Glycine betaine modulates extracellular polymeric substances to enhance microbial salinity tolerance

Abstract

High salinity inhibits microbial activity in the bioremediation of saline wastewater. To alleviate osmotic stress, glycine betaine (GB), an osmoprotectant, is added to enhance the secretion of extracellular polymeric substances (EPS). These EPS are pivotal in withstanding environmental stressors, yet the intricate interplay between GB supplementation and microbial responses through EPS modifications—encompassing composition, molecular architecture, and electrochemical features—remains elusive in hypersaline conditions. Here we show microbial strategies for salinity endurance by investigating the impact of GB on the dynamic alterations of EPS properties. Our findings reveal that GB supplementation at 3.5% salinity elevates the total EPS (T-EPS) content from 12.50 ± 0.05 to 24.58 ± 0.96 mg per g dry cell weight. The observed shift in zeta potential from −28.95 to −6.25 mV at 0% and 3.5% salinity, respectively, with GB treatment, indicates a reduction in electrostatic repulsion and compaction. Notably, the EPS protein secondary structure transition from β-sheet to α-helix, with GB addition, signifies a more compact protein configuration, less susceptible to salinity fluctuations. Electrochemical analyses, including cyclic voltammetry (CV) and differential pulse voltammetry (DPV), reveal GB's role in promoting the release of exogenous electron shuttles, such as flavins and c-type cytochromes (c-Cyts). The enhancement in DPV peak areas (Q_DPV) with GB addition implies an increase in available extracellular electron transfer sites. This investigation advances our comprehension of microbial adaptation mechanisms to salinity through EPS modifications facilitated by GB in saline habitats.

Graphical abstract

Highlights

• •Higher microbial viability was observed by betaine under high salinity conditions.
• •Betaine addition induced the contents of protein and humic acid by 0.56–2.68 times.
• •Betaine caused a transition of protein secondary structure from β-sheet to α-helix.
• •The sensitivity of protein structure to salinity was reduced by betaine.
• •Betaine maximally increased electron transfer sites by six orders of magnitude.

1. Introduction

In recent years, the development of some industrial production processes (e.g., pesticide, textile, and pharmaceutical production) has led to the discharge of large amounts of saline wastewater (>1% w/v NaCl) [1]. These industrial wastewaters always contain massive amounts of organic matter, anions, and cations [2], leading to the failure of biological treatment processes due to the inhibition of microbial activity and the toxicity of microbial cell structures. Numerous researchers have demonstrated the negative effects of salinity on microbes [3], and much effort has been applied to try to improve the negative effects of salt stress. Several of these studies have identified promoting the secretion of extracellular polymeric substances (EPS) as an effective approach to protecting microbes [4].

EPS are polymers consisting of polysaccharides (PS), proteins (PN), humic acids (HA), nucleic acids, etc., which are biosynthesized by several strains of microorganisms to resist adverse environmental change in the outside environment [5]. Specifically, hydrophobic groups in EPS significantly positively affect the interaction with hydrophobic and positively charged toxins to protect the microbes from toxic compounds [6]. However, these hydrophobic substances, such as proteins, are usually severely damaged by high salinity [7]. Additionally, EPS is a vital medium in the extracellular electron transfer (EET) process. It has been demonstrated that EPS stores large numbers of electrochemical active substances, such as flavins and c-type cytochromes (c-Cyts), which can mediate the EET process [8]. These electrochemical active substances related to EET are mainly embedded inside the PN in EPS as cofactors, and the main protein secondary structure that makes up these PN structures is α-helix [9]. These electron transfer properties of EPS pave the way for microbial degradation. Xu et al. [10] demonstrated that EPS could act as an electron donor and electron transfer medium, which could quickly reduce Cu(II) to Cu(I). The reduction rate accelerated when the EPS concentration was increased, and the redox-active substances in EPS (such as reducing sugars and c-type cytochromes) and O-/N groups (such as phenols and amides) were the cause of the copper reduction [10]. The elevated salinity levels may inhibit the secretion of redox substances and damage the secondary structures of proteins in EPS, thereby diminishing electron transfer efficiency. Hence, a feasible strategy for protecting protein structures and accelerating extracellular electron transfer in saline wastewater is needed.

Osmoprotectants, collectively known as compatible solutes, can protect microorganisms from harsh environmental conditions such as high osmotic pressures and low temperatures [11]. The accumulation of compatible solutes, such as glycine betaine (GB), helps cells cope with osmotic stress by maintaining physiologically adequate levels of expansion [12]. One notable function of compatible solutes is their ability to stabilize proteins and activate enzymes. Liu et al. [13] increased the rate of anaerobic ammonia oxidation by adding compatible solutes to high-salt wastewater. Their results showed that dehydrogenase activity significantly increased after the addition of compatible solutes. As an intracellular enzyme, dehydrogenase is closely related to the intracellular oxidative phosphorylation process and is an important indicator of microbial activity [14]. In addition, our previous study reported that the electron transfer system (ETS) increased by 41.5% following GB addition to high-salt nitrobenzene wastewater [15], suggesting GB's potential role in accelerating extracellular electron transfer in saline environments. However, there is still a lack of exploration of the complex and influential mechanisms related to GB addition on microbial salt-tolerance properties via EPS secretion under high-salt environments.

