© The Author(s) 2024. Published by Science Press and EDP Sciences.

INTRODUCTION

Due to their wide use in industries, agriculture, and daily used products, huge amounts of plastics are produced globally and their quantity is expected to rise to 33 billion tons by 2050 [1]. However, after their usage, high portion of plastics are not properly recycled, leading to the release of large amounts of plastics into the terrestrial, freshwater, and marine ecosystems [1,2]. By serving as habitats for the colonization of microorganisms such as bacteria and viruses, plastic debris in natural environments would form plastisphere, posing risks to ecosystem and human health [3–6]. The initial adhesion performance of microorganisms to plastics is crucial for the formation of plastisphere. Many studies thus have been devoting to understanding the factors controlling the initial microbial adhesion to plastics. Plastic properties (i.e. hydrophilicity/hydrophobicity, surface hardness, and roughness) [7–9], solution conditions (i.e. ionic strength, solution temperature, and copresence of humic acid) [10,11], and bacterial properties (i.e. bacterial size, surface charge, flagella, pili, and extracellular polymeric substances (EPS)) [10–12] have been shown to greatly affect initial bacterial adhesion to plastics.

Nutrient conditions for bacterial growth and survival vary greatly in natural environments with nutrient-rich in some places while nutrient-restricted in other places [13]. Bacteria grown/survived under different nutrient conditions (i.e. nutrient-rich condition, nutrient-restricted condition, or subjected to starvation) would differ in size, shape, zeta potentials, as well as the amounts, functional groups, and components of EPS [14–18]. Nutrient conditions thus significantly affected the adhesion performance of bacteria to solid surfaces. For instance, Mayton et al. [18] found that composition of EPS secreted by both E. coli O157:H7 and Salmonella Typhimurium grown under nutrient-restricted conditions greatly differed from those grown under nutrient-rich conditions. Both strains grown under these two different nutrient conditions thus exhibited different adhesion performance of bacteria onto spinach surfaces. By affecting the bacterial sizes and the hydrophobicity of EPS, starvation of bacteria also significantly impacted the bacterial attachment performance towards sand surfaces [14,15]. Previous studies showed that in order to maintain other more essential functions for survival, bacteria grown under nutrient-limited conditions or exposed to starvation process would lose their flagella [19,20]. As an important motility organelle, flagella could affect the moving trajectory of bacteria towards solid surfaces and impact their adhesion performance towards plastic/quartz sand surfaces [10,21]. The loss of flagella thus would alter the mobility of bacteria, changing the bacterial adhesion/attachment capability onto solid surfaces [10,21,22].

Clearly, via changing different properties of bacteria including their morphology (i.e. size, shape, and flagella) and physiochemical properties (i.e. the amounts, functional groups, and components of the secreted EPS), nutrient conditions would be highly likely to affect the initial bacterial adhesion to plastics in environment, impacting the formation of plastisphere. However, the effects of different nutrient conditions on bacterial adhesion capability towards plastics remain unclear. Moreover, the mechanisms controlling adhesion of bacteria to plastics under different nutrient conditions have not been well understood. Particularly, the respective contribution of different morphology and physiochemical properties of bacteria to the adhesion of bacteria grown/survived under different nutrient conditions onto plastics has not been systematically explored.

Herein, this research focused on investigating the effects and mechanisms of nutrient conditions especially the nutrient-rich and nutrient-restricted growth condition as well as the starvation condition on bacterial adhesion to plastics in both salt solution and actual river water. E. coli MG1655 and B. subtilis were used as the representative Gram-negative and Gram-positive bacteria, respectively. Six different types of commercial plastics (PP, PE, PVC, PU, PET, and PS) were employed as representative plastics widely present in natural environment. Via deep investigation of the respective contribution of bacterial shape, motility, flagella, and EPS via analyzing its overall amount, functional groups, proteins, and polysaccharides (major components of EPS) together with their corresponding secondary structure, the mechanisms of nutrient conditions affecting bacterial adhesion to plastics were systemically determined. The results of this study would be beneficial for understanding the formation of plastisphere in natural environments with different nutrient conditions.

