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.     

Development and application of an enzymatic and cell flotation treatment for the recovery of viable microbial cells from environmental matrices such as anaerobic sludge

Efficient dissociation of microorganisms from their aggregate matrix is required to study the microorganisms without interaction with their native environment (e.g., biofilms, flocs, granules, etc.) and to assess their community composition through the application of molecular or microscopy techniques. To this end, we combined enzymatic treatments and a cell extraction by density gradient to efficiently recover anaerobic microorganisms from urban wastewater treatment plant sludge. The enzymes employed (amylase, cellulase, DNase, and pectinase) as a pretreatment softly disintegrated the extrapolymeric substances (EPS) interlocked with the microorganisms. The potential damaging effects of the applied procedure on bacterial and archaeal communities were assessed by studying the variations in density (using quantitative PCR), diversity (using capillary electrophoresis single-strand conformation polymorphism fingerprinting [CE-SSCP]), and activity (using a standard anaerobic activity test) of the extracted microorganisms. The protocol preserved the general capacity of the microbial community to produce methane under anaerobic conditions and its diversity; particularly the archaeal community was not affected in terms of either density or structure. This cell extraction procedure from the matrix materials offers interesting perspectives for metabolic, microscopic, and molecular assays of microbial communities present in complex matrices constituted by bioaggregates or biofilms.     

The effects of extracellular polymeric substances on the formation and stability of biogranules

Biogranulation is a promising biotechnology developed for wastewater treatment. Biogranules exhibit a matrix microbial structure, and intensive research has shown that extracellular polymeric substances (EPS) are a major component of the biogranule matrix material in both anaerobic and aerobic granules. This paper aims to review the role of EPS in biogranulation, factors influencing EPS production, the effect of EPS on cell surface properties of biogranules, and the relationship of EPS to the structural stability of biogranules. EPS production is substantially enhanced when the microbial community is subject to stressful culture conditions, and the stimulated EPS production in the microbial matrix in turn favours the formation of anaerobic and aerobic granules. EPS can also play an essential role in maintaining the integrity and stability of spatial structure in mature biogranules. It is expected that this paper can provide deep insights into the functions of EPS in the biogranulation process.     

电活性微生物胞外聚合物的特征与应用

电活性微生物是一类能够通过直接接触、导电菌毛或氧化还原介质与电极或者其他细胞进行胞外电子传递的微生物。而在这个过程中,胞外聚合物(extracellular polymeric substances, EPS)扮演着重要的角色。EPS是微生物生长过程中通过细胞裂解、水解分泌的高分子聚合物的混合物,主要由蛋白质、多糖和腐殖质等物质组成。来自电活性微生物的EPS的不同组成成分和特性会对EPS的电活性以及电活性微生物胞外电子传递产生一定的影响,同时在环境应用方面发挥重要作用。因此,为了更全面了解电活性微生物EPS的电活性及其对电活性微生物胞外电子传递的作用,本文总体介绍了电活性微生物EPS的电活性的直接表征方法,再从组成成分、化学性质、物理性质和空间分布4个方面综述了其对EPS电活性的影响及其在电子传递中的作用,介绍了当前电活性微生物EPS在染料废水脱色、重金属吸附、有机污染物的生物转化和渗滤液管理等方面的环境应用,并从表征方法、试验规模和互作机理研究等角度展望了未来的研究方向。     

Microbial community developments and biomass characteristics in membrane bioreactors under different organic loadings

Microbial community developments and biomass characteristics (concentration, particle size, extracellular polymeric substances (EPS), and membrane fouling propensity) were compared when three MBRs were fed with the synthetic wastewater at different organic loadings. Results showed that the bacterial communities dynamically shifted in different ways and the EPS displayed dissimilar profiles under various organic loadings, which were associated with the ratios of food to microorganism and dissolved oxygen levels in the MBRs. The membrane fouling tendency of biomass in the low-loading MBR (0.57 g COD/L day) was insignificantly different from that in the medium-loading MBR (1.14 g COD/L day), which was apparently lower than that in the high-loading MBR (2.28 g COD/L day). The membrane fouling propensity of biomass was strongly correlated with their bound EPS contents, indicating cake layer fouling (i.e., deposition of microbial flocs) was predominant in membrane fouling at a high flux of 30 L/m² h.     

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.     

