Comparison of Bacterial Extracellular Polymer Extraction Methods

Five different bacterial extracellular polymer extraction methods and a combination of two of these methods were compared on cultures of activated sludge, synthetic activated sludge, and Klebsiella aerogenes. High-speed centrifugation was the most effective extraction method for the K. aerogenes culture, based on the comparatively small amount of cell disruption and the relatively high extracellular polymer yield. Steaming treatment was the most effective extraction method for the activated sludges, since it released a significant quantity of extracellular polymers from the flocs and caused less cellular disruption than ethylenediaminetetraacetic acid and sodium hydroxide treatments. Sodium hydroxide treatment caused extensive disruption in all cultures. Ultrasonication released low concentrations of extracellular polymers from all cultures. However, it caused no significant cell disruption and therefore may be useful as a preliminary treatment in conjunction with another extraction method.     

Structure of activated sludge floes

Relatively large activated sludge floes (larger than about 100 mum) were stabilized, using a histological tissue specimen preparation procedure, and then were sliced into sections of 3 to 6 mum thick. The study of these sections, after staining, revealed the internal structure of the activated sludge floes. No uniformity of this structure was found. The distribution of microorganisms and of extracellular polymers (EPs) in the floes varied randomly on the plane of the sections and along the dimension perpendicular to the plane, leaving large water channels and reservoirs in some of the floes. The lack of a characteristic size for the water gaps in the floes and a general self-similar appearance of the sections suggested that the activated sludge floes might be characterized by the fractal concept within a certain size limit. Direct observation of the interior of the floes indicated an abundant presence of extracellular polymers (EPs) in amorphous forms, surrounding microorganisms in most of the floes.     

Extracellular polymers in granular sludge from different upflow anaerobic sludge blanket (UASB) reactors

Thermal extraction was used to quantify extracellular polymers (ECP) in granules from anaerobic upflow reactors. The optimal time for extraction was determined as the time needed before the intracellular material gives a significant contribution to the extracted extracellular material due to cell lysis. ECP contents of 41 to 92 mg · g^–1 volatile suspended solids of granules were found depending on the type of granular sludge examined. The content of polysaccharides, protein and lipids in the extracted ECP was quantified. Furthermore, the different methyl esters of the lipids were determined and quantified. Lower amounts of polysaccharides and proteins were found in the extracellular material from granules grown on methanogenic and acetogenic substrates compared to granules grown on more complex substrates. In contrast, the lipid content was lower on complex substrates. Changing the feed of an upflow anaerobic sludge blanket reactor from a sugar-containing waste-water to a synthetic waste-water containing acetate, propionate and butyrate resulted in a decrease in both the protein and polysaccharide content and an increase in the lipid content of the extracellular material. Furthermore, the amount of protein and polysaccharides in the ECP found under mesophilic conditions was significantly higher than under thermophilic conditions, while the lipid content was lower.     

Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.     

Effect of sludge flocs on the aeration performance of suction-impellers

The authors have investigated a special aspect of the transfer of oxygen: the aeration performance of small-scale suction-impellers in a suspension of activated sludge floes and a performance comparison with clear water. The principle of the impellers is, that in order to disperse air bubbles into the aforementioned liquids, all that we need do is a simple stirring. Due to the impellers the authors could not show a reduction of the oxygen transfer coefficient in the presence of sludge flocs. Former comparative studies of oxygen transfer, using a fixed orifice, did show a reduction in the presence of sludge flocs. Therefore, it seems probable that the effect of activated sludge flocs on the transfer coefficient is linked with the manner in which air is dispersed, i.e., depends on whether an orifice is fixed or moved. The experimental results allow presumption that by developing a high intensity of shear near the location where air bubbles are produced, we are able to minimize the effect of sludge flocs on the process of bubble formation.     

Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.     

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.     