METHOD

Plastics prepared and characterization

Polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), polyurethane (PU), polyethylene terephthalate (PET), and polystyrene (PS), the most consumption plastic types in the world, were used as representative plastics in aqueous environment [10]. Plastic sheets with 2 cm length, 2 cm width, and 0.5 cm of height were used. SEM images, FTIR spectra, zeta potentials, and contact angles of plastics were provided in Figs S1 and S2, and Tables S1 and S2. The purchase information and washing method of plastics were reported in previous study [10] and also given in Supplementary Information.

Bacteria adhesion experiment

Batch experiments were employed to determine the initial adhesion performance of E. coli MG1655/B. subtilis grown either in nutrient-rich medium (LB) or nutrient-restricted medium (M9), or those being subjected to starvation of 2 and 7 days (25 °C and 200 r/min) to plastics in 25 mM NaCl solution (prepared by dissolving certain amount of NaCl into Milli-Q water). Additional batch experiments were conducted with using actual river water collected from Xiaojia River (Beijing, China). Detailed information about bacterial cultivation protocol, bacterial suspension preparation, and river water pretreatment protocols was provided in Supplementary Information. The characteristics of river water were given in Table S3. For a typical bacterial adhension batch experiment, 20 mL of bacterial suspensions (10⁸ cells/mL ± 10%) were mixed with plastics in 25 mM NaCl solution/river water for 4 h (at 25 °C and 200 r/min). After that, each plastic sheet was immersed in sterile phosphate buffer solution for three times to remove bacteria unabsorbed to plastic surfaces. The plastics were then transferred into centrifuge tubes with 5 mL NaOH solution (10 mM), which was shaken for 1 min using a vortex mixer to separate bacteria attached to plastic surfaces. The concentration of bacteria was detected via using a counting chamber (Marienfeld, Germany) with an inverted fluorescent microscope (Nikon, Japan). The adhesion density of bacteria to plastics was calculated by the total number of adhesive bacteria divided by the surface area of the plastic sheet. Moreover, to figure out the roles of flagella and EPS on bacterial adhesion, adhesion experiments of bacteria without flagella (by using E. coli MG1655 ΔfliC with the removal of flagellar genes) or with the removal of EPS (achieved by using extraction method of cation exchange resin) [23] were conducted. Zeta potentials, contact angles, and thermodynamic parameters of bacteria were given in Tables S4–S6.

Bacterial motility, movement speeds, hydrophobicity and viability assay

Bacterial motility was analyzed using semi-solid medium method [21]. Specifically, 2 μL of bacteria suspension (10⁸ cell/mL ± 10%) was injected into the center of culture dishes (diameter 95 mm) containing semi-solid mediums (consisting of culture medium for bacterial growth and additional 0.3% agar [wt/vol]), which was incubated at 37 °C for 24 h. Each experiment was replicated at least five times. The diameter of the coverage circle of bacteria represented bacterial motilities. Bacterial movement speeds were determined using ImageJ software (http://rsb.info.nih.gov) with video footage taken by an inverted fluorescent microscope (Nikon, Japan). Moreover, the hydrophobicity of bacteria was determined by microbial adherence to hydrocarbons (MATH) method. The viability of bacteria grown/survived under different nutrient conditions was also determined by using the Live/Dead BacLightTM bacterial viability kit (L7012, 107 Molecular Probes, Eugene, OR). Detailed measurement protocols of bacterial movement speeds, hydrophobicity, and viability were provided in previous studies [10,21] and also in Supplementary Information.

Extracellular polymeric substances preparation and characterization

EPS of bacteria either grown in nutrient-rich medium (LB) or nutrient-restricted medium (M9), or being subjected to starvation process were extracted using cation exchange resin (CER) [23]. Detailed information regarding the extraction and analysis of EPS was provided in Supplementary Information. The viscosity, types of function groups, and contents of hydrophobic functional groups of EPS were analyzed with details in Supplementary Information. BCA assay and Phenol-Sulfuric Acid method were used to determine the concentrations of proteins and polysaccharides in EPS, respectively [22]. Proteomics was used to characterize the difference in the expressed proteins among EPS from bacteria without/with starvation by using liquid chromatography with tandem mass spectrometry (LC-MS/MS, Hui Jun Biological Technology Company, Guangzhou, China). Amino acid compositions and secondary structure of proteins were analyzed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS, Majorbio Company, Shanghai, China) and FTIR spectra, respectively. Moreover, monosaccharide composition of exopolysaccharide in EPS was determined by ion chromatography (Kairui Company, Beijing, China).

