Active biomass in activated sludge: Comparison of respirometry with catalase activity measured using an on-line monitor

On-line measurements of the activity of activated sludge biomass may allow more efficient operation of treatment plants. A novel monitor for activity which can be used on-line follows oxygen evolution from hydrogen peroxide by catalase activity in activated sludge. Results from this monitor were compared with assays of oxygen uptake rate (OUR) by respirometry. Samples from the five activated sludge plants were assayed and showed, as expected, that the mixed liquor suspended solids (MLSS) and volatile suspended solids (VSS) levels were a poor measure of the activity of the sample determined by OUR. However, the activity of the biomass determined by the catalase activity biomass monitor correlated well with the respiration rate. This monitor should provide a simple and robust alternative to OUR measurements in activated sludge plants.     

Estimation of viable biomass in wastewater and activated sludge by determination of ATP, oxygen utilization rate and FDA hydrolysis

ATP content, oxygen utilization rate (OUR) and fluorescein diacetate (FDA) hydrolysis were tested for the ability to express the amount of viable biomass in wastewater and activated sludge.The relationship between biomass and these activity parameters was established in growth cultures made by inoculating a nutrient medium with either wastewater or activated sludge. Biomass was then determined directly by measurement of dry weight of growth culture (dw), and compared to data obtained by using the previously mentioned methods. In the exponential growth phase, ATP content showed the best correlation with biomass, while FDA hydrolysis in the sludge failed to show any such correlation. Conversion factors of 3 mg ATP/g dw, 300 mg O₂/h g dw and 0.4 A/h (mg dw/ml) for ATP, OUR and FDA methods, respectively, were calculated.When the methods were applied for in situ determinations in four different wastewater plants, it was found that ATP content and respiration rate estimated viable biomass to range from 81 to 293 mg dw/g SS for raw wastewater and from 67 to 187 mg dw/g SS for activated sludge with a rather weak correlation between ATP and respiration measurements. The FDA hydrolysis estimated viable biomass to be higher than suspended solids, for which reason this parameter could not be recommended for determination of viable biomass in wastewater and activated sludge.     

Sludge viability in a biological reactor

The volatile suspended solids (VSS) concentration cannot be used as a measure for the active biomass in a reactor which operates under a wide range of operating conditions since the viable organism content of the VSS is not constant. Using substrate saturation conditions the kinetic parameters maximum substrate removal rate ( ) and oxygen uptake rate (J)—both per mass of VSS—were determined in an experimental pulse fed batch biological reactor. It was found that and J both doubled during the experimental period (6 h). It was concluded that the increases in and J values were due to the increase in the sludge viability which are here defined as the percentage of VSS which is active biomass. Using the variations in and J values during each experiment, it was possible to calculate sludge viability. During a 6 h experimental period at substrate saturation level the sludge viability increased on average from 8.9 to 23.3%.In a loop type sewage conduits system operated as a plug flow reactor and enriched with biomass and air, it is possible to achieve high specific substrate removal rates when step feeding creates saturation conditions. This is further attenuated by a marked increase in the sludge viability.     

Effects of chromium (VI) addition on the activated sludge process

The effect of hexavalent chromium, Cr(VI), addition on various operating parameters of activated sludge process was evaluated. To accomplish this, two parallel lab-scale continuous-flow activated sludge plants were operated. One was used as a control plant, while the other received Cr(VI) concentrations equal to 0.5, 1, 3 and 5 mgl(-1). Cr(VI) concentrations of 0.5 mgl(-1) caused significant inhibition of the nitrification process (up to 74% decrease in ammonia removal efficiency). On the contrary, the effect of Cr(VI) on organic substrate removal was minor for concentrations up to 5 mgl(-1), indicating that heterotrophic microorganisms are less sensitive to Cr(VI) than nitrifiers. Activated sludge floc size and structure characterization showed that Cr(VI) concentrations higher than 1 mgl(-1) reduced the filaments abundance, causing the appearance of pin-point flocs and free-dispersed bacteria. Additionally, the variability of protozoa and rotifers was reduced. As a result of disperse growth, effluent quality deteriorated, since significant amounts of suspended solids escaped with the effluent. Termination of Cr(VI) addition led to a partial recovery of the nitrification process (up to 57% recovery). Similar recovery signs were not observed for activated sludge floc size and structure. Finally, shock loading to the control plant with 5 mgl(-1) Cr(VI) for 2 days resulted in a significant inhibition of the nitrification process and a reduction in filamentous microorganisms abundance.     