Determination of bulk mass transfer coefficient of biosorption on sludge granule based on liquid membrane mass transfer mechanism

A bulk mass transfer coefficient (BMTC) equation was derived from the mechanism of mass transfer in surface liquid membrane in this study, which was based on the analysis of biosorption process, conservation of mass in sludge granule and the unification of the dimension. A biosorption experiment was carried out in which anoxic sludge from an anoxic baffled reactor for printing and dyeing wastewater treatment was used to adsorb Acid Red GR dye. The results showed that there was a linear regression curve between ln[qe/(qe-q)] (qe and q were the amount adsorbed at equilibrium and at time t, respectively.) and time t. There was also a good agreement between the adsorbate amount measured and that predicted by the equation of BMTC. The BMTC of Acid Red GR dye adsorbed by anoxic sludge was 6.816 kgm(-3)min(-1). Experimental results indicated that the BMTC determined by a simple adsorptive experiment using this equation was credible. It could be a feasible and effective way to determine BMTC of activated sludge for biosorption performance.     

A standardized pre-treatment method of biofilm flocs for fluorescence microscopic characterization

The availability of epifluorescence microscopy opens the door for the visualization of biofilm flocs via the fluorescence staining technique. However, one of the main problems in the application of the method is a missing standardized protocol for sample preparation. In this study, different pre-treatment methods for the simultaneous staining of different sludge flocs components e.g. extracellular polymeric substances (EPS) and microorganisms (MO) were compared. A method was chosen, which remains the shape of sludge flocs from a municipal wastewater treatment plant and allows to stain major parts of the flocs thoroughly: For this the surrounding wastewater was removed by centrifugation. Then the flocs were resuspended in sterile tap water, immobilized by drying on a diagnostic slide and fixated with formaldehyde. The staining of the different compounds was performed with tetramethylrhodamine isothiocyanate-labeled lectin Concanavalin A (TRITC-Con A; glycoconjugates), fluoresceinisothiocyanate isomer I (FITC; amino groups) and 4,6-diamidino-2-phenylindoldihydrochloride (DAPI; nucleic acids). This staining method was successfully applied for protein- and carbohydrate-quantification using gelatin mixtures of dextran and bovine serum albumin (BSA). The pre-treatment and staining methods were appropriate to compare the composition of different biofilm flocs such as nitrifying activated sludge from a municipal wastewater treatment plant and a pure culture of the bacterial strain Pseudomonas putida (ATCC 17514) as revealed by the results of conventional analytical methods.     

A standardized pre-treatment method of biofilm flocs for fluorescence microscopic characterization

The availability of epifluorescence microscopy opens the door for the visualization of biofilm flocs via the fluorescence staining technique. However, one of the main problems in the application of the method is a missing standardized protocol for sample preparation. In this study, different pre-treatment methods for the simultaneous staining of different sludge flocs components e.g. extracellular polymeric substances (EPS) and microorganisms (MO) were compared. A method was chosen, which remains the shape of sludge flocs from a municipal wastewater treatment plant and allows to stain major parts of the flocs thoroughly: For this the surrounding wastewater was removed by centrifugation. Then the flocs were resuspended in sterile tap water, immobilized by drying on a diagnostic slide and fixated with formaldehyde. The staining of the different compounds was performed with tetramethylrhodamine isothiocyanate-labeled lectin Concanavalin A (TRITC-Con A; glycoconjugates), fluoresceinisothiocyanate isomer I (FITC; amino groups) and 4,6-diamidino-2-phenylindoldihydrochloride (DAPI; nucleic acids). This staining method was successfully applied for protein- and carbohydrate-quantification using gelatin mixtures of dextran and bovine serum albumin (BSA). The pre-treatment and staining methods were appropriate to compare the composition of different biofilm flocs such as nitrifying activated sludge from a municipal wastewater treatment plant and a pure culture of the bacterial strain Pseudomonas putida (ATCC 17514) as revealed by the results of conventional analytical methods.     

Kinetics of formation of humic substances in activated sludge systems and their effect on flocculation

Humic substances were isolated during batch aeration studies with activated sludge and a complex waste source, by using concentration and separation techniques that employ reverse osmosis, ultrafitration, and gel permeation chromatography. The study suggests that the formation of high molecular weight humic substances may occur after the removal of the readily available carbon source. The amount of refractory material finally present in the solution will depend on its adsorptive properties toward bacterial cells. The adsorptive characteristics may be determined by the magnitude of the carbohydrate fraction present in the humic substances. If the carbohydrate content decreases, adsorption onto the cells may decrease resulting in an impairment of both the floe formation and settleability of the sludge floes. Decreased adsorption will result in a higher total organic carbon content and an increase in color bearing materials in the effluent.     