RESULTS AND DISCUSSION

Effects of growth and starvation conditions on bacterial adhesion to plastics

The effects of growth and starvation conditions on bacterial adhesion to different types of plastic were investigated in 25 mM NaCl solution (to simulate environment-relevant solution condition). Regardless of plastic types, the adhesion density of both E. coli and B. subtilis grown in nutrient-rich conditions (LB) to all six plastics was higher than those grown in nutrient-restricted conditions (M9) under 25 mM NaCl solution condition (Figs 1 and 2). The results indicated that regardless of plastic types, bacteria grown in nutrient-rich conditions (LB) exhibited higher adhesion capability towards plastics than those grown in nutrient-restricted conditions (M9). Previous research has also reported that E. coli and Salmonella Typhimurium cultivated in LB and M9 media displayed different adhesion tendencies towards spinach surfaces [18].

Figure 1 Adhesive density of E. coli MG1655 grown in nutrient-rich conditions (LB) and nutrient-restricted conditions (M9) without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in 25 mM NaCl solution. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

Figure 2 Adhesive density of B. subtilis grown on LB and M9 culture medias without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in 25 mM NaCl solution. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

After 2 and 7 days of starvation, both E. coli and B. subtilis regardless grown in LB or M9 medium had lower adhesion density to six plastics compared to those without starvation under 25 mM NaCl solution condition (Figs 1 and 2). Furthermore, the adhesion density of both E. coli and B. subtilis starved for 7 days to all six types of plastics was lower than those starved for 2 days under the same solution condition. Clearly, regardless of growth condition, increasing duration of starvation could decrease bacteria adhesion to all six plastic surfaces. Previous studies also showed that bacteria with starvation (either short-term or long-term) displayed lower attach performance onto quartz sand surfaces than those without starvation [14,15,17].

Mechanisms for the altered bacterial adhesion to plastics with starvation

Altered viability, size, and zeta potential of bacteria

Viability tests (Table S7 and Fig. S3) showed that regardless of grown in nutrient-rich condition (LB) or in nutrient-restricted condition (M9), more than 99% of bacteria were viable. Moreover, even after being starved for 2 or 7 days, the viability of bacteria was over 98%. The observations indicated that nutrient conditions concerned in present study negligibly affected bacterial viability, which was consistent with previous study [24]. Clearly, the negligible change of cell viability would not cause the different bacterial adhesion performance towards plastics under different nutrient conditions.

The sizes and zeta potentials of bacteria have been shown to impact cell adhesion capability onto quartz sand and plastic surfaces [10,25,26]. Interestingly, bacteria grown in nutrient-rich condition (LB) and nutrient-restricted condition (M9) had similar sizes, while the zeta potentials of bacteria cultivated in LB medium were lower compared to those in M9 medium (Table S4). This suggests that the electrostatic repulsion between bacteria grown in LB medium and plastics was higher than that of bacteria grown in M9 culture medium (Fig. S4), which yet contradicted the observation of higher adhesion of bacteria grown in LB on plastic surfaces than those grown in M9. Moreover, both sizes and zeta potentials of E. coli and B. subtilis decreased with increasing starvation process from 2 to 7 days (Table S4). Regardless of growth conditions, E. coli and B. subtilis with starvation had more repulsion with plastics compared to those without starvation, which theoretically explained lower adhesion capability of starved bacteria on plastics than those without starvation. 2 days of starvation induced larger repulsion between bacteria and plastics than starvation for 7 days (Fig. S4a and b), which yet did not align with the decreased bacterial adhesion to plastics with increasing starvation duration. This suggested that starvation had more complex influence on bacteria rather than the change of sizes and zeta potentials.

