60 mg g−1 dry cell, respectively. Although EDTA treatment had a relatively lower yield of EPS (proteins and polysaccharides yields of 5.15 and 1.30 mg g−1 dry cell, respectively), cell lysis was barely found after EPS extraction. Three peaks were identified from the three-dimensional excitation–emission matrix spectrum of each EPS sample, suggesting the presence of protein-like substances. Furthermore, the peak intensity was in good accordance with protein concentration measured by the chemical analysis. In short, heating (40 °C) and EDTA treatments were found the most suitable methods for EPS extraction considering the cell lysis and EPS content, composition and functional groups together.

scholarly journals Examination of Extracellular Polymer (EPS) Extraction Methods for Anaerobic Membrane Bioreactor (AnMBR) Biomass

2021 ◽  
Vol 13 (22) ◽  
pp. 12584
Author(s):  
Mohammed A. Galib ◽  
Timothy Abbott ◽  
Hyung-Sool Lee
Keyword(s):  
Membrane Bioreactor ◽  
Extracellular Polymeric Substances ◽  
Ethylenediaminetetraacetic Acid ◽  
Complex Mixture ◽  
Oxygen Demand ◽  
Extraction Methods ◽  
Membrane Reactors ◽  
Anaerobic Microorganisms ◽  
Complex Process ◽  
Anaerobic Membrane Bioreactors

Membrane bioreactor fouling is a complex process, which is typically driven by extracellular polymeric substances (EPS), a complex mixture of polysaccharides, proteins, lipids, humic substances, and other intercellular polymers. While much is known about fouling in aerobic membrane reactors, far less is known about fouling in anaerobic membrane bioreactors (AnMBR). Much of this knowledge, including EPS extraction methods, has been extrapolated from aerobic processes and is commonly assumed to be comparable. Therefore, several extraction methods commonly used for aerobic EPS quantification, including ultrasonication, ethylenediaminetetraacetic acid (EDTA), and formaldehyde plus sodium hydroxide (CH2O+NaOH), were evaluated to determine the most suitable extraction method for EPS of anaerobic microorganisms in an AnMBR. To maximize EPS yields, each extraction was performed four times. Experimental results showed that the EDTA method was best for EPS quantification, based on chemical oxygen demand (COD), dissolved organic carbon (DOC), and protein yields: 1.43 mg COD/mg volatile suspended solids (VSS), 0.14 mg DOC/mg VSS, and 0.11 mg proteins/mg VSS. In comparison, the CH2O+NaOH method maximized the extraction of carbohydrates (0.12 mg carbohydrates/mg VSS). However, multiple extraction cycles with EDTA and ultrasonication exhibited lower extracellular adenosine triphosphate (ATP) concentrations compared to CH2O+NaOH extractions, indicating lower levels of released intracellular substances. Successive EPS extractions over four cycles are better able to quantify EPS from anaerobic microorganisms, since a single extraction may not accurately reflect the true levels of EPS contents in AnMBRs, and possibly in other anaerobic processes.

scholarly journals Optimising extraction of extracellular polymeric substances (EPS) from benthic diatoms: comparison of the efficiency of six EPS extraction methods

2009 ◽  
Vol 60 (12) ◽  
pp. 1201 ◽  
Author(s):  
Eri Takahashi ◽  
Jérôme Ledauphin ◽  
Didier Goux ◽  
Francis Orvain
Keyword(s):  
Extracellular Polymeric Substances ◽  
Cell Lysis ◽  
Extraction Methods ◽  
Benthic Diatoms ◽  
Universal Method ◽  
Intertidal Sediments ◽  
Electronic Microscopy ◽  
Glucose Content ◽  
Diatom Culture ◽  
High Yields