Bacterial survival and association with sludge flocs during aerobic and anaerobic digestion of wastewater sludge under laboratory conditions.

The fate of indicator bacteria, a bacterial pathogen, and total aerobic bacteria during aerobic and anaerobic digestion of wastewater sludge under laboratory conditions was determined. Correlation coefficients were calculated between physical and chemical parameters (temperature, dissolved oxygen, pH, total solids, and volatile solids) and either the daily change in bacterial numbers or the percentage of bacteria in the supernatant. The major factor influencing survival of Salmonella typhimurium and indicator bacteria during aerobic digestion was the temperature of sludge digestion. At 28 degrees C with greater than 4 mg of dissolved oxygen per liter, the daily change in numbers of these bacteria was approximately -1.0 log10/ml. At 6 degrees C, the daily change was less than -0.3 log10/ml. Most of the bacteria were associated with the sludge flocs during aerobic digestion of sludge at 28 degrees C with greater than 2.4 mg of dissolved oxygen per liter. Lowering the temperature or the amount of dissolved oxygen decreased the fraction of bacteria associated with the flocs and increased the fraction found in the supernatant.     

Use of a polarographic method to determine copper,nickel and zinc constants of complexation by extracellular polymers extracted from activated sludge

The constants of complexation of extracellular polymers (ECPs) extracted from sludges for three metals, Cu, Ni and Zn at pH 7, were determined using a polarographic method (stripping mercury dropping electrode mode (SMDE)). The curves obtained were exploited according to Chau's or Ruzic's method. The influence of the biochemical composition of ECP on the binding of Cu, Ni and Zn was investigated on six solutions of ECP. Polarography in SMDE mode is a simple method to determine complexation constants of ECP extracted from activated sludges. For each, the number of sites of complexation increased in the following order: Zn≪Ni<Cu. The low number of binding sites onto ECP for Zn did not allow the determination of a constant of complexation. For ECP studied, the constant of complexation was always higher for Cu than for Ni. A statistical study showed that the higher the content of proteins, humic acids and polysaccharides contained in ECP, the more they were able to bind high quantities of Cu. For Ni, the parameters of complexation were linked to the amount of uronic acid. These results suggest that carboxylic groups play a major role in Ni and Cu complexation by ECP at pH 7.     

Decolourisation of the textile dye Astrazon Red FBL by Funalia trogii pellets.

The effects of various conditions such as initial pH, dye concentrations, amount of pellet, temperature and agitation on decolourising activity of Funalia trogii were investigated. These, except initial pH, were all found to be important for dye decolourising activity of F. trogii. The decolourisation of the dye involved adsorption of the dye compound by fungal pellets at the initial stage, followed by the decolourisation through microbial metabolism. Heat-killed pellets were also tested for their ability to decolourise Astrazon Red dye. These pellets adsorbed the dye and 55% decolourisation was obtained in 24 h. But at the second cycle there was only 24% decolourisation. Our observation showed that Astrazon Red dye decolourisation by heat-killed pellets was mainly due to biosorption. The longevity of the decolourisation activity of F. trogii pellets was also investigated in repeated batch mode. Variations in the amount of pellet increased % decolourisation and stability of pellets.     

Bioflocculation as a microbial response to substrate limitations

Previous theories of nutrient supply to microbial floes assumed that transport within the flocs was by molecular diffusion, and they predict that overall nutrient uptake is reduced in floes compared to dispersed cells. Calculations, supported by recent advances in understanding fluid flow through suspended aggregates, however, have shown that substantial fluid flow may occur through highly permeable bacterial floes. Since bioflocculation of microorganisms in bioreactors is known to occur under conditions of low substrate availability, the rate of substrate uptake is assumed to be mass transfer limited. The hydrodynamic environment of a cell then determines cellular uptake rates. Through development of a relative uptake factor, the overall uptake by cells in flocs in sheared fluids and floes attached to bubbles are compared with the uptake by an identical quantity of dispersed cells. Bioflocculation is found to increase the rate of substrate transport to cells in permeable floes compared to dispersed cells, particularly for large-molecular-weight substrates and when bubbles are present.     

