Microbial Extracellular Polymeric Substances Research Articles

Going nano: A new step toward understanding the processes governing freshwater ooid formation

Marine and freshwater ooids were historically thought to form by purely physicochemical processes in turbulent environments. Recently, organomineralization has been identified as a key process for the initiation of freshwater ooid cortex formation, but the exact biochemical mechanism(s) involved and subsequent contribution to the development of the growing cortex remain unknown. Here, we show that photosynthetic microbes not only enhance early carbonate precipitation around the ooid nucleus but also control the formation of the entire cortex in freshwater ooids from Lake Geneva, Switzerland. Microbial extracellular polymeric substances are first permineralized as amorphous magnesium silicates ( am Mg-Si) before being calcified. An ∼5‰–6‰ depletion of 13 C in ooid cortices compared to both bulk values and carbonate nuclei supports this photosynthetic microbial mechanism and argues against contributions from sulfate-reducing bacteria or methanogens. These data have significant implications for paleoenvironmental studies since photosynthetic microbes now provide an alternative to turbulent hydrodynamic conditions in the formation of freshwater ooids.

PH Dependence of Structure and Surface Properties of Microbial EPS

The flocculation of microorganisms plays a crucial role in bioreactors, and is substantially affected by pH. However, the mechanism for such an effect remains unclear. In this work, with an integrated approach, the pH dependence of structure and surface property of microbial extracellular polymeric substances (EPS), excreted from Bacillus megaterium TF10, and accordingly its flocculation is elucidated. From the Fourier transform infrared spectra and acid-base titration test results, the main functional groups and buffering zones in the EPS responsible for the microbial flocculation are indentified. The laser light scattering analysis reveals that the deprotonated or protonated states of these functional groups in EPS result in more dense and compact structure at a lower pH because of hydrophobicity and intermolecular hydrogen bonds. The zeta potential measurements identify the isoelectric point and indicate that the electrostatic repulsion action of EPS is controlled by pH. The highest flocculation efficiency is achieved near the isoelectric point (pH 4.8). These results clearly demonstrate that the EPS structure, surface properties, and accordingly the microbial flocculation are dependent heavily on pH in solution.

Microbial Extracellular Polymeric Substances: Production, Isolation and Applications

Microbial Extracellular Polymeric Substances: Production, Isolation and Applications

Effects of Oxyanions, Natural Organic Matter, and Bacterial Cell Numbers on the Bioreduction of Lepidocrocite (γ-FeOOH) and the Formation of Secondary Mineralization Products

Microbial reduction of Fe(III) oxides results in the production of Fe(II) and may lead to the subsequent formation of Fe(II)-bearing secondary mineralization products including magnetite, siderite, vivianite, chukanovite (ferrous hydroxy carbonate (FHC)), and green rust; however, the factors controlling the formation of specific Fe(II) phases are often not well-defined. This study examined effects of (i) a range of inorganic oxyanions (arsenate, borate, molybdate, phosphate, silicate, and tungstate), (ii) natural organic matter (citrate, oxalate, microbial extracellular polymeric substances [EPS], and humic substances), and (iii) the type and number of dissimilatory iron-reducing bacteria on the bioreduction of lepidocrocite and formation of Fe(II)-bearing secondary mineralization products. The bioreduction kinetics clustered into two distinct Fe(II) production profiles. "Fast" Fe(II) production kinetics [19-24 mM Fe(II) d(-1)] were accompanied by formation of magnetite and FHC in the unamended control and in systems amended with borate, oxalate, gellan EPS, or Pony Lake fulvic acid or having "low" cell numbers. Systems amended with arsenate, citrate, molybdate, phosphate, silicate, tungstate, EPS from Shewanella putrefaciens CN32, or humic substances derived from terrestrial plant material or with "high" cell numbers exhibited comparatively slow Fe(II) production kinetics [1.8-4.0 mM Fe(II) d(-1)] and the formation of green rust. The results are consistent with a conceptual model whereby competitive sorption of more strongly bound anions blocks access of bacterial cells and reduced electron-shuttling compounds to sites on the iron oxide surface, thereby limiting the rate of bioreduction.

