Effects of Priestia megaterium A20 on the Aggregation Behavior and Growth Characteristics of Microcystis aeruginosa FACHB-912

Abstract

Microcystis aeruginosa formed in natural water bodies grow in aggregate particles, while Microcystis aeruginosa commonly used in scientific research grow in a single-celled discrete state during cultivation. To elucidate the factors and mechanisms of Microcystis aeruginosa entering the “cell-aggregate” survival state in the natural environment, we focused on studying the influence of biological factors in their living environment (coexisting bacteria) on the aggregation behavior and growth characteristics of Microcystis aeruginosa. The bacterial strain A20, which can promote the aggregative behavior of Microcystis aeruginosa, was isolated from the water of Taihu Lake, where a cyanobacterial bloom broke out. A20 was identified as Priestia megaterium. Results showed that A20 could significantly drive Microcystis aeruginosa to form sac-like aggregate structures and promote the increase of aggregate particle size from 3–7 μm to 180 μm. The coexistence of bacteria and algae exhibited a dynamic stage adaptation strategy, with A20 promoting the transition of Microcystis aeruginosa from “high-chlorophyll, low-photochemical efficiency growth and proliferation” to “stable survival and maintenance of chlorophyll and photochemical efficiency in fluctuating changes” adaptation strategies. The coexistence of bacteria and algae significantly intensified the release of humic acid-like, fulvic acid-like, and protein-like substances from Microcystis aeruginosa, with the most significant increase in small-molecule fulvic acid-like substances. This is probably related to the endogenous metabolic stress response of Microcystis aeruginosa during A20 invasion, as well as the utilization and transformation of autotrophic Microcystis aeruginosa metabolites by heterotrophic bacteria A20. This study contributes to the study of microbial interactions underlying bloom outbreaks and can be useful for developing community-targeted algal control technologies.

1. Introduction

The massive discharge of industrial wastewater and domestic sewage has exacerbated the eutrophication problem of water bodies in China, leading to the normal outbreak trend of harmful cyanobacterial blooms in lakes such as Taihu Lake and Chaohu Lake. Cyanobacterial blooms not only cause ecological damage to water bodies, but also pose a persistent challenge to drinking water treatment systems, seriously threatening the safety of the water supply [1].

Having group traits is the basic unit for cyanobacteria to exercise ecological functions [2]. Cyanobacterial blooms are actually the process by which cyanobacterial populations form protective barriers and gain population advantages while maintaining their aggregated state, and then float on the water surface and expand their growth [3,4,5]. During the outbreak of cyanobacterial blooms in Taihu Lake, Microcystis aeruginosa was the dominant algal species, which accounted for above 98% of total cell count [6]. Research has found that the aggregate form of Microcystis aeruginosa has multiple competitive advantages. For example, it has a significant advantage in inorganic carbon adsorption compared to its unicellular form [7]. In addition, increasing the size of Microcystis aeruginosa aggregates can effectively regulate their internal gas vesicle structure, prompting the aggregates to sink rapidly to avoid damage from high light intensity, or reducing cell density so that they can quickly float to the surface for photosynthesis [8,9]. A key manifestation of Microcystis aeruginosa aggregates is the highly organized gelatinous biofilm structure secreted outside of their cells, which plays an important role in the encapsulation, aggregation, and growth protection of Microcystis aeruginosa. However, it also poses a major technical challenge for algae control processes aimed at improving water quality [10,11]. Experiments have shown that, compared to single cells, Microcystis aeruginosa aggregates and their extracellular matrix substances can cause a series of disruptive effects on the operation of water treatment processes [12]. For example, the extracellular mucilage adhering to the surface of algae cells has anionic properties, which increase the colloidal stability of Microcystis aeruginosa, thereby increasing the treatment load of coagulation and reducing coagulation efficiency [13,14]; Furthermore, the extracellular substances of Microcystis aeruginosa aggregates are prone to combining with tiny inorganic particles in water during the flocculation process, forming enlarged organic colloids. This not only increases the amount of coagulants required in water treatment, but also leads to a poor formation of floc structure and a decrease in sedimentation performance [15]. The large amounts of organic components released by Microcystis aeruginosa aggregates are mostly colloidal or soluble molecules at the molecular scale, which are easily dispersed in the aqueous phase. This not only causes serious pollution of the water sources but also increases the risk of disinfection by-products during the treatment process [16,17].

