Kristian Keiding

Extraction of extracellular polymers from activated sludge using a cation exchange resin

Abstract The extraction of water soluble extracellular polymeric substances (EPS) from activated sludge was investigated. The extraction procedure was based upon cation exchange using a cation exchange resin (CER). Activated sludge from two different types of treatment plants responded very similarly to the extraction procedure. The EPS yield was enhanced by increasing the stirring intensity, the amounts of CER added and by increasing the extraction time. For the chosen extraction procedure the yield was twice as high as other commonly used procedures. The extract consisted mainly of protein but also humic compounds, carbohydrates, uronic acids and DNA were found in significant amounts. The extracted amounts and relative fraction of the individual compounds strongly depended on how the extraction was performed. The ratio between protein and carbohydrate was found in the range 3.9–5.1 depending on the extraction time. Humic compounds and DNA were the compounds most easily extracted. HPSEC investigation of the extract revealed that the extraction did not significantly degrade the EPS. Some cell lysis was identified during the extraction for extraction times greater than 1–2 h by observing a decrease in cell number (stained by DAPI, CTC and acridine orange). The lysis was not considered a significant problem for contaminating the EPS. Measurements of the cell number and cell size distribution in the sludge suggested that the cell mass did not account for more than approximately 10–15% of the total organic fraction of the investigated sludge. Two extraction strategies were formulated. One for a very gentle and one for a very effective method. Analytical methods for analysis of sludge and EPS extracts were compared and discussed. A corrected Lowry method for analyzing protein as well as humic compounds was implemented and found suitable.

Physico-chemical characteristics of full scale sewage sludges with implications to dewatering

An investigation was carried out for a variety of different sewage sludges in order to establish correlations between sludge composition, structure and dewatering properties. Results indicated that the fraction of extracellular polymeric substances (EPS) in sludges was the most important parameter with respect to sludge structure. With high EPS contents, sludges had a lower shear sensitivity and lower degree of dispersion. This in turn lead to better filterability in terms of low resistance to filtration (SRF). The floc stabilising role of EPS components was not consistent with DLVO-theory, as the zeta-potential increased with increased EPS content due to increased EPS charge content. This indicates that polymer entanglement is a key factor to stable floc structure. This does not rule out the possible change in dispersion due to changed electrostatic repulsion for a given EPS content. While EPS had a good effect on floc stability and filterability, the cake dry matter content decreased with large EPS contents. This could be due to an osmotic pressure related to the polymer charge quantity, or it could be caused by water entrapment in the floc structures. A high degree of sludge dispersion increased the cake dry matter content in filtration. This mechanism is, however, impractical due to high SRF and not important to conditioned sludge. In practice, dewatering also includes sludge expression. Taking this into account, osmosis related to EPS charges is likely to be increasingly important (increasing the negative effect of EPS content on cake dry matter).

On the stability of activated sludge flocs with implications to dewatering

Abstract It was shown that Ca2+ can be extracted from activated sludge from a plant with biological nitrogen and phosphorus removal either by an ion exchange process, where H+, Na+, K+ or Mg2+ served as counter ions, or by chelation by EGTA. The extraction of Ca2+ led to an increase in the number of small particles and subsequently an increase in the specific resistance to filtration. It was argued that approx. half of the total Ca2+ pool was associated with the exopolymers and thereby formed an alginate-like gel, which constituted the backbone of the floc structure. This notion was further emphasized by the observation that addition of Cu2+ which, along with releasing Ca2+, improved the dewaterability—probably because of the Cu2+ ions ability to maintain the three-dimensional structure of the exopolymers.

Desorption of organic macromolecules from activated sludge: Effect of ionic composition

Abstract Structural properties of activated sludge flocs were found to be sensitive to small changes in ionic strength and ionic composition. Removal of calcium from the bulk water by an ion exchange resin resulted in a release of calcium ions and organic macromolecules from the flocs. Dilution of the activated sludge with deionized water resulted in a release of calcium and organics to bulk water as well. In both types of experiments the small particles in the bulk water showed an increase in the negative surface charge density, as measured by zeta-potential or by colloidal titration. A change in the repulsive forces due to calcium desorption were possibly more important for the observed floc disintegration than were the effects of changes in the ionic strength. The kinetics of dissolution of calcium were shown to be controlled by Fickian diffusion. A model of a typical activated sludge floc, taking into account the colloidal stability, is suggested, where a “cloud” of organic macromolecules and single bacteria are attached with very weak forces to a more rigid backbone consisting of fibers, filamentous bacteria and bacterial colonies. The observed changes in stability of the flocs leading to increased turbidity have major practical implications in the solid-liquid separation, and effects on effluent quality and dewatering are discussed.

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.

Disintegration of activated sludge flocs in presence of sulfide

Addition of sulfide to activated sludge led to a change of floc structure due to specific reduction of Fe(III) to FeS. The change was observed as a weakening of the floc strength leading to an increased shear sensitivity of the flocs. The degree of disintegration thus depended on the shear rate in the system. The floc disintegration led to the dissolution of up to 10% of the total organic matter of the flocs when all Fe(III) was reduced. Bacteria, organic colloids and dissolved extracellular polymeric substances (EPS) were released from the activated sludge flocs. Laboratory-grown microbial flocs, grown without Fe(III), did not disintegrate upon addition of sulfide, which indicates that no direct bacterial response to sulfide occurred. Laboratory-grown microbial flocs grown in presence of Fe(III) showed some disintegration in the presence of sulfide, but not as much as was observed for activated sludge from a treatment plant. From these data and microscopical investigations it appears that the matter released was mainly EPS, colloids and loosely adhered bacteria, probably arising from the wastewater, while the bacterial microcolonies, particularly typical of the laboratory-grown flocs, were not affected by the sulfide treatment. The changes observed in the floc structure and floc stability have important implications for the solid-liquid separation at activated sludge treatment plants.

