Using event trees to quantify pathogen levels on root crops from land application of treated sewage sludge

AIMS: To quantify the incremental exposure of root crops, at point of harvest, to enteric pathogens from sewage sludge applied to agricultural land according to current regulations and guidance (Safe Sludge Matrix). METHODS AND RESULTS: A quantitative risk assessment based on the Source-Pathway-Receptor approach is developed for Cryptosporidium and salmonellas. Event trees are constructed to model the partitioning of pathogens present in raw sewage into sludge at the sewage treatment works and to model to the pathways by which root crops may be exposed to those pathogens after treatment and land application of the sludge. The main barriers are sewage sludge treatment, and decay and dilution of the pathogens in the soil. The exposures are expressed in terms of the arithmetic mean. This represents the total loading and accommodates fluctuations not only in the levels of pathogens present in sewage but also in the removal efficiencies by the various barriers. One source of uncertainty is the degree of by-pass of sludge treatment at operational scale. CONCLUSIONS: The models predict that land application of sewage sludge treated by conventional processes (achieving 2-log removal) increases the exposures of root crops to salmonellas and Cryptosporidium oocysts by counts of 0.070 and 0.033 kg(-1), respectively. These predictions are based on decay periods in the soil of 5 and 12 weeks, respectively, and are therefore worst case in not allowing for the full extent of no harvesting periods. A Monte Carlo simulation predicts that 0.01% of 1-kg batches contained > 50 salmonellas and demonstrates that, for risk assessment, it is acceptable to use the arithmetic mean exposure directly in the dose-response curve. SIGNIFICANCE AND IMPACT OF THE STUDY: The predicted numbers of pathogens on root crops at point of harvest provide a basis for modelling the excess risks to humans consuming such crops. The approach underpins scientifically the Safe Sludge Matrix.     

The Removal of Salmonellas in Conventional Sewage Treatment Processes

The numbers of salmonellas in raw sewage entering a treatment plant varied hourly and diurnally; their peak concentration preceded the peak influent waste water flow into the plant by about 2 hours. Salmonellas were detected in all raw influent samples collected from 2 sewage works and the mean population level at the daily peak period was 3000 organisms/1. On average. 70–80% of the salmonellas were removed during primary sedimentation when upwards of 74–84% of solids were removed. Biological treatment and secondary sedimentation removed a further 70–100% of the pathogen. Compared with the activated sludge process the trickling filters were less efficient in removing salmonellas and they were adversely affected by increased loading following rainfall. Considering the whole treatment process, the Guildford works with its activated sludge treatment removed an average of 99–83% salmonellas while the Woking works with its trickling filter plant removed an average of 93–04% of the organisms. The large variance in the numbers of salmonellas in the final effluent from the trickling filters suggest that greater emphasis should be placed on the actual quality of the effluent rather than on percentage removal efficiencies.     

Evaluation of Culture Media for the Isolation of Salmonellas from Sewage Sludge

Ten samples of sewage sludge were examined by various methods for the isolation of salmonellas using three types of enrichment broth: Muller-Kauffmann Tetrathionate Broth (MKTB), Selenite F Broth (SFB), and Brilliant Green Broth (BGB), two temperatures (37°C and 43°C) and three selective media: Deoxycholate Citrate Agar incubated aerobically (DCA), and anaerobically (DCA(N)), Brilliant Green Agar (BGA), and Bismuth Sulphite Agar (BSA). The results suggest that a combination of pre-enriched MKTB incubated at 37°C and plated on to BGA at 24 and 48 h was the best method, but when examining contaminated material such as sewage sludge, it appears unwise to rely on one single method.     

A note on the use of Rappaport-Vassiliadis medium (RIO/RV) for the isolation of salmonellas from sewage and sewage sludges

The use of a modified Rappaport broth for the selective enrichment of salmonellas in sewage sludge is described. Comparative trials were carried out using Muller Kauffman—Tetrathionate (MKT) broth and Rappaport-Vassiliadis (R10/RV) medium for selective enrichment. Results have indicated that R10/RV broth is more selective and is to be preferred for routine monitoring.     