This study investigated the effect of GB addition on EPS secretion in high-salt environments. Bacillus, known for its widespread presence in soil, water, and air, was selected as the model microorganism due to its exceptional EPS production capabilities and its robust tolerance to salt stress. This choice allowed for a detailed investigation into the salt-tolerance mechanism via the properties of its EPS. The main objectives of this work were: (1) to assess the effects of GB addition on microbial viability characteristics and EPS components, (2) to clarify the effects of GB addition on the structural properties of EPS, (3) to evaluate electrochemical activities of EPS and explore the relationship with the microbial degradation efficiency, and (4) to investigate the probable role of GB in salt resistance through EPS secretion.

2. Materials and methods

2.1. Strain isolation and EPS extraction protocols

Bacillus pumilus isolates selected for this study were obtained from a lab-scale up-flow anaerobic sludge bed (UASB) reactor exposed to high-salt environments. These isolates were termed Bacillus pumilus NJUST51. The details of the isolates were presented in Text S1 (Supplementary Information). The extracted bacterium NJUST51 was inoculated into a sterilized Luria-Bertani (LB) medium. The incubation lasted for 48 h at a constant temperature of 30 °C and a shaking speed of 180 rpm. Subsequently, the biomass was harvested through centrifugation at 6000 rpm for 10 min and then washed thrice with sterilized MSM. The bacterial pellet was inoculated into serum bottles containing MSM with different salinities to obtain the optical density at 600 nm (OD600) of 0.75 ± 0.02. This incubation was carried out at 30 °C for 24 h. The salt concentration in minimal salt medium (MSM) was settled as 0%, 1%, and 3.5% for fresh, saline, and hypersaline environments. After 24 h of incubation, the cells were collected for further EPS extraction analysis, with the specific measurement methods detailed in Text S2.

2.2. Quantitative and qualitative analysis of EPS components

The volumetric concentration of PS in EPS extractants was determined by the improved phenol-sulfuric acid colorimetric method, using glucose as the standard [16]. Proteins and humic acids were measured by a modified Lowry method [17]. The above parameters were quantified with an Ultraviolet (UV) spectrophotometer (Thermo Fisher Scientific, CN), and the concentrations of PS, PN, and HA were normalized to the biomass in units of mg per g dry cells.

EPS composition was analyzed by fluorescence spectroscopy (F7000, Hitachi, Japan) by three-dimensional excitation-emission matrix (3D-EEM) scanning. The excitation wavelength (Ex) varied in 2 nm increments from 200 to 400 nm, and the corresponding emission wavelength (Em) varied in 2 nm increments from 200 to 550 nm. The scanning speed for all measurements was set to 30,000 nm min⁻¹, and the spectral recording of Milli-Q water was used as a blank. The software package Origin 2021 was used to process the 3D-EEM data.

2.3. Structural characterization methods of EPS

The total extracellular polymeric substances (T-EPS) extracted from biomass was freeze-dried for 48 h, and the dry matter was analyzed by the Fourier transform infrared (FTIR) spectrometer (NICOLETIS10, Thermo Fisher Scientific, CN) to obtain information on organic matter in the EPS. In detail, the scanning frequency was 400–4000 cm⁻¹, with each sample scanned 64 times, and the resolution was 4 cm⁻¹ [18].

The protein spectrum was further analyzed in the amide I region of 1700−1600 cm⁻¹ to extract information about the secondary structure of the protein, which is recognized as a reliable region for studying protein structural changes. Increasing the spectral resolution, the amide I region was deconvolved to separate the overlapping peaks. In the protocol used, the second derivative 4 of the original amide I spectrum was obtained using a nine-point Savitzky-Golay derivative function. A method based on Noda's processes two-dimensional correlation spectroscopy (2D-COS) containing synchronous and asynchronous graphs with equilibrium concentrations as external perturbations were used to determine the effects of salt and betaine on the structure of the EPS in the amide I [19]. Ultraviolet–visible (UV–Vis) spectra with wavelengths from 200 to 550 nm were scanned using a spectrophotometer (Agilent 8453). Zeta potential was measured using a zeta potentiometer (ZetaPALS, Brookhaven, America) at room temperature and a pH of 7.

2.4. Electrochemical measurements of EPS and calculation of electron transfer sites

An electrochemical activity test was performed to explore the electrochemically active substances in the EPS, according to Xiao et al. [8]. All electrochemical data were recorded using an Electrochemistry Workstation (VMP3, Biologic, France), which had a three-electrode chamber containing platinum electrodes as the working electrode and counter electrode and an Ag/AgCl reference electrode saturated with KCl. In all electrochemical measurements, the T-EPS extracted under different environmental exposures was freeze-dried and re-dissolved in a phosphoric acid buffer (50 mM, pH = 7) as an electrolyte solution. The cyclic voltammetry (CV) electrochemical parameters were a scan rate of 10 mV s⁻¹, an Ei of −0.6 V, and an Ef of 0.6 V. The parameters of the differential pulse voltammetry (DPV) were an Ei of −0.6 V, an Ev of 0.6 V, pulse heights (P_H) of 60 mV, pulse widths (P_W) of 200 ms, step heights (S_H) of 25 mV, and a step time (S_T) of 500 ms. The peak area of the DPV was calculated using Origin software 2022b. The cytochrome c content in the EPS was quantitatively measured using a cytochrome c enzyme-linked immunoassay kit (48T, Shanghai Jingkang Biological Engineering Co., Ltd.).