Selection of commercial hydrolytic enzymes with potential antifouling activity in marine environments

In this work, the marine antifouling potential of some commercially available hydrolytic enzymes acting on the main constituents of extracellular polymeric substances (EPS) involved in bacterial biofilm formation was determined. The selected protease (i.e., alpha-chymotrypsin from bovine pancreas), carbohydrase (i.e., alpha-amylase from porcine pancreas) and lipase (from porcine pancreas) exhibited remarkable hydrolytic activities towards target macromolecules typically composing EPS under a wide range of pHs (6.5-9.0 for alpha-chymotrysin and alpha-amylase; 7.0-8.5 for the lipase) and temperatures (from 10 °C to 30 °C), as well as relevant half-lives (from about 2 weeks to about 2 months), in a marine synthetic water. The activity displayed by each enzyme was poorly affected by the co-presence of the other enzymes, thus indicating their suitability to be employed in combination. None of the enzymes was able to inhibit the formation of biofilm by an actual site marine microbial community when applied singly. However, a mixture of the same enzymes reduced biofilm formation by about 90% without affecting planktonic growth of the same microbial community. This indicates that multiple hydrolytic activities are required to efficiently prevent biofilm formation by complex microbial communities, and that the mixture of enzymes selected in this study has the potential to be employed as an environmental friendly antifouling agent in marine antifouling coatings.     

Bismuth dimercaptopropanol (BisBAL) inhibits formation of multispecies wastewater flocs

Aims: To determine the ability of a bismuth thiol to control floc formation in a multispecies population of micro‐organisms obtained from the activated sludge unit of a wastewater treatment plant. The molecular level mechanisms by which bismuth‐2‐3‐dimercapto‐1‐propanol (BisBAL) inhibits bioaggregation are also elucidated. Methods and Results: Micro‐organisms were grown over a 3‐day period in a batch system by adding glucose as an electron donor to stimulate short‐term heterotrophic activity. Extracellular polymeric substances (EPS) produced by activated sludge micro‐organisms during exponential and stationary growth phases in the presence and absence of BisBAL were characterized using colorimetry, X‐ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. BisBAL at its minimum inhibitory concentration (MIC, 10 μmol l^?1) was most effective in suppressing microbial floc formation. The principal effect of sub‐inhibitory concentrations of BisBAL was to decrease total EPS production while largely preserving homology. Conclusions: Antifouling and bactericidal properties of BisBAL arise from its ability to reduce EPS expression and preferentially suppressing acidic and O‐acetylated carbohydrates and certain protein secondary structures viz. β‐structures, random coils, and α‐and 3‐turn helices. As micro‐organisms exhibited a much weaker tendency to aggregate at lower concentrations of these specific EPS components, they also appear to be important for the formation of microbial flocs and bioaggregates. Significance and Impact of the Study: BisBAL was shown to be highly effective against multispecies microbial aggregation. Novel bismuth‐based biocides could also be potentially employed to control excess sludge production in wastewater treatment systems by inhibiting EPS expression.     

Overview of biopolymer-induced mineralization: What goes on in biofilms?

Bacteria are associated with mineralization and dissolution processes, some of which may enhance or compromise the physical stability of engineered structures. Examples include stabilization of sediment dikes, bioplugging, biogrouting, and self-healing of concrete and limestone structures. In contrast to ‘biologically controlled’ precipitation (e.g. shells) of eukaryote organisms, microbial precipitation primarily results from two major processes: (1) ‘biologically induced’ precipitation, where microbial activities generate biogeochemical conditions that facilitate precipitation; and (2) ‘biologically influenced’ precipitation, where passive interactions of extracellular biopolymers and the geochemical environment drive precipitation. A common location for such biopolymers is the microbial ‘biofilm’ (i.e. cells surrounded within a matrix of extracellular polymeric substances (EPS)). EPS biofilms occur commonly in both natural environments and many engineered surfaces. Emerging evidence now suggests that EPS inhibit, alter or enhance precipitation of calcium carbonate. Functional groups on EPS serve as initial nucleation sites, while other moieties function to control extent and types (e.g. crystals vs. amorphous organominerals) of precipitation. Understanding how to control, or even manipulate, precipitation/dissolution processes within the confines of EPS matrices will influence long-term structural integrities of materials. The present overview explores properties of EPS, and their potentially destructive (dissolution) and constructive (precipitation) effects on precipitation. Initial insight is offered for understanding how biopolymers might be controlled for applied purposes.     

Microbial transformation of biomacromolecules in a membrane bioreactor: implications for membrane fouling investigation

Background

The complex characteristics and unclear biological fate of biomacromolecules (BMM), including colloidal and soluble microbial products (SMP), extracellular polymeric substances (EPS) and membrane surface foulants (MSF), are crucial factors that limit our understanding of membrane fouling in membrane bioreactors (MBRs).

