Biogenic Nanomagnetic Carriers Derived from Magnetotactic Bacteria: Magnetic Parameters of Magnetosomes Inside Magnetospirillum spp.
, Denis Grouzdev 2, Veronika Koziaeva 3,4, Igor Kiselev 1, Ivan Larionov 1, Kamil Gareev 4,5, Nikita Sitkov 4,5, Tatiana Zimina 4,5, Yaroslav Marchenko 1 and Maxim Shevtsov 4,6,7,8,*
Round 1
Reviewer 1 Report
The manuscript entitled “Biogenic Nanomagnetic Carriers derived from Magnetotactic Bacteria: Magnetic parameters of magnetosomes inside Magnetospirillum spp.” by Ryzhov et al. provides interesting insights into the magnetic parameters of magnetosomes from different strains of the genus Magnetospirillum. In contrast to other studies that performed their investigations on isolated magnetosomes, here the data are collected from intact cells. Furthermore, potential effects on the magnetic characteristics of the cell suspensions upon long-term storage for one year are investigated. In particular for envisioned potential applications of magnetotactic bacteria and isolated magnetosomes, the study therefore provides important data on the (magnetic) “stability” of the particles upon storage.
The manuscript is well written, however, it addresses a rather specialized readership that is familiar with magnetism / magnetic measurements. I would therefore highly recommend providing more information on the applied methods. It should be stated more clearly which methods the authors used for which distinct parameter. Furthermore, no background information on the different bacterial strains is provided. For those who are not familiar with magnetotactic bacteria, more details on the strains should be provided. What are differences and similarities, and what is known so far about their magnetic characteristics?
In addition, the authors might consider shortening subsection 2.3 (“Magnetic measurements and data quantifying”) and provide a more detailed description of the method in the Supporting Information.
Overall, I can recommend publication in “Applied Sciences” after revision. Furthermore, there are many smaller mistakes and inaccuracies regarding the style and wording that should be corrected:
- In the article „the“ has been frequently omitted. Please check before resubmission.
- The authors use the old-fashioned unit "Oerstedt" for the magnetic fielt. Please change to the SI-units, which are "A/m" or "kA/m".
Abstract:
Page 1: “… in both the “new” and “old” samples. The EMR spectra were formed mostly by the “heavy” fraction for both samples. …”
Introduction:
Page 2 (first paragraph): “… MTB produce organelles …”
Page 2 (second paragraph): “… which produce magnetite-containing magnetosomes organized in a single chain. …” In many cases also the formation of two chains has been observed. The authors should rather state “organized in one or two chains”.
Methods section:
Page 3, subsection 2.1. “Cultivation of MTB“: As reference for FSM, rather cite Heyen and Schüler (2003), Applied microbiology and biotechnology, 61(5), 536.
Page 3, subsection 2.2. (second paragraph): It seems that this paragraph originates from the journals guidelines how to prepare the manuscript. This paragraph should be definitely removed.
In the methods section, the description of the performed TEM analysis is missing.
Results:
Page 5, subsection 3.1.: “The amount of magnetosomes in the chain as well as their size differs in different Magnetospirillum spp. strains [1-5].” Please specify the particle amounts produced by the different strains, and although the correct references are cited, why not provide own statistics on the particle amounts from TEM images?
Page 7/8: Please improve the resolution of Figures 2-5.
In Fig. 2 the labels of the y-axis are tiny. It is not discernible which exponent is plotted.
Fig. 2: Why are the signals for the fresh samples noisy, and the one for the old ones smooth?
Page 9, third paragraph: “The synthesis of magnetosomes and their chains in MTB is genetically controlled [1-5], …” Maybe add the following reference that describes the genetically controlled magnetosome synthesis? Lohße et al. (2014). Journal of bacteriology, 196(14), 2658.
Page 10: “Only one chain is synthesized in every cell [1-5] in agreement with the TEM image of Fig.1.” As mentioned above, partially also two chains are formed.
Page 10: “The high concentration of magnetosomes in the “light” fraction can be seen in AFM image in Fig. 1S. The biological role of this fraction is unclear.” Here, or in the conclusion, some hypotheses should be provided about the role of this “light” fraction (immature magnetosomes, particle precursors, …).
