Mechanisms of heavy-metal removal by activated sludge.

This paper investigates the chemical mechanisms operating in cadmium and lead removal by activated sludge in sequencing batch reactors. Selective extraction and acid digestion of sludge samples denoted that both Cd and Pb are mainly present as surface-bound metals. Characterisation of sludge samples by potentiometric titrations and IR spectra suggested that carboxylic and amino groups are the main active sites responsible for the binding properties of the biomass. Simulation of metal speciation implemented with complexation constants determined in biosorption tests, showed that cadmium predominates as biosorbed species, while lead was mainly removed by precipitation.

Role of biomass adaptation in the removal of formic acid in sequencing batch reactors.

This study deals with formic acid removal in activated sludge processes, in particular in the processes carried out in sequencing batch reactors (SBRs). Formic acid removal has been investigated in a SBR fed with acetic and formic acids at equimolar concentrations. Biomass performance in the reactor has been investigated both by the analysis of the removal of the two substrates and by batch tests. Regarding SBR process, the obtained results show that a relevant difference occurred between formic and acetic acid profiles. Acetic acid was never found in the effluent and was always completely removed during the reaction phase. On the other hand, formic acid removal was determined by biomass acclimation, which is in turn determined by sludge age imposed to the system. Batch tests confirmed that formic acid removal occurs only if biomass is acclimated. It has been shown that the minimal sludge age to obtain complete formic acid removal is much higher than those predictable with the classical models of microbial growth in wastewater treatment processes. The advantages of SBRs over continuous-flow systems in the removal of formic acid have also been highlighted.

Effect of feed length on settleability, substrate uptake and storage in a sequencing batch reactor treating an industrial wastewater.

The paper compares the performance of two Sequencing Batch Reactors (SBRs) treating the same industrial wastewater (composed of formic acid, ethylene glycol and methanol) operated at two different lengths of the feed. The two SBRs were operated in parallel under the same conditions of organic load (0.85 gCOD l(-1) d(-1)) and sludge age (about 10 d), the only difference being the length of the feed: less than 1 min vs. 5 h. In this way the conditions of a plug flow reactor and of a completely mixed reactor were simulated. The two systems were compared on sludge settleability (related to filaments abundance and floc morphology), substrate uptake rates and polyhydroxyalkanoates (PHAs) storage rates. The main difference between the two systems was in the settling properties of the sludge: both SVI and effluent solids were higher in the system with slow feed. With regard to filamentous microorganisms, even though both reactors were inoculated with the same sludge with high concentration of filaments, they were rapidly washed out from both systems. Microscopic observations showed that the reason for the different settling properties of the two sludges was in the floc structure, which was more compact in the system operated with fast feed. These data support the theory of the role of diffusion inside the flocs in determining the settling properties of the sludge. The maximum substrate uptake rates and PHA storage rates were similar in the two systems, showing that also the microorganisms grown at a constant and low substrate concentration were able to quickly increase their activity and to store PHAs when in the presence of a sudden change in substrate concentration.

Enrichment of activated sludge in a sequencing batch reactor for polyhydroxyalkanoate production.

The paper describes the start up of a process for the production of polyhydroxyalkanoates (PHAs) from activated sludge. The excess sludge from a wastewater treatment plant was inoculated in a lab-scale sequencing batch reactor (SBR) to be enriched under aerobic conditions through intermittent feeding with a mixture of organic acids. Enriching of activated sludge was monitored through the measurement of polymer concentrations either in the mixed liquor or in the microbial biomass. The bacterial population dynamics during the SBR start up was followed through denaturing gradient gel electrophoresis and the main species present at the steady state were identified. All the measured parameters significantly changed in the SBR during first two weeks after the inoculum was seeded into the reactor, they then stabilized. At the steady state, the SBR produced 2.6 gVSSl(-1) d(-1), with a PHA content of 11% (on a COD basis). The enriched microbial biomass was then transferred into a batch reactor where the bacterial polymer content was increased through a new feeding. In the final batch stage, maximum storage rate and maximum polymer content in the biomass were 405 mgCOD gCOD(-1) h(-1) and 44% (on a COD basis), respectively. The PHA storage from the enriched microbial biomass was about 20 times faster and the PHA content was about 4 times higher than that of the inoculated activated sludge. Observations by fluorescence microscopy showed that the majority of microorganisms in the enriched biomass could be stored. Among the numerically most representative genera in the enriched biomass, Thauera, Candidatus Meganema perideroedes, and Flavobacterium were identified.