The total formal charge in the EPS layer (Q_EPS), which was generally associated with electron transfer sites [20], was calculated according to Xiao et al. [8]:

$$\begin{array}{c}{Q}_{\text{EPS}}=\frac{\mathrm{v}}{\mathrm{\pi }{\mathrm{k}}_0\Delta \mathrm{E}}{Q}_{\text{DPV}}\end{array}$$ (1)

where v (mV s⁻¹) is the scan rate, set to 15 mV s⁻¹, and k₀ (s⁻¹) is the standard electrochemical rate constant of electron transfer, and its value is estimated from the CVs using 0.026 s⁻¹. ΔE (V) is the potential increment, with a value of 0.006 V. The summary peak areas of DPVs (Q_DPV, C) were obtained by integrating the peak areas using Origin software 2022b.

2.5. Other analysis methods

The viability of microbes evaluated using the ratio of live to dead cells under various salinities with/without GB addition was determined by staining with a Calcein AM·PI kit (Shanghai Xin Yu Biotech Co., Ltd, China) and visualized using a Confocal Laser Scanning Microscopy (CLSM) (A1+SIM S, Nikon, China). Image J software calculates the ratio between live and dead cells. The OD600 of the culture medium was measured with a UV spectrophotometer (Thermo Fisher Scientific, CN).

3. Results and discussion

3.1. Microbial viability regulation with GB addition

The viability of microbes under different salinity levels with GB addition was investigated using CLSM imaging (Fig. 1a–f). Live and dead cells were presented as green and red fluorescence, respectively. The ratios of live and dead cells to total cells were calculated using the green and red fluorescence to quantify the distribution of live and dead cells (Fig. 1g). The intensity of the red fluorescence was obviously strengthened, with a remarkable decrease in the ratio of live cells from 54.88 ± 6.00% to 31.69 ± 5.92% after the salinity increased from 0% to 3.5%. This was consistent with the previous study of Sierra et al. [21], indicating a visible increase of the compromised cells on exposure to salinity. This was also reflected in the OD600 value (Fig. 1h), which was 0.76 ± 0.007 at 0% salinity and 0.63 ± 0.003 at 3.5% salinity at 24 h. It has been reported that salinity could cause an imbalance of intracellular and extracellular osmosis, causing severe dehydration and rupture of cells [22]. In contrast, it was observed that the ratio of live cells increased significantly with GB addition under a saline environment by 22.45 ± 0.98% and 15.10 ± 1.16% when salinity increased from 1% to 3.5%. The OD600 values were simultaneously increased by 0.05 ± 0.005 and 0.10 ± 0.02 when salinity increased from 1% to 3.5%, respectively. Previous studies have found that GB could be synthesized or absorbed by microbes, which played a significant role in maintaining cellular osmotic pressure and alleviating salt stress [23]. Therefore, it can be concluded that exogenously added GB could be absorbed to reduce the difference between intercellular and extracellular pressure and maintain the viability of cells in high-salt environments [12].

Fig. 1.

a–f, The viability of microbes under different salinity levels in the presence/absence of glycine betaine (GB) using the Confocal Laser Scanning Microscopy (CLSM) imaging: live (green)/dead (red) bacteria without GB addition (a, c, e) and with GB addition (b, d, f) at saline contents of 0% (a, b), 1% (c, d), and 3.5% (e, f), respectively. The + symbol represents the group with GB addition. g, The ratio of live and dead cells to total cells based on three random fluorescence intensities under various salinities. Horizontal coordinates represent different salinities, and the + symbol represents the group with GB addition. h, The temporal variation on the growth of Bacillus pumilus NJUST51 characterized by optical density at 600 nm (OD600).

3.2. EPS component variations with GB addition

EPS has been demonstrated to play a vital role in the resistance of microbes to harsh environments due to its special composition and structure [24]. This study measured the variation of EPS content to elucidate the effect of GB addition on EPS secretion when microbes were exposed to high-salinity wastewater. These measurements included profiles of the T-EPS, the loosely-bounded extracellular polymeric substances (LB-EPS), and the tightly-bounded extracellular polymeric substances (TB-EPS) (Fig. 2).

Fig. 2.

The variation of extracellular polymeric substances (EPS) content demonstrates the effect of GB addition on EPS secretion when microbes are exposed to high salinity wastewater: a, 0% salinity; b, 1% salinity; c, 3.5% salinity.

T-EPS components. It is worth noting that a general increase of T-EPS was observed from 8.31 ± 0.21 to 12.50 ± 0.05 mg per g dry cell when the salinity increased from 0% to 3.5%. The measures taken by microbes in response to osmotic pressure could include the increase of endogenous respiration accompanied by EPS secretion [25]. Therefore, it is reasonable to speculate that increasing EPS content with increasing salinity might be a stress response of microorganisms to adverse environments.

