Interspecies ecological competition rejuvenates decayed Geobacter electroactive biofilm

Abstract Bioelectrochemical systems (BESs) exploit electroactive biofilms (EABs) for promising applications in biosensing, wastewater treatment, energy production, and chemical biosynthesis. However, during the operation of BESs, EABs inevitably decay. Seeking approaches to rejuvenate decayed EABs is critical for the sustainability and practical application of BESs. Prophage induction has been recognized as the primary reason for EAB decay. Herein, we report that introducing a competitive species of Geobacter uraniireducens suspended prophage induction in Geobacter sulfurreducens and thereby rejuvenated the decayed G. sulfurreducens EAB. The transcriptomic profile of G. sulfurreducens demonstrated that the addition of G. uraniireducens significantly affected the expression of metabolism- and stress response system-related genes and in particular suppressed the induction of phage-related genes. Mechanistic analyses revealed that interspecies ecological competition exerted by G. uraniireducens suppressed prophage induction. Our findings not only reveal a novel strategy to rejuvenate decayed EABs, which is significant for the sustainability of BESs, but also provide new knowledge for understanding phage–host interactions from an ecological perspective, with implications for developing therapies to defend against phage attack.


Introduction
Bioelectrochemical systems (BESs) utilize the ability of electroactive bacteria to synchronize intracellular biocatalytic redox reactions with extracellular electron exchange with an electrode and therefore hold great promise in wastewater treatment; green energy, such as electricity and hydrogen production; value-added chemical biosynthesis; and biosensing [1][2][3].In these BESs, electroactive bacteria usually attach to electrodes to form electroactive biofilms (EABs) [4].Moreover, various methods have been developed to induce the formation of EABs [5,6].This is because it is generally recognized that forming EABs is necessary to achieve efficient electron exchange between electroactive bacteria and electrodes.Furthermore, sustaining the activity of EABs determines the high performance and work efficiency of BESs [7].However, in most cases, EABs gradually decay with the operation of BESs and the performance of the BESs declines [8,9].Unfortunately, to date, no effective methods have been developed to rejuvenate decayed EABs, aside from the suggestion to regularly erase the decayed EABs from the electrodes to induce EAB regrowth [9,10].
Geobacter sulfurreducens is the type of electroactive bacterium that generates the highest current in BESs and widely exists in various EABs [11,12].It can reduce the anode directly by expressing abundant cytochromes and nanowires and thereafter form a thick biofilm of tens of micrometers on the anode [13].However, the G. sulfurreducens EAB does not always metabolize actively as a whole.During the operation of the BES, G. sulfurreducens EAB decays due to the accumulation of dead cells and, meanwhile, the current generation declines [14][15][16].Nutrient substance consumption and toxic metabolite accumulation have been suggested to be the reasons for the decay of G. sulfurreducens EAB [17][18][19][20].In addition, the attenuation of the redox gradient across G. sulfurreducens EAB during EAB development has also been thought to contribute to the decay [15].However, a recent study indicated that all of these factors might trigger the lysogenic to lytic transition of prophages in G. sulfurreducens cells, and phage attack is the primary and internal reason for G. sulfurreducens EAB decay [21].
Phages have both lysogenic and lytic life stages [22].The lysogenic stage can have neutral or beneficial effects on the host cell, but the lytic stage only has detrimental effects [23][24][25][26].Thus, blocking the lysogenic to lytic transition of prophages will prevent cell lysis triggered by phages.Primary efforts have been made to interfere with this lytic transition.For example, the genetically activating and controlling repressor protein CI or other cleavable repressors and the holin protein of the host cell impedes prophages from switching to the lytic cycle [27]; addition of protease inhibitors such as antipain inhibits prophage induction in microbes by blocking proteolytic inactivation of repressors [28]; increasing the amount of signal peptides, such as hexapeptide moieties, promotes the ability of phages to commit to the lysogenic cycle [29].However, these methods are only exclusive to some microbes and, to an extent, harm microbes in other physiological aspects.
The status of the host cell could also affect the prophage transition.For instance, when cells grow under optimal conditions, prophages can voluntarily transit to lytic cycles [30][31][32]; on the contrary, when hosts are under stress conditions, such as elevated or decreased temperatures (or pH) [33], overwhelming osmotic pressure [34], or nutrient deficiency [35], many prophages will predominantly stay in lysogenization.In this study, we found that introducing ecological competition via the addition of a competitive species, Geobacter uraniireducens, suppressed prophage induction in G. sulfurreducens and subsequently rejuvenated the decayed G. sulfurreducens EAB.Our findings provide the first strategy to revitalize a decayed EAB and raise new knowledge for understanding the phage life cycle and phage-host interactions.