There is no universal method that can be applied to extract bound extracellular polymeric substances (EPS) from benthic diatoms of intertidal sediments without causing cell lysis. Six extraction methods were tested on a diatom culture of Navicula jeffreyi to establish the best compromise between high yields of carbohydrate extraction and minimum cell lysis. Extraction with distilled water provoked cell lysis (as already known). The five other extraction methods (dowex resin, artificial seawater of half salinity and extractions after pretreatment with gluteraldehyde by three methods: water, dowex water and dowex buffer) did not provoke cell lysis as shown by transmission electronic microscopy. This result was confirmed by the minimum release of internal compounds (protein, ATP) and by the low proportions of glucose in dowex-extracted EPS compared with the water-extracted EPS, from which the high glucose content must be inferred as contamination by the chrysolaminaran. The extraction with dowex resin resulted in the second-highest concentration of carbohydrate after the water extraction and the EPS were especially rich in deoxy sugars, hence increasing the hydrophobic feature of these substances. For these reasons, we recommend extraction with dowex, which is also the best method for extracting bound EPS from other biofilms such as in activated sludges.

Comparison of extraction methods for quantifying extracellular polymers in biofilms

1999 ◽  
Vol 39 (7) ◽  
pp. 211-218 ◽  
Author(s):  
Xiaoqi Zhang ◽  
Paul L. Bishop ◽  
Brian K. Kinkle
Keyword(s):  
Extraction Method ◽  
Extracellular Polymeric Substances ◽  
Cell Lysis ◽  
Extraction Methods ◽  
The Other ◽  
Protein Yield ◽  
Total Carbohydrate ◽  
Extracellular Polymers ◽  
Sulfate Reducing ◽  
Edta Extraction

Five commonly used extraction methods - regular centrifugation, EDTA extraction, ultracentrifugation, steaming extraction and regular centrifugation with formaldehyde (RCF) - were selected to study their effectiveness and repeatability in extracting extracellular polymeric substances (EPS) from aerobic/sulfate reducing and nitrifying/denitrifying biofilm samples. Biofilm EPS extraction yields were represented by carbohydrate and protein concentrations; the amount of cell lysis during the extractions was indicated by DNA concentration. The results showed that analyzing wash waters is essential in quantifying biofilm EPS; the contribution of this step varied from 8-50% of the total carbohydrate yield, depending on the extraction method. Among the extraction methods, the RCF extraction gave the greatest carbohydrate yield, the steaming extraction gave the greatest protein yield, and the other three extraction methods gave approximately equivalent amounts of carbohydrate and proteins for both types of biofilm. DNA in the EPS was 27 times smaller than in the pellets, indicating no significant cell lysis occurred during the extractions.

Optimization for extracellular polymeric substances extraction of microbial aggregates

2015 ◽  
Vol 71 (7) ◽  
pp. 1106-1112 ◽  
Author(s):  
Liang Zhu ◽  
Haitian Yu ◽  
Yimei Liu ◽  
Hanying Qi ◽  
Xiangyang Xu
Keyword(s):  
Humic Acid ◽  
Extracellular Polymeric Substances ◽  
Three Dimensional ◽  
Interaction Mechanism ◽  
Extraction Methods ◽  
Aerobic Granules ◽  
Excitation Emission Matrix ◽  
Cation Exchange Resins ◽  
Heat Method ◽  
Exchange Resins

The extracellular polymeric substances (EPS) are important macromolecular components in microbial aggregates. The three EPS extraction methods – ultrasound + cation exchange resins (CER) + sulfide, ultrasound + formamide + NaOH, and ultrasound + heat – were investigated in the study, and the component differences of extracted EPS from the loose flocs and dense aerobic granules were compared using chemical analysis and three-dimensional excitation-emission matrix (3D-EEM). Results showed that the contents of EPS were extracted effectively by ultrasound + formamide + NaOH and ultrasound + heat methods, and the ultrasound + CER + sulfide method did not extract the polysaccharides (PS) or protein (PN) contents from the sludge samples. The 3D-EEM analysis indicated that the nature of peak B/D, peak C/E/F, and peak A/G were attributed to PN-like, humic acid-like and fulvic acid-like fluorophores. All fluorophores can be detected from the EPS extracted through the ultrasound + heat method. Hopefully this will provide more information about the EPS interaction mechanism of microbial aggregates.

