Sample Preparation Protocols for Sewage Sludge Microbiome Sequencing

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Sewage sludge is a known product of biological wastewater treatment. Its biological and chemical composition is based on the type of wastewater, as well as on the accomplished pre-treatment methods. It may contain pathogenic microorganisms and spores. Some of the common microbes found in such an environment are Salmonella, eggs of intestinal parasites such as Toxocara and Ascaris lumbricoides, and some coliforms such as Clostridium perfringens and Pseudomonas aeruginosa. Since sewage sludge poses potential harm to most living organisms, analyzing the present microbial community on it is important. Identifying these microorganisms typically using 16S rRNA gene sequencing, as well as understanding these microbes may serve as a key to improve both chemical and biological contamination in sewage sludges.


Since sewage sludge samples are potentially contaminated with hazardous and flammable substances and may contain pathogenic organisms increasing its liability to undergo biological action, several precautionary measures were established. First, remember that gas possibly generated by microorganisms are potentially flammable and may pressurize the sample container once generated in it. This may be potentially hazardous as the sample container might explode. This is also the reason why glass bottles must not be used as sample containers. Next, media, reagents, as well as bacteria used during the process must be covered by the Control of Substances Hazardous to Health Regulations. Also, necessary risk assessments should be accomplished before adopting any method. Lastly, standard laboratory microbiology safety procedures must be followed throughout the process.

Sample Requirements

Prior to the sampling process, there are important requirements for the samples to obtain more accurate results. First, the DNA sample must be greater than or equal to 300 ng, OD260/280 = 1.8 - 2.0, and a concentration of greater than or equal to 10 ng/μl. Next, the DNA must not degrade. Freezing and thawing cycles during the sampling period, as well as the shipment, must be repeated regularly. Enough amount of ice or dry ice is a must during the shipment. Lastly, samples should be stored at -20/-80 °C.

Sample Preparation

Initially suspend about 10 grams of wet sample in 90 milliliters of buffered peptone water. If there is a specified about of dried sludge required for the analysis, the weight of the wet sludge is equivalent to the weight of the required amount of dried sludge that must be obtained. After suspending the sample, homogenize, and process the sample depending on its characteristics. The homogenized solution must then be incubated in buffered peptone water at 37 °C for 21 ± 3 hours. After the incubation, obtain a 10 µl of the Rappaport Vassiliadis enrichment broth and plate it out in a petri dish of xylose lysine desoxycholate agar. After that, immediately return the broth to the incubator at 41.5°C for 24 hours while incubating the agar plate at 37°C for 21 ± 3 hours. Lastly, examine the selective agar and if there is no growth observed, plate out another loopful (10 µl) of the Rappaport Vassiliadis enrichment broth on another xylose lysine desoxycholate agar petri dish and incubate at 37°C for 21 ± 3 hours. Again, examine the selective agar for any microbial growth.


  1. Kantor RS, Miller SE, Nelson KL. The Water microbiome through a pilot scale advanced treatment facility for direct potable reuse. Frontiers in microbiology. 2019, 10:993.
  2. Nega, R.  Microbial Environmental Risks Associated Sewage Sludge Disposal. Open Access Journal of Microbiology & Biotechnology. 2018, 3(2).
  3. Walter A, Probst M, Franke‐Whittle IH, et al. H. Microbiota in anaerobic digestion of sewage sludge with and without co‐substrates. Water and Environment Journal. 2018, 33(2):214-222
* For Research Use Only. Not for use in diagnostic procedures or other clinical purposes.
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