Hygienization performances of innovative sludge treatment solutions to assure safe land spreading.

The present research aims at the evaluation of the hygienization performances of innovative sludge treatment processes applied for the separated treatment of secondary sludge. Namely, two digestion pretreatments (sonication and thermal hydrolysis) and two sequential biological processes (mesophilic/thermophilic and anaerobic/aerobic digestion) were compared to the mesophilic (MAD) and thermophilic anaerobic digestion (TAD). Microbial indicators (Escherichia coli, somatic coliphages and Clostridium perfringens spores) and pathogens (Salmonella and enteroviruses), which show different resistances to treatment processes, were monitored in untreated and treated sludge. Overall, microbial load in secondary sludge was shown to be similar or lower than previously reported in literature for mixed sludge. Notably, the anaerobic/aerobic digestion process increased the removal of E. coli and somatic coliphages compared to the simple MAD and always achieved the hygienization requirement (2-log-unit removal of E. coli) proposed by EU Commission in the 3rd Working Document on sludge (April 2000) for the use of treated sludges in agriculture with restriction on their application. The microbial quality limits for the unrestricted use of sludge in agriculture (no Salmonella in 50 g wet weight (WW) and E. coli <500 CFU/g) were always met when thermal digestion or pretreatment was applied; however, the required removal level (6-log-unit removal of E. coli) could not be assessed due to the low level of this microorganism in raw sludge. Observed levels of indicator removal showed a higher resistance of viral particles to thermal treatment compared with bacterial cells and confirmed the suitability of somatic coliphages as indicators in thermal treatment processes.

Quality assessment of digested sludges produced by advanced stabilization processes.

The European Union (EU) Project Routes aimed to discover new routes in sludge stabilization treatments leading to high-quality digested sludge, suitable for land application. In order to investigate the impact of different enhanced sludge stabilization processes such as (a) thermophilic digestion integrated with thermal hydrolysis pretreatment (TT), (b) sonication before mesophilic/thermophilic digestion (UMT), and (c) sequential anaerobic/aerobic digestion (AA) on digested sludge quality, a broad class of conventional and emerging organic micropollutants as well as ecotoxicity was analyzed, extending the assessment beyond the parameters typically considered (i.e., stability index and heavy metals). The stability index was improved by adding aerobic posttreatment or by operating dual-stage process but not by pretreatment integration. Filterability was worsened by thermophilic digestion, either alone (TT) or coupled with mesophilic digestion (UMT). The concentrations of heavy metals, present in ranking order Zn > Cu > Pb > Cr ~ Ni > Cd > Hg, were always below the current legal requirements for use on land and were not removed during the processes. Removals of conventional and emerging organic pollutants were greatly enhanced by performing double-stage digestion (UMT and AA treatment) compared to a single-stage process as TT; the same trend was found as regards toxicity reduction. Overall, all the digested sludges exhibited toxicity to the soil bacterium Arthrobacter globiformis at concentrations about factor 100 higher than the usual application rate of sludge to soil in Europe. For earthworms, a safety margin of factor 30 was generally achieved for all the digested samples.

Advanced anaerobic processes to enhance waste activated sludge stabilization.

The requirement for enhanced stabilization processes to obtain a more stable, pathogen-free sludge for agricultural use is an increasing challenge to comply with in the waste hierarchy. With this in mind, the Routes European project ('Novel processing routes for effective sewage sludge management') is addressed to assess innovative solutions with the aim of maximizing sludge quality and biological stability. In order to increase anaerobic stabilization performances, the sequential anerobic/aerobic process and the thermophilic digestion process, with or without integration of the thermal hydrolysis pre-treatment, were investigated as regards the effect on sludge stabilization, dewaterability and digestion performances. Thermal pre-treatment improved anaerobic digestion in terms of volatile solids reduction and biogas production, but digestate dewaterability worsened. Fluorescence in situ hybridization (FISH) quantification showed an increase of methanogens consistent with the increase of biogas produced. The aerobic post-treatment after mesophilic digestion had a beneficial effect on dewaterability and stability of the digested sludge even if was with a reduction of the potential energy recovery.