Bacterial strains and culture conditions
The G. sulfurreducens strain PCA (ATCC 51573), G. uraniireducens strain Rf4 (ATCC BAA-1134), Geobacter bemidiensis strain Bem (ATCC BAA-1014), and Escherichia coli DH5α were acquired from our laboratory culture collection.Geobacter sulfurreducens, G. uraniireducens, and G. bemidiensis were routinely cultured at 30 • C under strict anoxic conditions (80:20 N 2 /CO 2 ) in nutrient broth (NB) media supplemented with 15 mM sodium acetate as the electron donor and 40 mM fumarate as the electron acceptor, as previously reported [36].Escherichia coli was routinely cultured at 37 • C in Luria-Bertani medium.Cell growth was monitored by measuring optical density of the culture at 600 nm.

BES construction and electrochemical measurement
A three-electrode system consisting of a dual-chambered Hshaped bioelectrochemical reactor was constructed with the anode and the cathode chambers separated by a proton exchange membrane as previously described [37].In both chambers, polished graphite carbon plates with dimensions of 30 × 20 × 3 mm were used as electrodes, 100 mL of an anaerobic freshwater medium (FWNN) was used as the electrolyte, and 15 mM acetate was supplied in the anode chamber as the sole electron donor [38].The inoculum was prepared from a total of 10 mL of midexponential G. sulfurreducens culture grown in NBAF medium that was centrifuged at 8000 × g for 8 min and washed twice with FWNN.A constant voltage of +0.3 V (vs.Hg/HgCl sat.KCl) was applied to the working electrode, and the current was monitored continuously with a 1000C electrochemical workstation (CH Instrument Inc., China).The system was operated in batch mode.When the current decreased to less than 10 −3 mA, the electrolyte was replaced with fresh medium supplemented with 15 mM acetate.
Cyclic voltammetry was conducted with a CHI760E electrochemical workstation (CH Instrument Inc., Shanghai, China) by scanning the potential from −0.6 to 0.3 V with a gradient scanning rate.Electrochemical impedance spectroscopy was performed with a CHI760E electrochemical workstation at an open-circuit voltage with a sinusoidal perturbation amplitude of 5 mV over a frequency range from 10 000 to 0.1 Hz to analyze the internal impedances of the anode biofilms.All tests were performed under anoxic conditions (80:20 N 2 /CO 2 ) at 30 • C.

Biofilm viability analysis
The anodes were removed from the bioelectrochemical reactors, rinsed with 0.9% NaCl to wash out the f loating cells, and then stained with Live/Dead stain (Live/Dead BacLight bacterial viability kit; Thermo Fisher Scientific, USA) for 15 min in the dark following the manufacturer's instructions.Thereafter, the stained biofilm was visualized by a confocal laser scanning microscope (Carl Zeiss, Germany) equipped with a 20× objective lens at excitation wavelengths of 488 nm (SYTO 9, live cells) and 543 nm (propidium iodide, dead cells).The specific viability of each biofilm layer was determined by calculating the ratio of viable/total cells per area (obj/total) with Image-Pro Plus software as previously described [39].The total viability of an EAB was calculated as the average viability of each biofilm layer.

Bacterial quantification
The relative abundances of G. sulfurreducens and G. uraniireducens were quantified by quantitative PCR (qPCR).Cells were collected, and whole-genome DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, USA).Species-specific primers were designed for G. sulfurreducens and G. uraniireducens (Table S1).The DNA fragments amplified with the species-specific primers were TA-cloned and inserted into the T vector pMD19-T (Takara) and then verified by sequencing (Sango Biotech, Shanghai, China).The resulting plasmids were utilized for the preparation of the standard curves.The amounts of each species were determined by calculating against the standard curves.