Page 11, subsection 3.4: “EMR spectra were obtained from colloidal suspensions of BB-1, MSR-1 …”
Page 11, subsection 3.4: “Since the concentration of the bacteria is rather low (~107 1/mL), …” Please accurately state the concentration of the cells.
Page 11, last paragraph: “… colloidal aqueous suspensions of magnetite nanoparticles …”
Page 12, first paragraph: “… This suggests that the observed EMR signals should be attributed to the “heavy” fraction whose magnetosome chain is attached to the membrane [1-5, 19].” Please include some more recent references.
Page 12, legend to Figure 6: “… colloidal suspensions of BB-1, MSR-1 …”
Page 12, last paragraph: “…both for the “new” and “old” samples, as also seen from Table II.”
Page 13, first paragraph: “… “new” and “old” samples are found …”
Page 13, second paragraph: “In a magnetosome chain one can expect …”
Page 13, second paragraph: “… EMR signal from the Magnetospirillum ensemble to split into two peaks.” Magnetospirillum should be written in italics.
Page 13, second paragraph: “… as it is really observed for the main (magenta and blue) peaks of the “old” and “new” samples in all the strains”
Page 13: “… for the “new” and “old” samples of BB-1, MSR-1 and AMB-1 strains shows their increase in the “old” samples for all the strains”
Page 14, first paragraph: Rather use “magnetosome chain” instead of “magnetosomal” chain, and “samples” instead of “probes”.
Author Response
We would like to thank the reviewer for the provided comments. We have substantially revised the manuscript and have provided the answers to the raised comments below.
COMMENT 1: The manuscript is well written, however, it addresses a rather specialized readership that is familiar with magnetism / magnetic measurements. I would therefore highly recommend providing more information on the applied methods.
ANSWER 1: The NLR-M2 setup was described in Refs. 26, 32, 33, 34. We added some description of these setup peculiarities in the beginning of section 2.3. Some features of the EMR technique are added at the end of section 2.3.
The figure with the geometry of magnetic fields for NLR-M2 is added as Fig. 2.
COMMENT 2: It should be stated more clearly which methods the authors used for which distinct parameter.
ANSWER 2: The magnetic parameters were extracted solely from the NLR-M2 measurements and presented in Table 1 while the EMR data were analyzed phenomenologically with the presentation in Table 2 of the obtained qualitative information on the MTB strains complementing the quantitative data. We emphasized it in the Conclusion section.
COMMENT 3: Furthermore, no background information on the different bacterial strains is provided. For those who are not familiar with magnetotactic bacteria, more details on the strains should be provided. What are differences and similarities, and what is known so far about their magnetic characteristics?
ANSWER 3: This information is added in the 2nd paragraph of Introduction and in Section 3.1.
COMMENT 4: In addition, the authors might consider shortening subsection 2.3 (“Magnetic measurements and data quantifying”) and provide a more detailed description of the method in the Supporting Information.
ANSWER 4: We discussed this suggestion and tend to leave the content of this subsection in the main text due to its essential importance. It can be easily omitted by the reader as appropriate.
REVIEWER: Overall, I can recommend publication in “Applied Sciences” after revision. Furthermore, there are many smaller mistakes and inaccuracies regarding the style and wording that should be corrected:
COMMENT 5: In the article „the“ has been frequently omitted. Please check before resubmission.
ANSWER 5: The style and wording of the text are additionally revised.
COMMENT 6: The authors use the old-fashioned unit "Oerstedt" for the magnetic fielt. Please change to the SI-units, which are "A/m" or "kA/m".
ANSWER 6: This is done, additionally, also for the magnetization units.
COMMENT 7: Abstract:
Page 1: “… in both the “new” and “old” samples. The EMR spectra were formed mostly by the “heavy” fraction for both samples. …”
ANSWER 7: “probe” is substituted for “sample” through the whole text where needed.
COMMENT 8: Introduction:
Page 2 (first paragraph): “… MTB produce organelles …”
ANSWER 8: This was corrected.
COMMENT 9: Page 2 (second paragraph): “… which produce magnetite-containing magnetosomes organized in a single chain. …” In many cases also the formation of two chains has been observed. The authors should rather state “organized in one or two chains”.
ASNWER 9: In the bacteria of the genus Magnetospirillum, only one chain of magnetosomes is observed. Two or more chains of magnetosomes are found in the representatives of the lines Magnetococcales, phylum Termodesulfobacteriota, Nitrospirota, and Omnitrophota.