Altered shape and motility of bacteria

The colloidal shape has also been proven as an important factor affecting colloid adhesion onto solid surfaces [27]. Comparison of scanning electron microscope (SEM) images of E. coli MG1655 grown in M9 and LB media showed that cells grown under different nutrient conditons displayedsimilar rod-shape (Fig. 3a and b). This observation indicated that grown in different culture medium negligibly affected bacterial morphology. However, starvation induced the bacterial shape change from the rod-shape into spherical form (Fig. 3b–d). The shape of bacteria changed from rod to coccus after starvation has also been documented [13,28,29]. Due to surface heterogeneity, rod-shaped particles could attach more easily onto quartz and other surfaces relative to spherical particles [27]. The transformation of bacteria shape from rod to sphere under starvation condition might contribute to the decreased adhesion of bacteria with starvation (and with increasing starvation duration) to plastic surfaces.

Figure 3 SEM images of E. coli MG1655 grown in nutrient-restricted conditions (M9) without starvation (a) and nutrient-rich conditions (LB) with starvation of 0 (b), 2 (c), and 7 (d) days in 25 mM NaCl solution condition; migration diameter (e) and movement speeds (f) of E. coli MG1655 grown in nutrient-rich conditions (LB) and nutrient-restricted conditions (M9) without and with the starvation for 2 and 7 days in 25 mM NaCl solution. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

Through changing collision probability with the surrounding media, bacterial motility would influence adhesion performance of bacteria towards plastic and quartz sand surfaces. Migration diameters (representing the capability of bacteria to spread out from the central inoculation site) of E. coli MG1655 grown in LB were similar as those grown in M9 medium (Fig. 3e, P > 0.05). Consistently, the swimming speeds of E. coli MG1655 grown in LB and M9 media were also comparable (Fig. 3f, P > 0.05). These two mobility tests suggested that the less adhesion of bacteria grown in M9 to plastics than those grown in LB was not likely attributed to the bacterial motility. However, starvation reduced the migration diameters and swimming speeds of bacteria (Fig. 3e and f), which could result in inhibited bacterial adhesion to plastics after starvation. Note that no difference in migration diameters and swimming speeds was present between bacteria starvation of 2 and 7 days. The decreased adhesion of bacteria to plastics with increasing starvation period from 2 to 7 days indicated that other factors would also affect bacteria adhesion to plastics.

Loss flagella from bacteria

As important organelles of bacteria regulating bacterial swimming property, flagella could significantly influence the attachment performance of bacteria to quartz sand and plastic surfaces [10,30,31]. Owing to the lack of energy for assembling, bacteria would lose flagellar filaments when nutrients were limited [19,20]. We found that the amount of bacterial flagella was reduced or the loss of flagella occurred when bacteria were subjected to starvation process (as shown in Fig. 3b–d), which might impact bacterial adhesion capacity to plastics. To testify, adhesion experiments of E. coli MG1655 ΔfliC (without flagellar gene) without and with starvation of 2 and 7 days to six types of plastics thus were conducted. Comparing with E. coli MG1655 containing flagella without starvation, E. coli MG1655 ΔfliC without flagella exhibited lower adhesion to plastic surfaces (Fig. 4), which suggested that flagella could promote bacterial adhesion to plastics. Owing to the incomplete loss of flagella from bacteria after 2 days of starvation, the adhesion density of E. coli MG1655 with flagella after 2 days of starvation was still higher than that of bacteria without flagella with 2 days-starvation. Due to the compete loss of bacterial flagella after 7 days of starvation (Fig. 3d), the adhesion densities of E. coli MG1655 were comparable with those of E. coli MG1655 ΔfliC. The loss of flagella during starvation had some contribution to the inhibited bacterial adhesion to plastics after starvation. However, similar as strain with flagella, the adhesion density of E. coli MG1655 ΔfliC to plastics also decreased with increasing starvation duration (Fig. 4, P > 0.05). This observation suggested that besides loss of flagella, other factors would also contribute to the reduced adhesion of bacteria to plastic after starvation.

Figure 4 Adhesive density of E. coli MG1655 (with flagella) and E. coli MG1655 ΔfliC (without flagella) grown on LB culture medias without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics under 25 mM NaCl solution conditions. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