The content of T-EPS for the GB-addition group increased from 14.03 ± 1.08 mg per g dry cell (0% salinity) to 24.58 ± 0.96 mg per g dry cell (3.5% salinity), which was a remarkable increase compared to that of the group without GB addition. EPS secreted by microorganisms could act as a polymeric hydrogel matrix to form a rigid net for microbes and promote the sludge granulation process, which could protect microorganisms from harsh environments [26]. Hence, adding GB might have promoted the secretion of EPS, thus forming a thicker and tighter outer structure to protect cells.

LB-EPS components. The total content of the LB-EPS was the minimum between the LB-EPS and TB-EPS. The PN and HA contents slightly increased, with salinity increasing from 0% to 3.5%. Notably, PS content in LB-EPS did not increase significantly with salinity increasing in our study, contrary to what was observed by Zhang et al. [27]. This may be because LB-EPS is the outermost layer loosely wrapped around the microbial cell, where PS is preferentially used by the cell as a carbon source for metabolism against osmotic stress [28]. However, the LB-EPS content increased significantly with the addition of GB. Previous research suggests that the release of LB-EPS might be associated with extracellular redox activity, which was beneficial to the osmotic pressure balance between intracellular and extracellular [29]. This implies that the addition of GB could be an effective strategy to alleviate osmotic stress.

TB-EPS components. It could be seen that PN was the main component in the TB-EPS. When the salinity increased from 0% to 3.5%, the concentrations of PN in TB-EPS were increased from 3.86 ± 0.20 to 7.17 ± 0.14 mg per g dry cell, while the concentrations were remarkably increased from 7.27 ± 0.95 to 11.35 ± 0.93 mg per g dry cell with GB addition. Due to the numerous metal adsorption sites in PN-like aliphatic matters [30], which favored the relief of osmotic pressure, herein, microorganisms released osmotic pressure possibly through PN secretion [31]. In addition, PN containing a high level of amino acids could neutralize negative charges on the surface of microorganisms and promote bridging between cations and microorganisms [32], which could, in turn, promote microbial aggregation by reducing electrostatic repulsion between microbial cells and hence protecting microbes by reducing the contact between Na⁺ and microbial surfaces.

Compared to the group without GB addition, the HA content of the TB-EPS with GB addition increased by 0.73 ± 0.03 mg per g dry cell (0% salinity) and 2.69 ± 0.04 mg per g dry cell (3.5% salinity). As HA contains many metal-binding sites [28], GB could protect cells by promoting the secretion of EPS and further increasing the chance of intercepting Na⁺. Newly produced HA might also be utilized as a raw material for cell metabolism and further improve EPS secretion [26].

Noticeably, a low proportion of PS was also detected in TB-EPS. Although PS accounted for a relatively small proportion of the TB-EPS, PS secretion increased significantly after the addition of GB. A previous study demonstrated that PS could better protect cells by reducing Na⁺ stress, which might be another important regulatory mechanism promoted by GB in high-salt environments [33]. All these three components of TB-EPS (PN, HA, and PS) worked together to alleviate salt stress.

To further determine the protective mechanism influencing the reduction of Na⁺ stress with GB addition in EPS, the diffusion of GB and salt ions in the EPS was calculated using the formula presented in Text S3 (Supplementary Information). It was obvious from the results that the molarity of GB and salt ions diffusing into the EPS were inversely proportional. This suggested that the more GB accumulated in EPS, the fewer salt ions diffused into it, further relieving the osmotic pressure [34].

3D-EEM. The 3D-EEM EPS fluorescence spectra for various salinities were presented in Fig. S1. Two major peaks could be identified from the EEM fluorescence spectra. Peak A, identified at the excitation/emission wavelengths (Ex/Em) of 275–280/335–345 nm, was attributed to tryptophan [35]. Its fluorescence intensity increased with salt concentration, possibly resulting from the release of tryptophan substances in microbes as the increased salinity. Wang et al. [36] found that protein-like tryptophan was the primary substance released under low salinity (0.5%).

The fluorescence intensity of peak A became much stronger with GB addition, which might have resulted in a significant improvement in microbial stability due to the hydrophobic properties of the tryptophan [37]. However, there was no significant difference in location shift with GB addition, indicating little change in the conjugate bonds in the EPS components and the chain structure.

Another peak at Ex/Em values of 225–230/330–340 nm (peak B) was most likely associated with aromatic protein-like compounds [34], which had a similar trend to peak A. This was consistent with Huang et al. [38], who also found that GB could promote the release of protein-like substances. Aromatic protein-like EPS is important in facilitating the binding of microbial cells to form aggregates, which could have a significant protective effect against hyperosmotic environments [39].

