Bacteriophage as Biotechnological Tools to Improve the Effectiveness of Anaerobic Digestion Process

Abstract

Wastewater treatment plants (WWTPs) serve as habitats for diverse and densely populated bacterial communities, fostering intricate microbial interactions. Conventional treatment methods employed often fail to completely eliminate pathogens. Consequently, inadequate chemical treatments lead to the eventual release of waterborne bacterial pathogens into the environment through effluent water. Anaerobic digestion represents a biological treatment approach for organic waste and wastewater, providing cost-reduction benefits and enabling energy generation through biogas production from organic waste. However, the role of viruses-host interactions in anaerobic digestion and their effects on biological wastewater treatment (WWT) has been lacking and requires further research and attention. Bacteriophages (phages), viruses that target specific bacteria, are abundant within WWTPs and engage in diverse interactions with their host organisms. Also, there are reports indicating the presence of archaeal viruses capable of impacting crucial methanogenic organisms in anaerobic digestion, alongside phages. Despite their apparent lack of discernible metabolic functions, viral community have significant potential to influence WWT by shaping the structure of microbial communities, thereby impacting the efficiency of the processes. This chapter aims to explore the influence of reported viral communities, especially phages on shaping microbial communities; elucidate the dynamics and limitations of phage-host relationships; and evaluate their potential as biological tools for enhancing the anaerobic digestion process in WWT.

1. Introduction

Engineered biological systems comprise the basis of high-potential environmental processes such as wastewater treatment (WWT) and the production of bioenergy carriers. WWT is an application in which modified microbiological processes take a role in carbon and nutrient removal biologically, also termed biological wastewater treatment (BWT) systems such as anaerobic digesters. In this regard, anaerobic digestion (AD) is a useful and crucial process in which microbial communities comprise bacteria and archaea, which can degrade organic matter in anoxic conditions. AD is significantly employed in industrial processes besides occurring naturally in various environments including aquatic sediments and animal gut. It is a valuable process that plays an important role in reducing fossil fuel dependency and producing methane by transforming waste disposal into a useful process [1]. The anaerobic digestion process encompasses four primary stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. Acetogenic bacteria play a crucial role in this process by converting simple substrates, including acetate, H₂, CO₂, and various fermentation by-products such as propionate, butyrate, and alcohols. The preservation of a specific archaeal group, known as methanogens, is imperative to ensure the successful and stable operation of the AD process. This group is responsible for catalyzing the terminal and most delicate step of the anaerobic process, which is methanogenesis. Methanogens are generally categorized into two main groups based on their substrate conversion capabilities: acetotrophic and hydrogenotrophic methanogens, and of particular importance, acetotrophic methanogens significantly contribute to methane production, with approximately 70% of the generated methane originating from acetate conversion [2].

Structural changes in bacterial communities frequently cause variations in the effectiveness of biological wastewater treatment (BWT) systems. Therefore, the stabilization of microbial composition is very important for BWT. Although BWTs primarily use bacterial communities to decompose contaminants, the AD composition in the treatment is quite diverse, extending beyond bacteria. Although a diverse array of organisms contributes to the intricate dynamics of the microbial community in this process, there is an increasing recognition of the role that viruses, particularly bacteriophages, play in controlling bacterial populations. Moreover, the viral concentration is significantly higher in samples from WWTPs compared to in other aquatic environments, and the strong correlation detected between bacteriophages and bacteria in BWTs adds to this interest. This interest has moved toward the role of bacteriophages in assessing process efficiency and waste quality [3].

Bacteriophages, or simply phages, are viruses that target and infect specific bacteria and, like other viruses, are mostly obligate parasites, so they do not have an internal metabolism and need the metabolic mechanism of their hosts to maintain their life cycle [4]. It has been revealed that phage activity in various aquatic and terrestrial ecosystems, especially in the oceans, is a driving force in shaping microbial communities and in biodiversity through interspecies gene transfer [5]. Concentrations of phage are expressed to be approximately 108–109 particles per milliliter in BWT harboring wastewater from a wide variety of different sources, and this means a higher concentration than other ecosystems studied so far (Figure 1) [6]. In BWT, there is a significant increase in interest in phages in terms of their impact on the bacterial consortium and consequently process efficiency and effluent quality. It was revealed that there is a relationship between the viral community structure and methane production in the anaerobic digesters of WWTs, and it was found that the viral shunt was effective in methane production [7]. Even though anaerobic digesters have the potential to study the relationship of phages to the microbial community and the effects of these relationships on the system, as they are systems in which a large number and variety of phage and prokaryotic host interactions are associated with easy access and nutrient-rich environment, the effects of phages on the microbial community are still not fully understood. This is since classical methods used in phage studies were generally culture-dependent, and because of bacteria that are still unculturable or difficult to culture in BWT, it is difficult or impossible to detect phages specific to these bacteria by culture-dependent methods [8]. With the development of culture-independent methods, especially shotgun metagenomic with bioinformatic tools, novel phages are discovered in BWT systems and detailed information on phage-host dynamics is obtained [9]. The aim of this chapter is therefore to explain the role of phages in AD process by examining the effects of phages on anaerobic microbial community structure in the light of this new information.