Altered properties of extracellular polymeric substances of bacteria

Via affecting bacterial hydrophobicity, extracellular polymeric substances (EPS) could impact bacterial adhesion capability to quartz sand surfaces [32–34]. Relative expression levels of most genes of exopolysaccharides biosynthesis pathway in E. coli MG1655 decreased with the increase of starvation duration from 0 to 7 days (Fig. 5a), which indicated that starvation process might influence the secretion of EPS. Regardless of growth media, the quantities of both protein (PN) and polysaccharide (PS) contents of EPS decreased with increasing starvation duration (Fig. 5b), indicating the alteration in surface properties of bacteria with increasing starvation duration. The increased ratio of PN and PS of EPS indicated the increase of bacterial hydrophobicity [14,15,32]. The ratios of PN/PS of EPS extracted from E.coli MG1655 grown in LB (nutrient-rich condition) were significantly higher than those in M9 (nutrient-restricted condition) (Fig. 5c), which indicated that bacteria grown in LB culture medium were more hydrophobic than those grown in M9 culture medium. MATH tests also confirmed the larger hydrophobicity of bacteria grown in LB than those grown in M9 (Fig. 5d). Note that bacteria with relatively hydrophobic surfaces could more easily attach onto solid surfaces (e.g. quartz sand and PDMS surfaces) [32,35]. The larger hydrophobicity of bacteria grown in LB thus might have contribution to greater adhesion density of bacteria grown in LB to all six plastics than that of bacteria grown in M9.

Figure 5 Relative expression levels of six genes of exopolysaccharides biosynthesis pathway in E. coli MG1655 without and with the starvation for 2 and 7 days (a); the protein (blue columns) and polysaccharide (yellow columns) contents of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days (b); the ratio of protein (PN) and polysaccharide (PS) of EPS from E. coli MG1655 without and with the starvation for 2 and 7 days (c); surface hydrophobicity of E.coli MG1655 without and with the starvation for 2 and 7 days using MATH text (d); deposition efficiencies of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days (e); variations in dissipation as a function of frequency shifts of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days (f); high-resolution C 1s XPS spectra of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

Regardless of their growth conditions (originally grown either in LB or M9 culture medium), the ratios of PN/PS of EPS from E.coli MG1655 without starvation were higher than those from bacteria with starvation for 2 and 7 days. The observation suggested that bacteria without starvation had larger hydrophobicity than those with starvation, which was consistent with the results of MATH tests (Fig. 5d). The larger hydrophobicity of bacteria without starvation than that with starvation agreed with the greater adhesion density of bacteria without starvation to plastics relative to that with starvation. The PN/PS ratios of EPS from bacteria grown in LB medium with 2 days of starvation were comparable with those with 7 days of starvation (P > 0.05), which indicated that starvation duration of 2 and 7 days might induce comparable hydrophobicity of bacteria. MATH tests yet showed that increasing starvation duration from 2 to 7 days decreased the hydrophobicity of bacteria originally grown in LB, which was consistent with the reduced bacterial adhesion density to plastics with increasing starvation duration. Both PN/PS ratios versus starvation duration and MATH tests showed that the hydrophobicity of bacteria grown in M9 decreased with increasing starvation duration from 2 to 7 days, which agreed with lower bacterial adhesion density to plastics with longer starvation duration. The above observations indicated that the alteration in hydrophobicity of bacteria in different growth/starvation conditions overall was consistent with the changed adhesion density of bacteria to plastics obtained under different conditions.

The deposition rates (k_f) of EPS extracted from bacteria without and with starvation on polypropylene (PP) coated sensors in QCM-D experiments followed the order of k_f without starvation > k_f with 2-day starvation > k_f with 7-day starvation (Fig. 5e), which was consistent with the above results of bacterial adhesion density to plastics. Moreover, starvation and increasing starvation duration would induce softer and looser EPS layer on the PP-coated sensor (Fig. 5f). Clearly, the change of bacterial EPS hydrophobicity affected bacterial adhesion behaviors to plastic surfaces.

Removal of EPS from bacteria surfaces greatly inhibited the adhesion density E. coli MG1655 to six plastic surfaces (Fig. 6), which indicated that EPS had contribution to bacterial adhesion to plastics. Interestingly, when EPS was removed from bacteria, the adhesion density of bacteria without and with starvation of both 2 and 7 days to plastics was comparable, which was true for all six plastics. Similarly, removal of EPS from E. coli MG1655 ΔfliC without flagella led to the comparable adhesion density of bacteria without and with starvation of both 2 and 7 days to all six plastics. These results further confirmed the great contribution of EPS to the adhesion performance of bacteria to plastic surfaces. The comparable of adhesion density to plastics observed for E. coli MG1655 with EPS removal from surfaces and E. coli MG1655 ΔfliC lacking flagella yet with EPS removal from surfaces indicated the bacterial flagella loss during the EPS removal process due to long-term and high-intensity shear force and low temperature [21,22].