However, how is the aggregate of Microcystis aeruginosa formed? The production and driving mechanisms that promote the transition of Microcystis aeruginosa from cellular growth to aggregate growth are still poorly understood. It is worth noting that during the cultivation process of Microcystis aeruginosa in the laboratory, even with nutrient concentrations being far higher than those found in natural blooms and optimized physicochemical culture parameters, Microcystis aeruginosa still maintains a dispersed single-cell growth state and is unable to form the typical aggregate morphology found in actual aquatic environments. This suggests that in addition to physical and chemical factors, interspecies interactions between biological factors are highly likely to be the key factors in triggering the formation of Microcystis aeruginosa aggregates [18]. The environment surrounding the survival of Microcystis aeruginosa in natural water bodies contains a considerable abundance of bacteria [19]. The aggregates of Microcystis aeruginosa are closely related to their coexisting bacteria. The outbreak of dominant algal species can affect the composition and diversity of surrounding bacteria [20], while the abundance and type of bacteria can also affect the survival and growth patterns of different algal species [19,21,22]. However, what kind of bacteria drives or affects the formation of Microcystis aeruginosa aggregates in the complex microbial coexistence system? How does it affect the growth, metabolism, and developmental characteristics of Microcystis aeruginosa? These issues are of great significance for exploring the formation process and mechanism of Microcystis aeruginosa aggregates. They also play an important role in the prevention and treatment of cyanobacterial blooms. It should be noted that in the study of algal–bacterial interactions, using purified algal strains and purified bacterial strains to construct a stable co-culture system is an ideal experimental paradigm for eliminating interference from other environmental microorganisms and establishing direct causal relationships, which is crucial for revealing the specific mechanism of bacterial–algal action. Therefore, this study isolated and screened coexisting bacteria that can trigger the aggregation of Microcystis aeruginosa in the outbreak water environment in order to clarify the key factors for the “cell to population” survival transformation of Microcystis aeruginosa.

2. Materials and Methods

2.1. Algal Culture Conditions

Microcystis aeruginosa is a dominant algal species commonly found during cyanobacterial blooms. This research used Microcystis aeruginosa FACHB-912 as the experimental subject, which was provided by the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (Beijing, China), and its original source was isolated from Taihu Lake. Microcystis aeruginosa was inoculated into 500 mL of sterile BG-11 medium and cultured in a constant-temperature light-controlled incubator (GZP-300C, Ningbo Jiangnan, Ningbo, China). The cultivation conditions were as follows: temperature 25 ± 1 °C, light intensity 1000 lux, light–dark ratio 12 h:12 h, and the culture was shaken three times daily at regular intervals to ensure gas exchange in the algal suspension and prevent oxygen deficiency [23].

2.2. Screening Symbiotic Bacteria

2.2.1. Isolation and Purification of Bacteria Coexisting with Microcystis aeruginosa

During the cyanobacterial bloom season, a total of 3 L of water samples were collected at the sampling site (120°0′13″ E, 31°2′18″ N) from the surface, 0.5 m depth, and 1 m depth of Lake Taihu, and the water samples of each depth were mixed equally. To isolate the bacteria coexisting with Microcystis aeruginosa, a small amount of fresh Taihu Lake water sample was diluted 10¹, 10², 10³, 10⁴, and 10⁵ times, respectively, and aliquoted into five 10 mL sterile centrifuge tubes. In an ultra-clean workbench (SW-CJ-1F, Suzhou Jinghua, Suzhou, China), the water samples were spread onto beef extract peptone solid culture medium (BW143, Shanghai Bio-way, Shanghai, China), labeled, and incubated upside down in a biochemical incubator (SHP-250, Shanghai Jinghong, Shanghai, China) at 37 °C. After 24 h, the plates were removed for observation. Single colonies with clear growth were selected as the research objects. Colonies with different colors and shapes were picked from the plates and separately cultured on beef extract peptone solid medium for 2–3 generations to ensure the formation of pure strains. The different strains obtained were numbered accordingly.

