Polyhydroxyalkanoate (PHA) production from sludge and municipal wastewater treatment.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with comparable properties to some petroleum-based polyolefins. PHA production can be achieved in open, mixed microbial cultures and thereby coupled to wastewater and solid residual treatment. In this context, waste organic matter is utilised as a carbon source in activated sludge biological treatment for biopolymer synthesis. Within the EU project Routes, the feasibility of PHA production has been evaluated in processes for sludge treatment and volatile fatty acid (VFA) production and municipal wastewater treatment. This PHA production process is being investigated in four units: (i) wastewater treatment with enrichment and production of a functional biomass sustaining PHA storage capacity, (ii) acidogenic fermentation of sludge for VFA production, (iii) PHA accumulation from VFA-rich streams, and (iv) PHA recovery and characterisation. Laboratory- and pilot-scale studies demonstrated the feasibility of municipal wastewater and solid waste treatment alongside production of PHA-rich biomass. The PHA storage capacity of biomass selected under feast-famine with municipal wastewater has been increased up to 34% (g PHA g VSS(-1)) in batch accumulations with acetate during 20 h. VFAs obtained from waste activated sludge fermentation were found to be a suitable feedstock for PHA production.

Correlations between maximum reductive dechlorination rates and specific biomass parameters in Dehalococcoides mccartyi consortia enriched on chloroethenes PCE, TCE and cis-1,2-DCE.

One of the challenges to implementing the modeling of the biological reductive dechlorination (RD) process is the evaluation of biological parameters that represent the abundance/activity levels of the microorganisms involved in the biodegradation of chloroethenes. Here we report a combined analysis of kinetic and specific biomass parameters conducted on three dechlorinating consortia enriched on PCE, TCE and cis-1,2-DCE. In these consortia, Dehalococcoides mccartyi (Dhc) represented ≥70% of the bacterial population identified via 16S rRNA gene amplicon sequencing. Quantitative biomolecular methods were used to generate specific biomass parameters targeting either the Dhc population (16S rRNA genes or cells) or specific genes encoding RD process-involved reductive dehalogenases. The correlation factor between the abundance of active Dhc cells or tceA gene copies and maximum RD rates allowed to predict an increment of 7E+09 of active Dhc cells or 5E+09 tceA gene copies/L under controlled conditions. Diversely, the utilization of gene transcripts as biomass parameters for RD modeling did not provide reliable correlations with kinetic performances. This study provides valuable insights for further modeling of the RD process through the utilization of specific biomass parameters.

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.

Use of poly-beta-hydroxy-butyrate as a slow-release electron donor for the microbial reductive dechlorination of TCE.

In situ anaerobic reductive dechlorination, using slow-release electron donors, is emerging as an effective and sustainable (low-cost and low-maintenance) technology to remediate aquifers contaminated by chloroethenes. In the present study, we investigated the use of poly-beta-hydroxy-butyrate (PHB), a fully biodegradable polymer, as a slow-release source of hydrogen and acetate for the reductive dechlorination of trichloroethene (TCE). Results of this study indicated that TCE dechlorination in PHB-amended microcosms was 2.3-times higher than in non-amended controls. This higher activity was explained by a higher H(2) level in PHB-amended microcosms. As usual, acetate was the major sink (approximately 90%) of reducing equivalents available from PHB degradation, whereas no acetotrophic dechlorination was observed.

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.

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.

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.

Anaerobic transformation of tetrachloroethane, perchloroethylene, and their mixtures by mixed-cultures enriched from contaminated soils and sediments.

The focus of this research was to investigate the anaerobic transformation of tetrachloroethane (TeCA), perchloroethylene (PCE), and their mixtures by mixed cultures enriched from contaminated soils or sediments. Batch transformation studies were conducted using TeCA (60 microM), PCE (60 microM), or TeCA + PCE (each added at 60 microM) as electron acceptor(s) and H2 + acetate (each added at 3 mM) or butyrate (3mM) as electron donor(s). A Dehalococcoides spp.-containing, sediment-enrichment dechlorinated PCE rapidly to ethene (ETH) but slowly and incompletely dechlorinated TeCA. Moreover, when present in mixture with PCE, TeCA disrupted the ability of Dehalococcoides to dechlorinate vinyl chloride. In contrast, the soil-enrichment culture was able to completely dechlorinate TeCA and PCE to ETH, both when added as single contaminants and when added as a mixture.