Figure 6 Adhesive density of E. coli MG1655 and E. coli MG1655 ΔfliC grown on LB culture media without and with the starvation for 2 and 7 days without EPS on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in25 mM NaCl solution conditions. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

Altered properties of protein

EPS contained functional groups such as O–H, C–H, C=C, N–H, and C–O (Fig. S5). However, the proportion of hydrophobic functional groups (C–(C/H)) in EPS followed the order of 48.2% without starvation > 47.8% for 2 days-starvation > 43.8% for 7 days-starvation (Fig. 5g–l; Table S8). This indicated that starvation process would induce the decrease of hydrophobic components in EPS, affecting bacterial adhesion to plastic surfaces. As one major component of EPS, proteins play a crucial role in maintaining the structural integrity and stability of EPS. The proteomics analysis showed that the quantities of proteins in EPS decreased from 1584 in E. coli MG1655 EPS without starvation to 1556 in bacterial EPS with 2-day starvation and further to 1530 in bacterial EPS with 7 days of starvation (Fig. 7a). It is worth pointing out that 1381 proteins were present in all EPS extracted from bacteria either without or with starvation, while 63, 25, and 39 proteins were unique in EPS extracted from bacteria without starvation and with 2 and 7 days of starvation, respectively. The observation indicated that starvation process would change the amounts and the types of proteins present in the EPS, affecting the composition and properties of EPS.

Figure 7 Number of identified proteins of EPS from E. coli MG1655 without and with the starvation for 2 and 7 days (a); the number of upregulated and downregulated proteins in the EPS from E. coli MG1655 under different starvation durations, comparisons were made between 2 days vs. 0 day (b), 7 days vs. 0 day (c), and 7 days vs. 2 days (d) starvation; number of differential proteins assigned to Gene Ontology (GO) terms in comparisons of different groups (e).

Moreover, the number of downregulated proteins in the EPS became larger than the number of upregulated proteins in EPS extracted from bacteria after starvation and with longer starvation duration (Fig. 7b–d). This observation also confirmed that starvation process and increasing starvation duration decreased the quantities of proteins in EPS. The changed proteins in EPS extracted from E. coli MG1655 experiencing starvation process versus those without starvation were primarily involved in cellular processes, metabolic processes, binding, catalytic activity, and cellular anatomical entities (Fig. 7e). The ratio of hydrophilic amino acids/hydrophobic ones in EPS proteins extracted from bacteria without starvation was 1.7, which increased to 4.5 after starvation bacteria for 2 and 7 days (Fig. 8a and b, Table S9). This indicated that starvation process affected the composition of amino acids in proteins with the decreased amount of hydrophobic amino acids in EPS proteins from bacteria with starvation. However, further increasing starvation duration to 7 days did not lead to the change of the ratio of hydrophilic amino acids/hydrophobic ones in EPS proteins. Obviously, the decreased hydrophobicity of bacterial EPS after starvation could be attributed to the decreased amount of hydrophobic amino acids in EPS proteins. The further decreased EPS hydrophobicity with increasing starvation duration yet could not be explained by the unchanged amounts of hydrophobic amino acids in EPS proteins. Other mechanisms would drive to the decreased hydrophobicity of EPS with prolonged starvation duration.

Figure 8 Concentrations of hydrophilic amino acids (a) and hydrophobic amino acids (b) in EPS protein from E. coli MG1655 without and with the starvation for 2 and 7 days; Relative contents of each secondary structure in EPS proteins from E. coli MG1655 without and with the starvation for 2 and 7 days (c); Relative contents of monosaccharides in EPS polysaccharides from E. coli MG1655 without and with the starvation for 2 and 7 days (d).