Findings

In this study, the microbial transformation of BMM was investigated in a lab-scale MBR by well-controlled bioassay tests. The results of experimental measurements and mathematical modeling show that SMP, EPS, and MSF had different biodegradation behaviors and kinetic models. Based on the multi-exponential G models, SMP were mainly composed of slowly biodegradable polysaccharides (PS), proteins (PN), and non-biodegradable humic substances (HS). In contrast, EPS contained a large number of readily biodegradable PN, slowly biodegradable PS and HS. MSF were dominated by slowly biodegradable PS, which had a degradation rate constant similar to that of SMP-PS, while degradation behaviors of MSF-PN and MSF-HS were much more similar to those of EPS-PN and EPS-HS, respectively. In addition, the large-molecular weight (MW) compounds (>100 kDa) in BMM were found to have a faster microbial transformation rate compared to the small-MW compounds (<5 kDa). The parallel factor (PARAFAC) modeling of three-dimensional fluorescence excitation-emission matrix (EEM) spectra showed that the tryptophan-like PN were one of the major fractions in the BMM and they were more readily biodegradable than the HS. Besides microbial mineralization, humification and hydrolysis could be viewed as two important biotransformation mechanisms of large-MW compounds during the biodegradation process.

Significance

The results of this work can aid in tracking the origin of membrane foulants from the perspective of the biotransformation behaviors of SMP, EPS, and MSF.     

Microbial agents for decolorization of dye wastewater

Colored dye wastewater presents a formidable task for biological treatment. Depending on how it is generated, wide pH spans and high salt concentrations such as chloride ion often add to the difficulties. Systematic screening for dye decolorizing and/or degrading bioagents from soil and water samples discovered fungi which show dramatic color removal capability (Shen, et al., 1990). One example shows that up to 99% reduction of light absorption at characteristic wavelength of a red dye (200 mg/L) could be obtained within 48 hours. This ability does not appear to be specific toward dyes targeted for action. It clarifies, often beyond detection by naked eyes, a repertoire of colored wastewater samples. These results appeared to be insensitive to wide variations in pH and salt concentration and, they are not limited to one particular fungal species or genus either upon further investigation. This dye adsorption mechanism may be of great significance in uncovering new methods for bio-removal or bio-recovery of dye substances in wastewater.     

Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols

The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g⁻¹ of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.     

Enzymatic activity in the activated-sludge floc matrix

The enzymatic activity of activated sludge was investigated with special emphasis on the localization of the enzymes in the sludge floc matrix. Activated sludge from an advanced activated-sludge treatment plant, performing biological N and P removal, was used. An enzymatic fingerprint was established using a panel of six different enzymes. The fingerprint revealed peptidase as the most dominating specific enzyme tested. By monitoring sludge bulk enzymatic activity over a 3-month period using fluorescein diacetate as an enzyme substrate, considerable variations in activity were observed even over short periods (a few days). The variation in esterase activity was to some extent correlated to the presence of humic compounds in the sludge, but not to the sludge protein content. Comparison of full sludge enzyme activity to the activity of a batch-grown sludge culture indicated that enzymes accumulated in sludge flocs. A large proportion of the exoenzymes were immobilized in the sludge by adsorption in the extracellular polymeric substances (EPS) matrix. This was demonstrated by extraction of EPS from the activated sludge using cation exchange. Contemporary to the release of EPS a very large fraction of the exoenzymes was released into the water. This showed that the exoenzymes should be considered to be an integrated part of the EPS matrix rather than as direct indicators of the microbial activity or biomass.     

藻类胞外聚合物的产生因素及其在污水处理和生物絮凝方面的应用

藻类胞外聚合物(extracellular polymeric substances, EPS)是一种复杂的高分子聚合物,主要由多糖、蛋白质等物质组成。由于EPS具有独特的结构、大的比表面积及含有大量官能团等物理-化学特性,使其在污水处理及微藻生物质的絮凝回收等方面都有着非常重要的作用。本文系统介绍了EPS的组成及特性,重点论述了影响藻类EPS产生的生物因素及非生物因素,如光照、营养盐、pH及温度等,并对EPS在污水处理及生物絮凝方面的应用进行了总结。对藻类EPS产生机制及机理的深入研究有望为微藻提供更广阔的应用前景。     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.     