Comparison of extraction methods for the characterization of extracellular polymeric substances from aggregates of three biofilm-forming phototrophic microorganisms

2018 ◽  
Vol 64 (11) ◽  
pp. 887-899 ◽  
Author(s):  
Emilie Loustau ◽  
Jean-Luc Rols ◽  
Joséphine Leflaive ◽  
Claire-Emmanuelle Marcato-Romain ◽  
Elisabeth Girbal-Neuhauser
Keyword(s):  
Cation Exchange ◽  
Extracellular Polymeric Substances ◽  
Exchange Resin ◽  
Cation Exchange Resin ◽  
Cell Lysis ◽  
Extraction Methods ◽  
Size Exclusion ◽  
Tween 20 ◽  
Nitzschia Palea ◽  
Phototrophic Microorganisms

This paper aims to define a robust procedure to extract extracellular polymeric substances (EPS) from aggregates of three benthic phototrophic microorganisms: the cyanobacterium Phormidium autumnale, the diatom Nitzschia palea, and the green alga Uronema confervicolum. This study focuses on the extraction efficiency of polysaccharide and protein EPS by using two physical methods (sonication, cation exchange resin) and three chemical methods (formamide, EDTA, Tween 20) with minimum cell lysis. Cell lysis was evaluated by monitoring chlorophyll a release. The results indicated that sonication or incubation of the algae aggregates with 0.25% Tween 20 induced a high level of cell lysis. A combined extraction approach, with an initial dispersing pretreatment (Ultra-Turrax, 13 500 r·min–1, 1 min), followed by formamide addition (0.22%) and then incubation with Dowex cation exchange resin (50 g per g of dry biomass), provided the highest amount of extracted EPS (mostly proteins), with low cell lysis. Furthermore, extracted EPS were characterized by size exclusion chromatography, and the obtained fingerprints revealed similar profiles for the three benthic microorganisms with a majority of low molecular weight polymers (400 to 11 300 Da). However, additional EPS of high (>600 000 Da) and intermediate (20 000 to 80 000 Da) molecular sizes were specifically detected in the diatom extracts.

Process Description of an Unconventional Biofilm Formation by Bacterial Cells Autoagglutinating Through Sticky, Long, and Peritrichate Nanofibers

2019 ◽  
Author(s):  
Yoshihide Furuichi ◽  
Shogo Yoshimoto ◽  
Tomohiro Inaba ◽  
Nobuhiko Nomura ◽  
Katsutoshi Hori
Keyword(s):  
Formation Process ◽  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Waste Treatment ◽  
Large Cell ◽  
Dlvo Theory ◽  
Bacterial Cells ◽  
Chemical Production ◽  
Discontinuous Surface ◽  
Cell Cell

<p></p><p>Biofilms are used in environmental biotechnologies including waste treatment and environmentally friendly chemical production. Understanding the mechanisms of biofilm formation is essential to control microbial behavior and improve environmental biotechnologies. <i>Acinetobacter </i>sp. Tol 5 autoagglutinate through the interaction of the long, peritrichate nanofiber protein AtaA, a trimeric autotransporter adhesin. Using AtaA, without cell growth or the production of extracellular polymeric substances, Tol 5 cells quickly form an unconventional biofilm. In this study, we investigated the formation process of this unconventional biofilm, which started with cell–cell interactions, proceeded to cell clumping, and led to the formation of large cell aggregates. The cell–cell interaction was described by DLVO theory based on a new concept, which considers two independent interactions between two cell bodies and between two AtaA fiber tips forming a virtual discontinuous surface. If cell bodies cannot collide owing to an energy barrier at low ionic strengths but approach within the interactive distance of AtaA fibers, cells can agglutinate through their contact. Cell clumping proceeds following the cluster–cluster aggregation model, and an unconventional biofilm containing void spaces and a fractal nature develops. Understanding its formation process would extend the utilization of various types of biofilms, enhancing environmental biotechnologies.</p><p></p>