Effects of pH and oxidation state of chromium on the behavior of chromium in the activated sludge process

The behavior of chromium (Cr) in the activated sludge process (ASP) was evaluated in laboratory-scale, fill-and-draw activated sludge experiments. Both pH and the oxidation state of chromium were confirmed as critical parameters in the ASP for evaluating the behavior of chromium. More than 55% of chromium was removed when trivalent chromium [Cr(III)] was introduced into the influent while less than 60% was removed when hexavalent chromium [Cr(VI)] was added over a pH range from 5 to 9. As pH was increased, the removal increased when Cr(III) was introduced but the reverse occurred with Cr(VI). Introduction of Cr(VI) into the influent resulted in less than 80% of chromium associated with solids; however, with Cr(III), more than 90% of chromium was bound with solids. These results suggest that the ASP is capable of controlling the transport of Cr(III) to the environment but such is not case for Cr(VI).Theoretical consideration based on thermodynamics predicted that no reduction of Cr(VI) into Cr(III) should occur and the only redox reaction should be the oxidation of Cr(III) into Cr(VI). However, no oxidation of Cr(III) into Cr(VI) was observed; some Cr(VI) was reduced into Cr(III). Kinetic constraints may have impeded the oxidation of Cr(III). Under the conditions of this study, Cr(III) may have been removed through adsorption rather than precipitation as Cr(OH)₃. Cr(VI) might be adsorbed on the bacterial surface through specific adsorption.     

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 ± 2.4%), settled wastewater (10.7 ± 3.1%), activated sludge (11.1 ± 2.1%) and effluent (3.2 ± 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant.     

Biological Cr(VI) removal coupled with biomass growth, biomass decay, and multiple substrate limitation

In this work, a mathematical model for the biological reduction of Cr(VI), carbon and nitrogen sources consumption, and biomass growth under fully aerobic conditions was developed. The model comprises three types of aerobic heterotrophic cells (non-growing cells, growing cells with chromate reductase activity, and growing cells that have lost the chromate reductase activity), and five soluble compounds (organic substrate, ammonia nitrogen, non-metabolizable soluble products, dissolved oxygen, and hexavalent chromium). Two processes are considered responsible for the reduction of Cr(VI). The first one is the reduction of Cr(VI) coupled with growth, the second process is coupled with the endogenous decay of the biomass. The model was calibrated using the results obtained in batch cultures in the absence of carbon and nitrogen sources, using different initial Cr(VI) concentrations (0-100 mgCr L⁻¹), two carbon sources (cheese whey and lactose), and different initial nitrogen to carbon ratio (0-50 mgN gCOD⁻¹). The calibrated model was used to calculate steady-state values of TSS, soluble COD, TAN and Cr(VI) in continuous systems, obtaining a good agreement with the experimental data. The model also accurately predicted the transient concentration of Cr(VI) as a function of time in response to step changes of the inlet Cr(VI) concentration in continuous systems.     

Mechanism of hexavalent chromium removal by dead fungal biomass of Aspergillus niger

When synthetic wastewater containing Cr(VI) was placed in contact with the dead fungal biomass of Aspergillus niger, the Cr(VI) was completely removed from aqueous solution, whereas Cr(III), which was not initially present, appeared in aqueous solution. Desorption and X-ray photoelectron spectroscopy (XPS) studies showed that most of the Cr bound on the biomass was in trivalent form. These results indicated that the main mechanism of Cr(VI) removal was a redox reaction between Cr(VI) and the dead fungal biomass, which is quite different from previously reported mechanisms. The influences of contact time, pH, Cr(VI) concentration, biomass concentration and temperature on Cr(VI) removal were also evaluated. The Cr(VI) removal rate increased with a decrease in pH and with increases in Cr(VI) concentration, biomass concentration and temperature. Although removal kinetics was dependent on the experimental conditions, Cr(VI) was completely removed in the aqueous solution. In conclusion, a new mechanism of Cr(VI) removal by the dead fungal biomass has been proposed. From a practical viewpoint, this abundant and inexpensive dead fungal biomass has potential application in the conversion of toxic Cr(VI) into less toxic or nontoxic Cr(III).     