A note on the use of Rappaport-Vassiliadis medium (R10/RV) for the isolation of salmonellas from sewage and sewage sludges

The use of a modified Rappaport broth for the selective enrichment of salmonellas in sewage sludge is described. Comparative trials were carried out using Muller Kauffman--Tetrathionate (MKT) broth and Rappaport-Vassiliadis (R10/RV) medium for selective enrichment. Results have indicated that R10/RV broth is more selective and is to be preferred for routine monitoring.     

Inactivation of Salmonella during anaerobic digestion of sewage sludge

The inactivation of Salmonella duesseldorf in sewage sludge during anaerobic digestion was investigated at 35 and 48°C with mean retention periods of between 10 and 20 days. Digesters were fed daily with raw sludge containing added Salm. duesseldorf after removal of digested sludge. During steady operation, the levels of Salm. duesseldorf in the digested and the feed sludge were determined and their specific rates of decay were estimated. The latter were: (i) greater at 48°C than at 35°C for the same retention time; (ii) similar for retention periods greater than 15 d, but lower for 10 d; (iii) greater when the level of salmonellas in the feed was lower. Gas production, a measure of steady state, was gradually lost when the mean retention period was reduced to 6.7 d. In experiments in which a single dose of Salm. duesseldorf was added to digesting sludge, the inactivation appeared to follow first-order kinetics at 35°C and the decimal decay rate, 1.6/d, was similar to that in the daily feeding experiments (1.4/d) with larger and similar inocula of Salm. duesseldorf. At 48°C, however, the rate of inactivation declined with decreasing time from inoculation suggesting that the culture contained cells differing in thermal resistance. The degrees and rates of inactivation of salmonellas in those experiments were greater than in full-scale digesters, because the latter seldom operated under conditions ideal for inactivation or because indigenous salmonellas are more resistant.     

Observations on Pre-enrichment for Isolating Salmonellas from Sewage Polluted Natural Water Using Muller-Kauffmann Tetrathionate Broth Prepared with Fresh and Desiccated Ox Bile

Desiccated bile (Oxoid L50) was substituted for fresh bile obtained from the abattoir in Muller-Kauffmann Tetrathionate Broth. Two versions of the enrichment medium were prepared, one with desiccated bile the other with fresh bile. The efficiency of these two media was compared using 25 ml quantities of sewage polluted natural water as inocula. Direct enrichment and preenrichment techniques were used. With direct enrichment the Muller-Kauffmann Tetrathionate Broth prepared from fresh bile was more efficient for salmonella isolation. With the pre-enrichment technique using preliminary culture of the water sample in buffered peptone water prior to enrichment, there was no significant difference between the efficiency of the two versions of tetrathionate. Comparison of direct enrichment with the pre-enrichment method for isolating salmonellas from sewage polluted water clearly demonstrated the advantages of pre-enrichment. This would certainly have a bearing on the quantitative estimation of salmonellas in polluted water and might increase the number of serotypes isolated from a sample divided into subsamples.     

Molecular Analysis of a Bacterial Chitinolytic Community in an Upland Pasture

The effects of agricultural-improvement treatments on the chitinolytic activity and diversity of a microbial community were investigated within an upland pasture. The treatments of interest were lime and treated sewage sludge, both commonly applied to pasture land to improve fertility. Burial of chitin-containing litter bags at the field site resulted in enrichment of bacteria according to 16S rRNA fingerprinting. Chitinolytic-activity measurements showed that the highest activity occurred in those bags recovered from sludge-amended plots, which correlated well with increased counts of actinobacteria in samples from these chitin bags. Our findings suggest that sewage sludge increases the fertility of the soil in terms of chitinase activity. Ten clone libraries were constructed from family 18 subgroup A chitinases, PCR amplified from litter bags buried in soil in July 2000 or in September 2000, in a separate study. Analysis of these libraries by restriction fragment length polymorphism and sequencing showed that they were dominated by actinobacterium-like chitinase sequences. This suggests that actinobacteria have an important chitinolytic function in this soil ecosystem. Our findings showed that sludge application increased chitinolytic activity but decreased the diversity of chitinases present.     

