Elsevier

Waste Management

Volume 96, 1 August 2019, Pages 168-174
Waste Management

Assessment of the methodologies used in microbiological control of sewage sludge

https://doi.org/10.1016/j.wasman.2019.07.024Get rights and content

Highlights

  • Statistically significant differences between MPN and plate count have been found.

  • MPN underestimates the count of some bacterial genera.

  • Of the plate counting methods, membrane filtration is the most effective.

Abstract

Sewage sludge usually contains potentially polluting substances such as heavy metals, organic pollutants and various organisms including bacteria, protozoa, helminths, viruses and algae, some of which may be pathogenic. Certain of these pathogens could be transferred to the soil if the sludge is used on agricultural or land recovery applications. For its application on agricultural land, sewage sludge must comply with the limits established in the legislation, which in Europe does not include quality standards regarding microbiological parameters. Nevertheless, the presence of pathogens could limit its agricultural use, as it could pose a risk to human, animal and environmental health. This study compares 4 different methodologies used in microbiological analysis in order to identify the most efficient and reliable method on determining bacteria in sewage sludge. Escherichia coli and Enterococcus faecium are used as bacterial indicators. The results obtained in this work indicate that results obtained with three different plate count methods cannot be comparable with those obtained with the MPN method. The membrane filtration method is recommended for its high precision and sensitivity, both in low and high bacterial loads. It is also concluded that it would be necessary to establish the quality standard in concordance with the method used.

Introduction

In the circular economy framework, sewage sludge is considered as suitable for recycling, reversing the traditional view that it can only be destined for landfill disposal. The first alternative is its use on agricultural land (Lasheras, 2011). As an example, under Spanish waste and contaminated soils legislation, sewage sludge, which is treated in the installation itself and applied to the soil, is considered as a ‘by-product’ and not a ‘waste’.

The solid sludge content from Waste Water Treatment Plants (WWTP) ranges between 0.2 and 12% by weight, being mainly composed of organic matter in decomposition that makes it an important source of nutrients for agricultural soils. The quantity and quality of the sludge changes depending on its origin and age as well as the treatment conditions (pH, temperature, retention time, microbiological competition) and storage (Fytili and Zabaniotou, 2008, Eddy, 2002, Rajagopal et al., 2013, Sahlström et al., 2004, Smith et al., 2005a).

Sewage sludge can also contain other potentially polluting substances including heavy metals, such as cadmium, chromium, copper, mercury, lead or zinc, organic pollutants and various organisms, which include bacteria, protozoa, helminths, viruses and algae, some of which may be pathogenic. The concentration of metals detected in sludge depends in many cases on the industrial component of the sewage water. Some studies have reported that approximately half the amount of heavy metals in raw sewage water are precipitated in the sludge (Liu and tao, 2016, Tervahauta et al., 2014). Some heavy metals, depending on the pH and their oxidation state, can be assimilated by plants and accumulate in their issues, thus entering the food chain (Sobrados et al., 2010).

Moreover, certain pathogens present in the sludge could also be transferred to soil. The survival of microorganisms in the sludge applied to soil depends on the environmental conditions, pH, moisture, sludge retention time and the characteristics of the microbial community in the soil (Moynihan et al., 2015). Salmonella, Listeria, E. coli, Campylobacter, Mycobacterium, Clostridium, Yersinia, Cryptosporidium Giardia or norovirus are examples of pathogenic microorganisms detected in sewage sludge (Sahlström, 2003, Smith et al., 2005b, Sidhu and Toze, 2009, Hellmer et al., 2014). In certain conditions these microorganisms can contaminate raw vegetables or pastures when sludge is used in agriculture. In addition, animals may play an important role in the infectious cycles, since they can be infected or become carriers by contact with the sludge applied to land, and then transport the microorganisms to other areas (Huete, 2007).

The European Waste Framework Directive (2008/98/EC) (European Parliament and Council, 2008) on waste and contaminated soils sets out the basic concepts related to waste management and establishes objectives of prevention, re-use, recycling, recovery and disposal.

