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.

Fate and transport of faecal contamination microbial indicators, pathogenic protozoa and Campylobacter in the artificially recharged fractured aquifer of Salento, Italy.

The study investigates the fate and transport of microorganisms introduced by artificial groundwater recharge at the Nardò fractured aquifer in Salento, Italy. Microbial indicators of faecal contamination, parasitic protozoa (Giardia and Cryptosporidium) and pathogenic bacteria (Campylobacter spp.), were monitored into injected water and groundwater to test the efficiency of the "natural disinfection" into the fractured aquifer. A remarkable decrease of microbial indicators and pathogens was observed suggesting that pathogens removal or inactivation may be possible during water flow in fractured aquifer. The recently described PNA probe CJE195 (Lehtola et al. 2005) was utilised for the rapid and specific detection of Campylobacter spp. by fluorescence in situ hybridization (FISH) after enrichment. FISH results were consistent with those of traditional cultural method (ISO 17995) applied in parallel: time required for Campylobacter identification was reduced of 4 days.

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.

Description of filamentous bacteria present in industrial activated sludge WWTPs by conventional and molecular methods.

Conventional cultivation methods and molecular approaches were utilised to describe the filamentous bacterial population of industrial activated sludge WWTPs. In total 43 strains were isolated by micromanipulation and were affiliated with 12 different species, comprising two new species and a new genus. In particular, a new species of Microthrix, a new genus of a filamentous Alphaproteobacteria morphologically similar to Nostocoida limicola, and a new filamentous species closely related to the opportunistic pathogen Propionibacterium propionicum were obtained. Despite the high number of isolates, the cultivation approach was unable to describe the filamentous bacteria most common in industrial WWTP. A culture-independent approach, termed the cell sorting/RT-PCR method, was therefore applied to identify fastidious or non-culturable filamentous microrganisms from different industrial plants. By this method the relevant filaments were micromanipulated and their 16S rDNA genes were amplified by RT-PCR. This approach was highly efficient. In total 31 16S rRNA sequences were obtained and 16 of them were used for the design of new specific oligonucleotide probes that highlighted dominant filaments in industrial activated sludge plants.

Microbial community analysis with a high PHA storage capacity.

Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were affiliated to Azoarcus genus within Betaproteobacteria.

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.

Molecular monitoring of bulking sludge in industrial wastewater treatment plants.

Fluorescent In Situ Hybridisation (FISH) was used to monitor the presence of filamentous microorganisms in industrial wastewater treatment plants (WWTPs). Monitoring with a restricted set of FISH probes in WWTPs from potato industry showed growth and decline of Thiothrix populations that could be linked to operational procedures. In a follow up project new FISH probes were developed for filamentous bacteria in industrial WWTPs and 70 WWTPs were analysed for presence of these filaments. Several newly described species of filamentous bacteria appear to be common and dominant in industrial WWTPs. Monitoring of a WWTP from textile industry showed growth and decline of one of these organisms when operational conditions in the plant were varied. The present paper demonstrates that bulking sludge in industrial wastewater treatment plants can effectively be monitored using a combination of standard chemical analyses and the FISH technique.

"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.

Phylogenetic analysis and in situ identification of "Nostocoida limicola"-like filamentous bacteria in activated sludge from industrial wastewater treatment plants.

The diversity of filamentous bacteria present in industrial wastewater treatment plants was analysed by a combination of classical and molecular-biological approaches. Many unknown filamentous bacteria were observed in about 80 screened activated sludge samples from different industries with sometimes severe bulking sludge problems. A special focus was paid to filaments which resembled "Nostocoida limicola", a filamentous bacterium which was found to be present in many WWTPs. These filamentous bacteria are hardly cultivable and only one strain was obtained and maintained in co-culture with a yeast. The 16S rRNA sequences of several other "Nostocoida limicola"-like filamentous bacteria from different sludge samples were obtained by micromanipulation and different molecular-biological methods. The sequences were phylogenetically analyzed and specific molecular probes were developed and applied. The results clearly demonstrate that "Nostocoida limicola"-like filaments from industrial WWTPs are different from all other "Nostocoida limicola" types investigated so far. Our strains are affiliated to the alpha-subclass of Proteobacteria.

Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge.

AIMS: The study investigated the physiology of Amaricoccus kaplicensis to determine whether it could outcompete polyphosphate accumulating bacteria in activated sludge systems removing phosphorus, by preferentially assimilating substrates in the anaerobic stages of these processes. METHODS AND RESULTS: The storage processes were investigated under anaerobic, anoxic and aerobic conditions in both batch and periodically fed cultures in an aerobic sequencing batch reactor (SBR). Amaricoccus kaplicensis showed a high capacity for storing aerobically large amounts of acetate as poly beta-hydroxybutyrate (PHB) at high rates. However, no acetate assimilation under anaerobic conditions and very slow assimilation under anoxic conditions could be detected. CONCLUSION: Amaricoccus kaplicensis in pure culture does not behave as polyphosphate accumulating bacteria competitor; therefore it is difficult to understand why anaerobic/aerobic systems often contain such large numbers of Amaricoccus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Amaricoccus kaplicensis is probably not responsible for the failure of activated sludge systems removing phosphorus, and other organisms capable of anaerobic substrate assimilation should be sought.

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.