COMMENT 10: Methods section:
Page 3, subsection 2.1. “Cultivation of MTB“: As reference for FSM, rather cite Heyen and Schüler (2003), Applied microbiology and biotechnology, 61(5), 536.
ANSWER 10: The reference is corrected.
COMMENT 11: Page 3, subsection 2.2. (second paragraph): It seems that this paragraph originates from the journals guidelines how to prepare the manuscript. This paragraph should be definitely removed.
ANSWER 11: Yes, of course. It is removed.
COMMENT 12: In the methods section, the description of the performed TEM analysis is missing.
ANSWER 12: The corresponding paragraph is added in page 2, Section 2.2.
COMMENT 13: Results:
Page 5, subsection 3.1.: “The amount of magnetosomes in the chain as well as their size differs in different Magnetospirillum spp. strains [1-5].” Please specify the particle amounts produced by the different strains, and although the correct references are cited, why not provide own statistics on the particle amounts from TEM images?
ANSWER 13: This information is added in Sec. 3.1.
COMMENT 14: Page 7/8: Please improve the resolution of Figures 2-5.
In Fig. 2 the labels of the y-axis are tiny. It is not discernible which exponent is plotted.
ANSWER 14: The plots for “new” and “old” are combined in pairs for better comparison and the resolution is improved.
COMMENT 15: Fig. 2: Why are the signals for the fresh samples noisy, and the one for the old ones smooth?
ASNWER 15: Indeed, the smoothing of the signals for MSR-1 “old” (Fig. 3(d,e,f)) accompanied by the strong increase of the signal is, most likely, due to considerable aggregation of magnetic centers which does not occur for the other strains.
COMMENT 16: Page 9, third paragraph: “The synthesis of magnetosomes and their chains in MTB is genetically controlled [1-5], …” Maybe add the following reference that describes the genetically controlled magnetosome synthesis? Lohße et al. (2014). Journal of bacteriology, 196(14), 2658.
ANSWER 16: This and one more references are added.
COMMENT 17: Page 10: “Only one chain is synthesized in every cell [1-5] in agreement with the TEM image of Fig.1.” As mentioned above, partially also two chains are formed.
ANSWER 17: In Introduction the change is made: “Magnetospirillum spp. are basically spirilla-shaped bacteria, which produce magnetite-containing magnetosomes organized in a single chain” with the reference [Monteil, C.L., Grouzdev, D.S., Perrière, G. et al. Repeated horizontal gene transfers triggered parallel evolution of magnetotaxis in two evolutionary divergent lineages of magnetotactic bacteria. ISME J 14, 1783–1794 (2020). https://doi.org/10.1038/s41396-020-0647-x].
COMMENT 18: Page 10: “The high concentration of magnetosomes in the “light” fraction can be seen in AFM image in Fig. 1S. The biological role of this fraction is unclear.” Here, or in the conclusion, some hypotheses should be provided about the role of this “light” fraction (immature magnetosomes, particle precursors,…).
ANSWER 18: A possible explanation is provided below Table 1 in the second, third and fourth paragraphs.
COMMENT 19: Page 11, subsection 3.4: “EMR spectra were obtained from colloidal suspensions of BB-1, MSR-1 …”
ANSWER 19: Conventionally, the term “colloidal solution” refers to the case of the particles less than ~1 micron size whereas “suspension” belongs to larger particles which can precipitate, unlike the former. With account of your remark, we use through the whole text “colloidal solution” in case of magnetite 10-micron-sized nanoparticles while “suspension” is used for MTB systems.
COMMENT 20: Page 11, last paragraph: “… colloidal aqueous suspensions of magnetite nanoparticles …”
ANSWER 20: replied before
COMMENT 21: Page 11, subsection 3.4: “Since the concentration of the bacteria is rather low (~107 1/mL), …” Please accurately state the concentration of the cells.
ANSWER 21: It’s our mistake. Instead of concentration, the optical density was measured. Its value 0.06 - 0.09 indicates low concentration allowing us to neglect the interaction between bacteria.
COMMENT 22: Page 12, first paragraph: “… This suggests that the observed EMR signals should be attributed to the “heavy” fraction whose magnetosome chain is attached to the membrane [1-5, 19].” Please include some more recent references.