Environmental & Biochemical Effects of Microbial Extracellular Polymeric Substances on the Heavy Metals and Eutrophic Elements in Water Areas: A Review<SUP>*</SUP>

Environmental & Biochemical Effects of Microbial Extracellular Polymeric Substances on the Heavy Metals and Eutrophic Elements in Water Areas: A Review<SUP>*</SUP>

Ferrous phosphate surface precipitates resulting from the reduction of intragrain 6-line ferrihydrite by Shewanella oneidensis MR-1

The reductive biotransformation of 6-line ferrihydrite located within porous silica (intragrain ferrihydrite) by Shewanella oneidensis MR-1 was investigated and compared to the behavior of 6-line ferrihydrite in suspension (free ferrihydrite). The effect of buffer type (PIPES and NaHCO3), phosphate (P), and an electron shuttle (AQDS) on the extent of reduction and formation of Fe(II) secondary phases was investigated under anoxic conditions. Electron microscopy and micro X-ray diffraction were applied to evaluate the morphology and mineralogy of the biogenic precipitates and to study the distribution of microorganisms on the surface of porous silica after bioreduction. Kinetic reduction experiments with free and intragrain ferrihydrite revealed contrasting behavior with respect to the buffer and presence of P. The overall amount of intragrain ferrihydrite reduction was less than that of free ferrihydrite [at 5 mmol L−1 Fe(III)T]. Reductive mineralization was not observed in the intragrain ferrihydrite incubations without P, and all biogenic Fe(II) concentrated in the aqueous phase. Irrespective of buffer and AQDS addition, rosettes of Fe(II) phosphate of approximate 20–30 μm size were observed on porous silica when P was present. The rosettes grew not only on the silica surface but also within it, forming a coherent spherical structure. These precipitates were well colonized by microorganisms and contained extracellular materials at the end of incubation. Microbial extracellular polymeric substances may have adsorbed Fe(II) promoting Fe(II) phosphate nucleation with subsequent crystal growth proceeding in different directions from a common center.

Are preferential flow paths perpetuated by microbial activity in the soil matrix? A review

Recently, the interactions between soil structure and microbes have been associated with water transport, retention and preferential or column flow development. Of particular significance is the potential impact of microbial extracellular polymeric substances (EPS) on soil porosity (i.e., hydraulic conductivity reduction or bioclogging) and of exudates from biota, including bacteria, fungi, roots and earthworms on the degree of soil water repellency. These structural and surface property changes create points of wetting instability, which under certain infiltrating conditions can often result in the formation of persistent preferential flow paths. Moreover, distinct differences in physical and chemical properties between regions of water flow (preferential flow paths) and no-flow (soil matrix) provide a unique set of environmental living conditions for adaptable microorganisms to exist. In this review, special consideration is given to: (1) the functional significance of microbial activity in the host porous medium in terms of feedback mechanisms instigated by irregular water availability and (2) the related physical and chemical conditions that force the organization and formation of unique microbial habitats in unsaturated soils that prompt and potentially perpetuate the formation of preferential flow paths in the vadose zone.

Freshwater calcite precipitates from in vitro mesocosm flume experiments: a case for biomediation of tufas

