A recommended method for detecting salmonellae in composted biosolids

It has been found in Australia and in the United States that composting does not always result in the complete removal of salmonellae from biosolids. It is therefore likely that monitoring of composted Biosolids for salmonellae will be required in Australia to ensure the safety of biosolids products. At present rapid methods of detection such as PCR and ELISA are not sufficiently developed to monitor environmental samples. The relative efficiency of various culture methods for detecting salmonellae in composted biosolids was therefore investigated. On the basis of the results a presence/absence method is recommended for the detection of salmonellae in biosolids products. The recommended technique involves pre-enrichment of samples, followed by enrichment in Rappaport-Vassiliadis and mannitol selenite enrichment broths, and isolation on lysine mannitol glycerol agar

A risk assessment approach for setting criteria for pathogens in biosolids

Risks associated with pathogens in biosolids are uncertain and are potentially of major concern in the marketing of biosolids products. At present there are no formal National biosolids guidelines in Australia. US EPA regulations require the monitoring of biosolids for pathogens or indicator bacteria but criteria have been based on detectable limits for pathogens. Two problems with this approach are firstly that detection limits in microbiology are constantly changing, and secondly that in some cases criteria based on detection limits may result in unacceptable risk. For this reason a risk assessment approach has been developed and is described here. This risk assessment approach has been applied to setting criteria for pathogens in biosolids. This involved the steps of hazard identification, development of acceptable risk criteria, dose-response assessment, exposure assessment and calculation of limits. Based on this risk assessment procedure a limit of less than 1 Salmonella in 50 g of biosolids product is proposed for biosolids which are to have unrestricted distribution. It is recommended that limits for Giardia and enteric viruses should not be included in guidelines until methods for their detection in bisolids are more developed

Die-off of human pathogens in sludge amended soil

Anaerobically digested sludge is commonly used both in Australia and overseas as an agricultural soil amendment. Even though a number of studies have examined the die-off of faecal indicator bacteria in sludge amended soil, there is less information concerning the die-off of human pathogens. This study examined the die-off of enteroviruses, Salmonella and Giardia in sandy soil amended with sludge which had been treated by anaerobic digestion and mechanical dewatering. Enteroviruses were not detected in sludge amended soil even though they were present in sludge in low numbers. Giardia cysts were initially present but decreased in number and were not detected after 8 weeks. Salmonella decreased to non-detectable concentrations within 8 weeks but were then re-detected after 36 weeks. It appeared that Salmonella regrew as a result of an increase in soil moisture content at the beginning of winter. Faecal coliforms also appeared to regrow after a long period of non-detection. The potential regrowth of bacterial indicator organisms and human pathogens in soil amended with wastewater sludge is of concern

Pathogens in wastewater sludge

Information concerning pathogen densities in wastewater sludge is scarce, particularly for Australian sludges. As part of a project investigating the die-off of pathogens in stored sludge pathogen densities in sludge from Perth wastewater treatment plants were determined. Faecal coliform, faecal streptococci, enterovirus and Salmonella concentrations in sludge were quantified. Campylobacter, Shigella, Legionellaand helminths were not detected in sludge using qualitative tests. Giardia and Entamoeba were present in raw sludge but were not detected in digested and dewatered sludge. There was no consistent relationship between the die-off of indicator organisms and pathogens through sludge treatment. Pathogens generally survived treatment better than faecal coliforms and faecal streptococci. On the basis of these results the use of indicator bacteria to predict pathogen densities in wastewater sludge may not be appropriate

Selection of Salmonella typhimurium as an indicator for pathogen regrowth potential in composted biosolids.

In order to select a suitable indicator for monitoring the pathogen regrowth potential of composted biosolids, the growth kinetics of selected bacteria were investigated. Growth parameters of six serovars of Salmonella and three strains of Escherichia coli in sterilized compost were compared. Seeded Salmonella and E. coli grew rapidly, reaching population densities of more than 10(8) g-1 after 30 h of incubation. The specific growth rates of Salmonella serovars and E. coli strains were similar and varied from 0.49 to 0.55 h-1. The specific growth rate of the Salm. Typhimurium isolates was significantly higher than the other bacterial strains. It was concluded that an antibiotic-resistant strain of Salm. Typhimurium can be used as an indicator for a pathogen regrowth potential test

The role of indigenous microorganisms in suppression of salmonella regrowth in composted biosolids

Composting is commonly used as an effective means of stabilizing wastewater biosolids and reducing pathogens to very low concentrations. However, it has been shown that under certain conditions Salmonella can regrow in previously composted biosolids. Growth of seeded Salmonella typhimurium in composted biosolids ranging from two weeks to two years maturity was monitored. Results from sterile and non-sterile composted biosolids were compared. Seeded S. typhimurium colonized rapidly in sterilized biosolids reaching a maximum population density of more than 108g-1. Growth of seeded S. typhimurium was suppressed in non-sterilized compost with a maximum population density of less than 103g-1. There was a significant decline in the growth rate of seeded Salmonella in sterilized compost when the compost was stored, suggesting that bio-available nutrients declined with storage. However, in non-sterilized compost this was not the case. This suggests that the indigenous microflora play a significant role in suppression of Salmonella regrowth in composted biosolids. There was a strong negative correlation (-0.85) between the Salmonella inactivation rate and the maturity of compost in non-sterilized compost. The Salmonella inactivation rate was seven times higher in biosolids composting for two weeks as compared to compost stored for two years. This suggests that the antagonistic effect of indigenous microorganisms towards Salmonella declined with compost storage. It was concluded that all composted biosolids had a Salmonella regrowth potential. However, the indigenous microflora significantly reduced this regrowth potential. Long-term storage of compost is not recommended as this may increase the pathogen regrowth potential

Regrowth of faecal coliforms and salmonellae in stored biosolids and soil amended with biosolids

Stabilised wastewater sludge (biosolids) has beneficial re-use properties but these are limited by the presence of human pathogens. In this study soil amendment with biosolids and storage of biosolids prior to re-use were examined as disposal and treatment options. In a soil amendment trial biosolids were mixed with sandy soil and monitored for 37 weeks. In two storage trials biosolids were stored in piles 1m high and monitored for <60 weeks. Included in the monitoring programme were tests to determine the concentrations of faecal coliforms, faecal streptococci and salmonellae. In both the soil amendment trials and biosolids storage trials, concentrations of indicator organisms and salmonellae decreased through an extended hot, dry summer period. Although these organisms were not detected in the majority of samples taken during the summer, repopulation of faecal coliforms and salmonellae occurred in the trials following rainfall at the beginning of the winter. In the case of one of the storage trials repopulation occurred following a period of 50 weeks when salmonellae and faecal coliforms were not detected. When repopulation occurred, faecal coliform concentrations increased to higher than those at the beginning of the trials. These results suggest that faecal coliforms and salmonellae were at undetectable concentrations through the summer period but were able to grow when provided with favourable conditions. From this limited trial it was concluded that soil amended with biosolids could not be considered free from pathogens for at least one year following amendment