Olive oil mill effluents as a feedstock for production of biodegradable polymers.

The aim of the present paper was to study the feasibility of using olive oil mill effluents (OMEs) as a substrate in biodegradable polymer production. OMEs were anaerobically fermented to obtain volatile fatty acids (VFAs), which are the most highly used substrate for polyhydroxyalkanotes (PHAs) production. The anaerobic fermentation step was studied both without pretreatment and with different pretreatments (i.e., centrifugation, bentonite addition, and bentonite addition followed by centrifugation) and at various concentrations (28.5, 36.7 and 70.4 g CODL(-1)). During fermentation, VFA concentration was determined (7-16 g CODL(-1)) as well as the corresponding yield with respect to initial COD (22-44%). At all initial concentrations, centrifugation pretreatment (with or without previous addition of bentonite) significantly increased the final VFA concentration and yield, whereas the addition of bentonite alone had no influence. Moreover, centrifugation pretreatment led to a different acid distribution, which affected the hydroxyvalerate (HV) content within the obtained copolymer poly beta-(hydroxybutyrate-hydroxyvalerate) [P(HB-HV)]. OMEs were tested for PHA production by using a mixed culture from an aerobic SBR. Centrifuged OMEs, both with or without fermentation, were tested. PHAs were produced from both matrices, but with fermented OMEs PHA production was much higher, because of the higher VFA concentration. The initial specific rate of PHA production obtained with fermented OMEs was approximately 420 mg COD g COD(-1)h(-1) and the maximum HV content within the copolymer was about 11% (on a molar basis). The HV monomer was produced only until propionic acid remained present in the medium.

Storage of substrate mixtures by activated sludges under dynamic conditions in anoxic or aerobic environments.

In spite of the fact that in most activated sludge plants substrate complex mixtures are removed under alternating anoxic and aerobic conditions, most studies on the dynamic response of biomass are limited to feeding a single substrate (acetate or glucose) under a single redox condition (aerobic or anoxic). In this study, the dynamic response of biomass in a sequencing batch reactor is described in terms of substrate removal and related storage as internal polymers, as functions of single or simultaneous feed of several substrates (acetate, glucose, glutamic acid and ethanol) and of anoxic vs. aerobic conditions. Under anoxic conditions, the four substrates were simultaneously removed at a significantly greater nitrate removal rate than when single substrates were present, so showing that the simultaneous removal was partially due to independent metabolic activities. On the other hand, the removal of every substrate was affected (positively or negatively) by the presence of the others, demonstrating that the substrates can be also used by the same metabolism. As an exception, acetate removal was not affected by the presence of other substrates. As for the comparison of aerobic and anoxic conditions, the acetate uptake rate almost doubled moving from anoxic to aerobic conditions, whereas other substrates were only slightly affected. This difference was probably due to the additional presence of aerobic denitrification, which was much more important for acetate. This also confirmed that acetate removal was independent from other substrates. In all cases, storage was the main mechanism of solids formation, so confirming the general importance of such phenomenon under dynamic conditions, independently from feed complexity and redox conditions.

PHA storage from several substrates by different morphological types in an anoxic/aerobic SBR.

An activated sludge was cultivated on a mixture of several soluble substrates (acetate, ethanol, glucose, glutamic acid, peptone, Tween 80, starch, yeast extract) in an anoxic/aerobic SBR. Highly dynamic conditions in the SBR (feast famine regime) caused fast removal of most COD in the anoxic phase (in particular acetate, ethanol, glutamic acid and glucose were totally removed) and relevant contribution of storage. In spite of that, filament abundance was always high, as is typical of bulking sludges. Filaments which developed in the reactor were characterized on a morphological basis and on the basis of their ability to grow and to store polyhydroxyalkanoates (PHAs). Three main filaments prevailed in the biocenosis, whose relative abundance was varyng with time: Nostocoida limicola II, (two different morphological types), Haliscomenobacter hydrossis and an unidentified one. It was found that maximum growth rate was higher for flocformers than for filaments on each of the tested substrates. Epifluorescence showed that storage ability was more widespread among flocformers than in the filaments. Only one type of Nostocoida limicola II was able to store PHAs. The obtained data show that aerobic growth on the little residual fraction of COD from the anoxic phase was enough to support high abundance of filamentous microorganisms.

Gas chromatographic analysis of polyhydroxybutyrate in activated sludge: a round-robin test.

