Characterization of exopolymeric substances (EPS) produced by Aeromonas hydrophila under reducing conditions

The aim of this work was to investigate the production of extracellular polymeric substances (EPS) by Aeromonas hydrophila grown under anaerobic conditions. EPS composition was studied for planktonic cells, cells attached to carbon fibre supports using a soluble ferric iron source and cells grown with a solid ferric iron mineral (gossan). Conventional spectrophotometric methods, Fourier transform infrared (FTIR) and confocal laser scanning microscopy (CLSM) were used to determine the main components in the biofilm extracted from the cultures. The key EPS components were proteins, indicating their importance for electron transfer reactions. Carbohydrates were observed mostly on the mineral and contained terminal mannosyl and/or terminal glucose, fucose and N-acetylgalactosamine residues.     

Detection of heavy metals in bacterial biofilms and microbial flocs with the fluorescent complexing agent Newport Green

The complexing agent Newport Green fluoresces upon binding of nickel, zinc or cobalt. It was used to detect nickel or zinc in MOPS buffer, in gel-like matrices, and in natural biofilms and microbial flocs cultivated in the laboratory. The response curves for increasing nickel concentrations indicated an equimolar binding capacity of Newport Green for nickel in MOPS buffer, whereas zinc fluorescence reached saturation in the presence of a 10-fold excess of zinc ions relative to Newport Green molecules. The maximum fluorescence intensity as determined by luminometry was 8-fold and 4-fold above background for nickel and zinc, respectively. The response of Newport Green to either nickel or zinc in the presence of the other metal is consistent with a different binding affinity of Newport Green for the two metals. Zinc binds more strongly to the complexing agent than nickel but it leads to a weaker fluorescent signal which was detectable by luminometry but not by confocal laser scanning microscopy (CLSM). Newport Green was able to complex nickel in the presence of 1% gelatin or agarose as determined by CLSM and image processing. Its application to fully hydrated bacterial biofilms or microbial flocs revealed the presence of nickel outside of cells. The results suggest that in addition to cellular sorption, metals are bound extracellularly by extracellular polymeric substances in intact and undisturbed microbial aggregates. Journal of Industrial Microbiology & Biotechnology (2000) 24, 116–123. Received 11 June 1999/ Accepted in revised form 04 November 1999     

Effect of oxygen concentration on nitrification and denitrification in single activated sludge flocs

Simultaneous nitrification and denitrification (SND) was investigated in the single aeration tank of a municipal wastewater treatment plant. Microelectrode measurements and batch experiments were performed to test for the presence of SND. Microelectrodes recorded the presence of O(2) concentration gradients in individual activated sludge flocs. When the O(2) concentration in the bulk liquid was <45 microM, anoxic zones were detected within flocs with a larger diameter (approximately 3000 microm). The O(2) penetration depth in the floc was found to be dependent on the O(2) concentration in the bulk liquid. Nitrification was restricted to the oxic zones, whereas denitrification occurred mainly in the anoxic zones. The nitrification rate of the activated sludge increased with increasing O(2) concentration in the bulk liquid, up to 40 microM, and remained constant thereafter. SND was observed in the aerated activated sludge when O(2) concentration was in the range of 10 to 35 microM.     

Assessing the Importance of Organic Matrix Materials in Biofilm Chemical Reactivity: Insights from Proton and Cadmium Adsorption onto the Commercially Available Biopolymer Alginate

Biofilms coat the exterior of most water-exposed interfaces, from the surfaces of sediments and rocks to the interior walls of fluid transport systems and even medical and dental apparatus. Composed of a diverse assemblage of microbial species growing in a matrix of extracellular polymeric substances (EPS), biofilms are well-known for their ability to sorb metals and nucleate mineral phases. In this study, purified alginate, a major polysaccharide component of some algal and bacterial EPS, was studied to ascertain its chemical reactivity towards dissolved cadmium and protons, and thus better constrain its role in overall EPS reactivity. FTIR analysis and compositional constraints based on known molecular structure indicate that alginate’s geochemical behaviour is dominated by a single carboxyl functional group. Correspondingly, potentiometric titration data were best fit using a single functional group acidity constant (pK_a) and site concentration of 3.98 ± 0.01 and 1.728 ± 0.02 mol/kg, respectively, which are in agreement with typical carboxyl acidity (pK_a 3–6) and carboxyl functional group concentration based on alginate polymer composition. The logarithm of the Cd-carboxyl complexation constant (log K) was determined to be ?0.52 ± 0.22, lower than carboxyl-Cd stability constants reported from independent studies of isolated microbes. Together, these results place important constraints on organic matrix contributions to overall biofilm reactivity.     

