Can bacteriophages in wastewater treatment plants do more?


Biological processes for the decomposition of pollutants are the basis of municipal wastewater treatment. For this, they use the concentrated biomass of various microorganisms (activated sludge). Wastewater treatment plants play the role of bridges between environmental bacteria, the wastewater microbiome (including the human microbiome) and opportunistic pathogens. Treatment processes are centered around the removal of sludge and suspended solids, as well as phosphorus and nitrogen compounds. However, the more than century-old technology of using activated sludge is much worse at dealing with persistent industrial contaminants, pharmaceutical compounds or antibiotic-resistant microorganisms [1, 2]. Is the solution bacteriophages?

Why do modern wastewater treatment processes need to be modified on a micro scale?

Because microbe-based processes for removing phosphorus and nitrogen compounds are often exposed to variable conditions, their effectiveness can be compromised. For example, the process of nitrification, the oxidation of ammonium nitrogen to nitrate by microorganisms, requires maintaining a constant level of nutrients in the wastewater flowing into the treatment plant. However, their composition can change depending on the season or even the time of day. In addition, bacterial biomass grows slowly, and losses due to leaching or heavy rainfall can cause an imbalance in the transformation processes of nitrogen compounds. As a result, the efficiency of pollutant removal is reduced, but there is also an accumulation of chemical compounds to levels that are toxic to the microorganisms themselves [2].

In addition, according to recent studies, wastewater treatment plants are hot spots from where not only antibiotic-resistant microorganisms migrate into the environment, but where transfer of antibiotic-resistance genes between microbes also occurs [3]. Wastewater treatment processes are not designed to deal effectively with reducing the abundance of antibiotic resistance genes, and the impact of the methods used may only be indirect. The high density of various microorganisms, surfactants and disinfectants promote selection and horizontal gene transfer. In addition, microbes in treatment plants are stressed by exposure to microplastics and heavy metals. The new resistant pathogens are evolving and are different from the genotypes already circulating in the environment [3, 4].

Where will the microbiologist send the bacteriophages?

Solutions to at least some of the problems mentioned above are seen in the use of bacteriophages. Studies have shown that they can be used to optimize wastewater treatment and methane digestion processes. Treatment plants are particularly interesting microbial environments due naturally to the constant and large influx of microorganisms, including phages [5].

Bacteriophages are approximately 1 to 2 orders of magnitude smaller than bacterial cells, usually in the range of 20 nm to 200 nm. They have a simple structure – they consist of a protein capsid containing the phage genome, single- or double-stranded DNA or RNA, sometimes a lipid membrane in the capsid surrounding the genetic material. They make up the most numerous biological unit on Earth, outnumbering their hosts by up to 10 times. They can infect both bacteria (gram-negative, gram-positive, and even multidrug-resistant) and archaeons, usually starting the cycle by adhering to the bacterial cell wall and injecting their genome into it. Bacteriophage is simply a bacterial eater, which highlights its high bactericidal capacity, but also its specificity in action [5-7].

Most bacteriophages have high specificity against a particular type of bacteria and replicate at the site of infection. This means that the phage can take control of the bacterial machinery (e.g., transcription) and resources (e.g., nucleic base) for its own replication, which inhibits host growth and leads to host death (lysis) at rates as high as 1023 infections per second [5].

Bacteriophage-bacteria interactions can affect community composition, function and evolution of the microbiome. Thanks to these properties, they are increasingly being used in engineering, environmental and medical sciences.

The use of bacteriophages to modify the microbial community by eliminating undesirable strains may have the effect of enhancing aerobic processes in biological chambers and improving treatment efficiency [8]. One of the technological problems associated with activated sludge is the so-called “activated sludge”. swelling, which can lead to a reduction in sedimentation capacity and consequently to problems with the separation of sludge from the treated liquid. Swelling causes an increase in sediment volume and accumulation of extracellular polysaccharide substances with high viscosity. These substances increase water retention in the sludge, hindering its drainage and affecting floc stability [9].

Modern sequencing methods have enabled researchers to pinpoint the bacteria responsible for foaming and swelling. They account for between 1.86 and 9 percent. All microorganisms present in activated sludge. For example, Gordonia spp. has been identified as a species of filamentous bacteria that cause a number of problems in wastewater treatment: contaminant deposition, foaming or corrosion. Current chemical disinfection methods can lead to a number of toxic byproducts [8-10]. Reduction of bacteria responsible for technological problems can be achieved by using single phage species or phage cocktails (mixtures consisting of several species) [7].

Most of the bacteria in biological wastewater treatment systems are not suitable for culture under laboratory conditions (an estimated 99 percent), being part of the microbial dark matter, including many functional microbes that determine efficiency, such as Candidatus Accumulibacter phosphatis, ammonia-oxidizing bacteria and Microthrix parvicella. Therefore, the corresponding phages are difficult to detect by classical methods, a match is only given by advanced shotgun metagenomics methods combined with bioinformatics tools [11, 12].