Polyhydroxyalkanoates (PHA) and poly-beta-hydroxybutyrate (PHB) in particular have become compounds which is routinely investigated in wastewater research. The PHB analysis method has only recently been applied to activated sludge samples where PHA contents might be relatively low. This urges the need to investigate the reproducibility of the gas chromatographic method for PHB analysis. This was evaluated in a round-robin test in 5 European laboratories with samples from lab-scale and full-scale enhanced biological phosphorus removal systems. It was shown that the standard deviation of measurements in each lab and the reproducibility between the labs was very good. Experimental results obtained by different laboratories using this analysis method can be compared. Sludge samples with PHB contents varying between 0.3 and 22.5 mg PHB/mg sludge were analysed. The gas chromatographic method allows for PHV, PH2MB and PH2MV analysis as well.

Effect of different carbon sources on aerobic storage by activated sludge.

A study of substrate removal by real activated sludge with several synthetic substrates (acetate, ethanol, glutamic acid) and wastewater (raw and filtered) was carried out. Substrate, stored compounds (polyhydroxyalkanoates, PHA and internal carbohydrates), ammonia and oxygen uptake rate (OUR) were analytically determined. Polyhydroxybutyrate (PHB) was stored when the substrate was acetate or ethanol, while no appreciable formation of storage compound was detected using glutamic acid. A low amount of PHB was also formed in tests with raw and filtered wastewater which was probably mainly due to its acetate content. As far as the sum of storage and growth (indirectly estimated through ammonia consumption) did not match the overall solids formation, other unidentified mechanisms of substrate removal were likely to occur (biosorption, accumulation and/or storage of unidentified compounds). ASM3 and two derived models were used in the interpretation of experimental data with reference to synthetic substrates. With reference to synthetic substrates ASM3 can well describe the experimental data only assuming a stored product formation much higher than the analytically detected one, whereas the model that assumes a parallel growth and storage on the substrate can well describe the observed stored product profile only assuming a direct contribution of growth much higher than estimated from ammonia consumption. The model that assumes an accumulation/biosorption stage as first step of substrate removal can better describe the whole experimentally observed behaviour. However as well as in ASM3 this implies that some fraction of removed COD is still unidentified. With reference to real wastewater where the different phenomena were mixed up due to the presence of several substrates, the different models gave similar results.

The storage of acetate under anoxic conditions.

Till now the role of storage in activated sludge processes under transient conditions has been deeply investigated under anaerobic (EBPR processes) or aerobic (bulking control) environments. Little attention has been given to the role of storage in processes including anoxic environments. Hence, the aim of the present work was to investigate the anoxic storage along with other substrate removal mechanisms under transient conditions. Several mixed culture were ad hoc selected under anoxic environment and periodic feeding (acetate as carbon source) at different organic load rate (OLR) and feed length; then their transient response to substrate spike was investigated by batch tests under both anoxic and aerobic conditions. The relative role of different mechanisms in the substrate removal was established on the basis of COD balance assuming that the acetate COD removed from the liquid phase could be oxidised for energy needs or recovered into solids as poly-3-hydroxybutyric acid (PHB) (storage), other internal precursors or intermediates (accumulation) and active biomass (growth, as estimated by ammonium uptake). In all tested conditions, growth response was very little while PHB storage was prevailing. In some operating conditions, indirect evidence of accumulation (in forms still to be identified) was also found. The transient response was not affected by the presence of free amino acids, at least for the unacclimated mixed culture under observation. Transient response under aerobic condition was quite similar to the anoxic one.

Role of storage phenomena on removal of different substrates during pre-denitrification.

Removal mechanisms of different substrates during the pre-denitrification step of an anoxic/aerobic sequencing process are studied. Biomass was cultivated in an anoxic/aerobic SBR and fed with a mixture of low and high molecular weight compounds. Substrate removal mechanisms are studied by means of batch tests, performed under anoxic conditions. The dynamic response to a spike of four different substrates (acetate, glucose, glutamic acid and ethanol) is described by simultaneously considering substrate and electron acceptor removal, and PHB and carbohydrates storage. PHB storage is a relevant mechanism during the removal of acetate and ethanol, while glucose is removed mainly by carbohydrate storage.

Aerobic storage by activated sludge on real wastewater.

