En jämförelse mellan amplifierad singelmolekylanalys och selektiv agar vid kontroll av hygienisering av avloppsslam

Abstract: Bringing back the nutrients from the city to the country is becoming increasingly important, as the world’s stores of nutrients get more exhausted. Sewage sludge is an unapplied resource, rich in plant nutrients, which could replace parts of the commercial fertilizers used in Sweden today. Sewage sludge may contain large amounts of pathogens, such as Campylobacter and Salmonella, why use can cause severe spreading of infections, both to animals and humans. These pathogens have the ability to survive for a long time in soil and water and therefore some sort of treatment of the sludge is recommended before use. Sanitizing treatments used today includes pasteurisation, composting and addition of lime. These treatments are less suitable because of work environmental and economic reasons and therefore new methods are evaluated. One such method is ammonia sanitization. At higher pH the major part of the ammonia is in its uncharged form, NH3, which is the sanitizing agent, while at lower pH, it is ionised, NH4 +, and constitutes an important plant nutrient. Urea is the world’s most essential fertilizer and when in contact with the enzyme urease it decomposes into ammonia. The addition of ammonia or urea increase the sludge’s value as a fertilizer, because of the higher amount of nitrogen incorporated. Previous studies with ammonia sanitization in urine and faeces have shown effective inactivation of pathogens. The treatments consisted of additions of 0.5 - 2 % urea or ammonia at various temperatures (4-34°C), sanitizing the material from Salmonella, Enterococcus and Ascariseggs. Salmonella is the most studied organism in sewage sludge and may appear in concentrations of 107 colony forming units (CFU) /g dw sludge. For analysis of the effectiveness of the sanitization, the presence of microorganisms is measured in the material before, during and after the treatment. This is usually done by culturing on agar, a method that is highly time-consuming (often 3 days are required). Since the treatments are carried out in materials that are complex, it is difficult to find satisfactory alternative methods. One method that may be used is amplified single-molecule analysis. This study is a comparison of the the agar culturing and amplified single-molecule analysis as well as a study of how effective ammonia sanitization is when sanitizing sewage sludge. Therefore the experiment was divided into two parts; one where sanitization was measured by culturing Salmonella on agar and one where purified DNA from the samples were analysed with amplified single-molecule detection. After addition of Salmonella to the sewage sludge, 2 % urea or 0.5 % ammonia was added and then the samples incubated in 24 or 14°C respectively. Samples were analysed on day 0, 1, 2, 5, 8, 12, 15 and 29. Both treatments resulted in efficient sanitization of the sewage sludge and riddance of Salmonella. For the urea treatment, 2 days were enough to achieve satisfactory reduction, while for ammonia, 10 days were sufficient. In the part of the study with amplified single-molecule analysis, no bacteria were detected. The reason for this was that the padlock probes used did not fit the strain of Salmonella that was used. The method seems more suitable for analysis of more specific organisms, such as EHEC, and more research must be done on padlock probes for the method to work satisfactorily.