AbstractFreshwater carbonates (tufas) develop today from the Arctic to the tropics, many being localized about springs and upper water courses. Some Quaternary tufas, especially in the Mediterranean region, extend over tens of square kilometres and exceed 30 m in thickness. Radiometric dating of Holocene deposits shows that many have accumulated at an average rate of 1 mm year−1. However, local precipitation may be much faster and some Holocene deposits may even have outpaced their tropical marine carbonate counterparts. Recently, the study of active sites has attempted to quantify the precipitation mechanisms which lead to tufa deposition. However, field observation and sampling procedures suffer from the inherent disadvantages of uncontrolled fluctuations in environmental conditions during the study programme. These disadvantages compromise any interpretations, particularly where controls on spar versus micrite precipitation are concerned. Many of these problems have been overcome in the current study by the construction and operation of laboratory mesocosm flumes which simulate the natural conditions (e.g. pH, flow rate, ambient temperature and daylight) in which freshwater carbonate (tufa) is deposited. Three mesocosms were supplied with natural river water from tufa precipitating streams and two mesocosms were supplied with UV‐treated (sterile) river water from the same source. One of the untreated flume mesocosms was linked with a calcium reactor, which replaced calcium ions removed during the precipitation process in order to maintain tufa growth over extended experimental runs. Low‐magnesium calcite precipitates (both rhombic sparite grown from long‐crystallite dendrites and short‐crystallite dendrite triad precursors) and micrite peloids (grown from spherulitic precursors) were precipitated in intimate association with biofilm (extracellular polymeric substances) within the four mesocosms supplied with natural river water. Virtually, no tufa‐like precipitate was obtained from the flumes supplied with UV‐treated river water. A second extended run flume experiment was also carried out for comparison purposes using a calcium hydroxide solution in deionized water. Collectively, these experiments provide convincing evidence confirming that the presence of a microbial biofilm strongly influences the precipitation of carbonates in riverine freshwater settings. In particular, experimental results show that micro‐peloidal micrite and short‐crystallite calcite dendrites are only produced in the presence of microbial extracellular polymeric substances.

First evaluation of the applicability of microbial extracellular polymeric substances for corrosion protection of metal substrates

In order to investigate the suitability of extracellular polymeric substances (EPS) produced by bacteria for corrosion inhibition, a system for simulation of microbially influenced corrosion (MIC) was established. Afterwards, various strains of bacteria including sulphate-reducing bacteria (SRB) and Pseudomonas were cultivated and their EPS harvested. These substances were tested in respect to their intrinsic corrosiveness towards different metal substrates. Based on the different behaviour of the EPS concerning their corrosiveness and on electrochemical results a choice of EPS suitable for prospective examinations was made.

Analysis of microbial extracellular polysaccharides in biofilms by HPLC. Part I: development of the analytical method using two complementary stationary phases

The investigation of microbial extracellular polymeric substances (EPS) is helpful for the implementation of analytical methods which are suitable for biofilm analysis in order to understand the architecture and function of biofilms. A procedure for the qualitative and quantitative determination of various monosaccharides, oligosaccharides and uronic acids as important components of the carbohydrate fraction of microbial EPS by high-performance liquid chromatography (HPLC) and refractive index (RI)/UV detection is presented. Porous graphitic carbon and lead-form cation-exchanger have been examined as stationary phases. Therefore, two complementary HPLC methods are presented. To simulate the conditions of hydrolysis, the influences of various salts, acids and alkalis as matrix components have been investigated. Furthermore, the dependencies on the pH value and temperature of the mobile phase have been thoroughly studied. The results showed that the lead-form cation-exchanger is suitable for the separation of the neutral monosaccharides. However, for direct analysis after acidic hydrolysis with H(2)SO(4), HCl or trifluoroacetic acid, an additional purification step, e.g., precipitation or lyophilization, is necessary when the cation-exchanger is used. With the exception of hydrolysis with HCl, the porous graphitic carbon stationary phase can be used without any further purification step and is appropriate for the separation of uronic acids and their gamma-lactones. Additionally, the separation of a single monosaccharide and its derivatives is possible. Analytical parameters including the sensitivities, repeatabilities, limits of detection and limits of quantification of both HPLC methods using the RI detector are presented. The optimized method has been applied for the characterization of alginates and is also suitable for other extracellular polysaccharides in biofilms.

Microbial extracellular polymeric substances: central elements in heavy metal bioremediation.