In Europe, the use of sewage sludge on agricultural land must comply with the limits established in European Directive 86/278/EEC on the protection of the environment and, in particular, of the soil. In this directive, quality standards are established in order to regulate the content of heavy metals in sludge and soil in agricultural applications. However, the legislation does not establish limits regarding microbiological parameters even though the presence of pathogens in sludge could limit its agricultural use, as it could pose a risk to human, animal and environmental health (Erkan and Sanin, 2013, Guzmán et al., 2007, Levantesi et al., 2015, Lloret et al., 2012, Mininni et al., 2014, Pascual-Benito et al., 2014, Salsali et al., 2008, Sidhu and Toze, 2009, Zaleski et al., 2005). Spanish legislation (Order AAA/1072/2013) on the use of sewage sludge in the agricultural sector establishes the information that a WWTP has to provide in relation to sludge treatment (Ministerio de Agricultura Alimentación y Medio Ambiente, 2013). In this manner, the facilities that treat sludge and the managers responsible for its agricultural application must provide information including agronomic parameters together with metal and microbiological indicators, which are registered in the National Sludge Registry. Specifically, the presence/absence of Salmonella in 25 g of sample and the concentration of E. coli (CFU/g) are required. Other countries such as Denmark, Poland or Finland also establish regulations related to microbiological limits (Mininni et al., 2014).

In order to unify criteria, some institutions such as the United States Environmental Protection Agency (US EPA) and the European Union (EU), have proposed quality criteria and guidelines focused on reducing pathogenic microorganisms to an acceptable level so that they do not pose a risk to public health (Table 1) (Sobrados et al., 2010).

The detection and quantification of pathogens in sludge and biosolids is complicated, expensive, and requires long analysis times. Moreover, the final degree of sanitation degree difficult to determine (Guzmán et al., 2007). In addition, the disposable methods for microbiological analysis of sludge are not standardized, hindering its characterization (Guzmán et al., 2007, Pascual-Benito et al., 2014, Sidhu and Toze, 2009). As a consequence, few studies can be compared and the debate about the establishment of a new directive on sludge in the European Union has not progressed (Mininni et al., 2014). The EPA recommends the use of the standardized Most Probable Number (MPN) method to determine microorganisms in sludge. However, in Europe recommended quality criteria are based on plate count methodologies, where the results are expressed as CFU, per ml or per gram of sample. Therefore, in matrices such as sludge, where the composition of solids is variable, the misinterpretation of the results is possible.

To be able to use sewage sludge safely in agriculture and therefore to optimize the sanitation process, it is necessary to accurately quantify its microbiological content. This requires standardized methods that are sensitive, rapid, precise, simple, and efficient which can be used to detect and quantify pathogens in biosolids. In addition, the use of these methodologies will allow the comparison of the results obtained in different geographical areas, for different water and sludge treatment processes and for sludge obtained with different inlet water compositions, leading to unification in the criteria of the different regulations.

This study compares the main methodologies proposed for the bacteriological control of sewage sludge, the MPN and the plate count, and how the latter varies according to the procedure and the culture media used. The objective is to identify the most efficient method.

Section snippets

Bacterial strains

Enterococcus faecium ATCC® 19434™ and a wild environmental strain of Escherichia coli isolated from wastewater were used as bacterial strains.

Sewage sludge samples

Four sewage sludge samples were used to quantify E. coli. The samples were taken in December 2017 from a WWTP in the region of Navarra (Spain), located in the Ebro River Basin. Sludge from primary and secondary decanters was treated in an anaerobic reactor at thermophilic conditions and 15d of Hydraulic Retention Time (HRT). After the treatment, the

Results and discussion

Table 5 shows the concentration range of E. coli (log UFC/ml) and E. faecium in the sludge samples analysed.

The concentration of E. coli in the sludge samples was between 1.7 and 3.4 log CFU/ml for the plate count methods and between 2.0 and 2.2 log MPN/ml for the MPN method. The concentration of E. faecium in doped sludge samples was between 3.4 and 5.2 log CFU/ml and 0.6–2.2 log MPN/ml, respectively, according to the plate count methods and the MPN method.

In most cases, the values obtained by

Conclusions

The results obtained in this work indicate that there are significant differences between the two methods of microbiological control of sewage sludge, the MPN and the plate count, so it is necessary to select one as a reference method. The plate count methodologies are recommended, especially when low bacterial load are expected, due to their greater sensitivity and the fact that they provide results closer to the real ones, as can be seen with the doped samples. MPN could be used as an

Acknowledgments

This work was financed by the Gobierno de Aragon (Spain) (Research Reference Team Water and Environmental Health T51_17R) and co-financed by Feder 2014-2020 “Building Europe from Aragon” and the project “Research study for the improvement of the quality of effluents from urban wastewater treatment plants and landfills, located in the Community of Navarra (Spain)”, funded by NILSA (Navarra de Infraestructuras Locales, SA). The authors would like to acknowledge the assistance of NILSA.

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