Influence of microbial activity on the stability of activated sludge flocs

The influence of the microbial activity on the strength of activated sludge flocs was studied in short term experiments (0-3 h). Increased floc strength was generally obtained when the aerobic microbial activity was stimulated by adding substrate. Deflocculation was observed when the aerobic microbial activity was inhibited by (i) anaerobic conditions, (ii) addition of the metabolic inhibitors azide and chloramphenicol and (iii) reduction of the temperature to 4°C. Furthermore, addition of nitrate as electron acceptor under anaerobic conditions partly prevented deflocculation from taking place. These results strongly suggested that microorganisms using oxygen and/or nitrate as electron acceptors were important for maintaining the floc strength. The increase in turbidity under deflocculation was well correlated with the number of bacteria and concentration of protein, humic substances and carbohydrates in the supernatant. However, only approximately 1-2% of the total amount of sludge deflocculated, so the deflocculation could be understood as an erosion of small particles from the larger flocs. The extent of deflocculation under anaerobic conditions could be enhanced by stimulation of the anaerobic biological activity. When anaerobic conditions prevailed, a microbial iron reduction immediately started with iron reduction rates of 4-150 mmol/gVS×h. Subsequently, a decrease in floc strength was observed which could also be observed when the iron-reducing bacterium Shewanella alga BrY was added to the activated sludge. Furthermore, the importance of Fe(III) for the floc strength was illustrated by removal of Fe(III) from the sludge matrix by adding sulphide, which resulted in strong deflocculation. Thus, the deflocculation observed could be either directly due to lack of aerobic microbial activity or indirectly due to change in the local physico-chemical conditions mediated by anaerobic microbial activity.

Anaerobic deflocculation and aerobic reflocculation of activated sludge

Abstract Activated sludge deflocculated when it was exposed to anaerobic conditions and shear. The extent of deflocculation, measured as turbidity, varied with length of anaerobic period and sludge type (age). In order to restore the deflocculated sludge, oxygen was added, and subsequently, in less than 1 h, most of the deflocculated matter was reflocculated. A certain part of the deflocculated floc components remained, however, deflocculated even after several hours of aeration. As the sludge was exposed to repeated deflocculation–reflocculation conditions, the flocs were progressively damaged, and an accumulation of non-flocculated matter was observed. Measurements of the number of bacteria, the concentration of protein, humic substances and carbohydrate in the sludge and the supernatant indicated that the deflocculated material was mainly composed of bacteria and extracellular polymeric substances (EPS) attached to cells or floc fragments. The non-flocculated particles after reflocculation were enriched in bacteria and protein, compared to the average sludge composition. A reduced deflocculation was observed when nitrate acted as an electron acceptor (anoxic conditions), compared to anaerobic conditions. Increased shear and decreased temperature reinforced the deflocculation. Field experiments showed a temporal variation in floc strengths with the weakest flocs present in the winter period.

The shear sensitivity of activated sludge: an evaluation of the possibility for a standardised floc strength test

The presence of micron-sized primary particles is critical in many solid/liquid separation processes, and characterisation of stability with respect to the degree of dispersion is therefore relevant. Characterisation of floc strength so far has mainly dealt with the size of flocs and not addressed quantification with respect to primary particles. In the present work floc strength was quantified in terms of the shear sensitivity (kss) as a standardised parameter based on the recent adhesion-erosion model (AE-model). The shear sensitivity quantifies the degree of dispersion for low total solids contents and intermediate turbulent shear rates. It reflects the affinity of adhesion/erosion of primary particles to and from flocs and thus provides a means of characterising the interaction energy between the sludge colloids.

Observations on dewaterability and physical, chemical and microbiological changes in anaerobically stored activated sludge from a nutrient removal plant

Abstract Effects of anaerobic storage on physical, chemical and microbiological parameters in thickened activated sludge from a plant with biological N and P removal were examined. Short-term (0–1 day) as well as long-term (10–14 days) experiments were performed. The dewaterability of unconditioned sludge, measured as specific resistance to filtration (SRF), showed a steady decline and was strongly reduced after 10 days of anaerobic storage. An increase in turbidity in bulk water was observed and correlated well with the decreased dewaterability. Most of the turbidity could be explained by material not passing a 0.45 μm filter. Presence of free bacteria was observed of which many were motile and came from the flocs. The floc size distribution for flocs larger than 10 μm did not change during the storage. A significant increase in the conductivity of the sludge was observed as a result of a release of ions and other products from bacterial degradation processes. This increased conductivity coincided with an increased turbidity, and disintegration of the flocs was discussed in the context of the stability of the flocs under varying ionic strength. A more detailed study of the processes leading to the increased conductivity was performed. On a short term basis a bacterial release of ions (PO 4 3- , Mg 2+ and K + ) from Poly-P took place. After a few hours of anaerobic storage no more PO 4 3- was released but the levels of Mg 2+ , K 2+ and Ca 2+ were continuously observed to increase. Degradation of organic material was observed resulting in a production of dissolved organic carbon (DOC) and NH 4 + . The amount and the rate of ion release and mineralization were very constant during a year when expressed per amount of volatile solids.