Two-phase reactors applied to the removal of substituted phenols: comparison between liquid-liquid and liquid-solid systems.

In this paper, a comparison is provided between liquid-liquid and liquid-solid partitioning systems applied to the removal of high concentrations of 4-nitrophenol. The target compound is a typical representative of substituted phenols found in many industrial effluents while the biomass was a mixed culture operating as a conventional Sequencing Batch Reactor and acclimatized to 4-nitrophenol as the sole carbon source. Both two-phase systems showed enhanced performance relative to the conventional single phase bioreactor and may be suitable for industrial application. The best results were obtained with the polymer Hytrel which is characterized by higher partition capability in comparison to the immiscible liquid solvent (2-undecanone) and to the polymer Tone™. A model of the two systems was formulated and applied to evaluate the relative magnitudes of the reaction, mass transfer and diffusion characteristic times. Kinetic parameters for the Haldane equation, diffusivity and mass transfer coefficients have been evaluated by data fitting of batch tests for liquid-liquid and liquid-solid two phase systems. Finally, preliminary results showed the feasibility of polymer regeneration to facilitate polymer reuse by an extended contact time with the biomass.

Removal of xenobiotics in a two phase sequencing batch reactor: kinetics and modelling.

The objectives of the paper are to verify the potentialities of a sequential two phase partitioning bioreactor in degrading xenobiotics and to evaluate the kinetic parameters for modelling the system. The target compound investigated was the 4-nitrophenol. Preliminary tests were carried out to define the solvent most appropriate for the compound. Among the three investigated solvents 1-undecanol, 2-undecanon and oleyl alcohol, the 2-undecanon was chosen because of the higher partition coefficient of 30 and the negligible formation of emulsions. Moreover, the tested solvent showed satisfactory "biocompatibility" characteristics for the biomass with minor effects on the intrinsic kinetics. Parallel batch kinetic tests were then performed with the conventional one phase and the two phase systems. In the two phase system the biomass is exposed for all the time to 4NP concentrations that are significantly lower if compared to the conventional system and, for the highest concentration (450 mg/l) in the two phase system a reduction of the reaction time is observed depending on the biomass concentration. Kinetic parameters were also evaluated in both cases by fitting of the experimental data with a modified form of the Haldane equation.

Anaerobic degradation kinetics of particulate organic matter in untreated and sonicated sewage sludge: role of the inoculum.

Degradation kinetics of particulate matter in anaerobic digestion of secondary sludge, untreated and sonicated, was investigated by carrying out batch tests at different feed/inoculum ratio (F/I) (in the range of 0.1-4.0). Particulate COD degradation data were analysed using the four equations most widely utilized to model the hydrolysis process and the related kinetic parameters were evaluated. The increase of F/I results in a correspondent increase of the process rate up to one order of magnitude in the investigated interval for both untreated and sonicated sludge. The maximum step increase is observed in the range of 0.1-2.0 while for F/I varying from 2.0 to 4.0 only a modest enhancement of the process kinetics is detected. The effect of sonication on kinetics is not appreciable at low F/I, due to the low fraction of fed sludge and to the consequent strong substrate limitation, whereas at high F/I a slight increase is evidenced.

Bacterial growth kinetics estimation by fluorescence in situ hybridization and spectrofluorometric quantification.

AIMS: The aim of this study was to develop a specific and rapid method to identify and quantify relevant bacterial populations in mixed biomass by spectrofluorometric quantification (SQ) of whole cells hybridized with fluorescently labelled oligonucleotide probes targeting mature 16S ribosomal RNA (rRNA). Probe targeting the precursor of rRNA synthesis was also employed because it was being suggested as more indicative of the activity state of the micro-organisms. METHODS AND RESULTS: Original fluorescence in situ hybridization protocol was modified to be applied to liquid samples and the fluorescence emission from the Cy3-labelled cells was measured by spectrofluorometry. The method was calibrated on an exponentially growing cell suspension of Acinetobacter johnsonii and was successfully applied to generate kinetic data. No substantial difference in the estimated maximum specific growth rate (mu(max)) values was found between the SQ method and the classical method, using absorbance at 420 nm (6.2 d(-1)). The preliminary validation tests showed their direct applicability to target enriched cultures. CONCLUSIONS: This study demonstrated the validity of the SQ method to easily quantify the concentration and to determine the growth rate of specific micro-organisms present in mixed cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method can be directly utilized for quantification and kinetic characterization of microbial enrichments. It has the advantage of being easily applicable using simple, inexpensive equipment suitable for routine analysis.

Effect of feed/inoculum ratio on anaerobic digestion of sonicated sludge.

In recent years, relevant interest has been devoted to activated sludge disintegration and solubilisation techniques in order to cope with the biological limitations related to particulate degradation. Mechanical disintegration with ultrasound can efficiently transform insoluble organics into a soluble form: the solubilised organic matter is released from the cells to the bulk phase, thus accelerating the hydrolysis step in the digestion process. Experiments were carried out on bench scale anaerobic reactors fed with either untreated or disintegrated excess sludge, added with a biomass inoculum taken from a full scale anaerobic digester. Digestion tests have been carried out at different feed/inoculum ratios (F/I) in the range of 0.1-2, kinetics of VS reduction has been investigated and a beneficial effect of sonication is observed for all the experimental conditions. Similar beneficial results have also been found for biogas production with a maximum gain of 25% at 0.5 F/I ratio.

An innovative sludge management system based on separation of primary and secondary sludge treatment.

An innovative sludge management system based on separation of treatment and disposal of primary and secondary sludge is discussed with reference to a sewage treatment plant of 500,000 equivalent person capacity. Secondary sludge, if treated separately from primary sludge, can be recovered in agriculture considering its relatively high content of nitrogen and phosphorus and negligible presence of pathogens and micropollutants. One typical outlet for primary sludge is still incineration which can be optimised by rendering the process auto thermal and significantly reducing the size of the incineration plant units (dryer, fluidised bed furnace, boiler and units for exhaust gas treatment) in comparison with those required for mixed sludge incineration. Biogas produced in anaerobic digestion is totally available for energy conversion when sludge treatment separation is performed, while in the other case a large proportion may be used as fuel in incineration, thus reducing the net electric energy conversion from 0.85-0.9 to 0.35-0.4 MW for the plant considered.

Toxicity assessment of common xenobiotic compounds on municipal activated sludge: comparison between respirometry and Microtox.

The toxicity of four xenobiotic compounds 3,5-dichlorophenol, formaldehyde, 4-nitrophenol and dichloromethane, representative of industrial wastewater contaminants was evaluated by a simple respirometric procedure set up on the basis of OECD Method 209 and by the Microtox bioassay. Very good reproducibility was observed for both methods, the variation coefficients being in the range of 2-10% for the respirometric procedure and 6-15% for Microtox, values that can be considered very good for a biological method. Comparison of EC(50) data obtained with the two methods shows that in both cases 3,5-dichlorophenol is more toxic than other compounds investigated and dichloromethane has a very low toxicity value. Intermediate EC(50) values were found for the two other chemicals, formaldehyde and 4-nitrophenol. Moreover, the Microtox EC(50) values are generally lower (except for dichloromethane) than the respirometric ones: these differences could be explained by the fact that the Microtox method uses a pure culture of marine species and, therefore, should not necessarily be expected to behave like a community of activated sludge bacteria. In conclusion, both methods can be usefully applied for toxicity detection in wastewater treatment plants but it is advisable to take into account that Microtox is more sensitive than respirometry in estimating the acute toxicity effect on the biomass operating in the plant.