2.2.2. Co-Culture and Screening of Bacteria

This study used a single strain of Microcystis aeruginosa and a single strain of bacteria to construct a co-culture system in order to clarify that the changes and interactions between bacteria and algae in the co-culture system were entirely derived from this binary interaction, providing a pure excavator platform for complex microbial interactions in actual water environments. The isolated pure bacterial strains were separately inoculated into beef extract peptone liquid medium, shaken, and cultured at 37 °C for 24 h in a biochemical incubator. Once the bacterial culture medium showed granular bacterial structure and OD₆₀₀ reached about 1, the bacteria were co-cultured with Microcystis aeruginosa in the logarithmic growth phase at a ratio of 1:9. The bacteria and Microcystis aeruginosa were mixed thoroughly and incubated in a constant-temperature light incubator. The cultivation conditions were the same as when Microcystis aeruginosa was cultured alone. The conical flasks were shaken 2–3 times daily to ensure adequate contact between bacteria and Microcystis aeruginosa. In addition, the monoculture groups of Microcystis aeruginosa and bacteria were also set as controls. The monoculture group of Microcystis aeruginosa was composed of 90% Microcystis aeruginosa culture medium and 10% sterile deionized water to maintain the same amount of algae as in the co-culture system. The monoculture group of bacteria was composed of 10% bacterial culture medium and 90% BG11 medium to maintain the same amount of bacteria as in the co-culture system. Continuous microscopic monitoring was taken for several days to screen for bacteria strains that promote the aggregation behavior of Microcystis aeruginosa.

2.2.3. Molecular Biological Identification

After the co-cultivation with Microcystis aeruginosa, strains that can promote the aggregation of Microcystis aeruginosa were sent to Shanghai Ling’en Biotechnology Co., Ltd. (Shanghai, China) for 16S rRNA gene sequencing. The 16S rRNA gene sequence of the bacteria was imported into the NCBI database for comparison, and a phylogenetic tree was constructed using MEGA12 software and the N-J method.

2.3. Detection Indicators

2.3.1. Cell Morphology

A small amount of algae solution was taken and observed under an optical microscope (E100, Nikon, Tokyo, Japan).

2.3.2. Particle Size Distribution

A laser particle size analyzer (BT-9300ST, Bettersize, Beijing, China) was used to measure changes in the particle size distribution of Microcystis aeruginosa single cells and aggregates during the cultivation process. Before each measurement, the container and pipes were washed three times with pure water to prevent interference from impurities.

2.3.3. Chlorophyll-a

The Microcystis aeruginosa were filtered through an acetate fiber filter membrane with the pore size of 0.45 μm. After removing the filter membrane, 3–5 mL of 90% acetone and a small amount of magnesium carbonate powder were added to the membrane and subjected to multiple extractions with a small amount of acetone. The mixture was then transferred into a 10 mL centrifuge tube, diluted, and shaken well. Chlorophyll-a was extracted by soaking at 4 °C in the dark for 12 h. Then it was centrifuged at 3000× g for 15 min. The absorbance of the extraction solution at wavelengths of 630 nm, 645 nm, 663 nm, and 750 nm was measured using a UV-visible spectrophotometer (UV759S, Shanghai Jingke, Shanghai, China), with 90% acetone as the blank group absorbance for calibration. The final chlorophyll-a concentration (μg/L) of the water sample was calculated using Equation (1) [24].