ANSWER 22: The reference [Ben-Shimon, S., Stein, D., & Zarivach, R. (2021). Current view of iron biomineralization in magnetotactic bacteria. Journal of Structural Biology: X, 5, 100052] is added.
COMMENT 23: Page 12, legend to Figure 6: “… colloidal suspensions of BB-1, MSR-1 …”
ANSWER 23: replied above
COMMENT 24: Page 12, last paragraph: “…both for the “new” and “old” samples, as also seen from Table II.”
ANSWER 24: This was corrected.
COMMENT 25: Page 13, first paragraph: “… “new” and “old” samples are found …”
ANSWER 25: This was corrected.
COMMENT 26: Page 13, second paragraph: “In a magnetosome chain one can expect …”
ANSWER 26: This was corrected.
COMMENT 27: Page 13, second paragraph: “… EMR signal from the Magnetospirillum ensemble to split into two peaks.” Magnetospirillum should be written in italics.
ANSWER 27: This was corrected.
COMMENT 28: Page 13, second paragraph: “… as it is really observed for the main (magenta and blue) peaks of the “old” and “new” samples in all the strains”
ANSWER 28: This was corrected.
COMMENT 29: Page 13: “… for the “new” and “old” samples of BB-1, MSR-1 and AMB-1 strains shows their increase in the “old” samples for all the strains”
ANSWER 29: This was corrected.
COMMENT 30: Page 14, first paragraph: Rather use “magnetosome chain” instead of “magnetosomal” chain, and “samples” instead of “probes”.
ANSWER 30: This was corrected.
Reviewer 2 Report
The manuscript "Biogenic Nanomagnetic Carriers derived from Magnetotactic Bacteria: Magnetic parameters of magnetosomes inside Magnetospirillum spp." promises to report magnetic parameters of nanoparticle chains inside magnetotactic bacteria. While the manuscript is worth publishing, a few points have to be addressed before:
In the materials and methods section a text block seemingly originating from a Tex/Word template is included in the chapter, which makes me wonder, if the authors included all relevant information in the methods section, since they oversaw this remaining template text. In addition I'd like the authors to complement this section with general parameters, like pressures and temperatures for the (assuringly RT measurements) next to the 4C ones. I'd like the authors to recheck the materials chapter for completeness.
The results chapter on the TEM lacks precise information what was gained from the recorded images. Has there been a size distribution of the particles obtained from the analysis? Has there been an elemental analysis by EDX?
For the EMR results the authors state that the magnetosomes can be viewed due to the random orientation similar to a polycrystalline ferromagnet, with neglectable magnetic interactions between them due to the concentration. Did the authors check if there is influence of magnetic dipolar coupling between the magnetosomes, e.g. by TEM holography, or by creating a micromagnetic simulation approximation?
For the determination of the g-factor it is common practice to do a multi-frequency FMR analysis for samples exhibiting a magnetic order, which could also reveal magnetic damping (e.g. Gilbert damping factor alpha) for the samples. Did the authors consider to do such an analysis?
In addition the angular dependent FMR analysis of single particles, or particle chains, but also such a measurement of the complete sample with multiple chains could reveal magnetic anisotropy behaviour in terms of angular dependent resonance lines in the magnonic dispersion?
Finally a detailed measurement on the magnetic moment and the spin configuration could be revealed by scanning transmission X-ray microscopy employing the XMCD effect, which would give reliable information on spin and orbital momentum, as well as proof visually single domain states, or deviation from them.
I'd like to ask the authors to comment on the lack of usage of such standard techniques, especially since it is claimed to deliver magnetic parameters of the investigated samples, and if such measurements are planned, or have been done, but not reported here.
Author Response
We would like to thank the reviewer for the provided comments. We have substantially revised the manuscript and have provided the answers to the raised comments below.
COMMENT 1: In the materials and methods section a text block seemingly originating from a Tex/Word template is included in the chapter, which makes me wonder, if the authors included all relevant information in the methods section, since they oversaw this remaining template text.
ANSWER 1: The template paragraph appeared by an oversight and did not affect the relevant information. It is deleted.
COMMENT 2: In addition I'd like the authors to complement this section with general parameters, like pressures and temperatures for the (assuringly RT measurements) next to the 4C ones. I'd like the authors to recheck the materials chapter for completeness.