In wastewater treatment plants, pathogenic microorganisms are found in large numbers. The most common are E. coli, Salmonella spp., Staphylococcus aureus and Campylobacter jejuni. Although these pathogens can be adsorbed in flocs and then removed with excess sludge or ingested by protozoa, they can still pose a potential risk to the environment if biological processes are upset. Bacteriophages also increase the safety of wastewater reuse [5]. They are particularly promising in the fight against antibiotic resistance, reducing the number of organisms exposed to them or interrupting the chains of antibiotic resistance gene transfer [11, 12]. In Figure 1. The basic possibilities of using bacteriophages in wastewater treatment systems are presented.

Fig. 1. Biotechnological possibilities of phages in wastewater treatment plants

What are the opportunities and obstacles?

During phage infection, bacteria can develop defense mechanisms. They can change or lose their receptors, excreting substances that prevent bacteriophage adhesion to the host, inhibiting replication and phage release. Microorganisms organized in complex biofilms continue to pose a challenge to aggressors. In addition, they can recognize phage-specific nucleic acids and destroy them, preventing infection [6, 7]. Resistance to infection can be reduced by using phage cocktails. It is important to improve and develop strategies for predicting bacteriophage-host and bacteriophage-environment relationships. Undoubtedly, the development of molecular biology techniques can help track bacteria and bacteriophages. Which will help alleviate concerns about this still-new tool for controlling wastewater treatment biotechnology [5, 6].

The ecological role of bacteriophages is still being discovered. Phages appear to play an important role in regulating the structure of microbial communities in wastewater treatment plants [5-8]. Their predation can affect the efficiency of biological removal processes of nitrogen and phosphorus compounds by controlling swelling, foaming or eliminating certain pathogens.

While all studies to date have focused on the use of bacteriophages for biological control in laboratory systems, broader studies on the potential for full-scale use of phages in wastewater treatment plants are still lacking. This is primarily due to the variability of the activated sludge microbiome and the significant contribution of microbial dark matter, which continues to puzzle researchers [13]. Nonetheless, phages can provide an alternative to standard operational control (temperature, pH, oxygen concentration or retention time), becoming a valuable environmental and economic tool [5, 6].

Dr.-Ing. Edyta Łaskawiec – water and wastewater technologist, assistant professor at the Department of Environmental Biotechnology, Silesian University of Technology

In the article, I used, among others. From the works:

[1] Yuan L. et al. Pathogenic and Indigenous Denitrifying Bacteria are Transcriptionally Active and Key Multi-Antibiotic-Resistant Players in Wastewater Treatment Plants. Environ Sci Technol, 2021, 55(15), 10862-10874

[2] Hossein M. et al. Exploring eco-friendly approaches for mitigating pharmaceutical and personal care products in aquatic ecosystems: A sustainability assessment, Chemosphere, 2023, Vol 316, 137715

[3] Sun Sh. et al. High-risk antibiotics positively correlated with antibiotic resistance genes in five typical urban wastewater, J Environ Manag, 2023, Vol 342, 118296

[4] Mosaka T.B.M. et al. Inactivation of antibiotic-resistant bacteria and antibiotic-resistance genes in wastewater streams: Current challenges and future perspectives, Front Microbiol, 2023, Vol 16, 1-21

[5] Liu R. Bacteriophage ecology in biological wastewater treatment systems, Appl Microbiol Biotechnol, 2021, 105, 5299-5307

[6] Withey S. et al. Bacteriophages-potential for application in wastewater treatment processes, Sci Total Environ, 2005, Vol 339(1-3), 1-18

[7] Bolsan A.C. et al. Bacteriophages in wastewater treatment: can they be an approach to optimize biological treatment processes?, Environ Sci Pollut Res, 2022, 29, 89889-89898

[8] Runa V. et al. Bacteriophages in Biological Wastewater Treatment Systems: Occurrence, Characterization, and Function, Front Microbiol, 2021, Vol 12, 30, 1-19

[9] Shivaram K.B. et al. Bacteriophage-based biocontrol technology to enhance the efficiency of wastewater treatment and reduce targeted bacterial biofilms, Sci Total Environ, 2023, Vol 862, 160723

[10] Reisoglu Ş., Aydin S. Bacteriophages as a promising approach for the biocontrol of antibiotic resistant pathogens and the reconstruction of microbial interaction networks in wastewater treatment systems: A review, Sci Total Environ, 2023, Vol 890, 164291

[11] Sabatino R. et al. Bacteriophages limitedly cont ribute to the antimicrobial resistome of microbial communities in wastewater treatment plants, Microbiology Spectrum, 2023, Vol 11(5)

[12] Du B. et al. Responses of bacterial and bacteriophage communities to long-term exposure to antimicrobial agents in wastewater treatment systems, J Hazard Mater, 2021, Vol 414, 125486

[13] Zhang Y. et al. The microbial dark matter and “wanted list” in worldwide wastewater treatment plants, Microbiome, 2023, 11, 59

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