Measurement of niche and relative fitness differences
Mutual invasion experiments were conducted to determine the interspecies relationship of coexistence or competitive exclusion between G sulfurreducens and the other three species of G. uraniireducens, G. bemidiensis, and E. coli, respectively, as previously described [40,41].Specifically, one species acted as an invasive species was inoculated into a steady-state culture of the other species and was inoculated alone into a fresh culture medium, separately, and the growth of the invasive species was measured by qPCR.Thereafter, the growth rates of the invasive species in each situation (μ invading and μ alone ) over a 3-day period were calculated according to the following equation: where D 0 and D T are the cell densities of invasive species at Day 1 and 3, respectively, and T = 3.The sensitivity (S) of the invasive species to competition was calculated as follows: The niche difference (ND) and relative fitness difference (RFD) were calculated as follows: where S a and S b represent the sensitivity to competition of species a and b, respectively, when they are invaded mutually.

Flow cytometric enumeration of phage particles
The anolyte was harvested and centrifuged to remove cells.The supernatant was further filtered through a 0.22-μm filter membrane (Merck, Darmstadt, Germany).To remove extracellular vesicles, one volume of chloroform was added to the filtrate.The mixture was incubated at room temperature for 10 min and then centrifuged at 10 000 × g at 4 • C for 5 min.Further treatment of the sample for f low cytometric enumeration of phage particles was performed as described in a previous study [42].Brief ly, the supernatant was collected and further fixed with 0.5% v/v glutaraldehyde.After diluting 10 3 -fold with 0.1 M TE buffer (pH 8), the sample was stained with SYBR Green I (Sigma-Aldrich, St. Louis, USA) at 80 • C for 10 min in the dark and then cooled to room temperature prior to analysis via f low cytometry (Thermo Fisher Scientific, Waltham, USA).Phage particles were enumerated using a mixed function that included the cytometer f low rate, sample volume, sample dilution, and number of positive events within the gated region of interest.

Transcriptomic analysis
Anode biofilms were collected by scraping, and total RNA was extracted using the HiPure Universal RNA Mini Kit (Magen Biotechnology Co., Ltd., Guangzhou, China) according to the manufacturer's instructions.

Quantification of phage-related gene expression
Total RNA was treated with the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2 (Accurate Biology, Changsha, China) to remove genomic DNA, and reverse transcription was subsequently performed.Thereafter, qPCR was performed to quantify the expression of the gp17 (GSU2178) and gp19 (GSU2163) genes from prophage region 4 with gene-specific primers using the LightCycler 480 System (Roche Applied Science, Penzberg, Germany).The housekeeping gene rpoD was selected as the internal reference gene for G. sulfurreducens.All primers used are listed in Table S1.

Results
The addition of G. uraniireducens rejuvenates the decayed G. sulfurreducens EAB The BES for current generation was inoculated with G. sulfurreducens and subjected to batch mode via regular refreshing of the electrolytes over the long-term operation of 11 batches, and the metabolic activity of the anode biofilm was subsequently examined.As demonstrated, a decayed biofilm appeared with dead cells accumulated throughout the biofilm (Figs 1A and S1), and the current generation was impaired (Fig. 1D), as previously reported [21].In contrast, at this point, when Geobacter uraniireducens was added, the decayed biofilm was rejuvenated, showing an active metabolism wholly (Figs 1B and S2).In addition, the current generation recovered (Fig. 1D), the redox activity improved (Fig. S3), and the electron transfer resistance decreased (Fig. S4), thus indicating enhanced electrochemical activity.
Geobacter uraniireducens is also an electroactive bacterium.It can form a biofilm on the anode and reduce the anode directly for current generation, similar to G. sulfurreducens [43].However, it generates a much lower current than G. sulfurreducens.Unexpectedly, G. uraniireducens accounted for only a minor proportion (ca.0.1%) of the biofilm (Fig. S5).These results indicated that the rejuvenated biofilm was derived from the recovery of the decayed G. sulfurreducens in the biofilm and G. uraniireducens contributed to neither the metabolic activity of the biofilm nor the recovery of current generation.We further evaluated the possibility that G. uraniireducens might be a nutrient or provide some substrates for G. sulfurreducens and then stimulate rejuvenation.The cell-free lysate of G. uraniireducens was added to the decayed BES.Neither the biofilm rejuvenated ( Fig. 1C) nor the current generation recovered (Fig. 1D), suggesting that an active interaction between these two species is necessary for the rejuvenation of G. sulfurreducens.