Characterisation of the mineral fraction in extracellular polymeric substances (EPS) from activated sludges extracted by eight different methods

This work characterises the mineral fraction of EPS extracts obtained using eight different methods from two activated sludges by total mineral content determination, Fourier Transformed Infrared spectrometry and with scanning electron microscopy coupled with an EDX probe. Despite EPS dialysis, the EPS extracts displayed a mineral fraction between 2% and 40% of the EPS dry weight depending on the extraction method used. The main mineral elements found in the EPS extract were Ca, Mg, Na, K, Al, Fe, Mn, P, Si and S, but their contents were strongly affected by the extraction method used. Some of the minerals are associated with the organic molecules within the EPS. The presence of mineral particles of various compositions and structures (clays, quartz or carbonate) in the EPS extract with a wide range in size was clearly demonstrated. Moreover, the association of metallic elements with the mineral particles in the EPS extract was highlighted.     

Ultrastructural and genetic characteristics of endolithic cyanobacterial biofilms colonizing Antarctic granite rocks

The precise identification of the cyanobacteria that comprise an endolithic biofilm is hindered by difficulties in culturing the organisms found in these biofilms and a lack of previous molecular and ultrastructural data. This study characterizes, both at the ultrastructural and molecular level, two different cyanobacterial biofilms found in fissures of granite from continental Antarctica. Electron microscopy revealed structural differences between the two biofilms. One was only loosely adhered to the substrate, while the other biofilm showed a closer association between cells and rock minerals and was tightly attached to the substrate. Cells from both biofilms where ultrastructurally distinct, displaying, for instance, clear differences in their sheaths. The amounts of EPS and their organization associated with the cyanobacteria may determine the differences in adhesion and effects on the lithic substrate observed in the biofilms. By sequencing part of the 16S rRNA gene, the two cyanobacteria were also genetically characterized. The gene sequence of the cells comprising the biofilm that was tightly attached to the lithic substrate showed most homology with that of an endolithic cyanobacterium from Switzerland (AY153458), and the cyanobacterial type loosely adhered to the rock, clustered with Acaryochloris marina, the only organism unequivocally known to contain chlorophyll d. This study reveals the presence of at least two different types of endolithic biofilm, dominated each by a single type of cyanobacterium, able to withstand the harsh conditions of the Antarctic climate.     

Potential role of bacterial extracellular polymeric substances as biosorbent material for arsenic bioremediation

Carcinogenic effects of arsenic through consumption of contaminated water are an alarming threat and there is an emergent need to reduce extremely high levels of toxic arsenic from environment. Bacterial biofilms produce polyanionic extracellular polymeric substance (EPS) that is considered an excellent biosorbent material for the remediation of toxic metals and metalloids. This study was aimed to investigate the role of bacterial EPS in arsenic bioremediation. EPS was extracted from biofilm forming and arsenic reducer bacterial strains that were isolated from industrial waste water and characterized biochemically. Fourier transform infrared spectroscopy was also performed to study functional groups. Both Exiguobacterium profundum PT2 and Ochrobactrum ciceri SW1 exhibited enhanced EPS production in the presence of arsenic. Arsenic stress increased protein and carbohydrate contents in the EPS of both bacterial strains as indicated by the peaks of 1363 to 1613 and 1035 to 1218?cm^?1 wavenumbers, respectively to cope with arsenic present in the surroundings. Shifting of peaks in As⁵⁺ treated samples from 1363 to 1379, 847 to 800 and 1211 to 1134?cm^?1 demonstrated the involvement of proteins, carbohydrates and phosphates in the sequestration of arsenic. Scanning electron microscopic examination of EPS revealed structural alterations such as the presence of closely embedded large clumps with interstitial spaces between stacked layers of the EPS of E. profundum PT2 treated with As⁵⁺ displayed the enhanced polysaccharide content and arsenic sorption. Therefore, increased production of bacterial EPS with large number of polyanionic functional groups on its surface having tendency to sequester arsenic through electrostatic or covalent interactions presented EPS an excellent biosorbent material for arsenic bioremediation.     

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Aims: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Methods and Results: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR‐based denaturing gradient‐gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Conclusions: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. Significance and Impact of the Study: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.     