The ratios of α-helix/(β-sheet + random coil) in EPS proteins extracted from bacteria without starvation and those from bacteria starved for 2 and 7 days were 0.73, 0.56, and 0.55, respectively (Fig. 8c, Table S10). This suggested that after starvation, the proportion of α-helical structures in EPS proteins decreased and the overall conformation of proteins became looser, which increased the exposure probability of internal hydrophobic functional groups in proteins and thus increased the hydrophobicity of proteins. Clearly, the changed secondary structure of proteins during the starvation process could not explain the overall decreased hydrophobicity of EPS as well as the decreased adhesion of bacteria to plastics after starvation process.

Altered properties of polysaccharides

Besides proteins, polysaccharides are another important component of bacterial EPS [36–38]. Note that galacturonic acid (Gal-UA), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), and mannuronic acid (Man-UA) contained highly polar carboxyl groups, these monosaccharides thus were more hydrophilic and less hydrophobic than other monosaccharides [34,39]. Although the ratio of hydrophilic monosaccharides/hydrophobic monosaccharides in EPS extracted from 2 days starved bacteria was comparable as those from bacteria without starvation, increasing the starvation duration to 7 days yet greatly increased the corresponding ratio (Fig. 8d and Table S11). This observation indicated that short-term starvation (2 days) did not affect the proportion of hydrophobic monosaccharides, while increasing starvation duration to 7 days yet decreased the proportion of hydrophobic monosaccharides in EPS. The less hydrophobicity of monosaccharides in EPS after 7 days-starvation was consistent with the decreased overall hydrophobicity of EPS with increasing starvation from 2 to 7 days.

Based on the above analysis, we can conclude that the decreased hydrophobicity of EPS from bacteria without starvation to bacteria with 2 days-starvation could be mainly attributed to the decreased hydrophobicity of proteins (through altering amino acids composition), while the decreased hydrophobicity of EPS from bacteria with increasing starvation from 2 to 7 days was caused by the decreased hydrophobicity of monosaccharides in EPS. Clearly, the change of proteins and monosaccharides in EPS at different starvation duration respectively contributed to the overall changed hydrophobicity of bacterial EPS at varied starvation process, affecting the adhesion performance of bacteria to plastics.

Bacterial adhesion performance in actual river water sample

To determine whether the decreased bacterial adhesion to plastics after starvation achieved in salt solution would also apply to actual water sample, the adhesion performance of both E. coli MG1655 and B. subtilis without and with starvation (2 and 7 days) to six plastics in real river water was then investigated. Due to the presence of organic matter (2.68 mg /L TOC) in river water (Table S10), the adhesion density of bacteria (both E. coli MG1655 and B. subtilis) to six plastics in river water was lower than those in salt solutions (Fig. 9 vs. Figs 1 and 2). Unlike those observed from long-term effects of organic matter [40,41], the initial (short-term) bacterial adhesion to plastics was slightly inhibited by the organic substances existing in river water via increasing the repulsion between bacteria and plastic surface [10]. Similar to the results obtained in NaCl solutions (Figs 1 and 2), starvation process decreased the adhesion density of two bacterial strains to all six plastics in river water (Fig. 9). Moreover, increasing starvation duration from 2 to 7 days also could further decrease the adhesion of two strains to six plastics in river water. Clearly, the contribution of starvation to bacterial adhesion to plastics also held true in actual water sample.

Figure 9 Adhesive density of E. coli MG1655 and B. subtilis grown on LB culture medias without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in river water. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

CONCLUSION

This study investigated the impact of growth and starvation conditions on the adhesion performance of E. coli MG155 (Gram-negative bacteria) and B. subtilis (Gram-positive bacteria) to six types of plastics (PP, PE, PVC, PU, PET, and PS) in both salt solution and actual river water. We found that regardless of plastic types, bacteria grown in nutrient-rich culture medium (LB) exhibited better adhesion performance than those grown in nutrient-restricted culture medium (M9). Moreover, regardless of plastic types and growth conditions, starvation process could decrease bacteria adhesion to plastic surfaces in both in NaCl solutions and real river water. Via deep investigation the potential contributions from the bacteria shape, the migration and swimming property of bacteria, flagella, the overall quantity, functional groups, and composition of EPS, together with the secondary structure of proteins and polysaccharides (two main components of EPS), the mechanisms driving to the different adhesion performance of bacteria grown in different culture media and experienced starvation process to plastics were revealed. The larger hydrophobicity of bacteria contributed to the greater adhesion density of bacteria grown in LB to all six plastics than that of bacteria grown in M9. The transformation of bacteria shape from rod to sphere, the reduced the migration diameters and swimming speeds, the loss of flagella, especially the decreased hydrophobicity of EPS (via changing proteins and monosaccharides in EPS respectively at different starvation duration) all had contribution to the decreased adhesion of bacteria with starvation to plastics. Clearly, comparing with nutrient-restricted environment, bacteria are more easily to colonize onto plastics in nutrient-rich natural environments. After experiencing either short-term or long-term starvation process, the potential for bacteria to colonize onto plastics would greatly decrease. Obviously, the nutrient conditions would greatly influence the formation of plastisphere in natural environments, affecting the environmental risks of plastics.