An aerobic fixed-phase biofilm reactor system for the degradation of the low-molecular weight aromatic compounds occurring in the effluents of anaerobic digestors treating olive mill wastewaters.

An aerobic co-culture, prepared by combining Ralstonia sp. LD35 and Pseudomonas putida DSM1868, was recently found to be capable of extensively degrading many of the hydroxylated and/or methoxylated benzoic, phenylacetic and 3-phenyl-2-propenoic acids occurring in the olive mill wastewaters (OMWs). In the perspective of developing a biotechnological process for the degradation of low-molecular weight (MW) aromatic compounds occurring in the effluents of anaerobic digestors treating OMWs, the capability of this bacterial co-culture of biodegrading a synthetic mix of the above mentioned compounds and the aromatic compounds of an anaerobic OMW-treatment plant effluent in the physiological state of immobilised cells was investigated. Two aerobic fixed-bed biofilm reactors were developed by immobilising the co-culture cells on Manville silica beads and on polyurethane foam cubes. Both supports were found to give rise to a microbiologically stable and biologically active biofilm. The two biofilm reactors were found to be similarly capable of rapidly and completely biodegrading the components of a synthetic mix of nine monocyclic aromatic acids typically present in OMWs and the low-MW aromatic compounds occurring in the anaerobic effluent in batch conditions. However, in the same conditions, the silica bead-packed reactor was found to be more effective in the removal of high-MW phenolic compounds from the anaerobic effluent with respect to the polyurethane cube-packed reactor. These results are encouraging in the perspective of using the co-culture as immobilized cells for developing a continuous biotechnological process for the post-treatment of effluents with low-MW aromatic compounds produced by anaerobic digestors treating OMWs.

Behaviour of different eluents and stabilizing agents in the determination of sulphite in water by ion-chromatography.

Ion-chromatography has been used for the determination of sulphite in water. The eluents were solutions of Na(2)CO(3) (1.1mM)-NaHCO(3) (1.4mM) or NaHCO(3) (1.0mM)-formaldehyde (0.2% w/w), and formaldehyde, glycerol or fructose was used as stabilizing agent. With the first eluent, fructose or glycerol can be used to stabilize samples against sulphite oxidation, but formaldehyde affects the peak height. On the other hand, formaldehyde can stabilize sulphite in the presence of Fe(III), whereas glycerol and fructose can not. If Fe(III) is present, the second eluent is used and sulphite is eluted directly as hydroxymethanesulphonate; formaldehyde will not then affect the peak height. This eluent allows a good peak separation and is suitable for the sulphite concentration range 0.1-12.0 mg/l.

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.

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.

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.

Kaolinite sorption of Cd, Ni and Cu from landfill leachates: influence of leachate composition.

Heavy metal speciation in landfill leachates plays a significant role in determining the mobility during the percolation through soils. The complexation characteristics of landfill leachate directly affects heavy metal solubility and the extent of the interaction with soils, lowering or raising the sorbed amount depending on the relative affinity of the complexed metal and uncomplexed form to soil adsorption sites. In this paper, the adsorption of Cd, Ni and Cu onto kaolinite from three leachates (collected from landfill at different fermentation stage) is studied, also in comparison with metal speciation by two different operative procedures. The heavy metals, at their natural concentration, were divided into operational classes according to an exchange-based procedure and by fractionation on the basis of molecular weight (exchange onto Chelex100 resin and ultrafiltration, respectively). All the experiments were performed also on synthetic solutions designed according to leachate composition and theoretical speciation. The experimental results have shown leachate complexing capacity is strongly dependent on landfill age, and that broad parameters such as COD, DOC, pH, ionic strength and VFA concentration are not able to predict it. It is notheworthy that the strong complexing capacity of leachate can cause extraction of metals from the solid phase instead of adsorption from the liquid one.

