Response of Escherichia coli to hydrogen nanobubbles: an in vitro evaluation using synchrotron infrared spectroscopy

Response of Escherichia coli to hydrogen nanobubbles: an in vitro evaluation using synchrotron infrared spectroscopy 大肠杆菌对氢纳米气泡的响应：使用同步红外光谱进行体外评估 1 2 5 equal first-author 1 2 5 equal 3 2 4 1 2 1 2 corresp Bin LI, libin@sinap.ac.cn Bin LI, libin@sinap.ac.cn libin@sinap.ac.cn https://orcid.org/0000-0002-8348-7445 1 2 corresp Junhong LÜ, lujunhong@zjlab.org.cn Junhong LÜ, lujunhong@zjlab.org.cn lujunhong@zjlab.org.cn https://orcid.org/0000-0003-2873-3137 CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai201800, China CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai201800, China Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai201203, China Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai201203, China College of Pharmacy, Binzhou Medical University, Yantai264003, China College of Pharmacy, Binzhou Medical University, Yantai264003, China National Engineering Research Center for Nanotechnology, Shanghai201318, China National Engineering Research Center for Nanotechnology, Shanghai201318, China University of Chinese Academy of Sciences, Beijing100049, China University of Chinese Academy of Sciences, Beijing100049, China 目的 2 探究分子氢在细胞水平上的作用机制。 创新点 2 使用同步辐射红外光谱技术评估了氢纳米气泡在细胞水平上对大肠杆菌的影响，扩展了同步辐射红外光谱的应用范围。 方法 2 将大肠杆菌进行氢气纳米气泡处理后，使用同步辐射红外光谱测定其红外光谱，采用主成分分析等方法分析细胞成分的变化。 结论 2 氢纳米气泡处理后细胞的蛋白和脂肪酸成分发生较大改变，而核酸和多糖成分则没有明显变化。这些结果表明，氢气的作用靶点可能是细胞膜。此外，氢气分子的作用与传统的抗氧化剂（单宁）并不一致。 氢纳米气泡 同步辐射红外光谱 大肠杆菌 Hydrogen (H 2 )-rich water, an apparent source of molecular H 2 , is an emerging functional drink with many purported benefits for human health ( Yang et al., 2020 Yang et al., 2020 ; Ostojic, 2021 Ostojic, 2021 ). The preventive and therapeutic effects of H 2 on various pathological processes have been intensively investigated in numerous clinical trials; it is commonly believed that the beneficial effects are mainly attributed to its selective antioxidant and anti-inflammatory properties ( Lee et al., 2015 Lee et al., 2015 ; Ohta, 2015 Ohta, 2015 ; LeBaron et al., 2019 LeBaron et al., 2019 ; Qiu et al., 2020 Qiu et al., 2020 ). In recent years, a handful of rodent studies revealed that exogenous H 2 can affect the gut microbiota ( Sha et al., 2018 Sha et al., 2018 ; Valdes et al., 2018 Valdes et al., 2018 ). For example, H 2 was reported to induce a higher abundance of butyrate-producing bacteria in a rat model of Parkinson's disease ( Bordoni et al., 2019 Bordoni et al., 2019 ). Recent first-in-human trials have explored the effects of the long-term consumption of H 2 -rich water on antioxidant activity and the gut flora ( Sha et al., 2018 Sha et al., 2018 ; Suzuki et al., 2018 Suzuki et al., 2018 ). Although these promising results suggest that the intestinal microbiota may be another plausible target for molecular H 2 , more studies are highly warranted to explain the mechanism(s) of H 2 action on bacterial growth and functions. Fourier transform infrared (FTIR) spectroscopy can provide the chemical composition information of samples with the advantages of high sensitivity and good reproducibility ( Wang et al., 2017 Wang et al., 2017 ). With the technological advance of synchrotron light source, the higher brightness and smaller source spot size have allowed the FTIR spectra to have higher intensity and better signal-to-noise ratio. As the spectral bands contain molecular fingerprint information, FTIR spectroscopy exhibits sufficient specificity to establish the molecular differences of cells exposed to various types of stimulations or treatments. The changes of absorption bands, such as those in position or intensity, reflect the component differences. Thanks to this advantage, FTIR spectroscopy has been widely used in the field of microbiology, especially for studying the changes of microbial constituents under stress conditions ( Alvarez-Ordóñez et al., 2011 Alvarez-Ordóñez et al., 2011 ). For example, synchrotron infrared spectroscopy has been employed to study the response of Escherichia coli to heat, cold, and ethanol, as well as the interaction between bacteria and metal ions ( Hu et al., 2016 Hu et al., 2016 ). In this study, the effect of H 2 nanobubbles (NBs) on the viability of E. coli was first tested by measuring the bacterial growth curve. Compared with double distilled water (dd-water) and nitrogen (N 2 ) NBs, the growth curve of H 2 NB treatment group did not show any significant changes ( Fig. 1 Fig. 1 a). The colony number of live bacteria during the exponential growth phase was further counted on the culture plates. After culture for 8 h, the counts were 5.5×10 9 and 5.9×10 9 CFU/mL (CFU: colony forming units) for the H 2 NB treatment group and dd-water group, respectively ( Fig. 1 Fig. 1 b), indicating no noticeable