$$\mathrm{Chlorophyll-}\mathrm{a} (\mathsf{\mu }\mathrm{g}/\mathrm{L})=11.64\times ({OD}_{663}-{\mathrm{OD}}_{750})-2.16\times \left({\mathrm{OD}}_{645}-{\mathrm{OD}}_{750}\right)+0.10\times \left({\mathrm{OD}}_{630}-{\mathrm{OD}}_{750}\right)$$

(1)

2.3.4. Photochemical Efficiency (Fv/Fm)

The Fv/Fm ratio can be used to characterize the light energy conversion efficiency of algal cells and has been successfully employed as a sensitive indicator of photosynthetic performance [25]. The collected algal liquid samples were placed in a dark environment for 10 min, and then Fv/Fm was measured using a handheld chlorophyll fluorometer (AquaPen-C100, PSI, Drásov, Czech Republic) [25].

2.3.5. Dissolved Organic Carbon

Dissolved organic carbon (DOC) serves as a direct indicator of the total quantity of dissolved organic matter in a water body [26]. DOC content was measured using a total organic carbon analyzer (TOC-4200, Shimadzu, Kyoto, Japan).

2.3.6. UV₂₅₄

The UV₂₅₄ value reflects the amount of natural humic substances and aromatic compounds containing C=C and C=O double bonds present in water. Research has indicated that there is a certain correlation between UV₂₅₄ value and chromaticity, DOC, COD, etc., which can indirectly reflect the degree of organic pollution in water [27]. Measuring UV₂₅₄ is a typical method of characterizing organic matter with relatively simple and rapid advantages. The measurement of UV₂₅₄ was performed using pure water as control and measured using a UV-visible spectrophotometer (UV759S, Shanghai Jingke, Shanghai, China).

2.3.7. Fluorescence Spectroscopy Analysis of Component Structural Characteristics

Three-dimensional fluorescence spectroscopy was used to analyze changes in the structural composition of extracellular organic matter released by Microcystis aeruginosa. The samples were measured using a fluorescence spectrometer (F-7000, Hitachi, Tokyo, Japan) with the following parameters set: excitation wavelength range of 220–450 nm, emission wavelength range of 250–550 nm, excitation slit width of 5 nm, emission slit width of 1 nm, photomultiplier tube voltage of 700 V, and scanning speed of 2400 nm/min. Before sample analysis, ultra-pure water was used as a blank control. After analysis, the data were exported to an Excel spreadsheet, and contour color maps were plotted using Origin 2022 software. Based on the distribution positions and intensities of fluorescence peaks in the figure, the spectrum can be divided into 5 fluorescence regions. The division of each fluorescence region and the types of organic substances it represents are shown in Figure 1 [28,29,30].

2.4. Data Analysis and Visualization

All experiments were conducted in triplicate, and error bars in the plots represented the standard deviation (SD) values. All data were statistically analyzed using Student’s t-test, and differences were considered significant at p < 0.05.

3. Results and Discussion

3.1. Analysis of Bacterial Strain Causing Aggregation Behavior of Microcystis aeruginosa

The microscopic results of the co-culture of Microcystis aeruginosa and bacteria from Taihu Lake during a cyanobacteria outbreak period showed that strain A20 could promote the aggregation behavior of Microcystis aeruginosa. The effect of A20 on the growth and aggregation morphology of Microcystis aeruginosa cells is shown in Figure 2. When Microcystis aeruginosa was cultured alone, its growth and reproduction processes showed a uniform single-cell dispersed distribution, and its cell density increased with the growth time. After the addition of strain A20, the morphology of algal cells changed, showing obvious spherical sac-like aggregates and possessing a clear boundary-wrapped biofilm. As the co-culture time increased, the degree of aggregation became more and more obvious, indicating that the A20 strain could significantly promote the aggregation of Microcystis aeruginosa.