Figure 1.

Since wastewater treatment systems receive wastewater from different sources, various bacterial/viral/fungal microorganisms that come with these sources enter the treatment systems along with the wastewater.

2. Anaerobic microbial community dynamics and interaction

2.1 Phage-host dynamics in anaerobic digestion

The relationship of the phage with its host is based on the host density and infection frequency. Thus, these two parameters may be the driver of the evolution of phage-host dynamics in BWT. A study examining sequencing data from four anaerobic digesters in full-scale WWTPs revealed monthly fluctuations in phage and prokaryotic populations over a year, demonstrating significant correlations at both α- and β-diversity levels and supporting the notion that cell lysis operates in a density-dependent manner [7]. As to the other factor, high infection rates of virulent phages can result in excessive prokaryotic mortality, although the impact may vary due to differences in phage titers and infection cycles. In the context of BWT, an “arms race” ensues between virulent phages and their hosts, marked by increasing phage infectivity and host resistance over time. Prokaryotes employ various strategies to resist phage invasion, including protein-based defense mechanisms and emerging chemical antiphage defenses. Some of these mechanisms have been observed in WWTPs, demonstrating adaptation to local phage predation pressures. The “arms race” dynamics, characterized by ongoing host-phage interactions, incur costs for both bacterial resistance and phage infectivity resistance. As a result, these dynamics may eventually transition to fluctuating selection dynamics, where phage and host genotype frequencies oscillate due to negative frequency-dependent selection. This may provide an advantage to rare bacterial resistance alleles via phage evolution to infect common bacterial genotypes [10].

In addition to the host density and infection, the type of infection also has the capacity to seriously affect population dynamics and the functions of organisms. Phages generally affect their host in two ways: lytic and lysogenic. The lytic cycle results in the death (lysis) of the host bacteria following infection and the subsequent release of new phage virions, resulting in a drastic decrease in bacterial population density in this type of infection. In lysogeny, the phage cycle is continued by integrating phage genetic material within the genetic material of the host (prophage formation) without the lysis of the host cell. This means that the bacterial density in the medium is not changed by the phage, since there is no lysis of the host cells (Figure 2).

Figure 2.

The lytic and lysogenic life cycle of bacteriophages. When bacteria are infected by lytic phages, the bacterial cells undergo lysis, and the abundance of bacteria decreases while lysogenic infection does not decrease the infected bacterial abundance.

In a study showing that phages affect microbial community structure and performance of the process in BWT systems, Microlunatus phosphovorus density in an activated sludge reactor diminished with the addition of phages [11]. In another study conducted in an MBR system receiving industrial wastewater, phage abundance showed an inverse proportion to both the bacterial hosts and related bacteria [3]. Yang et al. [12] detected a simultaneous decrease in phage concentration and diversity with the increase in bacterial concentration in activated sludge in the municipal wastewater system. Brown et al. [13] stated that while there was a substantial relationship between increasing virus abundance and decrease in bacterial abundance in the nitrification process, the abundance of viral particles was notably affected by pH and magnesium ion exchange, which are effective in the attachment of phages to the host cell.

While lytic phage infection is characterized by a marked decrease in the number of host bacteria, the prophage incorporated into the bacterial genome rather than killing the bacterium may benefit the bacterium and promote its growth. Incorporation of the prophage into the host genome can protect the bacterial host by developing resistance to lytic phage infection, increase pathogenicity by promoting host growth and spread, and may provide the host with an advantage over the competition with other strains [14]. Prophages can support the resistance and bacterial stability in the microbial population, rather than cause the infected population to decline [15]. On the other hand, Heyer et al. [16] revealed that lytic phage infection can reduce the biogas production amount in the anaerobic digester while lysing the host cell, increasing nutrient cycling and promoting the growth of auxotrophic bacteria.