effects of H 2 on the vitality and proliferation of E. coli in vitro. Synchrotron FTIR spectroscopy was further conducted to explore whether H 2 NB treatment induces the biochemical component changes of bacterial cells. A total of 20 spectra obtained from the bacterial samples exposed to dd-water or H 2 NBs were collected and analyzed. As shown in Fig. 1 Fig. 1 c, the differences between the average spectra of the two groups of cells were negligible. To differentiate the component alterations, the second derivative spectra were processed and principal component analysis (PCA) was carried out. The PCA results and loading plots in the whole regions of 3000–2800 cm -1 and 1800–900 cm -1 are shown in Figs. 1d 1d and 1e 1e . The results showed a variance between the spectra of H 2 NB and dd-water treatment groups of E. coli within the 95% confidence interval (CI) ( Fig. 1 Fig. 1 d), which indicated that the chemical components of E. coli changed significantly with the existence of H 2 NBs. The loading plots of PC1 (61.72%) and PC2 (25.20%) showed that the bands 2920, 2850, 1626, 1109, 1097, and 1024 cm -1 had the largest contribution ( Fig. 1 Fig. 1 e). Notably, the cellular effect of H 2 NBs was also distinct with N 2 NBs, and the latter could not induce remark‍able spectral changes (data not shown), suggesting a specific effect and molecular mechanism of H 2 NBs on bacterial cells. In order to investigate which chemical component exhibited significant change, the infrared band shifts of second-derivative spectra were analyzed in detail. The significantly shifted spectral bands were marked with arrows in Fig. 2 Fig. 2 a and also listed in Table 1 Table 1 . The results showed that after the H 2 NB treatment, some infrared bands of the bacteria in the Amides I and II regions (1694.14, 1656.00, and 1544.85 cm -1 ) shifted to higher wavenumbers; several spectral bands corresponding to C–H vibrations of fatty acid components (2921.23, 2851.95, 1467.84, and 1452.17 cm -1 ) shifted to lower wavenumbers; and the P=O asymmetric stretching vibration band of nucleic acid at 1243.18 cm -1 also shifted, as well as the C‍–‍O‍–‍C vibrations of carbohydrates at 1123.29 and 1057.68 cm -1 . The band shifts of the two spectra indicated that chemical component differences in bacterial cells had taken place following exposure to H 2 NBs, and these differences could be detected by synchrotron FTIR spectroscopy. To intuitively reflect the bacterial component variations, PCA was further performed on the spectral protein region (1760–1480 cm -1 ; Fig. 2 Fig. 2 b), fatty acid region (3000–2800 cm -1 and 1480–1340 cm -1 ; Fig. 2 Fig. 2 c), and nucleic acid and polysaccharide region (1350‍–1000 cm -1 ; Fig. 2 Fig. 2 d). From the PCA diagrams, we could establish that both the protein and fatty acid compositions varied dramatically, while the nucleic acid and polysaccharide compositions showed no significant variations. These results indicated that the effects of H 2 NBs on bacterial cells mainly acted on the protein and fatty acid components. ν s : symmetric stretching vibration; ν as : asymmetric stretching vibration; dd-water: double distilled water; H 2 ‍NBs: hydrogen nanobubbles. The values are represented as mean±standard deviation (SD) for each group ( n =20). The up (down) arrow represents the increase (decrease) of the wavenumber of absorption peak, which corresponds to the blue (red) shift. Comparisons are performed by hypothesis testing through a two-sample t- test. ** and *** denote the degree of significance as P < 0.01 and P < 0.001, respectively. 2 Functional group 2 Wavenumber (cm -1 ) dd-water H 2 NBs    v as (C–H) of > CH 2 in fatty acids 2921.23±0.41 2920.66±0.52 *** ↓    v s (C–H) of > CH 2 in fatty acids 2851.95±0.22 2851.45±0.32 *** ↓ 3   Amide I 1694.14±0.23 1694.47±0.09 *** ↑ 1656.00±0.22 1656.45±0.49 *** ↑ 1627.19±0.23 1626.89±0.38 ** ↓   Amide II 1544.85±1.30 1546.81±0.50 *** ↑ 2   C–H deformation of > CH 2 > CH 2 bending 1467.84±0.21 1467.51±0.18 *** ↓ 1452.17±0.31 1451.79±0.21 *** ↓    v as (P=O) of > PO <math id="M1"><msubsup><mrow/><mrow><mn>2</mn></mrow><mrow><mo>-</mo></mrow></msubsup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27602052&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27602050&type= 1.18533325 2.53999996 phosphodiesters 1243.18±0.52 1241.89±0.96 *** ↓ 2   C–O–C and C–O dominated by ring vibrations of carbohydrates C–O–P and P–O–P 1123.29±0.88 1119.58±1.25 *** ↓ 1057.68±0.99 1056.58±0.56 *** ↓ Since H 2 gas is considered to act as a selective antioxidant, we investigated whether its cellular effect would be comparable with that of natural antioxidant tannins. To this end, bacterial cells were treated with different concentrations of tannins and measured by infrared spectroscopy. As shown in Fig. 3 Fig. 3 a, tannins