Studies on enhancement of Cr(VI) biosorption by chemically modified biomass of Rhizopus nigricans

This study reports the biosorption of Cr(VI) by chemically modified biomass of Rhizopus nigricans and the possible mechanism of Cr complexation to the adsorbent. The cell wall of this fungus possesses strong complexing property to effectively remove Cr(VI) anions from solution and wastewater. The mechanism of Cr adsorption by R. nigricans was ascertained by chemical modifications of the dead biomass followed by FTIR spectroscopic analysis of the cell wall constituents. Treatment of the biosorbent with mild alkalies (0.01 N NaOH and ammonia solution) and formaldehyde (10%, w/v) deteriorated the biosorption efficiency. However, extraction of the biomass powder in acids (0.1 N HCl and H2SO4), alcohols (50% v/v, CH3OH and C2H5OH) and acetone (50%, v/v) improved the Cr uptake capacity. Reaction of the cell wall amino groups with acetic anhydride reduced the biosorption potential drastically. Blocking of the-COOH groups by treatment with water soluble carbodiimide also resulted in initial lag in Cr binding. Biomass modification experiments conducted using Cetyl Trimethyl Ammonium Bromide (CTAB), Polyethylenimine (PEI), and Amino Propyl Trimethoxy Silane (APTS) improved the biosorption efficiency to exceptionally high levels. The FTIR spectroscopic analysis of the native, Cr bound and the other types of chemically modified biomass indicated the involvement of amino groups of Rhizopus cell wall in Cr binding. The adsorption data of the native and the most effectively modified biomass were evaluated by the Freundlich and the Langmuir adsorption isotherms and the possible adsorption phenomena are also discussed.     

Aerobic granulation in a sequencing batch airlift reactor

Aerobic granular sludge was cultivated in an intensely mixed sequencing batch airlift reactor (SBAR). A COD loading of 2.5 kg Acetate-COD/(m3 d) was applied. Granules developed in the reactor within one week after inoculation with suspended activated sludge from a conventional wastewater treatment plant. Selection of the dense granules from the biomass mixture occurs because of the differences in settling velocities between granules (fast settling biomass), and filaments and flocs (slow settling biomass). At 'steady state' the granules had an average diameter of 2.5 mm, a biomass density of 60g VSS/I of granules, and a settling rate of > 10 m/h. The biomass consisted of both heterotrophic and nitrifying bacteria. The reactor was operated over a long period during which the granular sludge proved to remain stable. The performance of the intermittently fed SBAR was compared to that of the continuously fed biofilm airlift suspension reactor (BASR). The most importance difference was that the density of the granules in the SBAR was much higher than the density of the biofilms in the BASR. It is discussed that this could be due to the fact that the SBAR is intermittently fed, while the BASR is continuously fed.     

Investigation of chromium phytoremediation and tolerance capacity of a weed,Portulaca oleracea L. in a hydroponic system

The present investigation was undertaken to study the growth, biomass, physiological‐biochemical responses and chromium (Cr) accumulation capacity of hydroponically grown Portulaca oleracea cuttings exposed to Hoagland solution supplemented with Cr(VI) (0.0, 0.1, 0.5, 1.0, 2.5, 5.0, 10 mg/L) for 30 days. The cuttings exhibited effective regeneration in Hoagland solution in comparison to deionized water. Plants demonstrated significant reduction in growth (root and shoot length, leaf area), biomass (root and shoot dry weight), pigments (total chlorophylls and carotenoids) and total soluble sugar content at higher concentration of Cr(VI) (10 mg/L). However, plants could tolerate Cr stress through significantly higher accumulation of proline and increased activity of peroxidase resulting in significant Cr accumulation (150–190 mg/kg dry weight) in harvestable parts of Portulaca. Thus, the results suggest application of P. oleracea for phytoremediation of Cr‐contaminated sites for the protection of environment.     

Biosorption of chromium(VI) from aqueous solutions by green algae Spirogyra species.

Biosorption of heavy metals is an effective technology for the treatment of industrial wastewaters. Results are presented showing the sorption of Cr(VI) from solutions by biomass of filamentous algae Spirogyra species. Batch experiments were conducted to determine the adsorption properties of the biomass and it was observed that the adsorption capacity of the biomass strongly depends on equilibrium pH. Equilibrium isotherms were also obtained and maximum removal of Cr(VI) was around 14.7 x 10(3) mg metal, kg of dry weight biomass at a pH of 2.0 in 120 min with 5 mg/l of initial concentration. The results indicated that the biomass of Spirogyra species is suitable for the development of efficient biosorbent for the removal and recovery of Cr(VI) from wastewater.     

