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.

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.

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.

Field distribution and activity of chlorinated solvents degrading bacteria by combining CARD-FISH and real time PCR.

Nowadays several advanced molecular techniques are applied for quantifying bacteria involved in contaminant degradation processes. However, despite the fact that significant efforts have been taken to make these tools more reliable and specific, their application for the analysis of field samples is hardly ever applied. In this study, a combination of three methods (CARD-FISH, qPCR and RT-qPCR) was successfully applied to evaluate the distribution and the activity of known chlorinated solvent dechlorinating bacteria in a contaminated site where no remedial actions have been undertaken. CAtalysed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) specifically provided the cell densities of known dechlorinating bacteria and was found to be more sensitive than quantitative PCR (qPCR) for the quantification of 'Dehalococcoides' cell numbers in the aquifer. Among the screened dechlorinators, 'Dehalococcoides' spp. were mainly found and nearly homogenously distributed in the aquifers at concentrations ranging from 8.1×10(5)±1.2×10(5) to 2.5×10(7)±5.6×10(6)cells per liter of groundwater (with a relative abundance out of the total Bacteria of 0.7-15%). Further, the dechlorination potentialities of 'Dehalococcoides' species living in the aquifer were evaluated by analyzing the abundance and the expression of 16S rRNA genes and reductive dehalogenase (RDase) encoding functional genes by qPCR and Reverse Transcription qPCR (RT-qPCR). 'Dehalococcoides'tceA gene, known to be associated to strains capable of reducing chlorinated solvents beyond cis-DCE, was found and expressed in the field. Overall, this study proved the existence of a well-established dechlorinating microbial community able to use contaminants as substrates for their metabolic activity and indicated the occurrence of reductive dechlorination at the site.

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.

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.

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.

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.

Biological treatment of tannery wastewater in the presence of chromium.

Experiments were carried out to determine the feasibility of treating tannery wastewater containing chromium, an inhibiting compound, with sequencing batch reactors (SBR). The maximum chromium concentration tolerated by microorganisms was determined through aerobic and anoxic batch experiments, and the biomass inhibition process was analyzed in a lab scale reactor at increasing chromium concentrations. The results obtained, in batch experiments and in the SBR reactor, have demonstrated that chromium addition had less influence on the denitrification bacteria than on the nitrification bacteria. In addition, it was observed that nitrification and denitrification rates, at the same chromium concentration, were higher in the SBR reactor than in batch experiments with unacclimated biomass. Experimental results confirm that sequencing batch reactors are able to produce a more resistant biomass, which acclimates quickly to inhibiting conditions. A large amount of chromium was found in the sludge from the reactor, while the effluent was devoid of the inhibiting metal.

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.

Detection and quantitative estimation of Dehalococcoides spp. in a dechlorinating bioreactor by a combination of fluorescent in situ hybridisation (FISH) and kinetic analysis.

The unique capacity of Dehalococcoides ethenogenes of completely dechlorinating the common groundwater pollutant tetrachloroethene (PCE) to the harmless ethene makes this microorganism very attractive for application in natural or engineered bioremediation systems. In this study, the qualitative and quantitative determination of Dehalococcoides spp. in a lab-scale bioreactor was performed based on the combination of fluorescent in situ hybridisation (FISH) for specific detection, and kinetic batch tests at non-limiting hydrogen and PCE concentration for quantitative determination. The dechlorinating bioreactor was operated at a high and constant PCE loading rate of 255 micromol PCE [g volatile suspended solids (VSS)](-1) day(-1). Pale coccoid cells resembling the distinctive morphotype of D. ethenogenes were present in the microbial culture. These cocci hybridised with both eubacterial probes and the Dhe1259t probe recently designed for detecting Dehalococcoides spp. Positive hybridisation was also observed when the DHC1377 reverse primer was used as a specific probe and applied to the dechlorinating microbial consortium. The maximum dechlorination rate obtained under non-limiting hydrogen and PCE concentrations was 3.22 +/- 0.08 mmol Cl(-) l(-1 )day(-1). From the specific activity of D. ethenogenes [i.e. 0.055 +/- 0.008 mmol Cl(-) (mg VSS)(-1) day(-1)], as reported from pure culture study, this observed maximum rate corresponded to a concentration of this bacterium in the mixed liquor of the bioreactor of 59.0+/-10.4 mg VSS.l(-1) (41.5+/-11.2% of overall VSS). This calculated relative abundance of D. ethenogenes was in agreement with the percentage of methanol (in terms of reducing equivalents) channeled to reductive dechlorination (approximately 30%) supporting the assumption that most reductive dechlorination was actually due to this microorganism.

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.

Removal of molecular weight fractions of COD and phenolic compounds in an integrated treatment of olive oil mill effluents.

Previous works (Beccari et al. 1999b; Beccari et al. 2001a; Beccari et al. 2001b) on the anaerobic treatment of olive oil mill effluents (OME) have shown: (a) a pre-treatment based on the addition of Ca(OH)2 and bentonite was able to remove lipids (i.e. the most inhibiting substances present in OME) almost quantitatively; (b) the mixture OME-Ca(OH)2-bentonite, fed to a methanogenic reactor without providing an intermediate phase separation, gave way to high biogas production even at very low dilution ratios; (c) the effluent from the methanogenic reactor still contained significant concentrations of residual phenolic compounds (i.e. the most biorecalcitrant substances present in OME). Consequently, this paper was aimed at evaluating the fate of the phenolic fractions with different molecular weights during the sequence of operations (adsorption on bentonite, methanogenic digestion, activated sludge post-treatment). The results show that a very high percentage (above 80%) of the phenolic fraction below 500 D is removed by the methanogenic process whereas the phenolic fractions above 1,000 D are significantly adsorbed on bentonite; the 8-day activated sludge post-treatment allows an additional removal of about 40% of total filtered phenolic compounds. The complete sequence of treatments was able to remove more than the 96% of the phenolic fraction below 500 D (i.e. the most toxic fraction towards plant germination). Preliminary respirometric tests show low level of inhibition exerted by the effluent from the methanogenic reactor on aerobic activated sludges taken from full-scale municipal wastewater plants.

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.

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.

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.

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.