at 0.5 g/L had no noticeable effect on bacterial growth (indicated in purple), similarly to the effect of H 2 . These cells treated with tannin and H 2 NB-tannin mixture were used to obtain FTIR spectra. The averaged FTIR absorption spectra of these four groups of bacteria are shown in Fig. 3 Fig. 3 b, which showed that the band positions and strength of tannin and H 2 NB-tannin treatment groups were significantly different from the untreated and H 2 NB treatment groups of bacteria. The corresponding second-derivative spectra are shown in Fig. 3 Fig. 3 c, where the significantly shifted bands of the tannin or H 2 NB-tannin treatment group were labeled with arrows. PCA on full spectral regions (3000 – 2800 cm -1 and 1800 – 900 cm -1 ) could not well discriminate the tannin treatment and the H 2 NB-tannin co-treatment groups ( Fig. 3 Fig. 3 d). The loading plots of PC1 and PC2 are presented in Fig. 3 Fig. 3 e. However, PCA on the protein region showed significant differences between the tannin treatment group (purple) and the co-treatment group (blue) ( Fig. 3 Fig. 3 f), while the fatty acid and nucleic acid and polysaccharide components were not clearly distinguishable (Figs. S1–S3). These results suggested that H 2 might have a distinct mechanism of action beyond acting as an antioxidant. In conclusion, we have expanded the application of FTIR to evaluate the cellular effects of H 2 NBs on the model bacterium E. coli . The obtained results revealed that molecular H 2 mainly induces the cellular changes in protein and fatty acid compositions in vitro, indicating that the possible direct target of H 2 lies on the membrane. Given the fact that different effects of H 2 NBs on bacterial cells were observed compared with natural antioxidant tannins and the results of our previous report on the binding of H 2 gas to the hydrophobic sites of proteins ( Cheng et al., 2020 Cheng et al., 2020 ), we believe that the cellular mechanism of H 2 molecule and/or NB is beyond being an antioxidant, i.e., H 2 might have other effects besides the widely described antioxidant effect. Thus, our findings provide a novel insight into the possible mechanism of action of molecular H 2 , as well as other gases, at the cellular level. To our knowledge, this is the first report on the effects of H 2 on bacterial cells at the chemical molecular level in vitro, and in turn justifies further scientific endeavors in this direction. Materials and methods 1 Detailed methods are provided in the electronic supplementary materials of this paper. References Alvarez-Ordóñez A , Mouwen DJM , López M , et al. , 2011 . 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BMJ , 361 : k2179 . https://doi.org/10.1136/bmj.k2179 https://doi.org/10.1136/bmj.k2179 Role of the gut microbiota in nutrition and health Wang YD , Li XL , Liu ZX , et al. , 2017 . Discrimination of foodborne pathogenic bacteria using synchrotron FTIR microspectroscopy . Nucl Sci Tech , 28 : 49 . https://doi.org/10.1007/s41365-017-0209-8 https://doi.org/10.1007/s41365-017-0209-8 Discrimination of foodborne pathogenic bacteria using synchrotron FTIR microspectroscopy Yang ML , Dong YM , He QN , et al. , 2020 . Hydrogen: a novel option in human disease treatment . Oxid Med Cell Longev , 2020 : 8384742 . https://doi.org/10.1155/2020/8384742 https://doi.org/10.1155/2020/8384742 Hydrogen: a novel option in human disease treatment Junhong LÜ designed the experiments and co-supervised the project with Bin LI and Jun HU. Jinfang LU, Jin ZHENG, Yadi WANG, and Jie CHENG performed the experiments. Junhong LÜ, Bin LI, and Xueling LI analyzed the data. Junhong LÜ, Jinfang LU, Jin ZHENG, and Yadi WANG wrote the manuscript. All authors have read and approved the final version of the manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Natural Science Foundation of China (No. U1732130), the Key Research Program of Frontier Sciences of the Chinese Academy of Sciences (No. QYZDJSSW-SLH019), and the Yantai Region and College Integration Development Project (No. 2021XDRHXMPT28). We thank all the staff at the BL01B beamline of the National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility for the assistance during data collection. 10.1631/jzus.B2100407 1673-1581（2021）11-0966-05 This work was supported by the National Natural Science Foundation of China (No. U1732130), the Key Research Program of Frontier Sciences of the Chinese Academy of Sciences (No. QYZDJSSW-SLH019), and the Yantai Region and College Integration Development Project (No. 2021XDRHXMPT28). 2021-05-09 2021-08-07 2021-11-15 2022-05-09 2021-10-14 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2021 2021 浙江大学出版社 Jinfang LU, Jin ZHENG, Yadi WANG, Jie CHENG, Xueling LI, Jun HU, Bin LI, and Junhong LÜ declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 11 22