The use of Magallanic peat as non-conventional sorbent for EDTA removal from wastewater

Kraft mills are responsible for large volumes discharges of highly polluted effluents. Application of new bleaching processes (i.e. total chlorine-free (TCF) process) is already a feasible option to reduce environmental impacts. The current trend in the increase in the production of TCF pulp will proportionally increase the consumption of chelating agents. The most commonly used chelants, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DPTA) are supposed to be relatively persistent substances, poorly degradable in biological treatment facilities and are subsequently considered as environmentally critical compounds. Adsorption could be used as a treatment technique to remove recalcitrant compounds from wastewaters. However, in most cases, sorbent and regeneration costs can make the whole process not economically feasible. The goal of this study was to evaluate the use of Magallanic peat as non-conventional sorbent for EDTA removal from wastewater. Adsorption studies were carried out considering a 2(3) factorial design. pH, temperature and sorbent/sorbate (S/S) relationship effects were evaluated in EDTA adsorption onto Magallanic peat. In addition, adsorption isotherm constants were determined according to the Langmuir and Freundlich models. The results showed that the optimal conditions for EDTA adsorption onto Magallanic peat were 20 degrees C, acid pH (4.0) and a low sorbent/sorbate ratio (0.1/100). At these conditions Magallanic peat showed an adsorption capacity for EDTA (Cs(sat)) of 128.2mg/g, comparable and even better than activated carbon (Cs(sat) 56.5mg/g). EDTA adsorption data at 60 degrees C obtained are not shown due to Magallanic peat degradation phenomena.     

Microbe-surface interactions in biofouling and biocorrosion processes.

The presence of microorganisms on material surfaces can have a profound effect on materials performance. Surface-associated microbial growth, i.e. a biofilm, is known to instigate biofouling. The presence of biofilms may promote interfacial physico-chemical reactions that are not favored under abiotic conditions. In the case of metallic materials, undesirable changes in material properties due to a biofilm (or a biofouling layer) are referred to as biocorrosion or microbially influenced corrosion (MIC). Biofouling and biocorrosion occur in aquatic and terrestrial habitats varying in nutrient content, temperature, pressure and pH. Interfacial chemistry in such systems reflects a wide variety of physiological activities carried out by diverse microbial populations thriving within biofilms. Biocorrosion can be viewed as a consequence of coupled biological and abiotic electron-transfer reactions, i.e. redox reactions of metals, enabled by microbial ecology. Microbially produced extracellular polymeric substances (EPS), which comprise different macromolecules, mediate initial cell adhesion to the material surface and constitute a biofilm matrix. Despite their unquestionable importance in biofilm development, the extent to which EPS contribute to biocorrosion is not well-understood. This review offers a current perspective on material/microbe interactions pertinent to biocorrosion and biofouling, with EPS as a focal point, while emphasizing the role atomic force spectroscopy and mass spectrometry techniques can play in elucidating such interactions.     

细菌生物被膜基质的研究进展

细菌生物被膜(biofilm)附着在生物或者非生物表面,由细菌及其分泌的糖、蛋白质和核酸等多种基质组成的细菌群落,是造成病原细菌持续性感染、毒力和耐药性的重要原因之一.细菌的生物被膜基质由复杂的胞外聚合物(extracellular polymeric substances,EPS)构成,影响生物被膜的结构和功能.本文...     

Integrating microbial ecology in bioprocess understanding: the case of gas biofiltration

Biofilters are packed-bed bioreactors where contaminants, once transferred from the gas phase to the biofilm, are oxidized by diverse and complex communities of attached microorganisms. Over the last decade, more and more studies aimed at opening the back box of biofiltration by unraveling the biodiversity-ecosystem function relationship. In this review, we report the insights provided by the microbial ecology approach in biofilters and we emphasize the parallels existing with other engineered ecosystems used for wastewater treatment, as they all constitute relevant model ecosystems to explore ecological issues. We considered three characteristic ecological indicators: the density, the diversity, and the structure of the microbial community. Special attention was paid to the temporal and spatial dynamics of each indicator, insofar as it can disclose the potential relationship, or absence of relation, with any operating or functional parameter. We also focused on the impact of disturbance regime on the microbial community structure, in terms of resistance, resilience, and memory. This literature review led to mitigated conclusions in terms of biodiversity–ecosystem function relationship. Depending on the environmental system itself and the way it is investigated, the spatial and temporal dynamics of the microbial community can be either correlated (e.g., spatial stratification) or uncoupled (e.g., temporal instability) to the ecosystem function. This lack of generality shows the limits of current 16S approach in complex ecosystems, where a functional approach may be more suitable.     

生物膜形成机理及影响因素探究

生物膜是一种依附于载体材料的特殊微生物聚集体,其大量存在于自然环境中,并在水质净化、废水处理等领域广泛应用.本文介绍了生物膜形成基本原理,综述了有关载体界面性质、胞外多聚物(EPS)关键组分对生物膜形成及其稳定性的影响,并对各学科交叉研究生物膜提供技术思路.