Ruthenium red adsorption method for measurement of extracellular polysaccharides in sludge flocs

A nondestructive method of measuring extracellular polysaccharides (ECP) in activated sludge floes using Ruthenium Red dye adsorption was developed at the Environmental Engineering Laboratory at the University of Colorado at Boulder. The effects of pH, buffer solution, dye concentration, sludge mass, temperature, and incubation time on dye adsorption was determined. Ruthenium Red dye adsorption to bacterial floes was found to fit a Brunauer-Emmett-Teller (BET) isotherm model. Of the other environmental conditions in the system, pH was found to have the strongest effect on dye adsorption to bacterial flocs. The amount of extra cellular polysaccharides (ECP) measured by Ruthenium Red adsorption was compared with extracellular polysaccharides measured by two chemical extraction methods. Of all methods considered Ruthenium Red dye adsorption measured the highest amount of extracellular polysaccharide with the lowest amount of bacterial cell disruption. Thus, Ruthenium Red dye adsorption was more effective than extraction procedures for measurement of extracellular polysaccharides in activated sludge flocs.     

Adaptation of anaerobic biomass to saline conditions: Role of compatible solutes and extracellular polysaccharides

This study investigated the role of compatible solutes, extracellular polysaccharides (EPS), and nutrients on anaerobic biomass when stressed with salinity. When 1 mM of osmoregulants glycine betaine, α-glutamate and β-glutamate were added separately to serum bottles containing biomass not adapted to sodium, and fed with glucose and 35 g NaCl/L, all the compatible solutes were found to alleviate sodium inhibition, although glycine betaine was found to be the most effective. The effect of glycine betaine on different anaerobic bacterial groups under salinity stress was monitored using VFAs, and showed that methanogens were more protected than propionate utilisers. Moreover, the addition of 1 mM of glycine betaine to anaerobic biomass not adapted to salinity resulted in significantly higher methane production rates compared with anaerobic biomass that was exposed for 4 weeks to 35 g NaCl/L. Interestingly, under saline batch conditions when the medium was replaced totally the culture produced less methane than when only new substrate was added due to compatible solutes cycling between the media and the cell. The elimination of macronutrients from the medium was found to have a more pronounced negative effect on biomass under saline compared with nonsaline conditions, and because of the synthesis of N-compatible solutes sufficient nutrients should always be present. On the other hand, the absence from the medium of micronutrients did not further reduce biomass activity under salinity. Finally, a higher production of EPS was obtained from biomass exposed to higher salt concentrations, and its composition was found to change under different saline conditions and time. As a result, biomass under saline conditions had a slightly higher mean flock size compared with the biomass that was not subjected to salt.     

Distribution and behavior of sludge in upflow reactors for anaerobic treatment of wastewater

A model has been developed and experimentally checked for the physical behavior of sludge in the blanket in upflow reactors. The model is based on the mass balance for the sludge in the blanket, and can be used to predict the distribution of sludge in an upflow reactor in relation with the gas production, sludge settling characteristics, and the linear fluid velocity in the reactor. The quantitative values of the transport factors that are a measure of the efficiency of the transport of sludge by the fluid streams occurring in the reactor were determined experimentally in reactors of 30- and 200-m(3) volumes. As this was done for wastewater containing lower fatty acids as the main organic pollutants and for sludge with good settling characteristics, the predictive value of the model is limited. It may be used for the second (methane forming) step of anaerobic treatment of wastewater.     

Changes in the composition of extracellular polymeric substances in activated sludge during anaerobic storage