Funding

This work was supported by the National Natural Science Foundation of China (42025706 and 42207424), and Science Foundation of China University of Petroleum-Beijing (2462023YJRC020).

Supplementary information

Supplementary file provided by the authors. Access here

The supporting information is available online at https://doi.org/10.1360/nso/20240002. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

References

All Figures

	Figure 1 Adhesive density of E. coli MG1655 grown in nutrient-rich conditions (LB) and nutrient-restricted conditions (M9) without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in 25 mM NaCl solution. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.
In the text

	Figure 2 Adhesive density of B. subtilis grown on LB and M9 culture medias without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in 25 mM NaCl solution. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.
In the text

Figure 3 SEM images of E. coli MG1655 grown in nutrient-restricted conditions (M9) without starvation (a) and nutrient-rich conditions (LB) with starvation of 0 (b), 2 (c), and 7 (d) days in 25 mM NaCl solution condition; migration diameter (e) and movement speeds (f) of E. coli MG1655 grown in nutrient-rich conditions (LB) and nutrient-restricted conditions (M9) without and with the starvation for 2 and 7 days in 25 mM NaCl solution. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

In the text

Figure 4 Adhesive density of E. coli MG1655 (with flagella) and E. coli MG1655 ΔfliC (without flagella) grown on LB culture medias without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics under 25 mM NaCl solution conditions. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

In the text

Figure 5 Relative expression levels of six genes of exopolysaccharides biosynthesis pathway in E. coli MG1655 without and with the starvation for 2 and 7 days (a); the protein (blue columns) and polysaccharide (yellow columns) contents of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days (b); the ratio of protein (PN) and polysaccharide (PS) of EPS from E. coli MG1655 without and with the starvation for 2 and 7 days (c); surface hydrophobicity of E.coli MG1655 without and with the starvation for 2 and 7 days using MATH text (d); deposition efficiencies of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days (e); variations in dissipation as a function of frequency shifts of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days (f); high-resolution C 1s XPS spectra of EPS from E.coli MG1655 without and with the starvation for 2 and 7 days. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.

In the text

	Figure 6 Adhesive density of E. coli MG1655 and E. coli MG1655 ΔfliC grown on LB culture media without and with the starvation for 2 and 7 days without EPS on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in25 mM NaCl solution conditions. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.
In the text

Figure 7 Number of identified proteins of EPS from E. coli MG1655 without and with the starvation for 2 and 7 days (a); the number of upregulated and downregulated proteins in the EPS from E. coli MG1655 under different starvation durations, comparisons were made between 2 days vs. 0 day (b), 7 days vs. 0 day (c), and 7 days vs. 2 days (d) starvation; number of differential proteins assigned to Gene Ontology (GO) terms in comparisons of different groups (e).

In the text

Figure 8 Concentrations of hydrophilic amino acids (a) and hydrophobic amino acids (b) in EPS protein from E. coli MG1655 without and with the starvation for 2 and 7 days; Relative contents of each secondary structure in EPS proteins from E. coli MG1655 without and with the starvation for 2 and 7 days (c); Relative contents of monosaccharides in EPS polysaccharides from E. coli MG1655 without and with the starvation for 2 and 7 days (d).

In the text

	Figure 9 Adhesive density of E. coli MG1655 and B. subtilis grown on LB culture medias without and with the starvation for 2 and 7 days on PP (a), PE (b), PVC (c), PU (d), PET (e), and PS (f) plastics in river water. Error bar indicates standard deviation of measurements (n ≥ 3). The significant differences are indicated with *P < 0.05, **P < 0.01.
In the text