In addition to the other microbial communities in wastewater treatment, archaeal community dynamics have crucial importance for improving the anaerobic treatment and waste reclamation via biogas. In a study conducted by Aydin et al. [9], the density of the archaeal community and structure were evaluated via Illumina Next-Generation Sequencing. Before the implementation of phage cocktails in the reactor, the most abundant archaeal genera were found to be Methanothermobacter and Methanosaeta; however, microbial dynamics and archaeal community underwent alterations with the addition of the phage cocktail. While the dominant genus of the archaeal composition in phage-added reactor was Methanoculleus with a 43% ratio, both Methanosaeta and Thermoplasmatales followed with 22% relative abundance. Even though abundances of methanogens show alterations in the AnMBRs, the main pathway in the production of biogas continued as hydrogenotrophic methanogenesis.

2.2 Archaeal viruses in anaerobic digestion

Viruses exhibit a ubiquitous presence within the biosphere, exerting significant influence upon the hosts they infect. A widely accepted concept is that viruses can target members of virtually every microbial taxon, with an extensive catalog of bacterial viruses, namely, phages, already well-documented. In addition to phages, there are viruses known to infect archaeal cells, termed archaeal viruses or archeoviruses. The ramifications of viral interactions for the structural composition and population dynamics of archaeal communities within anaerobic digesters have only recently emerged as a focal point of scientific inquiry, representing a field that remains largely uncharted [17]. Investigations into archaeal viruses, in contrast to phages, reveal relatively limited information; nevertheless, various archaeal viruses have been reported to infect microorganisms associated with anaerobic digesters. In a study conducted by Wolf et al., a lytic archaeal virus named “Drs3” was detailed alongside its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its corresponding virus were both obtained from the anaerobic digester of an experimental biogas plant [18].

Based on the current scientific literature, the existence of mycoviruses, viruses that target fungal cells, within anaerobic digesters has not been ascertained. However, the absence of reports does not preclude the possibility of mycoviruses inhabiting these environments. Indeed, it is plausible that mycoviruses remain largely unexplored and yet to be discovered, representing a promising frontier in the quest to unravel the intricate dynamics governing microbial communities within anaerobic digestion systems. Drawing from the widely accepted concept that viruses have the capacity to infect members of virtually every microbial taxon, the potential existence of mycoviruses underscores the imperative need for advanced research endeavors to extend the comprehension of this intricate ecosystem [17]. Utilizing isolation-independent approaches, like metagenomics and genome-based searches, has the potential to augment the catalog of viruses linked to methanogenic archaea in anaerobic digesters. Through the detection of viral communities and their interplay with methanogens, there is the prospect of improvement of the stability of anaerobic digesters. By unraveling the presence and dynamics of individual viral communities in conjunction with methanogens, there is a potential avenue to bolster the stability of anaerobic digesters [19].

3. Pathogen biocontrol

WWTPs receive wastewater from different sources such as municipal, industrial, and livestock wastewater in addition to hospitals, which include highly opportunistic pathogenic bacteria. Therefore, the influent water of BWT generally involves pathogens including opportunistic Escherichia coli strains, Salmonella spp., Staphylococcus aureus, and Acinetobacter baumannii. Despite using different biological or chemical water treatment methods, WWTPs are not fully capable of eliminating pathogens, so pathogenic species can often be found in the effluent, threatening environmental and public health through release in the aquatic environments [20]. For instance, the opportunistic pathogen A. baumanni was detected in each stage of WWTP and eventually released from the treatment plant via effluent water to the aquatic media without lysis [21]. Also, it has been indicated by Oliveira et al. [22] that the conventional treatment process for carbapenem-resistant pathogen removal was not adequate with the discovery of this pathogen in effluent water samples. The effluent water, which is reused as drinking water or for recreational purposes, may contain microbial contamination due to the lack of adequate and effective treatment. If wastewater discharges contain fecally transmitted pathogens originating from humans and livestock, diseases can spread even in developed countries with the reuse of this treated water [23]. Considering the high lethal levels of pathogenic bacteria found in discharged effluent samples without any degradation and their dissemination into the environment through aquatic resources, it is obvious that pathogen removal must be ensured as much as possible with the most efficient approaches in the BWT process.