Dewatering of Extracellular Polymer and Activated Sludges by Thermochemical Treatment

Of the dewatering characteristics of activated sludges in our previous paper, the dewatering rate of sludge decreased in proportion to increasing amounts of extracellular polymer. As extracellular polymer in activated sludge was one of the important factors in the dewatering process, the change of the dewatering characteristics of thermochemically treated sludge (containing extracellular polymer) were compared with that of extracellular polymer extracted from sludge. The ultrafiltration rate of extracellular polymer flocculated by thermochemical treatment was much faster than that without treatment, indicating improved dewatering characteristics. Under the same treatment conditions, the dewatering characteristics of sludge were also much improved. The addition of the extracellular polymer treated thermochemically below pH 3 had no effect on the dewatering characteristics of the sludge. The addition of a flocculant to the thermochemically treated sludge was found to further improve the dewatering characteristics. The thermochemical treatment under low pH condition facilitated the flaking of the cake from the filter.     

Analysis of the microbial community structure in a membrane bioreactor during initial stages of filtration

Membrane biofouling was investigated during the early stages of filtration in a laboratory-scale membrane bioreactor operated on molasses wastewater. The bacterial diversity and composition of the membrane biofilm and activated sludge were analyzed using terminal restriction fragment length polymorphism coupled with 16S rRNA clone library construction and sequencing. The amount of extracellular polymeric substances produced by bacteria was investigated using spectroscopic methods. The results reveal that the bacterial community of activated sludge differs significantly from that of the membrane biofilm, especially at the initial phase. Phylogenetic analysis based on 16S rRNA gene sequences identified 25 pioneer OTUs responsible for membrane surface colonization. Also, the relationship between the identified bacterial strains and the system specifications was explored.     

Polysaccharides connected with microtubules in the lipotubuloids of Ornithogalum umbellatum L.

Summary The presence of polysaccharides connected with microtubules in the lipotubuloids of Ornithogalum umbellatum L. was detected by the silver hexamine method and Ruthenium Red staining. Polysaccharides are localized on the outer surface of microtubules. Staining of polysaccharides by Ruthenium Red causes an increase in the microtubule walls thickness by 2.5 nm and in their diameter by 4.5 nm.     

Does a change in reactor loading rate affect activated sludge bioflocculation?

A collection of particles held together by different interparticle forces might eventually give rise to the formation of activated sludge flocs. This process is known as bioflocculation and is crucial for both conventional activated sludge systems and membrane bioreactors. Since industrial wastewater treatment plants generally face varying reactor loading rates due to varying production schemes in the facility, this paper investigates the impact of reactor loading rates on activated sludge bioflocculation. For this purpose, two reactors were initially operated at a nominal reactor loading rate (RLR) and afterwards changed to a high and low RLR. Based on the obtained results, it can be observed that sludge under low RLR conditions is prone to floc fragmentation due to an increase in water-soluble extracellular polymeric substances (EPS). The reactor under high RLR indicated increased floc erosion as a result of increased biomass concentration, which might imply more collisions between sludge flocs, releasing small sludge particles from the floc. In the high RLR reactor, no significant increase in EPS was observed. A distinction between the different (de)flocculation phenomena was made based on sludge volume index, effluent suspended solids and EPS data supplemented with microscopic image analysis.     

Application of semi-continuous culture on membrane filters for the study of activated sludge bacteria

Bacterial population of activated sludge flocs from the petrochemical wastewater purification plant was studied by semi-continuous culture on membrane filters. Almost all (90%) of bacterial cells in the flocs were metabolically active. Only a small fraction of bacteria in the flocs was able to use phenol or ethylene glycol as the sole source of carbon. At higher concentrations of these compounds growth of the bacteria was strongly retarded.