The changes in particle size distribution in the co-culture system of Microcystis aeruginosa and A20 are shown in Figure 3. Compared to the control group of Microcystis aeruginosa alone, the proportion of particles with sizes in the 3–7 μm range was significantly reduced in the co-culture system, while the proportion of larger particle was significantly higher. As the culture time increased, the proportion of large particles continued to rise, indicating that the addition of A20 significantly promoted the aggregation of Microcystis aeruginosa cells into clusters, and the aggregation state continued to increase and persisted. In the co-culture system, particles larger than 180 μm even appeared in the later stage, indicating that A20 also stimulated the continuous expansion of alginate clusters, forming larger Microcystis aeruginosa aggregates.

The characteristics of strain A20 (Figure 4) showed that its colony structure was circular, with neat edges, smooth and bright surface, moist and viscous texture, white color, raised center, and 2–4 mm colony diameter. The staining result of strain A20 was purple, which conformed to the characteristics of Gram-positive bacteria. According to the sequencing results analysis, the 16S rRNA gene sequence of strain A20 was most closely related to Priestia megaterium NBRC 15308 ^T. Strain A20 was identified as Priestia megaterium, and the bacteria isolated in this study have not been previously reported to promote aggregation behavior of Microcystis aeruginosa. Raw sequencing reads of Priestia megaterium A20 have been deposited in the NCBI Sequence Read Archive (SRA) with the BioProject accession PRJNA1354831.

3.2. Effect of A20 on the Growth Trends of Microcystis aeruginosa

The changes in chlorophyll-a concentration and photochemical efficiency Fv/Fm of Microcystis aeruginosa during co-cultivation with A20 are shown in Figure 5. Compared to the control group of Microcystis aeruginosa alone, the addition of A20 caused significant fluctuations in chlorophyll-a concentration, with a trend of increasing, decreasing, and then increasing again. The concentration of chlorophyll-a increased to 1455.24 μg/L at 0–3 d, which differed significantly from that of the control group (1161.65 μg/L of Microcystis aeruginosa alone, with p < 0.05). This may be related to the ability of Priestia megaterium to produce siderophore [31,32]. Siderophore can rapidly supply iron to Microcystis aeruginosa in the early stage, and iron is a key cofactor for chlorophyll synthesis (e.g., ferrochelatase), directly stimulating the accumulation of chlorophyll precursors [33]. In addition, studies showed that some phycospheric bacteria could assist Microcystis aeruginosa in obtaining P nutrition, thereby stimulating its growth. For example, some heterotrophic phosphonate-degrading phycospheric bacteria could assist cyanobacteria against phosphorus scarcity by facilitating phosphonate availability [34]; some phycospheric bacteria could secrete organic acids and alkaline phosphatase to accelerate the solubilization and transformation of phosphorus in the coexistence system of bacteria and algae and stimulate the synthesis of more photosynthetic pigments by Microcystis aeruginosa [35]. After 3–5 d, the chlorophyll-a concentration in the co-culture system rapidly decreased to 998.41 μg/L, reaching a level significantly lower than that in the control group of Microcystis aeruginosa alone (1422.93 μg/L, p < 0.01). This suggested that, with the development of a co-culture system and the consumption of limited resources by bacteria, there might also be a lack of raw materials for chlorophyll synthesis in Microcystis aeruginosa. For example, excessive chelation of iron by siderophores could potentially create an “iron trap,” which is an inhibitory effect of siderophores, and lead to insufficient iron acquisition for Microcystis aeruginosa [36,37,38]. The recovery and increase of chlorophyll-a in the co-culture system after 5–7 d also indicated that the symbiotic relationship between Microcystis aeruginosa and A20 had a mutually beneficial and competitive coexistence fluctuation regulation mechanism.

At the same time, changes in Fv/Fm indicated that the addition of A20 led to a fluctuating upward trend in the photochemical efficiency of Microcystis aeruginosa. Combined with the analysis of chlorophyll-a concentration, when the chlorophyll-a concentration of Microcystis aeruginosa increased rapidly (e.g., at 3 d), the Fv/Fm actually decreased to 0.23. This indicated that the coexistence of A20 led to a temporary imbalance of “high chlorophyll and low photochemical efficiency” in the growth process of Microcystis aeruginosa. The rapid increase of chlorophyll in the short term might result in insufficient assembly efficiency of the PSII photosystem reaction center, changes in the spatial conformation of phycobilisomes, weakening of light energy transfer efficiency, congestion of the electron transport chain, disorder of thylakoid membrane structure, and mismatch between light energy capture and electron transfer, thereby causing a decrease in Fv/Fm [39]. Afterward, there was a rapid decrease in chlorophyll-a concentration and a rapid recovery of photochemical efficiency (Fv/Fm increased to 0.35 at 5 d), demonstrating the stress adaptation strategy of Microcystis aeruginosa: shifting from “quantity growth” to “quality maintenance,” optimizing light energy utilization through energy redistribution, reducing chlorophyll synthesis to decrease light capture, and concentrating resources to maintain the efficiency of existing photosynthetic apparatus. Furthermore, the recovery of chlorophyll and the stable fluctuation of photochemical efficiency in the later stage of the co-culture system indicated that Microcystis aeruginosa and A20 gradually entered into environmentally adaptive coexistence through a staged symbiotic strategy adjustment.

3.3. Effect of A20 on the Metabolic Release Capacity of Microcystis aeruginosa

The changes in organic content released by the co-culture system of Microcystis aeruginosa and A20 are shown in Figure 6. The trends of changes in DOC and UV₂₅₄ were basically the same, both showing a gradual increase in organic release over time, with UV₂₅₄ rapidly increasing to 0.2983 cm⁻¹, and DOC rapidly increasing to 69.85 mg/L. Moreover, the growth rate of the co-culture system was significantly faster than that of the control group cultured with Microcystis aeruginosa alone or A20 alone (the difference in UV₂₅₄ was significant at p < 0.01, and the difference in DOC was significant at p < 0.001). This showed that the addition of A20 significantly promoted the release of extracellular organic matter from Microcystis aeruginosa. Based on the previous analysis, Microcystis aeruginosa capsule aggregates appeared in the co-culture system, and after aggregation, the cells of Microcystis aeruginosa still maintained their survival form and good photochemical efficiency, indicating that the organic matter release capacity of Microcystis aeruginosa was enhanced after forming aggregates. This might be a means for Microcystis aeruginosa to resist bacterial invasion. Research has shown that algal cells can respond by increasing the production of extracellular organic polymers to resist external stimuli [40,41,42,43], and that these extracellular polymeric substances have significant adhesive and film-forming ability, which are the direct material basis for cell aggregation and the formation of alginate clusters [3,44]. Therefore, we hypothesized that Microcystis aeruginosa triggered a cooperative defense mechanism when faced with invasion from other bacterial species or changes in the microenvironment. It physically reduced its contact area with bacteria by producing more viscous extracellular organic matters to form cell aggregates and adjusted its physiological state by utilizing the released substances and other nutrients (such as siderophore, vitamin B12, etc.) produced by bacteria A20 [45,46,47,48], transitioning from “high-speed quantity reproduction” to “survival quality maintenance”. In this state, the normal metabolic homeostasis of Microcystis aeruginosa cells might also be disrupted, leading to an imbalance in cell osmotic pressure regulation and changes in some cell membrane permeability, resulting in further expansion and release of metabolic leaks from surviving but stressed cells and further increasing their stress response to the environment. Of course, the increase in organic matter content was also related to the secretion and release of bacteria added to the system. Overall, the symbiotic system of Microcystis aeruginosa and A20 could significantly increase the organic matter content in its environmental water body.

