# Improving biocide evaluation using propidium monoazide (PMA) viability staining technique

## TL;DR

This study shows that using PMA staining improves the accuracy of measuring biocide effectiveness by distinguishing live and dead microbes in energy sector applications.

## Contribution

The study demonstrates the feasibility and benefits of integrating PMA staining into standard DNA-based methods for biocide evaluation.

## Key findings

- PMA reduced microbial abundance estimates by at least an order of magnitude compared to standard methods.
- PMA results correlated with reduced hydrogen sulfide production, unlike standard methods.
- PMA revealed significant changes in microbial diversity and composition after biocide treatment.

## Abstract

Chemical biocides are commonly employed to manage problems caused by microbial processes. In the energy sector, for example, engineered systems are often treated with biocides to control microbiologically influenced corrosion (MIC), biofouling, and the biological generation of hydrogen sulfide. Standard DNA-based methods that are widely used to assess biocide effectiveness often cannot distinguish between live and dead microorganisms, potentially leading to inflated estimates of living cell populations. Incorporating propidium monoazide (PMA) viability staining technique offers a promising solution to this limitation. In this study, we explored the application of PMA within a standard DNA-based workflow to evaluate biocide performance more accurately. A model sulfate-reducing microbial consortium, derived from oilfield produced water, was exposed to widely used biocides including glutaraldehyde (Glut) and tetrakis(hydroxymethyl)phosphonium sulfate (THPS). PMA was applied prior to standard DNA extraction and subsequent qPCR and amplicon sequencing procedures. We observed PMA-derived microbial abundance at least an order of magnitude lower compared to that without PMA. The reduced PMA-derived microbial abundance correlated with the lower ability of the model microbial communities to produce hydrogen sulfide – an association that was absent based on the usual approach without PMA. Biocide-treated communities, in comparison to untreated controls, displayed significant alterations in their microbial ecological properties, such as alpha diversity, beta diversity, and taxonomic composition, as determined through 16S rRNA gene sequencing – differences that were only apparent when PMA was applied. These results confirm that incorporating PMA into standard DNA-based biocide assessment protocols is both feasible and beneficial. Since PMA implementation requires minimal additional effort, we advocate for its adoption in future biocide performance studies, in particular for engineered systems in the energy industry.