Changes in the chemical composition of organic compounds in total activated sludge, activated sludge extracellular polymeric substances (EPS), and sludge bulk water during anaerobic storage (12 days) were studied. The background for the study was that anaerobic storage of activated sludge, which often takes place at wastewater treatment plants before dewatering, causes a deterioration of the dewaterability. The reasons are not known at present, but may be related to changes in exopolymer composition of the flocs. The results showed that a fast decrease in total sludge protein and carbohydrate took place within 3 days of anaerobic storage as a result of degradation processes, which accounted for approximately 20% of the organic fraction. The amount of uronic acids and humic compounds remained almost constant in the sludge. The EPS were extracted from the floc matrix using a cationexchange resin. In the EPS matrix a similar initial (2–3 days) degradation of proteins and carbohydrate took place, whereas the content of DNA and uronic acids showed minor changes. The extractability of humic compounds increased during the first 3 days of storage. No changes in extractability of the carbohydrate were observed. A fraction of the EPS protein was found to be difficult to extract but was observed to be degraded during the anaerobic storage. The EPS composition was further characterized by high-performance size-exclusion chromatography analysis obtained by on-line UV detection and post-column detection of proteins, carbohydrates, humic compounds and DNA. Four fractions of polysaccharides were found, of which only one was responsible for the decrease in the carbohydrate content observed with storage time. The fraction was presumably of low molecular mass. Humic compounds and volatile fatty acids (acetate and propionate) were released to the bulk water from the flocs during the storage. A possible mechanism to explain the reduced dewaterability developed during anaerobic storage, partly because of the observed changes in EPS, is discussed.     

Distribution, localization, and phylogeny of abundant populations of Crenarchaeota in anaerobic granular sludge

Eight anaerobic granular sludges were surveyed for Crenarchaeota using rRNA gene cloning. Microbial arrangement and substrate uptake patterns were elucidated by fluorescent in situ hybridization and beta imaging. Group 1.3 Crenarchaeota represented up to 50% of Archaea and 25% of the total microbiota in five sludges. Crenarchaeota were localized in close association with methanogenic Archaea.     

Microbial community and extracellular polymeric substances analysis of anaerobic granular sludge exposed to selenate,cadmium and zinc

The microbial community and extracellular polymeric substances composition of anaerobic granular sludge exposed to selenate (~10 mg/L), cadmium (Cd) and zinc (Zn) (~2 and 5 mg/L) were investigated by high-throughput sequencing and fluorescence excitation emission matrix (FEEM) spectra, respectively. As a response to selenate, Cd and/or Zn exposure, significant fluorescence quenching of fulvic-like acids and humic-like substances was observed. With selenate, Cd and/or Zn in the influent with respective concentrations of 10, 5 and 5 mg/L, the abundance of the phyla Proteobacteria, Firmicutes, Spirochaetae, Cloacimonetes and Synergistetes increased significantly, and the dominant taxa in the anaerobic granular sludge exposed to Se, Cd and/or Zn were Halothiobacillaceae (10.2%), Pseudomonas (8.8%), Synergistaceae (7.7%), Spirochaetaceae (7.2%), Blvii28 wastewater sludge group (6.7%), Telmatospirillum (4.6%), Veillonellaceae (4.3%), Geobacter (4.0%) and Enterobacteriaceae (3.0%). Compared with the inoculum, the abundance of the archaea Methanobacterium and Methanosaeta decreased to below detection limit in the UASB reactor after 116 days exposure to Se, Cd and Zn.     

Response of extracellular polymeric substances and enzymatic activity to salinity for the waste activated sludge anaerobic fermentation process

Bioprocess and Biosystems Engineering - Salinity (NaCl) was used in waste activated sludge (WAS) anaerobic fermentation system which had been presented to greatly enhance the extracellular...     

Preservation characteristics of anaerobic granular sludge

Summary The preservation characteristics of granular sludges were investigated in terms of storing age and environmental condition. As a preservative index, physicochemical, microbiological, and morphological parameters were measured in order to quantify the change in granular sludge. Results from this research indicated that, as the anaerobic granular sludge had an outstanding preservation characteristic, UASB system could be effectively applied to the industries operating seasonally or intermittently.     

Composition of extracellular polysaccharides ofRhizobium trifolii

Analysis of the extracellular polysaccharides of some related strains ofRhizobium trifolii indicated the presence of glucose, galactose, glucuronic acid, pyruvate and acetate. Small differences in composition between strains were not related to the ability to nodulate or to the capacity of the symbiotic organisms to fix nitrogen. I wish to thank R. D. Woods for sedimentation analyses, R. J. Avery and E. Bird for nitrogen and ash determinations and Miss P. Watson for technical assistance.     