Soluble microbial products (SMP) and soluble extracellular polymeric substances (EPS) from wastewater sludge

Laspidou and Rittmann (Water Research 36:2711–2720, 2002) proposed that the soluble extracellular polymeric substances (EPS) are identical to soluble microbial products (SMP) in sludge liquor. In this paper, we compared the physicochemical characteristics of the SMP and soluble EPS from original and aerobically or anaerobically digested wastewater sludge. The surface charges, particle sizes, residual turbidities of polyaluminum chloride (PACl) coagulated supernatant, and chemical compositions of the SMP and soluble EPS containing suspensions were used as comparison index. Experimental results revealed that the particles in SMP and soluble EPS fractions extracted from original wastewater sludge, before and after digestion, were not identical in all physicochemical characteristics herein measured. The current test cannot support the proposal by Laspidou and Rittmann (Water Research 36:2711–2720, 2002) that SMP is identical to the soluble EPS from a wastewater sludge.     

Anions effects on biosorption of Mn(II) by extracellular polymeric substance (EPS) from Rhizobium etli

Microbial extracellular polymeric substances (EPS) are potential biosorbents for metal remediation and recovery. The Langmuir and Freundlich kinetics of Mn(II) binding by the EPS from a novel Mn(II) oxidising strain of Rhizobium etli were determined. Maximum manganese specific adsorptions (q _max) decreased in the sequence: sulphate (62 mg Mn per g EPS) > nitrate (53 mg g^–1) > chloride (21 mg g^–1). Consideration of the anion during kinetic studies is usually neglected but is important in providing more practical and comparable data between different biosorbent systems.     

Responses of estuarine intertidal microphytobenthic algal assemblages to enhanced ultraviolet B radiation

As a result of ozone depletion, ground doses of ultraviolet B (UVB) radiation in the mid latitudes of the Northern Hemisphere have increased since the 1980s, and current predictions indicate no possible alleviation until at least post 2020. Mudflats and sandflats are important coastal-zone habitats, and support extensive biofilms of benthic microalgae (microphytobenthos). In intertidal situations, these assemblages are exposed to high levels of UVB radiation during periods of tidal exposure. Exposure of intertidal biofilms dominated by epipelic (mud-inhabiting) diatoms to 0, 0.18 or 0.35 W m⁻² UVB radiation for between 4 and 10 days resulted in no significant decreases in the maximum PSII quantum efficiency (F_v/F_m) throughout diel exposure periods. Although the quantum efficiency of electron transport (F_q′/F_m′) showed significant reductions early in some experiments, the major response was an increase in F_q′/F_m′ in UVB exposed biofilms. This increase in F_q′/F_m′ was suggestive of a protective vertical migration down into the sediment. Single-cell and whole biofilm fluorescence imaging demonstrated, for the first time, that motile diatoms are able to detect UVB radiation independently of UVA or photosynthetically active radiation (PAR) and migrate rapidly down (within 15 min) into the sediments to avoid it. This behavioural acclimation mechanism appears to prevent significant accumulation of UVB induced damage to the algae. UVB exposure had no significant effect of biofilm photosynthesis (measured by ¹⁴C carbon fixation), but did alter organic carbon allocation patterns, with significantly less new carbon allocated to intracellular storage (glucan) and extracellular colloidal carbohydrate fractions. Significant reductions in the sediment standing stocks of chlorophyll a (Chl a), colloidal carbohydrates, extracellular polymeric substances (EPS) were seen after 7 days of UVB exposure. This study showed that marine intertidal benthic diatoms use a behavioural strategy to avoid exposure to UVB and that this response is effective as a short-term protection mechanism against UVB damage. However, altered carbon allocation patterns feed forward over time into changes in biofilm biomass and sediment carbohydrate dynamics. This suggests that continual long-term exposure to UVB may impact on sediment carbon cycling and trophic interactions and on the stabilization of sediments by microalgal biofilms through their production of extracellular carbohydrates.     

Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges

The extracellular polymeric substances (EPS) extracted from three granular and one flocculant anaerobic sludges were characterised by size exclusion chromatography (SEC) using two serially linked chromatographic columns in order to obtain more detailed chromatograms. A Superdex peptide 10/300 GL (0.1–7 kDa) and Superdex 20010/300GL (10–600 kDa) from Amersham Biosciences were used in series with a mobile phase at pH 7 with an ionic strength of 0.223 M (phosphate buffer 50 mM and NaCl 150 mM). A part of the EPS molecules displays hydrophobic and/or ionic interactions with the column packing. Interactions could be modified by changing the mobile phase ionic strength or polarity (addition of acetonitrile). The detection wavelength (210 or 280 nm) affects strongly the EPS chromatogram. For a sludge originating from the same type of biofilms (i.e., anaerobic granules), the differences in EPS fingerprints are mainly due to differences in the absorbance of the chromatographic peaks, linked to EPS molecules content and composition. The EPS fingerprint changes significantly when the EPS originate from another type of anaerobic sludges. In addition, EPS fingerprints were affected by the extraction method used (centrifugation only; heat and centrifugation or cationic exchange resin and centrifugation). This phenomenon was observed mainly for the largest and smallest molecules and molecules which display interactions with column packing.     

Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review

A review concerning the definition, extraction, characterization, production and functions of extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment reactors is given in this paper. EPS are a complex high-molecular-weight mixture of polymers excreted by microorganisms, produced from cell lysis and adsorbed organic matter from wastewater. They are a major component in microbial aggregates for keeping them together in a three-dimensional matrix. Their characteristics (e.g., adsorption abilities, biodegradability and hydrophilicity/hydrophobicity) and the contents of the main components (e.g., carbohydrates, proteins, humic substances and nucleic acids) in EPS are found to crucially affect the properties of microbial aggregates, such as mass transfer, surface characteristics, adsorption ability, stability, the formation of microbial aggregates etc. However, as EPS are very complex, the knowledge regarding EPS is far from complete and much work is still required to fully understand their precise roles in the biological treatment process.     

Effect of carbon source on the formation of polyhydroxyalkanoates (PHA) by a phosphate-accumulating mixed culture

In the present work, attention was devoted to understand how different carbon substrates and their concentration can influence the production of PHA by polyphosphate-accumulating bacteria. Acetate, propionate, and butyrate were tested independently. The composition of the polymers formed was found to vary with the substrate used. Acetate leads to the production of a copolymer of hydroxybutyrate (HB) and hydroxyvalerate (HV) with the HB units being dominant. With propionate, HV units are mainly produced and only a small amount of HB is synthesized. When butyrate is used, the amount of polymer formed is much lower with the HB units being produced to a higher extent. The yield of polymer produced per carbon consumed (Y_P/S) was found to diminish from acetate (0.97) to propionate (0.61) to butyrate (0.21). Using a mixture of acetate, propionate, and butyrate and increasing the carbon concentration, although maintaining the relative concentration of each substrate, propionate is primarily consumed and consequently, PHA synthesized was enriched in HV units. The polymers obtained in all experiments were copolymers with the average molecular weight of the most representative fraction higher when hydroxybutyrate units were present in considerable amounts. All the polymers synthesized were found to be quite homogeneous and their average molecular weight is of the same order of magnitude as the ones commercially available.     

Characteristics of extracellular polymeric substances (EPS) fractions from excess sludges and their effects on bioflocculability

Extracellular polymeric substances (EPS) of biological origin are ubiquitous in excess sludges and can be applied as an underlying bioflocculant, owing to their high content of macromolecules and cations. However, low flocculating activity limits the feasibility of their practical applications. This study provides a novel EPS fractionation approach to improve their flocculability by extracting an active EPS fraction and removing the others with low flocculability. The results showed that for two excess sludges (called sludge A and sludge B), the tightly bound EPS (TB-EPS) fraction possessed a high flocculating rate to kaolin suspension compared with the other EPS fractions [i.e., supernatant, slime, and loosely bound EPS (LB-EPS) fraction] (>54.1 ± 1.4% vs <7.8 ± 1.6%). High bioflocculability of TB-EPS fraction could be attributable to high contents of macromolecules (330–1200 kDa) and trivalent cations (Fe³⁺ and Al³⁺). Further investigation reveals that the TB-EPS fraction caused aggregation of particles by bridging and sweep flocculation.     

New insight into the effects of Ca(II) on cake layer structure in submerged membrane bioreactors

The effects of Ca(II) on the structure of the cake layer in submerged membrane bioreactors (SMBRs) were investigated in this study. Three parallel laboratory-scale SMBRs were operated with synthetic municipal wastewater with three Ca(II) levels (82, 208 and 410?mg?l^?1). As the Ca(II) concentration increased, the sludge floc size increased and the molecular weight of the soluble microbial products (SMP) in the bulk liquid decreased. These observations were attributed to the neutralization and bridging function of Ca(II). Furthermore, Ca(II) addition did not change the thickness of the cake layer, but inhibited the deposition of other elements, such as Al, Si, Mg, and Fe. As a result of Ca(II) addition, the cake layer became less compact and more porous. The interspaces among the flocs in the cake layer helped to reduce the membrane fouling potential.     

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na₂CO₃) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca²⁺). From the six extraction methods used, only the Na₂CO₃ extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca²⁺. The Ca²⁺-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.