In recent years, the phage application for pathogen biocontrol has been gaining substantial attention instead of the current physicochemical methods since phages can be active until the last target bacterial cell is eliminated [24]. The use of antibiotics in the treatment of pathogens from wastewater may not give the desired result, since most pathogens have developed resistance to antibiotics. Phages have several properties that make them appealing as therapeutic or biocontrol agents. While phages have an antibacterial effect in removing antibiotic-resistant pathogens, they are equally influential for antibiotic-resistant bacteria. Phages, which target and lysis only bacteria specific to them through special receptors, can increase in number depending on the density of pathogens and can easily adapt to the environment [25]. Self-limiting phages hardly affect the native flora. Being naturally found in any environment, they are generally easily discovered and are easy to isolate, especially with culture-independent methods [26].

Recently, pathogen-specific phage isolation has been made and some of these isolated phages have been found to be successful agents of pathogen biocontrol in BWT. In a study conducted by Dhevagi and Anusuya [27], the application of E. coli and Salmonella phages resulted in a significant reduction in the abundance of these pathogens in sewage sludge. In addition to single phage application, the application of a phage cocktail, in which more than one species-specific phage is combined, is a popular phage application method since it expands the host range and eliminates the development of phage resistance and the pathogen recovery after phage implementation [28]. In this regard, the efficiency of a single phage, a combination of a two-phage, and a cocktail of three phages on the removal of Salmonella in wastewater were tested. The phage cocktail provided the most efficient removal of pathogens compared to the other two [29]. In addition, phage cocktail consisting of polyvalent phages with a wider host range can exhibit more successful results. For example, in a study conducted in activated sludge systems, it was determined that the cocktail consisting of polyvalent phages was more effective than the cocktail consisting of phages with narrow host ranges in reducing the antibiotic-resistant E. coli strain [30]. It is worth mentioning that a careful selection of phages to be used in pathogen biocontrol is crucial to targeting and eliminating bacterial populations. Because lysogenic phages do not always degrade the bacterial cell and may undesirably transfer genetic information via horizontal gene transfer, therefore lysogenic phages may not be suitable for pathogen biocontrol. The phages to be used for this purpose must have a lysis cycle [31].

4. Biofilm disaggregation

Biofilm is a complex matrix structure that bacteria produce in their environments and infections in order to survive. Bacteria forming the biofilm are embedded in the extracellular polymeric substance (EPS) matrix. These EPSs are typically polymeric substances consisting of polysaccharides, proteinaceous substances, glycopeptides, lipids, and lipopolysaccharides. The biofilm, which develops as bacteria adhere to the surface and form colonies, is usually a complex structure consisting of the combination of more than one bacterial species and acts as a protective shield for bacteria against antibacterials and other bacteria. Through this protective structure, the stability of the bacteria increases while at the same time reducing the effectiveness of traditional antibacterial agents. For membrane bioreactors, which is a novel approach in wastewater treatment, biofilm structures form challenges, because the biofilm layer significantly affects the normal operation of the device by continuously reducing the net wastewater flow rate passing through the membrane surface. In recent years, phage-based applications as a solution to the biofilm-based blockage and foaming problem have attracted intense interest from researchers due to the unresponsiveness of resistant bacteria to existing antibiotics [32]. In this context, there are studies in which phages are used in the disaggregation of biofilms, which are formed by pathogenic bacteria in wastewater treatment and limit the progress and effect of the process. By using the lytic phage isolated from the wastewater treatment system to eradicate the biofilm formed by the Delftia tsuruhatensis pathogen on the membrane filter, the membrane flow was improved by 70% and the success of the isolated phage as a biocontrol agent was demonstrated [33]. In the context of an urban wastewater treatment facility, approximately 48 bacteriophages specific to Proteus mirabilis were isolated, thereby inhibiting the development of biofilm formation caused by this strain [34]. Ayyaru et al. [35] stated that the E. coli phage they used to clean the nanocomposite membrane contaminated by antibiotic-resistant pathogens solved the contamination problem and increased membrane flow. Another illustration of biofilm disaggregation was provided in a study conducted by [36], which demonstrated the inhibitory activity of bacteriophage MA-1 against various strains of Pseudomonas aeruginosa.