To further investigate the structural components of extracellular organic substances secreted during the growth and reproduction of Microcystis aeruginosa under the influence of A20, three-dimensional fluorescence spectroscopy was employed to analyze the fluorescent organic components in the systems, as shown in Figure 7. It was observed that compared to the single culture system of Microcystis aeruginosa or A20, the fluorescence spectrum of the co-cultured system showed a significant enhancement of the fluorescence peak signals in region D (tryptophan-like proteins and soluble microbial metabolites), region C (fulvic acid-like substances), and region E (humic acid-like substances), indicating that the addition of A20 promoted the release of various substance components in the system environment. In terms of signal intensity, substances in the D, C, and E regions increased at the same frequency at 0–5 d, while the signal peaks of substances in the C and E regions showed a more significant increase at 5–9 d, with the most significant increase in the release of small molecule fulvic acid-like substances in the C region. Research [32] has shown that Priestia megaterium is heterotrophic bacteria, which can degrade and convert large organic molecules in their living environment into small organic molecules. There was also a study showing that some heterotrophic phycospheric bacteria can degrade inert organic phosphorus compounds, providing a bioavailable phosphorus source for Microcystis aeruginosa [34]. Therefore, we speculated that in the coexistence system with Microcystis aeruginosa and A20, the organic matters released by Microcystis aeruginosa provided the necessary carbon sources for Priestia megaterium A20, and during the degradation and utilization of these organic substances, A20 might assist Microcystis aeruginosa in obtaining more beneficial P nutrients or other nutrient substrates for its survival. Additionally, A20 accelerated the chemical transformation and re-polymerization of some organic matter in the system, promoting the accumulation of more fulvic-like substances or their precursors. The accumulation of small molecule organic matter signal peaks in region C were highly likely related to the utilization and degradation of metabolites released by autotrophic Microcystis aeruginosa through heterotrophic Priestia megaterium A20. Overall, Priestia megaterium A20 not only promoted the aggregation behavior of Microcystis aeruginosa but also stimulated or assisted in the production of more humic acid, furanic acid, and protein-like substances by Microcystis aeruginosa, which were continuously released into the aquatic environment, resulting in a severe overload of organic matter in the environment.

4. Conclusions

Priestia megaterium A20 could promote obvious aggregative behavior of Microcystis aeruginosa, causing them to form spherical sac-like aggregates with particle sizes exceeding 180 μm, surrounded by biofilm layers with clear boundaries.

Priestia megaterium A20 and Microcystis aeruginosa gradually entered into environmentally adaptive coexistence growth through a staged symbiotic strategy adjustment. The co-growth of Priestia megaterium A20 and Microcystis aeruginosa led to a temporary imbalance of high chlorophyll and low photochemical efficiency of Microcystis aeruginosa. In response, Microcystis aeruginosa showed corresponding stress adaptation strategies, shifting from quantity growth to quality maintenance. They optimized light energy utilization through energy redistribution, reduced chlorophyll synthesis from 1455.24 to 998.41 μg/L to minimize light capture, and concentrated resources to maintain the efficiency of the existing photosynthetic apparatus. These processes resulted in chlorophyll and photochemical efficiency tending toward stability in fluctuating changes.

The coexistence of Priestia megaterium A20 stimulated or assisted Microcystis aeruginosa in producing more organic metabolites, with DOC increasing to 69.85 mg/L and UV₂₅₄ increasing to 0.2983 cm⁻¹. The organic contents were significantly higher than those of Microcystis aeruginosa or Priestia megaterium A20 systems, with significant differences at p < 0.001 for DOC and p < 0.01 for UV₂₅₄. This may lead to a severe overload of organic content in the environmental water bodies. The addition of Priestia megaterium A20 significantly enhanced the fluorescence peak signals of tryptophan-like proteins, soluble microbial metabolites, furfuric acid-like substances, and humic acid-like substances of Microcystis aeruginosa. Among them, the increase and release of small molecule fulvic acid-like substances were the most obvious, which may be related to the utilization and degradation of autotrophic Microcystis aeruginosa metabolites by heterotrophic Priestia megaterium A20. During the degradation and utilization of organic metabolites from Microcystis aeruginosa, A20 bacteria accelerated the chemical transformation and re-polymerization of some organic compounds, promoting the accumulation of more fulvic acid-like substances or their precursors.