Extracellular polymeric substances (EPS) of microbial origin are a complex mixture of biopolymers comprising polysaccharides, proteins, nucleic acids, uronic acids, humic substances, lipids, etc. Bacterial secretions, shedding of cell surface materials, cell lysates and adsorption of organic constituents from the environment result in EPS formation in a wide variety of free-living bacteria as well as microbial aggregates like biofilms, bioflocs and biogranules. Irrespective of origin, EPS may be loosely attached to the cell surface or bacteria may be embedded in EPS. Compositional variation exists amongst EPS extracted from pure bacterial cultures and heterogeneous microbial communities which are regulated by the organic and inorganic constituents of the microenvironment. Functionally, EPS aid in cell-to-cell aggregation, adhesion to substratum, formation of flocs, protection from dessication and resistance to harmful exogenous materials. In addition, exopolymers serve as biosorbing agents by accumulating nutrients from the surrounding environment and also play a crucial role in biosorption of heavy metals. Being polyanionic in nature, EPS forms complexes with metal cations resulting in metal immobilization within the exopolymeric matrix. These complexes generally result from electrostatic interactions between the metal ligands and negatively charged components of biopolymers. Moreover, enzymatic activities in EPS also assist detoxification of heavy metals by transformation and subsequent precipitation in the polymeric mass. Although the core mechanism for metal binding and / or transformation using microbial exopolymer remains identical, the existence and complexity of EPS from pure bacterial cultures, biofilms, biogranules and activated sludge systems differ significantly, which in turn affects the EPS-metal interactions. This paper presents the features of EPS from various sources with a view to establish their role as central elements in bioremediation of heavy metals.

Side effects of flux enhancing chemicals in membrane bioreactors (MBRs): study on their biological toxicity and their residual fouling propensity

Soluble and colloidal materials like soluble microbial products (SMP) or extracellular polymeric substances (EPS) are considered to be major foulants in membrane bioreactors (MBRs). Removing these fouling causing substances is thus thought to reduce the fouling of the membrane in general. In addition to traditional strategies for fouling prevention which mostly try to remedy the effects of fouling by air scour, etc., the new and promising method of adding chemicals is being investigated here. Previous tests with 30 different substances have shown that several of these reduce SMP concentration in the supernatant and enhance filtration. Nevertheless, additive dosing might have unknown side effects in filtration systems. Results presented in this study indicate that these additives may themselves cause severe fouling on different membranes if they remain unbound in the liquid phase. Therefore, the thorough control of the dosing rate of these chemicals will be of paramount importance in full scale applications. Biological toxicity of additives was measured in terms of respiration. OUR tests did not show inhibiting effects for most additives. Chitosan even showed an enhanced OUR due to biodegradability. Oxygen transfer could be enhanced for 25% with the addition of a polymer.

Controls on the precipitation of barite (BaSO 4) crystals in calcite travertine at Twitya Spring, a warm sulphur spring in Canada's Northwest Territories

Twitya Spring discharges warm (24 °C), anoxic, sulphide-, calcium- (65 ppm) and barium- (≥ 0.78 ppm) rich spring water to a steep flow path that is inhabited by streamer and mat-forming microbes ( Thiothrix, Beggiatoa, Oscillatoria, Spirulina, diatoms, rod shaped bacteria). Oxidation and CO 2 degassing drive precipitation of elemental sulphur, barite, opaline silica, and calcite. A mound of travertine at the base of the flow path, dominantly composed of bedded barium-enriched crystallographic and noncrystallographic dendritic calcite crystals and calcite cements, hosts three types of barite crystals: type 1 (T1) intergrown tabular crystals that formed in solution, type 2 (T2) tabular and rhombic crystals that nucleated on calcite, and type 3 (T3) subhedral and anhedral microcrystals that nucleated on microbial cell surfaces and in microbial extracellular polymeric substances. The formation and distribution of T1, T2, and T3 barite in the Twitya Spring flow path are controlled by physiochemical gradients, calcite precipitation rates, and adsorption of barium to microbial biomass, all of which vary seasonally and episodically at Twitya Spring. The complex physiochemical and biological controls on barite formation at Twitya Spring both suggest that the classification of biogenic or inorganic sedimentary barite on the basis of crystal size and morphology may be oversimplified. There is also the potential that primary and authigenic barite crystals hosted in carbonates may yield information about the microbial ecology and ambient physiochemistry of their depositional environments.