The interaction between the bacteria in the biofilm and the related phages of these bacteria is seen as an important tool in the fight against biofilms formed by bacteria. While bacteria use EPSs to protect them from attack by phages, it becomes easier for phages that can express polysaccharide depolymerase enzymes to find the appropriate host receptor (Figure 3) [38]. In this context, genetically engineered phages can provide a high rate of removal in biofilm degradation. It has been shown that the modified T7 phage shows very high success in biofilm removal by overexpressing DspB, a polysaccharide depolymerase enzyme [39]. As in eliminating the formation of pathogenic bacteria in complex structures, the application of a phage cocktail containing more than one type of phage may be more beneficial than the application of a single phage in biofilm removal. Because biofilm structures are usually complex structures formed by more than one type of bacteria, treatment with a wide host range is essential to interfere with this structure. Wide-host-range polyvalent phages and phage cocktails are an important strategy in biofilm disaggregation, offering high efficiency in recent years [40].

Figure 3.

Bacterial biofilm disaggregation through phage-mediated disruption. As depicted in the figure, the sequential steps involved in biofilm biocontrol using bacteriophages comprise (1) phage application, (2) initiations of EPS disruption through enzymes such as dispersion B (DspB) or EPS depolymerizes, (3) biofilm disaggregation through the disintegration of EPS and exposure of the phage infection, and (4) completion biofilm disaggregation and cell lysis [37].

5. Reconstruction of microbial community structure

The microbial community’s composition is influenced by various factors, including both biotic and abiotic elements. However, our understanding of biotic control remains limited due to the lack of suitable tools for studying biological interactions. Recent studies have highlighted the significant presence of phages, which are recognized as the most abundant biological entities on Earth [41]. Consequently, there has been growing interest in investigating the impact of bacteriophage predation on microbial communities. For instance, in the oceans, phages are responsible for lysing more than 25% of microbial cells. Similarly, in natural freshwater environments, phage lysis accounts for up to 71% of microbial mortality, exerting a profound influence on microbial food webs and the cycling of aquatic carbon [42].

In the context of WWTPs, anaerobic BWT represents a highly dynamic process wherein the symbiotic relationship between bacteria and archaea leads to the production of methane, a significant bioenergy source. The utilization of phages and their beneficial application in the augmentation of anaerobic bioreactor systems emerges as a promising strategy for enhancing performance, mitigating membrane biofouling, and influencing community shape, as illustrated in Figure 3. By selectively influencing specific bacteria within the community using phages, it is plausible to enhance methane production by modulating the proportion of archaea. The relationship between bacteria and archaea in anaerobic environments, such as anaerobic bioreactors, is vital for methane production. Bacteria break down complex organic compounds into simpler forms, which are then utilized by archaea to produce methane. Through the introduction of bacteriophages, which target and control the growth of specific bacterial strains, it is possible to manipulate the composition and dynamics of the microbial community. By reducing the abundance of certain bacteria through phage-mediated mechanisms, it is conceivable to shift the balance within the community toward favoring specific archaeal populations that are more efficient in methane production [43]. This targeted modulation of the microbial community can optimize metabolic processes and potentially enhance methane production rates.

The outcome of phage-host interactions and their influence on the coexistence dynamics of bacterial hosts within microbial communities depend on the nature of these interactions as well as external conditions. The nature of phage-host interactions refers to factors such as the specificity of phages toward certain bacterial strains, the efficiency of phage infection and replication within hosts, and the ability of hosts to develop resistance mechanisms against phages. These factors determine whether phages act as facilitators of coexistence or promoters of competitive exclusion. External conditions, including environmental factors and resource availability, also play a significant role. Furthermore, the presence of other species within the community can interact with phage-host dynamics, influencing the outcome. Interactions between phages, bacterial hosts, and other community members can create complex ecological networks that shape coexistence patterns [44]. Overall, the coexistence or competitive exclusion effects mediated by phages within microbial communities are contingent upon the specific phage-host interactions and the prevailing external conditions. Understanding these factors is essential for comprehending the dynamics and stability of microbial communities. Every instance of phage infection introduces novel genetic information to the specific bacteria it targets, establishing a close and interdependent relationship between bacteriophages and their host bacteria. This dynamic connection between phages and hosts plays a pivotal role in shaping the evolution and ecology of microbial communities [45].

The interaction between phages and bacteria can reshape bacterial communities through various mechanisms. It can alter the dynamics of bacterial competition, drive bacterial diversity, or facilitate horizontal gene transfer between different species within the microbial community [46]. Scientists have proposed several approaches to explain this coevolutionary relationship. One of these approaches is the Red Queen hypothesis. This hypothesis, originally proposed by Van Valen [47], suggests that the successful adaptation of one organism can reduce the adaptability of other species that inhabit the same environment and interact with it. In the context of phages and bacteria, this hypothesis involves a coadaptation process referred to as an “arms race,” wherein these entities constantly engage in defensive strategies against each other [48]. However, when the level of resistance between phages and their host bacteria reaches a high threshold, the ongoing arms race between them may deteriorate, ultimately leading to the emergence of fluctuating selection dynamics. This phenomenon can create an opportunity for rare bacterial resistance alleles to persist and exploit the evolutionary dynamics of phages, enabling them to infect more prevalent bacterial genotypes [49].

The second hypothesis, Kill-the-Winner, proposes that the dynamics between phages and bacterial hosts are driven by negative frequency-dependent selection within the microbial population. This hypothesis suggests that phages selectively target and infect the most abundant bacterial strains, often referred to as the “winners” in the population. By reducing the population size of these dominant strains, phages alleviate competition and create opportunities for less abundant strains, or “losers,” to proliferate. The negative frequency-dependent selection arises from the fact that the efficacy of phages in infecting bacterial hosts depends on the relative abundance of the target strains. The more abundant a particular bacterial strain becomes, the higher its susceptibility to phage infection is due to the increased encounter rate between the phages and their hosts. As a result, the growth of dominant strains is suppressed, allowing less abundant strains to catch up and contribute to the overall diversity of the microbial community. This mechanism of negative frequency-dependent selection promotes the coexistence of multiple bacterial strains by preventing any one strain from achieving long-term dominance. Instead, it maintains a dynamic equilibrium where the relative abundance of bacterial strains fluctuates over time [50]. The Kill-the-Winner hypothesis highlights the role of phages in shaping the structure and diversity of microbial communities by balancing the competitive interactions among bacterial strains. It emphasizes the importance of considering the ecological and evolutionary dynamics between phages and their bacterial hosts in understanding the stability and functioning of microbial ecosystems. In resource-limited environments, phages may act as “Kill-the-Winner” agents, targeting and reducing the population of dominant bacterial strains, which allows less abundant strains to thrive and coexist. In contrast, under resource-rich conditions, phages may selectively infect and eliminate specific strains, leading to competitive exclusion. This recurring rise and fall of specific microbial populations can contribute to bacterial diversity by regulating the competitiveness of different species and facilitating the coexistence of diverse organisms [51].

The third explanation for phage-bacteria interactions is the concept of polyvalency. It is commonly observed that phages exhibit a narrow host range, meaning they can only infect specific bacterial species or strains. However, there are exceptions to this pattern, as certain phages known as polyvalent phages possess a broad host range, enabling them to infect multiple bacterial species. Polyvalent phages have the ability to recognize and bind to a wider range of host receptors, allowing them to infect diverse bacterial hosts. This broader host range is advantageous for phages in environments where multiple bacterial species coexist, as it increases their potential targets for infection [52]. The existence of polyvalent phages has been documented in various studies, and their broad host range has significant implications for microbial communities. By infecting multiple bacterial species, polyvalent phages can influence community dynamics, including competition and coexistence patterns, as well as the overall stability and diversity of the microbial community. Understanding the prevalence and impact of polyvalent phages adds a layer of complexity to the interplay between phages and bacterial hosts within microbial communities [53].

The impact of phage-driven changes in microbiota on BWT performance has been observed in WWTPs. Liu et al. [54] addressed the knowledge gap regarding phage population dynamics during sludge bulking. They noted a substantial reduction in the abundance of nitrifying bacteria during sludge bulking. Moreover, viral contigs linked to nitrifiers were more frequently identified and found in greater abundance in viromes from bulking sludge samples. These findings suggest that phages may contribute to the decline of autotrophic nitrifiers under bulking conditions. Additionally, through the utilization of advanced sequential methodologies, a significant revelation emerged regarding the phages isolated from activated sludge. It was discerned that a subset of these phages demonstrated a notable interspecies infection capability, signifying their capacity to infect bacteria across diverse species boundaries [30]. In the study of Aydin et al. [9], the effects of a phage cocktail on the treatment of pharmaceutical wastewater using an anaerobic bioreactor were investigated. The implementation of the phage cocktail resulted in significant changes in the bacterial and archaeal community in both the biofilm and sludge. Notably, there was a transition from Methanothermobacter to Methanoculleus as the dominant archaeal community, accompanied by a syntrophic interaction between the bacterial genera Macellibacteroidetes-Desulfovibrio and the archaeal genus Methanoculleus. These findings suggest that harnessing bacteriophages could be a promising strategy for regulating bacteria within anaerobic microbial communities and restoring the balance between bacterial and archaeal populations in a rapid manner. However, it is important to note that there is a limited number of experimental studies exploring the effects of different bacteriophage species and combinations on microbial community structure and activity within anaerobic bioreactors. Hence, conducting comprehensive studies over extended operational periods is strongly recommended to assess the influence of different phage species and combinations on the performance of anaerobic reactors and the dynamics of microbial communities. In conclusion, this study highlights the potential of phage-based approaches in enhancing the treatment of pharmaceutical wastewater in anaerobic bioreactors. Further research is needed to explore a broader range of bacteriophage species and combinations, as well as to assess their long-term effects on reactor performance and microbial community dynamics. Such studies will contribute to advancing our understanding and application of phage-mediated strategies in anaerobic wastewater treatment.

6. Limitations and perspectives

Typically, phages are thought to target specific bacterial species or strains. However, polyvalent phages with a broad host range have been frequently found in wastewater treatment systems. Additionally, multiple phages may infect hosts that are more abundant in the environment. Consequently, phage-host interactions in wastewater treatment ecosystems often form intricate networks. Yet it remains challenging to identify phage-host relationships under in situ conditions [53]. Traditional host-range assessments rely on available hosts in laboratory settings. However, in complex ecosystems like wastewater treatment systems, the diversity and number of potential hosts far exceed those found in labs, and phages may also compete for hosts. While modern computational methods based on sequencing can predict host interactions, these methods typically establish one-to-one relationships [55]. Therefore, they may not fully capture the complex phage-host infection dynamics in wastewater treatment systems, necessitating the development of innovative approaches to assess host ranges under in situ conditions.

Furthermore, unlike virulent phages, temperate phages have the ability to make a pivotal decision shortly after infecting a host cell: they can opt for lytic growth, causing the host cell to burst, or they can enter the lysogenic cycle, integrating their genetic material into the host cell as a prophage. Given that free phages are consistently flushed out with the effluent, prophages might have a higher likelihood of persisting in BWT systems. Prophages can play a direct role in host cell survival in unfavorable conditions by suppressing unnecessary metabolic activities. However, they also carry the potential to act as genetic bombs that can lead to host cell lysis, or even the demise of the entire host population, under specific environmental circumstances [56]. Extensive prophage induction followed by the sudden lysis of a substantial portion of the microbial community can contribute to issues like foaming, bulking, or reduced process efficiency in BWT systems [57]. Nevertheless, temperate phages in BWT environments have not been comprehensively investigated. The factors that influence the choice between lytic and lysogenic infections remain poorly understood, and the triggers for prophage induction are yet to be explored.

7. Conclusion

Phages possess the capacity to influence the diversity and arrangement of microbial communities within anaerobic digesters of wastewater treatment plants (WWTPs). The occurrence of frequent fluctuations in both phages and prokaryotes has the potential to impact the stability of the microbial composition and evolutionary processes. However, it is important to note that phages are not the sole viral entities present in anaerobic digesters. There have been reports of archaeal viruses with the potential to influence the methanogenic organisms critical to the anaerobic digestion process. Considering the possibility that each species may be susceptible to one or more viruses, it is reasonable to assume that the current knowledge of viruses affecting methanogens is merely the tip of the iceberg. Therefore, it is imperative to conduct further investigations into the genetic diversity of viruses targeting methanogens.

Of equal importance is a comprehensive understanding of the dynamics governing virus-host interactions throughout the AD process. Furthermore, the deliberate implementation of host-based treatments, tailored to regulate the prevalence of distinct microbial groups, holds promise for effectively addressing challenges like bulking and foaming issues in the process. These strategic interventions can guide the system toward its intended operational objectives. Enhancing the understanding of how these viruses impact the microbial community of AD and its dynamics will play a pivotal role in evaluating the efficiency and stability of the entire biogas production process.