Activated sludge processes are often operated under dynamic conditions, where the microbial response can include, besides of growth, several COD removal mechanisms, and particularly the storage in form of polymers. While abundant evidence of aerobic storage under dynamic conditions with synthetic substrates can be found (Majone et al., Water Sci. Technol. 39(1) (1999) 61), there is still little knowledge about COD removal mechanisms with real activated sludge and wastewater. The aim of the present paper is therefore to give a direct evidence of storage phenomena occurring when a real sludge is mixed with influent wastewater and of their influence onto OUR profiles in typical respirometric batch tests. For this purpose, respirometric batch tests were performed on the same sludge by using acetate, filtered wastewater and raw wastewater as carbon source along with determination of acetate uptake and storage polymer formation. Comparison of results obtained has shown that poly-3-hydroxybutyrate (PHB) storage gives always the main contribution to acetate removal and that in the case of wastewater PHB is also formed from other substrates. PHB formation clearly occurs during the high-rate RBCOD-phase, however for wastewater it accounts for only a fraction (18-22%) of overall RBCOD removal, so calling for other unidentified storage compounds or other non-storage phenomena. In the low-rate SBCOD phase of respirogram PHB is clearly utilised in tests with acetate as internal reserve material once the acetate is depleted. In tests with filtered and raw wastewater the PHB concentration decreases much slower, probably because more PHB is formed due to the availability of external SBCOD (soluble and not). Moreover, reported OUR in the SBCOD-phase from filtered or raw wastewater are quite higher than those reported in batch tests with acetate, so confirming a main contribution of external SBCOD. However, the respective contributions for utilisation of previously stored compounds and of external SBCOD cannot be easily separated by the comparison of tests on filtered and raw wastewater, because both substrates are simultaneously present also in tests with the filtered wastewater. As a side consequence, the chemical-physical method for evaluation of true soluble and biodegradable COD tends to overestimate the respirometry-based RBCOD, at least for the wastewater under observation. Even though modelling by ASM3 (Gujer et al., Water Sci. Technol. 39(1) (1999) 183) makes it possible to well describe the whole experimental behaviour, it requires that much more storage compounds are formed than the experimentally observed PHB. These compounds have still to be identified and quantified in order to confirm the conceptual structure of ASM3.

[Recent progress in the stimulation of the histiocyte system and the anti-infection defences. Research into the effect of stanozolol treatment on the phagocyte activity of human macrophages]. / Recenti progressi nella stimolazione del sistema istiocitario e delle difese anti-infettive. Ricerche sugli effetti del trattamento con stanozololo sull'attivltà fagocitaria del macrofagi umani.

A group of patients was treated with stanozolol. A modified version of Roebuck's skin window technique was used to obtain macrophages. These were then fixed in vitro with a suspension of Paracolonbacter aerogenoides. Comparison with a group of control subjects showed that the macrophages from the treated patients appeared to take up and destroy germs more effectively and that this could be potentiated by prior opsonisation with serum from the treated subjects. These results are seen as an expression of functional activation of the macrophages and an increase in serum opsonin content.

[Appearance of proliferative activity in peritoneal macrophages cultured in vitro. Mitotic index values and percent frequency of frequency of mitosis phase]. / Sulla comparsa di attività riproduttiva nei macrofagi peritoneali coltivati in vitro. Indici mitotici e frequenza delle fasi mitotiche.

In vitro culture of rat peritoneal macrophages, cells generally considered devoid of proliferative activity, was found to lead to the appearance of mitosis. Mitotic index and percent phase incidence values were determined for these cells.

[Appearance of proliferative activity in peritoneal macrophages cultured in vitro. Stathmokinetic index and proliferative volume]. / L'attività riproduttiva dei macrofagi peritoneali in vitro. Indici statmocinetici e gettito proliferativo.

In the course of further studies of mitotic activity in vitro cultures of rat peritoneal macrophages, an increment in proliferative volume between the 2nd and 6th day was observed, followed by a decrease. The relationships between these variations and those of the mitotic index (previously reported) are discussed.

[Appearance of proliferative activity in peritoneal macrophages cultured in vitro. Karyokinetic chronology and phases]. / Attività mitotica dei macrofagi peritoneali del ratto. Cronologia della cariogenesi e delle sue fasi.

Contrast phase microscopy was employed to determine mitosis phase duration values in rat peritoneal macrophages in which proliferative activity had appeared following culture in vitro. Mitosis duration values were considerable and tended to increase in function of increases in culture time. These data are discussed in a general account of in vitro reproductive activity in peritoneal macrophages.