Antibiotics Application Strategies to Control Biofilm Formation in Pathogenic Bacteria

2020 ◽  
Vol 21 (4) ◽  
pp. 270-286 ◽  
Author(s):  
Fazlurrahman Khan ◽  
Dung T.N. Pham ◽  
Sandra F. Oloketuyi ◽  
Young-Mog Kim
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Extracellular Polymeric Substances ◽  
Quorum Quenching ◽  
Resistance Mechanisms ◽  
Bacterial Biofilm ◽  
Bacterial Cells ◽  
High Concentration ◽  
Biofilm Inhibition ◽  
Abiotic Surfaces

Background: The establishment of a biofilm by most pathogenic bacteria has been known as one of the resistance mechanisms against antibiotics. A biofilm is a structural component where the bacterial community adheres to the biotic or abiotic surfaces by the help of Extracellular Polymeric Substances (EPS) produced by bacterial cells. The biofilm matrix possesses the ability to resist several adverse environmental factors, including the effect of antibiotics. Therefore, the resistance of bacterial biofilm-forming cells could be increased up to 1000 times than the planktonic cells, hence requiring a significantly high concentration of antibiotics for treatment. Methods: Up to the present, several methodologies employing antibiotics as an anti-biofilm, antivirulence or quorum quenching agent have been developed for biofilm inhibition and eradication of a pre-formed mature biofilm. Results: Among the anti-biofilm strategies being tested, the sub-minimal inhibitory concentration of several antibiotics either alone or in combination has been shown to inhibit biofilm formation and down-regulate the production of virulence factors. The combinatorial strategies include (1) combination of multiple antibiotics, (2) combination of antibiotics with non-antibiotic agents and (3) loading of antibiotics onto a carrier. Conclusion: The present review paper describes the role of several antibiotics as biofilm inhibitors and also the alternative strategies adopted for applications in eradicating and inhibiting the formation of biofilm by pathogenic bacteria.

Mechanism of Cell Lysis in Microfluidic Channel With Integrated Nanocomposite Electrodes

2013 ◽  
Author(s):  
Madhusmita Mishra ◽  
Anil Krishna Koduri ◽  
Aman Chandra ◽  
D. Roy Mahapatra ◽  
G. M. Hegde
Keyword(s):  
Electric Field ◽  
Microfluidic Channel ◽  
Cell Lysis ◽  
Bacterial Cells ◽  
Nano Composite ◽  
Time Resolved ◽  
Ac Electric Field ◽  
Cell Dispersion ◽  
Electrical Lysis ◽  
Nanocomposite Electrodes

This paper reports on the characterization of an integrated micro-fluidic platform for controlled electrical lysis of biological cells and subsequent extraction of intracellular biomolecules. The proposed methodology is capable of high throughput electrical cell lysis facilitated by nano-composite coated electrodes. The nano-composites are synthesized using Carbon Nanotube and ZnO nanorod dispersion in polymer. Bacterial cells are used to demonstrate the lysis performance of these nanocomposite electrodes. Investigation of electrical lysis in the microchannel is carried out under different parameters, one with continuous DC application and the other under DC biased AC electric field. Lysis in DC field is dependent on optimal field strength and governed by the cell type. By introducing the AC electrical field, the electrokinetics is controlled to prevent cell clogging in the micro-channel and ensure uniform cell dispersion and lysis. Lysis mechanism is analyzed with time-resolved fluorescence imaging which reveal the time scale of electrical lysis and explain the dynamic behavior of GFP-expressing E. coli cells under the electric field induced by nanocomposite electrodes. The DNA and protein samples extracted after lysis are compared with those obtained from a conventional chemical lysis method by using a UV–Visible spectroscopy and fluorimetry. The paper also focuses on the mechanistic understanding of the nano-composite coating material and the film thickness on the leakage charge densities which lead to differential lysis efficiency.

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