Prophage induction is inhibited after the addition of G. uraniireducens
To identify the possible interaction between G. sulfurreducens and G. uraniireducens, we further analyzed the transcriptome profiles of G. sulfurreducens EABs, and 492 genes of G. sulfurreducens were significantly affected in the presence of G. uraniireducens (Fig. 2A).Among them, 160 genes were upregulated, and 332 genes were downregulated.Specifically, two-thirds of the upregulated genes belonged to metabolic pathways (Fig. 2B), encompassing amino acid metabolism, carbohydrate metabolism, energy metabolism, lipid metabolism, and cofactor and vitamin metabolism.This result is consistent with the high viability of G. sulfurreducens after treatment with G. uraniireducens (Fig. 1B).In contrast, those phagerelated genes were downregulated, particularly those genes in prophage region 4, which encodes G. sulfurreducens phage (Fig. 2C) [21], and the amount of phage particles secreted into the culture medium decreased (Fig. 2D) correspondingly.Thus, the presence of G. uraniireducens restrained the prophage induction of G. sulfurreducens.Prophage induction, particularly the activation of prophage region 4, has been shown to be the primary cause of G. sulfurreducens EAB decay [21].The inhibited expression of phagerelated genes must suspend the decay and then contribute to the rejuvenation of G. sulfurreduces biofilm.
Most of the rest prominently affected genes were related to stress-response systems, including upregulated genes related to genetic information processing (Fig. S6), such as genes encoding DNA and RNA polymerases, sigma factors [44], and cold shock-domain proteins (Csps) [45,46], all of which are known to confer stress tolerance and protect cells by facilitating resistance to stress and repairing cellular damage; genes encoding OmpA proteins [ 47], HgtR-related proteins [48], and stress-responsive proteins [49], all of which could contribute to cellular adaptation to stresses by stabilizing the outer membrane; as well as downregulated genes mainly enriched in the functions of cell motility, signal transduction, cellular community, and membrane transport and are under the control of stress response systems.These results suggest that the presence of G. uraniireducens likely stresses G. sulfurreducens, thereby triggering its global stress response.

Ecological competition causes the suppression of prophage induction
Geobacter uraniireducens and G. sulfurreducens are closely related electroactive Geobacter species that utilize similar electron donor and acceptor spectra.Thus, they should compete for the same resources when cocultured.We speculate that the addition of G. uraniireducens introduced competition stress to G. sulfurreducens.To test this hypothesis, we calculated the ND between G. uraniireducens and G. sulfurreducens.The ND measures the proportion of resources not shared by two species and ref lects the level of niche overlap.In general, a smaller ND represents a greater overlap of niches, which indicates greater interspecies competition, and an ND value smaller than 0.1 usually indicates significant interspecies competition [40].The results showed that the ND between G. uraniireducens and G. sulfurreducens was very minimal close to 0.02 (Fig. S7), corroborating the intense competition between G. uraniireducens and G. sulfurreducens.Furthermore, we measured the RFD, which represents the difference in competitive ability among species, and thereafter evaluated the competition outcome by comparing RFD with (1-ND) −1 .According to modern coexistence theory, competitive exclusion occurs when RFD > (1-ND) −1 ; otherwise, coexistence dominates (RFD < (1-ND) −1 ) [50].Between G. uraniireducens and G. sulfurreducens, the RFD was greater than that of (1-ND) −1 (Fig. 3A) indicating competitive exclusion.Thus, G. sulfurreducens must experience competition stress when G. uraniireducens is present.This result raises the question of whether the introduction of competition suppresses prophage induction in G. sulfurreducens.In order to test the possibility that interspecies competition affects prophage induction, we tuned the intensity of competition by introducing two other bacterial species, Geobacter bemidjiensis and E. coli, with different NDs of 0.04 and 0.33, respectively, to the decayed G. sulfurreducens EAB.Thus, in theory, G. bemidjiensis and E. coli will exhibit high and low competition with G. sulfurreducens, respectively.In addition, G. bemidjiensis should undergo competitive exclusion from G. sulfurreducens, whereas E. coli should coexist with G. sulfurreducens ( Fig. 3A).We also treated the decayed G. sulfurreducens EAB with metabolically active G. sulfurreducens, which theoretically should not introduce any competition.Similar to the G. uraniireducens treatment, the addition of G. bemidjiensis led to the downregulation of phage-related genes (Fig. 3B) and a decrease in phage secretion by G. sulfurreducens (Fig. 3C), indicating the suppression of prophage induction.Correspondingly, the biofilm viability (Fig. 3D) and current generation (Fig. S8) recovered significantly.In contrast, neither the addition of E. coli nor G. sulfurreducens treatment affected prophage induction (Fig. 3B, C) or had a significant impact on the biofilm viability (Fig. 3D) or current generation (Fig. S6) of G. sulfurreducens.Collectively, these results demonstrated that interspecies competition contributes to the suppression of prophage induction in G. sulfurreducens.