Viability of microbial mass in compartmentalised single activated sludge process

Investigations were carried out for steady-state conditions on laboratory-scale model of an anaerobic-aerobic-anaerobic-aerobic configuration of the activated sludge process, for hydraulic detention time of 9.13 h and sludge age of 9, 16.4 and 28.3 days. Data was obtained on volatile suspended solids (MLVSS), adenosine triphosphate (ATP), oxygen uptake rate (OUR) and relative dehydrogenase activity (RDA) on the laboratory model and the extended aeration biological treatment unit at the ARAMCO treatment plant in Dhahran. A mathematical model was obtained based on the laboratory data, using a calibration method to estimate MLVSS for given values of OUR, RDA and ATP. Regression coefficients for MLVSS and OUR. MLVSS and RDA and MLVSS and ATP were determined.It was found that the mathematical model obtained does not adequately describe the inter-relationship between various parameters. The inter-relationship is a function of operational conditions occurring in the plant and too complex to be described by a mathematical model. MLVSS offers the best estimate and cannot be related by an equation with OUR, ATP and RDA.     

A new interpretation of endogenous respiration profiles for the evaluation of the endogenous decay rate of heterotrophic biomass in activated sludge

In current activated sludge models aerobic degradation, resulting in loss of activity and mass of activated sludge is expressed with only one process called decay. The kinetics of this process is regarded to be first order and constant with respect to the loading conditions. In this work twelve aerobic digestion batch experiments were conducted for the activated sludge of seven different water resource recovery facilities (WRRFs). An analysis of the obtained respirograms shows three clearly distinguishable phases. The first phase is assumed to be due to the degradation of stored material (X_STOR) and active biomass simultaneously. The second phase is exclusively due to the degradation of active biomass that is regarded to consist mainly of ordinary heterotrophic biomass (X_OHO). The first order decay rate is slower than the degradation rate in phase 1 and varies between samples. The decay rate correlates with the activity of the activated sludge expressed as the ratio of initial heterotrophic OUR and the initial organic fraction X_ORG of the activated sludge. This second phase was detectable until day 5 of most of the experiments. After that time within phase 3 the OUR decrease slows down and the OUR even increased for short intervals. This behaviour is thought to be due to the activity of higher organisms and the adaptation of microorganisms to starvation.     

Modelling of the activated sludge process for microprocessor-based state estimation and control

A simple dynamic model of the activated sludge process including the volatile suspended solids (VSS) concentration in the aeration basin, the VSS or suspended solids (SS) concentration in the recycle flow and the SS concentration in the effluent was obtained by simplifying a comprehensive model using empirically verified assumptions. The model can be used for on-line estimation of the influent BOD-load and the effluent BOD, in combination with a recursive algorithm for oxygen uptake rate (OUR) and k_La estimation requiring only dissolved oxygen and air flow rate measurements. The estimation procedure has been implemented and tested at a real plant using a microprocessor. Control of the activated sludge process is discussed and concluded to be a hierarchical two-level problem. The upper level control actions are aimed at bringing the process to an optimal state of operation. For this purpose verbally formulated control laws are used. On the lower level the control task is to maintain the process in the optimal state.     

Simultaneous removal of phenol and ammonia by an activated sludge process with cross-flow filtration

Attempts were made for removing ammonia from synthetic wastewater under the presence of phenol, which is inhibitory to nitrification, by using a single-stage activated sludge process with cross-flow filtration. Activated sludge biomass which had been acclimated with phenol for over 15 years was used for the inoculum, and synthetic wastewater was continuously supplied to the process retaining biomass at 8000 mg VSS l(-1). Phenol was completely removed, and ammonia was simultaneously nitrified to nitrate; nitrification rate reached 200 mg N l(-1) d(-1) when phenol was removed at a rate up to 300 mg l(-1) d(-1). It was observed that 0-13% of the ammonia was removed via denitrification. Intermittent aeration enhanced the denitrification rate to 160 mg N l(-1) d(-1) by utilizing phenol. and approximately 24% of the denitrified nitrogen was recovered as nitrous oxide. Methanol, which is the most commonly used electron donor in conventional nitrogen removal processes, did not enhance the denitrification rate of the phenol-acclimated activated sludge used in this study, however phenol did. The results suggest that this process potentially works as a space- and energy-saving nitrogen removal process by utilizing substances inhibitory to nitrifiers as electron donors for denitrification.     

Earthworm-microorganism interactions: A strategy to stabilize domestic wastewater sludge

The performance of a conventional biofilter (BF) and a vermifilter containing the earthworm, Eisenia foetida, (VF) for the treatment of domestic wastewater sludge were compared with the earthworm-microorganism interaction mechanisms involved in sludge stabilization. The results revealed that the presence of earthworms in the VF led to significant stabilization of the sludge by enhancing the reduction in volatile suspended solids (VSS) by 25.1%. Digestion by earthworms and the earthworm-microorganism interactions were responsible for 54% and 46% of this increase, respectively. Specifically, earthworms in the VF were capable of transforming insoluble organic materials to a soluble form and then selectively digesting the sludge particles of 10-200 μm to finer particles of 0-2 μm, which led to the further degradation of organic materials by the microorganisms in the reactor. Additionally, denaturing gradient gel electrophoresis (DGGE) profiles showed that there was an intensified bacterial diversity in the vermifilter due to the presence of earthworms, especially in response to the nutrients in their casts.     