Enhancement of anaerobic treatability of olive oil mill effluents by addition of Ca(OH)2 and bentonite without intermediate solid/liquid separation.

Previous work on the anaerobic treatment of olive oil mill effluents (OME) have shown: (a) lipids, even if more easily degraded than phenols, were potentially capable of inhibiting methanogenesis more strongly; (b) a pretreatment based on addition of Ca(OH)2 and bentonite removed lipids almost quantitatively; (c) preliminary biotreatability tests performed on the pretreated OME showed high bioconversion into methane at very low dilutions ratios, especially when the mixture (OME, Ca(OH)2 and bentonite) was fed to the biological treatment without providing an intermediate phase separation. This paper was directed towards two main aims: (a) to optimize pretreatment: the best results in terms of methane production were obtained by addition of Ca(OH)2 up to pH 6.5 and of 10 g L-1 of bentonite; (b) to evaluate the enhancement of anaerobic treatability of OME pretreated under optimized conditions in a lab-scale continuous methanogenic reactor fed with the substrate without intermediate solid/liquid separation: very satisfactory performances were obtained (at an organic load of 8.2 kg COD m-3 d-1 and at a dilution ratio of 1:1.5 total COD removal was 91%, biogas production was 0.80 g CH4 (as COD)/g tot. COD, lipids removal was 98%, phenols removal was 63%). The results confirm the double role played by bentonite (adsorption of the inhibiting substances and release of the adsorbed biodegradable matter in the methanogenic reactor).

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.

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.

[Arterial embolism, venous thrombosis, pulmonary embolism: a suggestive triad]. / Embolia arteriosa, trombosi venosa, embolia polmonare. Una triade suggestiva.

The aim of this study is to present a relatively rare case of paradoxical arterial embolism, found in a patient who was sent to us for serious pulmonary embolism. Taking into account that the foramen ovale, despite being functionally competent, remains anatomically patent in 30% of the adult population, we cannot neglect the possibility of a paradoxical embolism, in the presence of a sudden embolic limb ischemia unless heart pathology or aortic lesions can be held responsible. Furthermore it must not be forgotten that deep venous thrombosis in the lower limbs or in the pelvic plexus may go unobserved on a purely clinical evaluation.

Lead adsorption onto "red soils" as function of environmental conditions.

The adsorption characteristics of the "Red Soil" with respect to lead were studied as function of different experimental conditions. Lead adsorption was investigated as function of the complexing capacity of the liquid phase (background electrolyte NaClO4, NaCl, CH3COONa e EDTA), pH (experimental range 4-7) and ionic strength (experimental range 0.001-0.35 M), by determining the adsorption isotherms at the different conditions. Experimental results allowed to identify the presence of different sorption sites, acting on lead removal through different mechanisms (ion exchange and surface complexation). These sorption sites are differently affected by changing the experimental conditions. Adsorption representation in terms of free metal was not able to describe the experimental behaviour, especially when different charged species can be formed and might be sorbed at the surface with different affinities. Particular attention was given to the optimisation of the experimental system based on the flow-through reactor set-up, in order to carry out adsorption tests more representative of the field situation.

Model evaluation of starch utilization by acclimated biomass with different culture history under pulse and continuous feeding.

The study involved model evaluation of the fate and utilization of starch by microbial culture acclimated to different growth conditions and feeding regimes. For this purpose, parallel sequencing batch reactors were operated with pulse and continuous feeding of soluble starch at sludge ages of 8 and 2 days. High-rate adsorption was identified as the initial process for starch utilization under all operating conditions. Hydrolysis mechanism acted as the rate limiting mechanism for different substrate removal/storage modes sustained under pulse and continuous feeding at different sludge ages. Together with variable growth kinetics, faster growth conditions also triggered high-rate hydrolysis and relatively slower storage kinetics to ensure the level of substrate supply for faster microbial growth. Model evaluation indicated the presence of particulate sugar adsorbed, especially under continuous feeding. It enabled accurate interpretation of observed particulate sugar values and this way, differentiating glycogen from the adsorbed starch remaining on the biomass.