Discussion
During the long-term operation of BESs, prophage induction in G. sulfurreducens causes the decay of G. sulfurreducens EAB and subsequently impairs current generation.Our study illustrated a therapy in which G. uraniireducens was introduced into the system to cure the phage attack and subsequently rejuvenated the decayed G. sulfurreducens EAB.Specifically, G. uraniireducens acted as a competitive species and probably applied competitive stress toward G. sulfurreducens, which triggered the suppression of prophage induction in G. sulfurreducens and thereafter allowed the metabolic activity of the G. sulfurreducens EAB to recover (Fig. 4).However, G. uraniireducens could not sustain proliferation in the system, possibly because it could not outcompete G. sulfurreducens for anode reduction, and during subsequent further operation, the abundance of the G. uraniireducens community declined gradually (account for less than 0.01%), and the G. sulfurreducens EAB started to decay again (Fig. S9).Therefore, it might be reasonable to regularly treat the G. sulfurreducens EAB with G. uraniireducens for maintaining long-term vitality.Even though G. bemidjiensis also exhibits competitive exclusion with G. sulfurreducens, it could not fully recover the biofilm vitality and current generation as G. uraniireducens (Figs 3D and S8).The reason should be that compared with G. uraniireducens and G. sulfurreducens, the competition between G. bemidjiensis and G. sulfurreducens is lower ( Fig. 3A).
Our results provide the evidence that interspecies competition can restrain prophage induction in bacterial cells.This finding is contrary to previous studies showing that interspecies competition generally stimulates lysogenized bacteria to evoke prophage induction, by which released phage particles kill or outcompete susceptible competitors [51,52].Even so, in either case, the competitive species acts as an external stressor on the cell.Previous studies have indicated that external stressors can trigger the host cell stress response to regulate the lyticlysogenic switch of prophages [53][54][55].Therefore, the different prophage decisions might be due to the different stress responses of the host cell.Even though, in either case, the host cell received competitive advantages, and in return, the phage achieved an increased level of fitness, suggesting symbiosis between phages and bacteria.Previous studies have also indicated that microbial stress response systems are the primary mechanism by which cells sense and respond to external stressors [56,57].Similarly, in our study, G. sulfurreducens activated the stress response system when competitive species were introduced, suggesting that the suppression of prophage induction in G. sulfurreducens was probably due to the inhibitory effect of the stress response system.
The finding that introducing competitive species could enhance the current generation of G. sulfurreducens sheds new light on the construction or design of synthetic microbial communities for BESs.In previous studies, the philosophy of "division of labor" guided the selection or engineering of functional bacterial species for constructing an efficient EAB [58].Typically, one fermentative species is usually incorporated to incharge of decomposing complex organics into small molecular organic acids, and another electroactive bacterium is selected to oxidize those organic acids for current generation.Thus, synergistic cooperation occurs, and interspecies competition has generally been considered to harm this process and therefore should be avoided.Importantly, our findings challenge this notion and reveal the advantage of "interspecies competition" in sustaining the performance of EABs.Actually, it has been supported by previous reports that mixed-species EABs usually perform better than pure culture biofilms [59], and coculturing competitive Geobacter species generated a greater current than single species [60].Thus, our findings also provide new knowledge that can be used to help understand or explain the ecology of interspecies interactions in EABs.
Although mixed-species EABs are usually more sustainable than single Geobacter EABs, they always exhibit a much lower coulombic efficiency, which limits their application in BESs.In contrast, Geobacter species are the most efficient electroactive bacteria and generate the highest reported current; thus, these bacteria show the greatest promise in BESs.However, Geobacter EABs will decay during long-term operation.Previous studies have suggested applying external forces, such as ultrasound treatment and periodic polarization, to dislodge those decayed biofilms and thereafter to regrow fresh biofilms to make BESs sustainable [10,61].Apparently, these strategies have taken drastic measures and are sophisticated and energy-consuming.In addition, regrowing a new functional biofilm takes days.In contrast, our study provides a novel strategy involving treatment with competitive species to rejuvenate the decayed biofilm, which is time-saving (only requires several hours) and maneuverable.In particular, the competitive species could also be inoculated at the very beginning together with the working Geobacter species to sustain the longevity of the biofilm and thereafter enhance the performance of the BES (Fig. S10).Furthermore, considering the universality of prophages in bacterial species and their detrimental effects on biofilm decay, our study also has implications for defending against phage attack in other biofilm-based reactors.