Zeta potential. The zeta potential of the extracted EPS with GB addition under various salinities was measured to assess its surface electronegativity (Table S1). The extracted EPS was negatively charged with a zeta potential of −29.54 mV under 0% salinity due to the structural compound of ionizable functional groups such as –COOH and –NH₂ in the EPS [40]. When the salinity increased to 3.5%, the zeta potential was −14.16 mV, which was possibly related to the change in PN and PS contents. Additionally, Zeng et al. [41] have demonstrated that zeta potential could be neutralized by released metal ions and amino groups which intrinsically carried positive charges. Thus, the change in zeta potential may be due to the neutralization of negatively charged functional groups with Na⁺ or stimulation of positively charged PN in EPS. The zeta potential in the group with GB addition was −28.95 and −6.25 mV at 0% and 3.5% salinity, respectively. A decrease in negative charges suggested a weakened electrostatic repulsion and compression effect [42], which promoted the formation of a tighter EPS protective layer. Additionally, GB, as a zwitterion, consists of quaternary ammonium salt type cation and carboxyl anion, which could be attracted by the negative surface charges of EPS [43]. Thus, it might be speculated that Na⁺ contributed by the salinity and positively charged PN neutralized with negatively charged functional groups in EPS, while a weakened electrostatic repulsion and compression effect was observed with GB addition to promote a tighter EPS protective layer formation.

3.3. Changes of EPS structural properties with GB addition

3.3.1. Molecular structure from UV–Vis spectra

UV–Vis absorption spectroscopy can evaluate the aromatic structure and unsaturated fat chains with various functional groups in EPS [44]. This study observed two obvious absorption bands at 210 and 260−265 nm (Fig. 3a). The band at 210 nm is commonly assigned to the n→π∗ electron transition of amide bonds in proteins [45]. The band at 260–265 nm can be attributed to π→π∗ electron transitions in the C O and C C structures of aromatic compounds, such as tryptophan, tyrosine, phenylalanine, and humic compounds [6].

Fig. 3.

a, Comparison of ultraviolet–visible (UV–Vis) absorption spectra. The figure on the right shows the localized detail of the wavelength around 250 nm b, Fourier transform infrared (FTIR) spectra of EPS at different salinities with GB addition.

In detail, the wavelength, as well as intensity of the peaks at 210 nm peaks, remained stable when exposed to salinity, indicating that there was almost no significant attack of salinity on the amide bond in the polypeptide chain [45]. Nonetheless, there was a pronounced red shift in the peak at 260−265 nm as the salinity increased from 0% to 3.5%, indicating a change in the molecular structure for the increased energy requirement for electron transition [46]. Normally, an increase in PN content might lead to a red shift for a PN molecule containing many aromatic and multi-aromatic fractions belonging to strong electron-donating groups [47]. This was consistent with our findings, i.e., that the content of PN increased as the salinity increased from 0% to 3.5%.

It was noteworthy that the intensity of peaks at 260−265 nm with GB addition obviously increased compared to the group without GB addition under various salinities, which might have been due to changes in the conformations and inter-chromophores (such as humic compounds) of EPS molecules with GB addition [48]. Besides, compared to the group without GB addition, the peaks at 260−265 nm under various salinities exhibited a blueshift in the group with GB addition, representing the elimination of specific functional groups (like hydroxyl and amine) and the reduction in the degree of π-electron systems [49].

3.3.2. Functional groups of EPS based on FTIR spectra

To better understand the effect of GB dosing on EPS structure change in high-salt environments, FTIR spectra were obtained (Fig. 3b). It was found that the intensity of an absorption band near 1650 cm⁻¹, which was associated with the C O stretching vibration of β-sheets in proteins, showed a slight increase in the GB-addition group [36]. The intensity of the absorption band was based on the y-axis “transmittance” values in FTIR. This finding confirmed that GB could stimulate the secretion of PN in EPS. The adsorption band near 1402 cm⁻¹ represents the absorption of aliphatic C–H or the functional group of COO⁻ [50,51]. The intensity due to these electron transport enzyme-related functional groups increased with 1% salinity and GB addition.

It was possible to conjecture that 1% salinity, or the addition of GB, could accelerate electron transport activity. This can also provide a chemical structural explanation for the hydrophobicity of the extracted EPS with GB addition due to increased secretion of polar functional groups such as acylamino and carboxyl, which was consistent with the zeta potential value [52].

A distinct and sharp absorption band near 1065 cm⁻¹ was observed associated with phosphates in nucleic acids [53]. Previous studies have demonstrated that monovalent ions such as Na⁺ are attracted to phosphate groups [54]; thus, it can be speculated that based on the stronger peak intensity in FTIR, GB addition could stimulate phosphate secretion in EPS to further enhance the absorption capacity of EPS for Na⁺.

Other main functional groups were as follows. The peak near 1241 cm⁻¹ corresponded principally to C–O–C stretching, and the peak at 879 cm⁻¹ can be assigned to the out-of-phase ring stretching of aromatics [55,56]. These peaks barely changed with GB addition, while they exhibited distinct change with increasing salinity.

3.3.3. Protein secondary structure based on FTIR spectra

Second-derivative spectroscopic observations. Second-derivative spectra obtained from curve fitting of T-EPS in amide I (1700−1600 cm⁻¹) were used to further understand the protein secondary structure (Fig. 4). The main protein secondary structure categories were β-sheet (1640−1610 cm⁻¹), random coil (1650−1640 cm⁻¹), α-helix (1660−1650 cm⁻¹), and β-turn (1695−1660 cm⁻¹) [11].