Novel Steel Corrosion Protection by Microbial Extracellular Polymeric Substances (EPS) – Biofilm-Induced Corrosion Inhibition

Microbially influenced corrosion (MIC) of steel has gained increasing attention in recent years because the damage caused by this process is a significant cost factor for industry. Consequently, inhibition of corrosion and especially the development of corrosion protective films is an important present-day research topic. In this connection, application of microbially produced EPS for mitigating steel corrosion is an innovative idea. However, observations of ”protective” biofilms on metallic surfaces have been previously reported. Their inhibiting effect is generally thought to be caused by oxygen depletion or the formation of passivating layers. In contrast to many conventional corrosion protective methods, EPS or EPS-derived agents would be a cheap and environmentally friendly solution. Extensive research activities are still required, before biofilms or cell-free EPS can be used for corrosion protection on larger scale. In this study, we are developing a novel EPS-based corrosion protection method for unalloyed and corrosion resistant steel in aqueous media, which is based upon the application of microbial metabolic products. EPS of various sulfatereducing bacteria and other microorganisms are investigated for their inhibiting effect. The extent of such inhibition is evaluated in a model test system, in which different steels are subjected to corrosive conditions under sulfate-reducing conditions. To elucidate the protective mechanisms, comparative analyses of the chemical composition of the applied EPS are performed.

Tomographic study of Paleoproterozoic carbonates as key to understanding the formation of molar-tooth structure

X-ray computed tomographic studies of relatively pure Paleoproterozoic limestones from the George Formation, Muskwa Assemblage, northern British Columbia, Canada indicate that molar-tooth structures developed along linked fractures in gel-like semi-plastic carbonate mud, with a high organic content. Where pore fluid and/or gas pressures matched confining loads, MT blobs developed. Where pressure exceeded loads, cracks propagated into adjacent semi-elastic sediment and were rapidly filled by clusters of uniform, equant, microcrystalline carbonate. Where abundant carbonate was not precipitated, incipient cracks and sheets collapsed leaving residual trains of microcrystalline carbonate with similar density to the molar-tooth carbonate. Tomographic studies show that the density of calcite domains within petrographically uniform sheets of MT void-filling calcite is uneven, suggesting that precipitation was not instantaneous, but was propagated from discrete centres. It is here suggested that carbonate production and sediment rheology were both strongly influenced by organic matter. During early sea-floor diagenesis microcrystalline carbonate precipitated within organic-rich sediment with high water content, possibly within decomposing mats of microbial extracellular polymeric substances (EPS). When pore pressures in the host sediment increased in response to cyclic loading by long-period waves, pore fluids containing EPS were injected into newly created fractures, allowing rapid precipitation of molar-tooth carbonate. Because tomographic studies allow detailed resolution of minor density differences, they provide a useful method of evaluating structures in relatively uniform carbonate rocks of any age.

Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge

Laboratory experiments on the activated sludge (AS) process were carried out to investigate the influence of microbial extracellular polymeric substances (EPS), including loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS), on biomass flocculation, sludge settlement and dewaterability. The heat EPS extraction method was modified to include a mild step and a harsh step for extracting the LB-EPS and TB-EPS, respectively, from the sludge suspension. Six lab-scale AS reactors were used to grow AS with different carbon sources of glucose and sodium acetate, and different sludge retention times (SRTs) of 5, 10 and 20 days. The variation in the bioreactor condition produced sludge with different abundances of EPS and different flocculation and separation characteristics. The sludge that was fed on glucose had more EPS than the sludge that was fed on acetate. For any of the feeding substrates, the sludge had a nearly consistent TB-EPS value regardless of the SRT, and an LB-EPS content that decreased with the SRT. The acetate-fed sludge performed better than the glucose-fed sludge in terms of bioflocculation, sludge sedimentation and compression, and sludge dewaterability. The sludge flocculation and separation improved considerably as the SRT lengthened. The results demonstrate that the LB-EPS had a negative effect on bioflocculation and sludge–water separation. The parameters for the performance of sludge–water separation were much more closely correlated with the amount of LB-EPS than with the amount of TB-EPS. It is argued that although EPS is essential to sludge floc formation, excessive EPS in the form of LB-EPS could weaken cell attachment and the floc structure, resulting in poor bioflocculation, greater cell erosion and retarded sludge–water separation.