Molecular analysis of a bacterial chitinolytic community in an upland pasture

The effects of agricultural-improvement treatments on the chitinolytic activity and diversity of a microbial community were investigated within an upland pasture. The treatments of interest were lime and treated sewage sludge, both commonly applied to pasture land to improve fertility. Burial of chitin-containing litter bags at the field site resulted in enrichment of bacteria according to 16S rRNA fingerprinting. Chitinolytic-activity measurements showed that the highest activity occurred in those bags recovered from sludge-amended plots, which correlated well with increased counts of actinobacteria in samples from these chitin bags. Our findings suggest that sewage sludge increases the fertility of the soil in terms of chitinase activity. Ten clone libraries were constructed from family 18 subgroup A chitinases, PCR amplified from litter bags buried in soil in July 2000 or in September 2000, in a separate study. Analysis of these libraries by restriction fragment length polymorphism and sequencing showed that they were dominated by actinobacterium-like chitinase sequences. This suggests that actinobacteria have an important chitinolytic function in this soil ecosystem. Our findings showed that sludge application increased chitinolytic activity but decreased the diversity of chitinases present.     

The removal of Salmonella enteritidis in activated sludge

Salmonella destruction efficiencies of 99% were obtained after 10 h aeration at 15°C in a laboratory model of the activated sludge process. This study demonstrated that, in a batch process, the removal of salmonellas occurred in three phases. (i) By 4 h, 90% of the original inoculum had disappeared from the activated sludge, probably due mainly to predation by ciliated protozoa. The remaining 10% was distributed between the liquid phase (approximately 90%) and the sludge floc (approximately 10%). (ii) During the next 2 h this situation was inverted so that, by 6 h, more than 80% of the remaining salmonellas were then adsorbed to floc, leaving less than 20% in liquid suspension. (iii) From 6 h onwards there was a much slower decline of the remaining salmonellas attached to floc. The addition of dioctyl sodium sulphosuccinate after 4 h, inactivated the ciliated protozoa populations and completely eliminated the continued reduction of salmonellas from activated sludge observed previously.     

Can Organic Amendments Be Useful in Transforming a Mediterranean Shrubland into a Dehesa?

Transforming a shrubland into a dehesa system may be useful for recovering certain productive and regulatory functions of ecosystems such as grazing potential, soil erosion control, and also for reducing the risk of wildfire. However, the productivity of the herbaceous cover and tree development in the transformed system may be limited by soil fertility, especially after wildfire events. Previous studies have shown that adequate doses of sewage sludge may improve soil fertility and facilitate plant recovery, but few studies have focused on plant biodiversity assessment. Here, we compare the effects of sewage sludge that has undergone different post‐treatments (dewatering, composting, or thermal drying) as a soil amendment used to transform a fire‐affected shrubland into a dehesa, on tree growth and pasture composition (vegetation cover, species richness, and diversity). In the short term, sewage sludge causes changes in both pasture cover and tree growth. Although no major differences in vegetation species richness and composition have been detected, fertilization using sewage sludge was shown to modify the functional diversity of the vegetation community. Rapid replacement of shrubs by herbaceous cover and ruderal plants (e.g. Bromus hordeaceus and Leontodon taraxacoides) and of the three grass species sown (Festuca arundinacea, Lolium perenne, and Dactylis glomerata) was observed, whereas N‐fixing species (leguminous) tended to be more abundant in nonfertilized soils and soils amended with composted sludge. These results indicate that sewage sludge modifies the functionality of vegetation when applied to soils, and that the response varies according to the treatment that the sludge has undergone.     

A rapid luminescent-phage based MPN method for the enumeration of Salmonella typhimurium in environmental samples

Materials such as soils, waters, sewage sludges and foods can contain low numbers of salmonellas. A most-probable-number (MPN) method that utilized a bioluminescent-bacteriophage is described that allowed the specific determination of as few as one Salmonella typhimurium cell/100 ml of material within 24 h. The method was developed with soil, lake water and sewage sludge inoculated with Salm. typhimurium and had an efficiency of 100% when tested against a traditional MPN method. The protocol is rapid, sensitive, inexpensive, has a low operator time compared to the traditional MPN method, allows for the repair of injured cells and is amenable to automation.     