Fig. 4.

Second derivative resolution enhanced and curve-fitted amide I region (1700−1600 cm⁻¹) spectra of total extracellular polymeric substances (T-EPS) under various salinities: a, 0% without GB; b, 0% with GB; c, 1% without GB; d, 1% with GB; e, 3.5% without GB; f, 3.5% with GB.

The α-helix ratio has been reported to be attributed to the aggregation and adsorption of bacteria [57,58], which showed a severe decreasing trend from 20.18% to 17.89% when salinity increased from 0% to 3.5% (Table 1). This was consistent with Li et al. [59], who found that the decreased ratio of α-helix led to the failure of auto-aggregation of microbe strains. The value of α-helix dropped from 21.57% to 19.86% with GB addition, suggesting that GB addition promoted cell aggregations by mitigating the reduction of the α-helix protein structure ratio. At the same time, hydrogen bonding between water molecules involved in protein-hydrophilic group interactions and water molecules involved in protein-hydrophobic group interactions could be significantly enhanced by GB addition. This helped the formation of a strong ice-like hydrated sphere of water molecules around the protein molecules [60]. The tightness of the formation of a hydrated layer around the protein can be characterized by the parameter Nw, which is the number of water molecules in solution that are bound to ions. It could be calculated by the formulas presented in Text S4 (Supplementary Information). A higher value of Nw with GB addition indicated that the volume reduction caused by the electrostriction of water molecules had increased [61]. This suggested that the presence of GB could form a tighter hydrated shell on the protein's surface to protect the protein from salt.

Table 1.

Secondary protein structures in total extracellular polymeric substances (T-EPS) under various salinities with glycine betaine (GB) addition obtained from derivative spectra and curve fitting.

Groups	α-helix (%)	β-sheet (%)	β-turn (%)	Random coil (%)
0% salinity	20.18	30.86	26.22	22.75
0% salinity with GB	21.57	28.87	27.14	22.42
1% salinity	20.48	31.41	25.50	22.61
1% salinity with GB	20.63	30.59	26.90	21.88
3.5% salinity	17.89	38.01	22.20	21.90
3.5% salinity with GB	19.86	30.06	29.29	20.78

According to current research, the ratio of α-helix/(β-sheet + random coil) could represent the protein structure [62]. Specifically, the lower the ratio, the looser the protein structure [62]. In this study, the ratio of α-helix/(β-sheet + random coil) was 0.38, 0.38, 0.29 for salinities of 0%, 1%, and 3.5%, respectively, while it was 0.42, 0.39, 0.39 for the same salinities but with GB addition. This suggested that higher salinity resulted in a looser protein structure, while GB addition mitigated the occurrence of this undesirable phenomenon. At the same time, the increased content of β-sheet induced the formation of a loose molecular EPS structure [63]. This outcome also indicated that adding GB promoted the forming of a tighter protein structure in EPS.

In conclusion, the addition of GB enhanced the hydrogen bond interaction. It induced a shift of the secondary structure of the protein from β-sheet to α-helix, prioritizing the formation of a denser hydrated shell around the protein to further protect the cell.

2D-COS analysis. 2D-COS of amide I bands (1700−1600 cm⁻¹) was used to further obtain functional group complexing with salinity variations and covariations with GB addition (Fig. 5). Two automatic peaks in the synchronous fluorescence spectra centered at 1652 cm⁻¹ (α-helix) and 1606 cm⁻¹ (β-sheet) along the diagonal line were observed without GB addition (Fig. 5a), and peak at 1652 cm⁻¹ had a higher intensity than peak at 1606 cm⁻¹ (Fig. 5a). It implied that α-helix was more susceptible to increased salinity than β-sheet due to the intensity of an automatic peak could reflect the degree of response of the protein secondary structure to changes in external conditions [64]. Compared to the group without GB addition, a significant decrease in peak intensity at 1652 cm⁻¹ (reflected by fluorescence intensity) indicated that GB greatly decreased the sensitivity of α-helix to salinity.

Fig. 5.

Two-dimensional correlation spectroscopy (2D-COS) demonstrating functional group complexing with salinity variations and covariations with GB addition. a–b, Synchronous (a) and asynchronous (b) graphs were generated from 2D-COS based on the FTIR of T-EPS without GB addition. c–d, Synchronous (c) and asynchronous (d) graphs were generated from 2D-COS based on the FTIR of T-EPS with GB addition.