Microscopic calcite dendrites in cold‐water tufa: implications for nucleation of micrite and cement

AbstractDendritic calcite forms in an active cold‐water tufa system in association with extracellular polymeric substances (EPS) that discontinuously coat bryophytes and cyanobacteria. Dendrites consist of 100–200 nm thick calcite fibres that form 3D lattice‐like domains. In each dendrite domain, fibres have three structurally equal orientations, which correspond in disposition to radii from the centre of a calcite unit cell to the convex triple face junctions on its surface. Fibres do not form in the orientation of the c‐axis. The external form of each dendrite has the shape of half of a shortened octahedron, with an upper triangular surface parallel to the substrate. Dendrite nucleation takes place on or in microbial EPS, whether microbial cells are present or not, and is probably effected by attraction of Ca2+ cations to negatively charged EPS, together with CO2‐degassing and concomitant pH increase of supersaturated spring water in stream splash zones. Ensuing dendrite growth is abiogenic and controlled by diffusion. Dendrite c‐axes are perpendicular to the substrate, probably because the negative charge of EPS forces the orientation of Ca2+ and CO planes within the developing dendrite crystal to be parallel to the EPS film surface. Dendrites are eventually filled and overgrown by solid, syntaxial calcite, which gradually and completely obliterates the dendrites as more familiar calcite crystal forms develop. No trace of the dendritic nucleus remains in the rock record. Calcite crystal nucleation may take place by this mechanism in many marine and meteoric settings, given that microbial EPS is now assumed to be virtually ubiquitous in these environments. This phenomenon could contribute to the development of familiar fabrics such as marine micrite cement and fibrous calcite cement, radial ooids, peloids, ‘abiogenic’ stromatolites, sea floor precipitates, microbialites, tufa, travertine, speleothems, and some meteoric cements. It may also contribute to the substrate‐normal orientation of c‐axes of common cement fabrics.

Organotemplate structures in sedimentary manganese carbonates of the Neoproterozoic Penganga Group, Adilabad, India

Manganese carbonates interstratified with bedded chert in the Chanda Limestone of the Neoproterozoic Penganga Group at Adilabad, south India, have been studied for possible evidence that microbiota played a role in the mediation of early diagenetic Mn-carbonate formation in Precambrian marine sedimentary successions. The manganese carbonate and chert beds occur within a below wave base, deep-water distally steepened ramp succession. High resolution SEM petrography of the manganese carbonates revealed two basic morphologies-spherical to oval-cylindrical shaped microconcretions, and tubular to irregular, elongated, film-like microstructures. Infolded filmy to hollow tubular strand-like internal morphologies of the spherical to oval-cylindrical shaped microconcretions suggest their microbial affinity. The tubular and film morphologies with mesh-like interconnections closely resemble architectures of microbial extracellular polymeric substance (EPS). Mineralization took place on these organotemplates by the process of permineralization as well as replacement in an early diagenetic pore-water environment with reduction of higher manganese oxy-hydroxides by organic matter and consequent increase in dissolved carbonate.

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.

The measurement of microbial carbohydrate exopolymers from intertidal sediments

Measurements of microbial extracellular polymeric substances (EPS) of natural field populations are required to understand the processes of biogenic stabilization and the microbial ecology of intertidal sediments. EPS in sediments can be measured by the phenol‐sulfuric acid assay to measure carbohydrate concentration in sediment samples. We conducted comparative studies of storage and extraction methods for carbohydrate fractions on intertidal estuarine sediments. Measurements of both total and colloidal (material remaining in suspension after aqueous extraction and centrifugation) carbohydrate concentrations were highly dependent on storage conditions, sample size, extraction media, and time. Precipitation (in 70% ethanol) of EPS from saline (25‰) extractions of colloidal carbohydrate (colloidal S) from four different sediment microbial assemblages showed that 20% of colloidal carbohydrate present in extracts consisted of EPS. Concentrations of colloidal S were highly correlated with diatom biomass as determined by chlorophyll a concentrations. Extractions that used 100 mM Na2EDTA as the extracting medium (colloidal EDTA) increased the concentrations of colloidal carbohydrate obtained and, in sediments dominated by bacteria and cyanobacteria, increased the percentage of EPS in the material to 38%. Uronic acids comprised 20% of the total amount of carbohydrate measured in both total sediment and colloidal‐S extracts but increased to 65% in colloidal‐EDTA extracts, suggesting that colloidal‐EDTA extractions remove bacterial capsular EPS and EPS more closely associated with sediment particles.