"Microthrix parvicella": a new approach for kinetic and physiological characterization.

Two strains of "Microthrix parvicella" (RN1 and 4B strains) have been physiologically and kinetically characterized by utilizing a new technique particularly suitable for slow growing bacteria. The proposed method is based on the Total Extended Filament Length (TEFL) measure on filaments grown on agar surfaces. This allows us to study more easily and rapidly slow growing bacteria under many different conditions without altering the growth environment during the test. "Microthrix parvicella" appears to have versatile physiological capabilities allowing effective competition against other bacteria in the complex biocenosis of activated sludge. Main peculiarities of this organism are its storage capabilities under an aerobic/anoxic/anaerobic reaction environment, the resistance to long periods of anoxic/anaerobic conditions, the possibility of growing with appreciable kinetics at very low temperatures. In the kinetic characterization low Ks values have been observed; this finding supports the assumption that "Microthrix parvicella" is a "Ks- strategist" with high substrate affinity. Kinetics tests performed in a mixed population of activated sludge bacteria and on pure culture of filaments isolated from the same sludge sample showed that no beneficial effect on the growth rates of "Microthrix parvicella" seems attributable to the presence of activated sludge. This last result confirms the validity of pure culture studies for this microorganism and the reliability of physiological and kinetic data so evaluated in describing real systems.

Inhibiting effects of chloroform on anaerobic microbial consortia as monitored by the Rantox biosensor.

The Rantox biosensor was designed for anaerobic wastewater treatment process control, and detects modifications of the feed based on the response of the acetoclastic methanogens contained in the sensor to periodic pulses of a concentrated organic substrate. The biosensor was tested under various operating conditions at the laboratory scale, in parallel with a digester under control fed on the same substrate. The aim was to evaluate the response of the biosensor in the presence of an incoming organic toxic compound (CHCl3). The experimental set-up, i.e. the biosensor and the digester, was connected to an automated control system developed under LabVIEW environment for data acquisition and operational sequence programming (the Rantox Virtual Instrument). Biomasses with different activities were used as inocula, and inhibition was induced by dosing chloroform according to two different procedures. The results showed good sensitivity and rapid response of the biosensor to feed intoxication. The presence of chloroform was detected by the Rantox with a rapid and visible response, and well in advance with respect to the digester.

Monitoring toxicity in anaerobic digesters by the rantox biosensor: theoretical background.

Process control of anaerobic reactors is difficult due to the complexity of the methabolic pathways in the microbial consortium and to the difficulty of detecting and monitoring process instability in short time, before the biomass is poisoned by incoming toxicants. Process control based on the Rantox biosensor is based on the following principle: the wastewater that can potentially induce an overload or contains a toxicant is first tested on a small "upstream" digester (the Rantox). This reactor makes possible to detect the potential instability and, if necessary, to divert the concentrated and/or contaminated wastewater to a buffer tank and consequently to protect the active biomass of the full-scale reactor. It is generally accepted that methanogens are the most sensitive microorganisms in anaerobic digestion. Among these bacteria, the acetoclastic methanogens are of primary importance because some 70% of the converted chemical oxyen demand (COD) mass flow passes through acetic acid. Therefore the first objective in the development of the Rantox biosensor has been to monitor the metabolism of acetoclastic methanogens in the presence of toxicants. This article presents the theoretical background required to evaluate the toxicity effects by determining the kinetic constants of the considered microorganisms from experimental data. The results of two series of calibration tests, performed in order to obtain a preliminary evaluation of the biosensor response to overload and toxicity conditions, are reported. In a second article, calibration tests will be described which refer to two prototypes of the biosensor tested in different operating conditions. The crucial point related to the Rantox, i.e., its comparison with a "normal" laboratory-scale digester to simulate a full-scale plant, will be the subject of the third (and last) article, which is planned to describe the development of this instrument.