Additional information about the sequential relationship was provided by the asynchronous map obtained when salinity increased with GB addition (Fig. 5b and d). The order of the protein secondary structure interacting with salinity was reflected in the signs of the cross-peaks in the asynchronous spectra. As could be seen in Fig. 5b–a principal negative cross-peak (λ₁/λ₂: 1661/1618 cm⁻¹) was observed, which was related to β-turn (1661 cm⁻¹) and β-sheet (1618 cm⁻¹). According to Noda's rule, the relative sequential order information can be probed by signs of asynchronous spectra [19], which suggested that salinity bound to EPS protein fractions in the following sequence: β-sheet → β-turn. The sensitivity of β-sheet in the interaction with salinity might lead to the collapse of the EPS matrix due mainly to the fact that β-sheet was of great importance in building structural function in the EPS matrix [65]. Compared to the group without GB addition, more cross-peaks were exhibited with GB addition (Fig. 5d), including a major positive cross-peak (λ₁/λ₂: 1653/1600 cm⁻¹) and several small negative cross-peaks (λ₁/λ₂: 1646/1653, 1670/1646, and 1669/1655 cm⁻¹). These suggest a more active interaction between molecular structures [66]. Besides, the sequence order of the protein secondary structure involved in binding with salt can be established as: α-helix → random coil → β-sheet → β-turn. This indicated that β-sheet and β-turn had greater reaction activity with salinity after GB addition [64]. Accordingly, we speculated that GB protected protein structure by reducing the sensitivity of α-helix to salinity and enhancing the activity of β-sheet.

3.4. Electrochemical characteristics of EPS with GB addition

Many redox-active and electrochemically active substances were found in the EPS matrix, indispensable in cell metabolism processes and electron transfer [66]. Here, the redox properties of the T-EPS under various salinities with GB addition were further characterized using the CV test (Fig. 6a–c). A more rectangular CV shape represented improved capacitive behavior [67], and it could be observed that the CV shape with GB addition was more rectangular than the one without GB addition. EPS extracted under 0% and 1% salinity showed two reductive peaks with potentials of −398 and −227 mV (0%, Fig. 6a) and −407 and −276 mV (1%, Fig. 6b), respectively. However, only one reductive peak at around −278 mV was observed under 3.5% salinity (Fig. 6c).

Fig. 6.

a–c, cyclic voltammetry (CV) curves of T-EPS under 0% (a), 1% (b), and 3.5% (c) salinities. d–f, pulse voltammetry (DPV) analysis for 0% (d), 1% (e), and 3.5% (f) salinities with GB addition.

A previous study demonstrated that the peak potentials around −210 and −380 mV were probably attributed to c-Cyts and flavins, which were important in interspecies electron transfer [68]. Notably, in this study, apart from the increasing salinity, the CV curves were not re-shaped with GB addition, which suggested that the dominant redox substances of c-Cyts and flavins might not be affected by GB. However, the addition of GB stimulated the secretion of c-Cyts, which manifested as a stronger peak strength.

The more sensitive DPV analysis was used to detect other electrochemically active substances to illustrate the role of EPS in the electron transfer process. Only two pairs of DPV peaks under potentials around −400/−350 mV and −75/−126 mV (anodic/cathodic), which could be attributed to flavins and c-Cyts, could be seen in Fig. 6d and e. In the group without GB addition, the peak associated with flavin almost disappeared after the salinity was increased, indicating that the flavin-dependent electron transfer process was reduced in response to the stimulation by salt. The increasing strength of the c-Cyts peak and the decreasing strength of the flavins peak could be because the c-Cyts-dependent electron transfer process was chosen as a more effective strategy in a high-salt environment. The contents of c-Cyts were quantitatively examined, consistent with the CV and DPV results (Fig. S3). The results indicated that more c-Cyts were released with GB addition than without GB addition, which increased by 0.00179, 0.00179, and 0.00339 nmol per g dry cell at 0%, 1%, and 3.5%, respectively. A previous study demonstrated that the contribution of c-Cyts was much higher than that of flavins in the electron transfer process [69]. Moreover, it was observed that the currents of both flavins and c-Cyts increased with GB addition, indicating that GB could effectively enhance the content of extracellular electroactive secreta to facilitate electron transfer further [70].

GB addition could increase methyl compounds, which enhances the concentration of redox substances [71]. Thus, it was reasonable to infer that the addition of GB could provide more electron transfer sites in EPS from redox secreta, which accelerated electron transfer rates.

To verify our above inference, the total formal charge in the EPS layer Q_EPS, which was generally associated with electron transfer sites [20], was calculated according to formula 1, the summary peak areas of DPVs (Q_DPV, C) are presented in Table S2. According to this model, Q_EPS was proportional to Q_DPV, meaning the larger the Q_DPV value, the more electron transfer sites were generated within the EPS layer. As seen in Table S2, a remarkable increment in the Q_EPS value was observed following the addition of GB. This suggested that GB addition might increase the number of electron transfer sites for EET by stimulating the secretion of redox substances, which acted as an electron shuttle in EPS. Additionally, Jiang et al. [72] found that the C-terminal of α-helix peptide provided more effective electron transfer sites, which could further increase the electroactivity of EPS. Given the important role of EPS in the electron transfer process, nitrobenzene (NB) reduction was carried out after removing the EPS of Bacillus pumilus NJUST51 (Fig. S3 and Table S3). Results showed that the NB reduction rate decreased almost 3.5-fold at 3.5% salinity after removing EPS; this was attributed to the loss of electrochemical active substances in EPS [73].