Figure 1 .
Figure 1.The metabolic activity and current generation of G. sulfurreducens biofilms; confocal laser scanning microscopy images and depth-related viability profiles of G. sulfurreducens biofilms before (A) and after treatment with active G. uraniireducens (B) or cell-free lysate of G. uraniireducens (inactive) (C); 5 mL of G. uraniireducens culture with an OD600 of 0.35 was added.Geobacter sulfurreducens biofilms growing in the 10th batch were tested, and biofilms from the 11th batch were examined when a peak current was generated, and biofilms were stained with live/dead stain; live cells were imaged as green, dead cells were imaged as red, and representative images were selected from three independent experiments, and the scale bars represent 30 μm, and for the viability calculation, at least five different sites of the anode biofilm were evaluated; (D) peak current profiles of G. sulfurreducens during 11 batches of operation; the arrows indicate the addition of G. uraniireducens (+ G.u) and the addition of cell-free lysate of G. uraniireducens (+ inactivated G.u); the error bars represent the standard deviations of three replicate samples.

Figure 2 .
Figure 2. Transcriptomic profile and phage secretion in G. sulfurreducens biofilms after the addition of G. uraniireducens; (A) volcano plot illustrating the overall differences in gene expression between G. sulfurreducens biofilms treated with and without G. uraniireducens; genes with upregulated expression are represented by red dots, genes with downregulated expression are represented by blue dots, and genes not significantly differentially expressed (P > .05)are represented by gray dots; (B) the top 20 enriched KEGG pathways of differently expressed genes; (C) heatmap analysis of phage-related genes in prophage region 4, and red represents high gene expression abundance (Z_Score from 0 to 1.5), and blue represents low gene expression abundance (Z_Score from 0 to −1.5), and (D) the concentration of phage particles released in G. sulfurreducens biofilms; each data point represents one independent sample, and the horizontal and vertical bars stand for the mean of the data and the standard error of mean, respectively, and statistical analysis was performed by a two-tailed Student's t test.* * P < .005,n = 3.

Figure 3 .
Figure 3. Ecological competition evaluation and prophage induction analyses; (A) the outcome of competition between G. sulfurreducens and three other species, G. uraniireducens, G. bemidjiensis, and E. coli; the curve RFD = (1-ND) −1 demarcates the boundary between competitive exclusion (light orange area) and coexisting (light gray area), and competition exclusion occurs when the calculated RFD is located in the light orange area (where RFD > (1-ND) −1 ), whereas coexisting occurs when RND is located in the light gray area (where RFD < (1-ND) −1 ), and (B) the relative fold change in the levels of the phage-related genes gp17 and gp19; (C) the phage particle concentrations; the dots represent the separate individual data, whereas the horizontal and vertical bars stand for the mean of the data and the standard error of mean, respectively; (D) the total biofilm viability after the introduction of different bacterial species to G. sulfurreducens EAB, and statistical analysis was performed by a two-tailed Student's t-test.* P < .05,* * P < .005,n = 3.

Figure 4 .
Figure 4.The model that introducing G. uraniireducens rejuvenates the decayed G. sulfurreducens EAB; prophage induction, a lysogenic to lytic switch, causes the decay of G. sulfurreducens EABs, and the addition of competitive species of G. uraniireducens exerts competition stress on G. sulfurreducens, which suppresses prophage induction to trigger the lytic to lysogenic transition in G. sulfurreducens and subsequently rejuvenates the decayed biofilm.