Microbial Ecology of Activated Sludge: I. Dominant Bacteria

Over 300 bacterial strains were isolated from seven samples of activated sludge by plating on sewage agar. Gram-negative bacteria of the genera Zoogloea and Comamonas predominated. Many isolates (51%) showed sudanophilic inclusions of poly-β-hydroxybutyric acid, whereas 34% accumulated iodophilic material on media containing starch. A large number required either vitamins or amino acids, or both, for growth. None of the isolates tested for their ability to bring about changes in autoclaved sewage produced an effluent comparable in quality to the activated sludge control, although the Zoogloea did produce activated sludgelike flocs. A study of 150 bacterial strains isolated from raw sewage revealed that they differed from the sludge isolates in several respects. Coliforms, which constitute nearly a quarter of the sewage isolates, were rarely encountered in sludge.     

Enumeration of pathogenic bacteria from sewage sludge in Kuwait

Domestic sewage in Kuwait is mainly treated by an activated sludge process. Sewage sludges at different treatment stages and the digested sludge were studied for their content of pathogenic bacteria. Two Gram-positive cocci and eight Gram-negative bacilli were isolated and enumerated in the sewage sludges and in raw sewage. During March, Aeromonas hydrophila was the dominant bacterium isolated. Serratia liquefaciens in raw sewage and sludge-2, and Salmonella species in digested sludge were dominant during April. Enterobacter aerogenes was dominant in sewage samples in May. Various types of bacteria isolated were tested for their sensitivity to a wide range of antibiotics.     

Salmonella isolated in sewage sludge traced back to human cases of salmonellosis

AIM: The main aim of this study was to investigate a possible connection between the Salmonella content in sewage sludge and human cases of salmonellosis. An additional aim was to survey the antimicrobial resistance situation in Salmonella isolated from Swedish sewage sludge. METHODS AND RESULTS: The Salmonella strains were compared by restriction enzyme analysis combined with pulsed field gel electrophoresis and antimicrobial susceptibility testing. This study suggests a link between Salmonella isolated from sewage sludge and human Salmonella isolates. CONCLUSIONS: This study demonstrates that Salmonella spp. isolated in sewage treatment plants (STP) originate from infected humans and survive treatment at STP. It also highlights the risk of spreading resistant Salmonella strains from sewage sludge to the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: As Salmonella spp. originating from infected humans can survive the treatment at STP, the risk of Salmonella spp. being spread with sewage sludge to the environment and then to people and animals is enhanced. The threat to society is even worse if the bacteria are resistant to antimicrobial agents.     

Investigation of Salmonella infection in goats fed corn silage grown on land fertilized with sewage sludge

A total of 36 goats were fed for 17 months with corn silage grown on land fertilized with human sewage sludge. These animals were investigated for salmonella infections. Salmonellae were not detected in cultures of fecal or silage samples. No significant agglutination titers were detected in goat sera examined. Salmonella newport C2 was isolated from the sludge used as fertilizer on the cornfields. The public health aspects of the findings are discussed as they relate to the increasing use of sewage sludge for agricultural fertilizers, as well as to the resultant effects on human food and livestock feed.     

Investigation of Salmonella infection in goats fed corn silage grown on land fertilized with sewage sludge.

A total of 36 goats were fed for 17 months with corn silage grown on land fertilized with human sewage sludge. These animals were investigated for salmonella infections. Salmonellae were not detected in cultures of fecal or silage samples. No significant agglutination titers were detected in goat sera examined. Salmonella newport C2 was isolated from the sludge used as fertilizer on the cornfields. The public health aspects of the findings are discussed as they relate to the increasing use of sewage sludge for agricultural fertilizers, as well as to the resultant effects on human food and livestock feed.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42 degrees C) were higher even when MKT (43 degrees C) performance was improved by the use of brilliant green sul-phamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42°C) were higher even when MKT (43°C) performance was improved by the use of brilliant green sulphamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Mycoflora of activated sewage sludge

Thirty-eight species of fungi were identified in pure culture after isolation from activated sewage sludge by serial dilution. Nine species and genera were identified that had not been previously reported.In 1963, Cooke (1) published an excellent laboratory guide on the identification of fungi from polluted water, sewage, and sewage treatment systems; of approximately 30 papers cited only one (2) dealt with fungi from activated sewage sludge. Later (1970), Cooke & Pipes (3) enumerated 47 fungi consisting of 4 genera of yeasts and 33 genera of filamentous fungi that had been isolated from activated sludge. This paper reports the mycoflora of anaerobically digested sludge from a residential area in Auburn, Alabama.