In summary, the addition of GB facilitates the secretion of exogenous electron mediators, such as flavins and c-Cyts, involved in extracellular respiration, and these accelerate the process of EET. Additionally, changes in the molecular structure of proteins in EPS, such as the increased ratio of α-helix, suggested that GB addition results in more electron transfer sites being revealed, which provides more possibilities for EET.

3.5. Possible mechanism of GB addition on salt-tolerant properties of microbes via EPS secretion

Compatible solutes such as GB were considered an effective bioaugmentation additive in relieving osmotic stress, and their addition was usually accompanied by the secretion of EPS [74]. However, the complex characteristics of EPS have hindered investigations into the detailed response of microbes under high-salt environments. In this study, the recognition of components, molecular structure, and electrochemical characteristics of EPS advanced the understanding of the complex, influential mechanism of GB addition on microbes under high-salt environments. On the one hand, the addition of GB promoted microbial metabolism to stimulate PN and HA secretion, which contained a large number of Na⁺-binding sites (Fig. 2) and facilitated the formation of a tighter EPS protective layer by weakening the electrostatic repulsion and compression effect. On the other hand, the more GB accumulated in EPS, the less Na⁺ diffused into it, which further relieved cellular osmolality [34].

The severe damage to PN structures in EPS due to salinity was also significantly improved by the addition of GB. For instance, the increasing content of α-helix facilitated the formation of a more stable protein structure (Fig. 4). Additionally, the hydrogen bonding between water molecules involved in the protein-hydrophilic group interaction and water molecules involved in the protein-hydrophobic group interaction could be remarkably enhanced by GB addition [60]. This helped the formation of a strong ice-like hydrated sphere around protein molecules by water molecules. Besides this, the volume reduction caused by the electrostriction of water molecules increased with GB addition, which indicated a tighter hydrated shell was formed on the surface of the PN to protect the PN from salt. Therefore, GB reduced the sensitivity of the EPS protein structure to salinity, which enhanced the salt tolerance of microorganisms.

The reduction of the electron transfer efficiency resulting from high salinity was also improved by the addition of GB. It effectually enhanced the content of extracellular electroactive secreta such as flavins and c-Cyts to further facilitate electron transfer (Fig. 6). In addition, GB addition revealed more electron transfer sites, which provided more possibilities for EET mediation by the α-helix in PN, which was related to molecular structural changes of EPS proteins such as the increased ratio of α-helix (Table S2) [72].

Overall, the addition of GB facilitated salt tolerance of microorganisms in EPS via the protection of protein secondary structures and the acceleration of electron transfer. Thus, such protective manipulation of microbes by GB addition could significantly relieve osmotic pressure. This provided an improved understanding of microbial salt-tolerance properties due to EPS secretion.

4. Implication

As an effective osmoprotectant, GB is a universally applied microbial protection measure in relieving osmotic pressure [12]. GB has been reported to be absorbed through the Opu system to facilitate microbial adaptation to high osmolality [74] and promote methane production in saline environments [75]. Besides, the quaternary structure of proteins and membrane structures were stabilized with GB addition against the adverse effects of high salinity and extreme temperatures [76]. However, the effect of GB addition on microbes has been well studied, but the mechanism of salt tolerance through EPS properties has not been fully explored. Previous research has primarily focused on EPS composition analysis [38], with a notable lack of in-depth investigation into EPS structure and electron transfer properties. This study revealed that GB stimulated the secretion of EPS and especially increased the content of hydrophobic substances such as PN and HA. The improved stability of EPS structure and a tighter EPS layer formation were demonstrated with GB addition. Additionally, more redox substances (such as flavins and c-Cyts) and electron transfer sites enhance microbial degradation efficiency in a high-salt environment. This study provides a new perspective to investigate the biological adaptation mechanism of hypersaline wastewater.

5. Conclusion

This study explored the regulating influence of microbial salt-tolerance mechanisms via EPS secretion with GB addition under high-salt environments. It focused on examining changes in microbial activity, EPS composition, EPS structural characteristics, and EPS electrochemical characteristics. The main conclusions are as follows.

• (1)Exogenously added GB could reduce cell damage from salinity and maintain the viability of cells in high-salt environments.
• (2)The presence of GB stimulated EPS secretion, especially increasing the content of hydrophobic substances such as PN and HA.
• (3)A remarkable contribution of GB to the shift from β-sheet to α-helix and the decreased sensitivity of α-helix to salinity ultimately stabilizes the PN structure in EPS.
• (4)More extracellular electroactive secreta (i.e., flavins and c-Cyts) and electron transfer sites could be effectually increased with GB addition.

This study gives deep insights into microbial salt-tolerance mechanism via EPS properties, spurring further development and application of biotreatment of high-salt wastewater.

CRediT authorship contribution statement

Yan Xia: Conceptualization, Investigation, Writing - Original Draft. Xinbai Jiang: Funding Acquisition, Supervision, Writing - Review & Editing. Shuaishuai Guo: Formal Analysis, Data Curation. Yuxuan Wang: Investigation, Data Curation. Yang Mu: Writing - Review & Editing. Jinyou Shen: Conceptualization, Project Administration, Funding Acquisition, Writing - Review & Editing. All authors read and approved the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: