Lipase and protease extraction from activated sludge

In the process of wastewater treatment hydrolysis of polymeric substances is the first and rate-limiting step. A closer study of the enzymes catalysing these reactions is essential for a better understanding of the microbial activity in the wastewater treatment process. Therefore, development of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination for the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase activity and negligible protease activity was extracted in the presence of buffer alone, indicating that enzyme extraction was not due to shear force alone. The highest lipase activity was extracted using 0.1% Triton X-100 above which the activity was gradually decreasing. For proteases, the highest activity was obtained in the presence of 0.5% Triton X-100 and no decrease in activity was observed. Differences observed in the extraction efficiency of the two enzymes indicate the need for optimisation of the extraction process for the different enzymes or the extracellular polymeric substances from activated sludge.     

Toward understanding the mechanism of improving the production of volatile fatty acids from activated sludge at pH 10.0

A well-defined fractionation approach for sludge flocs was applied to a better understanding of the underlying mechanism of improving the production of volatile fatty acids (VFA) in the hydrolysis and acidification processes at pH 10.0. Specifically, sludge flocs were fractionated through centrifugation and ultrasound into four fractions: (1) slime, (2) loosely bound extracellular polymeric substances (LB-EPS), (3) tightly bound EPS (TB-EPS) and (4) pellet. Result showed that during 20 days of fermentation, the total VFA production at pH 10.0 was higher, from 2 to 34 times, than that at pH 5.5. At pH 10.0, however, the enzyme activities (i.e. protease, alpha-amylase, alkaline phosphatase and acid phosphatase) in all fractions of sludge flocs were all lower than pH 5.5, which strongly suggests that the biotic effect was not the leading cause of the VFA improvement. Further investigation suggests that pH 10.0 could significantly improve the VFA production through the break of sludge matrix which is usually hydrolyzed by the extracellular enzymes embedded in itself, increase the effective contact between extracellular organic matters and enzymes, and create a favorable environment for microbes to accumulate VFA. Hydrolysis and acidification at pH 10.0 can be considered as part of an appropriate solution for tertiary treatment and contribute to the headway toward the goal of sustainable water treatment technologies.     

Kinetic model of excess activated sludge thermohydrolysis

Thermal hydrolysis of excess activated sludge suspensions was carried at temperatures ranging from 423 K to 523 K and under pressure 0.2-4.0 MPa. Changes of total organic carbon (TOC) concentration in a solid and liquid phase were measured during these studies. At the temperature 423 K, after 2 h of the process, TOC concentration in the reaction mixture decreased by 15-18% of the initial value. At 473 K total organic carbon removal from activated sludge suspension increased to 30%.It was also found that the solubilisation of particulate organic matter strongly depended on the process temperature. At 423 K the transfer of TOC from solid particles into liquid phase after 1 h of the process reached 25% of the initial value, however, at the temperature of 523 K the conversion degree of ‘solid’ TOC attained 50% just after 15 min of the process.In the article a lumped kinetic model of the process of activated sludge thermohydrolysis has been proposed. It was assumed that during heating of the activated sludge suspension to a temperature in the range of 423-523 K two parallel reactions occurred. One, connected with thermal destruction of activated sludge particles, caused solubilisation of organic carbon and an increase of dissolved organic carbon concentration in the liquid phase (hydrolysate). The parallel reaction led to a new kind of unsolvable solid phase, which was further decomposed into gaseous products (CO₂). The collected experimental data were used to identify unknown parameters of the model, i.e. activation energies and pre-exponential factors of elementary reactions. The mathematical model of activated sludge thermohydrolysis appropriately describes the kinetics of reactions occurring in the studied system.     

The function of cation-binding agents in the enzymatic treatment of municipal sludge

Treatment of sludge with enzymes has previously been shown to efficiently release organic matter. However, the added enzymes were partially adsorbed to, entrapped by or bound to the sludge structure. Simultaneous decrease of enzymes activities was observed. Reduced adsorption and more effective, lower, enzyme dose was achieved in sludge pre-treated with three cation-binding agents. The enzymatic solubilisation of sludge was improved by 150%, 240% and 290%, by 50mM sodium tripolyphosphate (STPP), 25mM citric acid (CA) or 50mM ethylenediaminetetraacetate (EDTA), respectively. With cation binders, the lower relative enzyme dose 0.2 (13.7mg/g total solids (TS)) released 3.5 times higher COD than enzyme dose 1 (68.5mg/g TS) alone. In the presence of 25mM CA, 75% added protease remained soluble. In the presence of 50mM CA, EDTA or STPP, 50% of alpha-amylase and cellulase remained soluble. At 200mM STPP, alpha-amylase was inactive, and the efficiency of enzymatic sludge hydrolysis decreased. CA was the most effective of the three cation-binding agents tested. It is biodegradable and can be produced endogenously by the microorganisms in sludge. CA has the greatest potential for the practical application to enhance biogas production. This paper reports on the possible mechanisms of enzymes adsorption to the sludge matrix and possible methods of decreasing the adsorption. We suggest that steric hindrances were responsible for the decreased enzymatic sludge solubilisation and that polyvalent metal ions were directly involved in adsorption of enzymes to sludge matrix. The addition of cation binders eliminated both phenomena and thereby improved the enzymatic solubilisation of sludge.     

Waste activated sludge hydrolysis and short-chain fatty acids accumulation under mesophilic and thermophilic conditions: Effect of pH

The effect of pH (4.0–11.0) on waste activated sludge (WAS) hydrolysis and short-chain fatty acids (SCFAs) accumulation under mesophilic and thermophilic conditions were investigated. The WAS hydrolysis increased markedly in thermophilic fermentation compared to mesophilic fermentation at any pH investigated. The hydrolysis at alkaline pHs (8.0–11.0) was greater than that at acidic pHs, but both of the acidic and alkaline hydrolysis was higher than that pH uncontrolled under either mesophilic or thermophilic conditions. No matter in mesophilic or thermophilic fermentation, the accumulation of SCFAs at alkaline pHs was greater than at acidic or uncontrolled pHs. The optimum SCFAs accumulation was 0.298 g COD/g volatile suspended solids (VSS) with mesophilic fermentation, and 0.368 with thermophilic fermentation, which was observed respectively at pH 9.0 and fermentation time 5 d and pH 8.0 and time 9 d. The maximum SCFAs productions reported in this study were much greater than that in the literature. The analysis of the SCFAs composition showed that acetic acid was the prevalent acid in the accumulated SCFAs at any pH investigated under both temperatures, followed by propionic acid and n-valeric acid. Nevertheless, during the entire mesophilic and thermophilic fermentation the activity of methanogens was inhibited severely at acid or alkaline pHs, and the highest methane concentration was obtained at pH 7.0 in most cases. The studies of carbon mass balance showed that during WAS fermentation the reduction of VSS decreased with the increase of pH, and the thermophilic VSS reduction was greater than the mesophilic one. Further investigation indicated that most of the reduced VSS was converted to soluble protein and carbohydrate and SCFAs in two fermentations systems, while little formed methane and carbon dioxide.     

Anaerobic stabilisation and conversion of biopolymers in primary sludge--effect of temperature and sludge retention time

The effect of sludge retention time (SRT) and process temperature on the hydrolysis, acidification and methanogenesis of primary sludge was investigated in completely stirred tank reactors (CSTRs). The CSTRs were operated to maintain SRTs of 10, 15, 20 and 30 days at process temperatures of 25 degrees C and 35 degrees C. The rates of hydrolysis and the biodegradability of primary sludge were assessed in batch reactors incubated at 15 degrees C, 25 degrees C and 35 degrees C. The results revealed that the major amount of sludge stabilisation occurred between 0 and 10 days at 35 degrees C and 10 and 15 days at 25 degrees C. Hydrolysis was found to be the rate limiting-step of the overall digestion process, for the reactors operated at 35 degrees C and 25 degrees C, except for the reactor operated at 10 days and 25 degrees C. At the latter conditions, methanogenesis was the rate-limiting step of the overall digestion process. Proteins hydrolysis was limited to a maximum value of 39% at 30 days and 35 degrees C due to proteins availability in the form of biomass. The biodegradability of primary sludge was around 60%, and showed no temperature dependency. The hydrolysis of the main biopolymers and overall particulate COD of the primary sludge digested in CSTRs were well described by first-order kinetics, in case hydrolysis was the rate-limiting step. Similarly, the hydrolysis of the overall particulate COD of the primary sludge digested in batch reactors were described by first-order kinetics and revealed strong temperature dependency, which follows Arrhenius equation.     

Characterization and distribution of esterase activity in activated sludge.

The location and activity of esterase enzymes in activated sludge from three municipal wastewater treatment plants were characterized using model substrates and denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) of particulate, freeze-thaw (primarily periplasmic enzymes and those associated with outer cell surfaces) and extracellular fractions of activated sludge bacteria. Particulate and freeze-thaw fractions had a similar spectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C2-C18 monoesters of p-nitrophenol and C2-C8 diesters of fluorescein. Esterase activity was highest with substrates that had short alkyl chains (C4) and decreased as the chain lengths increased beyond C8. Extracellular fractions contained very low levels of protein (<0.1 mg/l) and showed no esterase activity against any of the model substrates tested. Multiple bands were observed upon analysis of particulate and freeze-thaw fractions by non-denaturing PAGE in combination with activity staining using various alpha-naphthol ester substrates (C2-C8). Our results indicate that esterase enzymes in activated sludge are fairly diverse from a structural standpoint but exhibit a high level of functional redundancy, with different enzymes catalyzing the same reactions in different sludges. Extracellular esterase activity was totally absent for the substrates we tested and the esterase activity that we observed was closely linked to a particulate floc or cellular material.     

Stability and maturity of thickened wastewater sludge treated in pilot-scale sludge treatment wetlands

Thickened wastewater activated sludge was treated in 13 pilot-scale sludge treatment wetlands of various configurations that operated continuously for three years in North Greece. Sludge was loaded for approximately 2.5 years, and the beds were left to rest for the remaining period. Three different sludge loading rates were used that represented three different population equivalents. Residual sludge stability and maturity were monitored for the last year. Sludge was regularly sampled and microbial respiration activity indices were measured via a static respiration assay. The phytotoxicity of sludge was quantified via a seed germination bioassay. Measurements of total solids, organic matter, total coliforms, pH and electrical conductivity were also made. According to microbial respiration activity measurements, the sludge end-product was classified as stable. The germination index of the final product exceeded 100% in most wetland units, while final pH values were approximately 6.5. The presence of plants positively affected the stability and maturity of the residual sludge end-product. Passive aeration did not significantly affect the quality of the residual sludge, while the addition of chromium at high concentrations hindered the sludge decomposition process. Conclusively, sludge treatment wetlands can be successfully used, not only to dewater, but also to stabilize and mature wastewater sludge after approximately a four-month resting phase.     

The chemistry of heavy metals in digested sewage sludge—I. Copper(II) complexation with soluble organic matter

Characterisitcs of copper-binding, in the soluble fraction of a digested sewage sludge, were determined using ion-selective electrodes. Results showed that ion exchange with protons is a principal component in copper binding and that the soluble organic matter has a cation exchange capacity of 8.86 m-equiv g⁻¹ at pH 6.5. A pH-dependent exchange constant, defined in terms of mole fractions of exchange components, describes the data in the pH range 2–8 and is independent of both pH and the extent of exchange in the range 5.5–8. Supplementary data include acid/base titration curves and simple kinetic measurements. Anion uptake is insignificant in the pH range 2.5–7. A model of the aqueous phase of a sewage sludge, containing soluble organic matter with only copper and protons as competing ions, predicts that at pH 7 over 99% of the copper present will be bound to the soluble organic matter.     

Extracellular enzyme activities during regulated hydrolysis of high-solid organic wastes

The hydrolysis process, where the complex insoluble organic materials are hydrolyzed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Recirculating the leachate from hydrolysis reactor and recycling the effluent from methanogenic reactor to hydrolysis reactor in the two-stage solid-liquid anaerobic digestion process could accelerate degradation of organic solid wastes. To justify the influencing mechanism of recirculation and recycling on hydrolysis, the relationship of hydrolysis to the synthesis and locations of extracellular enzymes was evaluated by regulating the dilution rate of the methanogenic effluent recycle. The results showed that the hydrolysis could be enhanced by increasing the dilution rate, resulting from improved total extracellular enzyme activities. About 15%, 25%, 37%, 56% and 92% of carbon, and about 9%, 18%, 27%, 45% and 80% of nitrogen were converted from the solid phase to the liquid phase at dilution rates of 0.09, 0.25, 0.5, 0.9 and 1.8d(-1), respectively. The hydrolysis of organic wastes was mainly attributable to cell-free enzyme, followed by biofilm-associated enzyme. Increasing the dilution rate afforded cell-free extracellular enzymes with more opportunity to access the surface of organic solid waste, which ensured a faster renewal of niche where extracellular enzymes functioned actively. Meanwhile, the increment of biofilm-associated enzyme was promoted concomitantly, and therefore improved the hydrolysis of organic solid wastes.     

Soil organic matter-hydrogen peroxide dynamics in the treatment of contaminated soils and groundwater using catalyzed H2O2 propagations (modified Fenton's reagent)

The interactions between catalyzed H(2)O(2) propagations (CHP-i.e. modified Fenton's reagent) and soil organic matter (SOM) during the treatment of contaminated soils and groundwater was studied in a well-characterized surface soil. The fate of two fractions of SOM, particulate organic matter (POM) and nonparticulate organic matter (NPOM), during CHP reactions was evaluated using concentrations of hydrogen peroxide from 0.5 to 3M catalyzed by soluble iron (III), an iron (III)-ethylenediamine tetraacetic acid (EDTA) chelate, or naturally-occurring soil minerals. The destruction of total SOM in CHP systems was directly proportional to the hydrogen peroxide dosage, and was significantly greater at pH 3 than at neutral pH; furthermore, SOM destruction occurred predominantly in the NPOM fraction. At pH 3, SOM did not affect hydrogen peroxide decomposition rates or hydroxyl radical activity in CHP reactions. However, at neutral pH, increasing the mass of SOM decreased the hydrogen peroxide decomposition rate and increased the rate of hydroxyl radical generation in CHP systems. These results show that, while CHP reactions destroy some of the organic carbon pools, SOM does not have a significant effect on the CHP treatment of soils and groundwater.     

Modeling copper partitioning in surface waters

Suspended particulate matter from the surface waters of the Susquehanna, Christina and Brandywine Rivers was collected by tangential flow filtration to study copper partitioning. Copper adsorption increased with an increase of suspended particles and decreased with low pH values or with an increase of dissolved organic matter. Effects of particulate organic matter on copper distribution between suspended particulate and solution phases were studied using modified aluminum oxide. An increase of particulate organic matter enhanced copper adsorption. Copper adsorption per mass of particulate organic carbon was similar for the different sources of suspended particles. A model, based on copper adsorption on particles and complexation with dissolved organic matter, was developed to assess copper partition coefficient as a function of the easily measurable water quality parameters: pH, alkalinity, dissolved organic carbon and particulate organic matter. The partitioning model was calibrated on pH edge data using suspended particles collected from the three rivers. The model was verified using partitioning data as a function of dissolved organic matter. The model adequately describes the system containing natural suspended solids collected from the surface waters.     

The chemistry of heavy metals in digested sewage sludge—II. Heavy metal complexation with soluble organic matter

Characterisitcs of copper-binding, in the soluble fraction of a digested sewage sludge, were determined using ion-selective electrodes. Results showed that ion exchange with protons is a principal component in copper binding and that the soluble organic matter has a cation exchange capacity of 8.86 m-equiv g⁻¹ at pH 6.5. A pH-dependent exchange constant, defined in terms of mole fractions of exchange components, describes the data in the pH range 2–8 and is independent of both pH and the extent of exchange in the range 5.5–8. Supplementary data include acid/base titration curves and simple kinetic measurements. Anion uptake is insignificant in the pH range 2.5–7. A model of the aqueous phase of a sewage sludge, containing soluble organic matter with only copper and protons as competing ions, predicts that at pH 7 over 99% of the copper present will be bound to the soluble organic matter.     

Degradation of parathion in seawater

The decomposition of parathion (O,O-diethyl-O-p-nitrophenylthiophosphate) in seawater has been followed in the laboratory. The influence of temperature, pH, salinity, and particulate matter on the degradation time and products is shown. Chemical hydrolysis as well as biodegradation occurred. The ratio of the two depends on the pretreatment of the water. During chemical hydrolysis dearylation and dealkylation occur. Biodegradation was observed through the disappearance of the organic nitro compounds. Among the degradation products dealkylated parathion was found to be stable against further hydrolysis and microbial degradation under environmental conditions.     

Operating strategies for acid phase digestion: an industrial case study

Acid phase digestion was investigated for enhanced operation in an industrial wastewater treatment plant. In particular, sludge retention time (SRT), temperature and pH were assessed for determining optimal conditions under operating constraints. Volatile fatty acid (VFA) production and soluble chemical oxygen demand (SCOD) were the key process parameters used to assess system performance. Increase in SRT from 0.8 to 1.4 days (at 22°C) had moderate effect on VFA production (approximately 15% increase), achieving a maximum VFA production of 3600 mg/L. High VFA production rate (0.101 ± 0.035 mg/mgVS.d) was obtained at the highest operating retention time during summer (27°C). The degree of solubilisation of particulate organic matter increased with temperature and retention time. Despite an increase in SCOD (6472 ± 873 mg/L max.), a corresponding increase in VFA was not observed. SCOD showed a linear correlation with decrease in pH, while acidogenesis (SCOD conversion to VFA) was found to be favourable at a pH of 4.5.     

Characterisation of riverine particulate organic matter by pyrolysis-gas chromatography-mass spectrometry

Particulate matter samples were collected in the mountainous section and river mouth at the Tech River basin, south France, during flood (December 1996) and summer (September 1997) periods. Suspended material was analysed by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) and 23 identified pyrolysis products were divided into five main fractions: aminosugars, aromatic hydrocarbons, polysaccharides, phenols and nitrogenous compounds. Analysis of relative amounts of these fractions revealed neither significant spatial nor temporal changes in the particulate organic matter composition. Nevertheless, their specific composition showed that during flooding there is a certain homogeneity in the composition of the riverine particulate organic matter, with more degraded material of pedogenic (allochthonous) character, and during the summer the results suggest the presence of two components, allochthonous and autochthonous.     

Enzymatic extraction of activated sludge extracellular polymers and implications on bioflocculation

This study examines enzyme hydrolysis, a mild, effective, but a rarely used method of extracellular polymer extraction, in removing polymers from mixed culture activated sludge flocs. Two carbohydrate specific enzymes (alpha-amylase and cellulase) and a protein specific enzyme (proteinase) are used during the study. First, the kinetic aspect is investigated, then enzyme dose optimization is carried out on laboratory grown activated sludge samples cultured at solids retention times (SRT) of 4 and 20 days. A more commonly used cation exchange resin (CER) extraction technique is also employed for comparison purposes. Results indicate that the extraction of extracellular polymers by enzymes is a rather quick process reaching equilibrium within only a few hours. As the doses of enzymes are increased, the extracted polymer quantities increase up to a certain dose, beyond which not much extraction is observed. The method does not cause any significant cell lysis as measured by the viable cell counts. Carbohydrate-hydrolyzing enzymes extract small amount of proteins along with the carbohydrates and protein-hydrolyzing enzyme extracts some carbohydrates together with the proteins, indicating that proteins and carbohydrates exist bound to each other in the extracellular polymer network of sludge. Enzyme extraction generally gives a lower estimate of polymers compared to the CER method, but correctly detects the trends in the polymer quantity.     

Composition of particulate and dissolved organic matter in a disturbed watershed of southeast Brazil (Piracicaba River basin)

The elemental and isotopic composition of particulate and dissolved organic matter was investigated in the Piracicaba River basin, S?o Paulo State, Brazil. Comparison of riverine organic matter from the Piracicaba River basin, a region where rivers and streams receive urban sewage and industrial effluents, with data reported for the pristine Amazon system revealed significant differences associated with anthropogenic impacts. One important difference was N enrichment in the particulate organic material of the Piracicaba basin rivers, due to (a) urban and industrial effluents, and (b) enhanced phytoplankton growth, which results from the combination of nutrient enrichment and damming of sections of the rivers. Radiocarbon concentrations were overall more depleted (older 14C age) in the Piracicaba basin rivers than in the Amazon, which may reflect the importance of soil erosion in the former. Analyses of stable and radioactive carbon isotopes and lignin-derived compounds indicated that coarse particulate organic material is composed of a mixture of soil particles and degraded organic matter from C3 and C4 vascular plants. Fine particulate organic material was composed mainly of soil particles and phytoplankton cell remains, the latter especially during low water. Ultrafiltered dissolved organic matter was the most degraded fraction according to its lignin oxidation products, and showed the greatest influence of C4 plant sources.     

Hydrolysis and acidification of waste activated sludge at different pHs

The effect of pH from 4.0 to 11.0 on the hydrolysis and acidification of waste activated sludge (WAS) was investigated. Experimental results showed that at room temperature the sludge hydrolysis was in the following order: alkaline>acidic>(neutral and blank test), and between pH 6.0 and 11.0 the sludge hydrolysis increased with pH. The three main components, soluble protein, carbohydrate and volatile fatty acids (VFAs) in the hydrolytic product were analyzed. It was observed that both the soluble protein and carbohydrate increased with pH in the pH range 7.0-11.0, but also increased to a smaller extent with pH from 7.0 to 4.0. The VFAs concentration was also affected by pH. Under alkaline conditions, the VFAs production was significantly higher than under other conditions. The concentration of VFAs on the 8th day of fermentation at pH 4.0, 7.0 and 10.0 was, respectively, 354.49, 842.00 and 2708.02 mg/L, while VFAs in the blank test was only 633.59 mg/L. The VFAs consisted of acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids, but acetic, propionic and iso-valeric were the three main products. Also, the release of soluble phosphorus and ammonia and the production of methane was studied during WAS fermentation at different pHs.     

Speciation of aluminum in an acidic mountain stream

Using a chelating resin a practical method was developed for speciating aluminum in those oligotrophic streams impacted by acid rain. The four categories operationally defined were rapidly exchangeable (monomeric and small polymeric cationically charged species), moderately fast exchangeable (principally inorganically complexed species), slowly exchangeable (principally organically complexed species), and nonexchangeable or inert aluminum (strong alumino-organic complexes, colloidal, or crystalline forms). Applying the procedure to unfiltered samples collected from a mildly acidic mountain stream, it was found that in upstream samples, where pH was low, rapidly exchangeable aluminum species dominated, while in the downstream samples, where pH was higher, the moderately fast exchangeable, slowly exchangeable, and inert aluminum species were the major ones.Two principal forms of particulate aluminum were also identified and determined: that which dissolves and is taken up by the resin (principally inorganic) and that which remains relatively inert (principally associated with humic material and/or other organic matter).