Biodegradation of the endogenous residue of activated sludge

This study evaluated the potential biodegradability of the endogenous residue in activated sludge subjected to batch digestion under either non-aerated or alternating aerated and non-aerated conditions. Mixed liquor for the tests was generated in a 200 L pilot-scale aerobic membrane bioreactor (MBR) operated at a 5.2 days SRT. The MBR system was fed a soluble and completely biodegradable synthetic influent composed of sodium acetate as the sole carbon source. This influent, which contained no influent unbiodegradable organic or inorganic materials, allowed to generate sludge composed of essentially two fractions: a heterotrophic biomass X_H and an endogenous residue X_E, the nitrifying biomass being negligible (less than 2%). The endogenous decay rate and the active biomass fraction of the MBR sludge were determined in 21-day aerobic digestion batch tests by monitoring the VSS and OUR responses. Fractions of X_H and X_E: 68% and 32% were obtained, respectively, at a 5.2 days SRT. To assess the biodegradability of X_E, two batch digestion units operated at 35 °C were run for 90 days using thickened sludge from the MBR system. In the first unit, anaerobic conditions were maintained while in the second unit, alternating aerated and non-aerated conditions were applied. Data for both units showed apparent partial biodegradation of the endogenous residue. Modeling the batch tests indicated endogenous residue decay rates of 0.005 d⁻¹ and 0.012 d⁻¹ for the anaerobic unit and the alternating aerated and non-aerated conditions, respectively.     

Occurrence of methanogenesis during start-up of a full-scale synthesis gas-fed reactor treating sulfate and metal-rich wastewater

The start-up of a full-scale synthesis gas-fed gas-lift reactor treating metal and sulfate-rich wastewater was investigated. Sludge from a pilot-scale reactor was used to seed the full-scale reactor. The main difference in design between the pilot- and full-scale reactor was that metal precipitation and sulfate reduction occurred in the same reactor. After 7 weeks the full-scale reactor achieved the sulfate conversion design rate of 15 kg/m3day. Zinc sulfide precipitation inside the reactor did not interfere with obtaining a high rate of sulfate reduction. 16S rRNA gene analysis demonstrated that the bacterial communities in both reactors were dominated by the sulfate-reducing genus Desulfomicrobium. Archaeal communities of both reactors were dominated by the methanogenic genus Methanobacterium. Most Probable Number (MPN) counts confirmed that heterotrophic Sulfate-Reducing Bacteria (SRB) were dominant (10(11) -10(12) cells/g VSS) compared to homoacetogens (10(5) -10(6) cells/g VSS) and methanogens (10(8) -10(9) cells/g VSS). Methanogenesis was not suppressed during start-up of the full scale-reactor, despite the predominance of SRB, which have a lower hydrogen threshold. Due to the short sludge retention time (4-7 days) competition for hydrogen is determined by Monod kinetics, not hydrogen thresholds. As the kinetic parameters for SRB and methanogens are similar, methanogenesis may persist which results in a loss of hydrogen.     

Bio-reduction of soluble chromate using a hydrogen-based membrane biofilm reactor

Hexavalent chromium (Cr(VI)) is a mutagen and carcinogen that is a significant concern in water and wastewater. A simple and non-hazardous means to remove Cr(VI) is bioreduction to Cr(III), which should precipitate as Cr(OH)3(s). Since Cr(VI)-reducing bacteria can use hydrogen (H2) as an electron donor, we tested the potential of the H2-based membrane biofilm reactor (MBfR) for chromate reduction and removal from water and wastewater. When Cr(VI) was added to a denitrifying MBfR, Cr(VI) reduction was immediate and increased over 11 days. Short-term experiments investigated the effects of Cr(VI) loading, H2 pressure, and nitrate loading on Cr(VI) reduction. Increasing the H2 pressure improved Cr(VI) reduction. Cr(VI) reduction also was sensitive to pH, with an optimum near 7.0, a sharp drop off below 7.0, and a gradual decline to 8.2. Cr(III) precipitated after a small upward adjustment of the pH. These experiments confirm that a denitrifying, H2-based MBfR can be used to reduce Cr(VI) to Cr(III) and remove Cr from water. The research shows that critical operational parameters include the H2 concentration, nitrate concentration, and pH.