Methanogenic and perchloroethylene-dechlorinating activity of anaerobic granular sludge

The biodegradation and toxicity of tetrachloroethylene (C₂Cl₄) and trichloroethylene (C₂HCl₃) were studied with different anaerobic enrichment cultures using the following electron donors: acetate, propionate, butyrate, methanol, formate and hydrogen. All of them sustained dechlorination except propionate, for which C₂Cl₄ biodegradation rates were not significant. The best results were obtained with butyrate. Hydrogen appeared to be a relevant electron donor for dechlorination with the present cultures. In the presence of specific inhibitors such as bromoethanesulphonate or molybdate, a slight inhibition of dechlorination was observed. According to dechlorination kinetics, Monod-type behaviour was observed up to 120 μM C₂Cl₄ or 200 μM C₂HCl₃ with K _s values around 7 μM for both compounds. Dechlorination was partially inhibited at higher concentrations. In contrast, methanogens, or at least methane production, were more sensitive to the presence of chlorinated ethylenes and inhibition of methanogenesis was observed to different extents over all the C₂Cl₄/C₂HCl₃ concentration range tested, even at the lowest concentrations. Received: 17 April 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Antibody recognition of bacterial surfaces and extracellular polysaccharides

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Nitrogen availability of anaerobic swine lagoon sludge: sludge source effects

Increased numbers of swine producers will be removing sludge from their anaerobic waste treatment lagoons in the next few years, due to sludge exceeding designed storage capacity. Information on availability of nitrogen (N) in the sludge is needed to improve application recommendations for crops. The objective of this study was to investigate possible effects of different companies and types of swine operations on the availability of N in sludge from their associated lagoons. A laboratory incubation study was conducted to quantify the availability of N (i.e. initial inorganic N plus the potentially mineralizable organic N) in the sludge. Nine sludge sources from lagoons of sow, nursery and finishing operations of three different swine companies were mixed with a loamy sand soil (200 mg total Kjeldahl N kg(-1) soil) and incubated at a water content of 0.19 g. water g(-1) dry soil and 25+/-2 degrees C for 12 weeks. Samples were taken at eight times over the 12-week period and analyzed for inorganic N (i.e. NH(4)-N and NO(3)-N) to determine mineralization of organic N in the sludge. Company and type of swine operation had no significant effects (P < 0.05) on the pattern of inorganic N accumulation over time. Thus, inorganic N accumulation from all sludge sources was fit to a first order equation [Nt = Ni + No (1-e(-kt)]. This relationship indicated that of the 200 mg of total sludge N added per kg soil, 23.5% was in the form of potentially mineralizable organic N (No) and 17.5% was in the form of inorganic N (Ni). The sum of these two pools (41%) represents an estimate of the proportion of total N in the applied sludge in plant available form after the 12 week incubation. While plant N availability coefficients were not measured in this study, the lack of significant company or type of swine operation effects on sludge N mineralization suggests that use of the same plant N availability coefficient for sludge from different types of lagoons is justifiable. The validity of this interpretation depends on the assumption that variation in other components of different sludge sources such as Cu and Zn does not differentially alter N uptake by the receiver crops.     

Physical characterization of a phytodewatered anaerobic sludge

Some physical parameters were determined on an organic product derived from a new phytodewatering process of anaerobic sludge, in order to verify its possible use as a substrate. Wetting-drying cycles were carried out with a sandy soil, and with mixtures of this soil with the organic product taken before and after the phytodewatering process. The phytodewatering process decreased bulk density while raising aggregate stability and friability.     

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.     

Kinetics and mass-transfer phenomena in anaerobic granular sludge

The kinetic properties of acetate-degrading methanogenic granular sludge of different mean diameters were assessed at different up-flow velocities (V(up)). Using this approach, the influence of internal and external mass transfer could be estimated. First, the apparent Monod constant (K(S)) for each data set was calculated by means of a curve-fitting procedure. The experimental results revealed that variations in the V(up) did not affect the apparent K(S)-value, indicating that external mass-transport resistance normally can be neglected. With regard to the granule size, a clear increase in K(S) was found at increasing granule diameters. The experimental data were further used to validate a dynamic mathematical biofilm model. The biofilm model was able to describe reaction-diffusion kinetics in anaerobic granules, using a single value for the effective diffusion coefficient in the granules. This suggests that biogas formation did not influence the diffusion-rates in the granular biomass.