ANSWER 2: We have made the appropriate corrections:
Section 3.3, first paragraph: …The measurements were carried out at room temperature.
Section 3.4, first paragraph: …the samples in a flat cylindrical cuvette 0.15 mm high and a diameter of 20 mm, filled and sealed under nitrogen, were placed…; … signals were recorded at room temperature.
COMMENT 3: The results chapter on the TEM lacks precise information what was gained from the recorded images. Has there been a size distribution of the particles obtained from the analysis? Has there been an elemental analysis by EDX?
ANSWER 3: Brief information on the size distribution of magnetosomes in various strains of Magnetospirillum spp. obtained from the analysis of the TEM data is added in Section 3.1.
COMMENT 4: For the EMR results the authors state that the magnetosomes can be viewed due to the random orientation similar to a polycrystalline ferromagnet, with neglectable magnetic interactions between them due to the concentration. Did the authors check if there is influence of magnetic dipolar coupling between the magnetosomes, e.g. by TEM holography, or by creating a micromagnetic simulation approximation?
ANSWER 4: Magnetic dipolar coupling between the magnetosomes in the chains was estimated using the known distance between them from TEM data and their magnetic moments corresponding to their size. The estimation of the dipolar field is presented in the fourth paragraph of Sec. 3.4. The dipolar interaction between the bacteria was neglected due to their small concentration as seen from the optical density measurements (the second paragraph in Sec. 3.4).
TEM holography is applicable for dehydrated fixed bacteria samples rather than for suspensions. The micromagnetic modeling assumes lyophilized, fixed samples and the availability of the information about the spatial location of magnetic centers in them while the purpose of this study was the investigation of magnetosomes inside the cells in the nutrient medium in the liquid phase.
COMMENT 5: For the determination of the g-factor it is common practice to do a multi-frequency FMR analysis for samples exhibiting a magnetic order, which could also reveal magnetic damping (e.g. Gilbert damping factor alpha) for the samples. Did the authors consider to do such an analysis?
ANSWER 5: At the moment we don’t have this technical possibility. Alternatively, we are working on the application of the FP formalism to the FMR spectra analysis, hopefully enabling us to obtain the whole scope of magnetic parameters including the data on the magnetization dynamics. From M2 measurements, we estimated the Gilbert damping factor to be of the order 10 manifested in quite small ImM2 components compared to the ReM2 parts for all samples (Figs. 3-6).
COMMENT 6: In addition the angular dependent FMR analysis of single particles, or particle chains, but also such a measurement of the complete sample with multiple chains could reveal magnetic anisotropy behaviour in terms of angular dependent resonance lines in the magnonic dispersion? Finally a detailed measurement on the magnetic moment and the spin configuration could be revealed by scanning transmission X-ray microscopy employing the XMCD effect, which would give reliable information on spin and orbital momentum, as well as proof visually single domain states, or deviation from them.
ANSWER 6: The main purpose of the study was to obtain information about the magnetic characteristics of some strains of Magnetospirillum spp. in liquid phase and we employed our NLR-M2 and EMR setups adapted for the study of liquid samples. The single domain state of the magnetosomes in the studied strains was evidenced definitely by the dependence of the magnetic hysteresis on the H-scan frequency in M2-response (Figs. 3-6). The estimation of the anisotropy energy Ea ~ 10^3 K was obtained from the M2 data fitting by the numerical solutions of the FP equation based on the Gilbert-Landau-Lifshitz formalism (the paragraph preceding Table 1).
COMMENT 7: I'd like to ask the authors to comment on the lack of usage of such standard techniques, especially since it is claimed to deliver magnetic parameters of the investigated samples, and if such measurements are planned, or have been done, but not reported here.
ANSWER 7: The investigations by other magnetic techniques with freeze-dried, fixed samples are beyond the scope of this study and now under consideration for future development of this work.
Reviewer 3 Report
These are my comments to the authors to further improve their manuscripts.
1- Sample preparation and process for TEM is not explained. authors should add to their materials and characterization methods.
2- Second paragraph at part 2.2 doesn't make any sense. Authors must clarify that the paragraph is a mistake or they are trying to explain sth.
3- Figure legends for fig. 2 are not unified. For all the charts / figure legends same font and same location should be used with proper size and readability to the reader.
4- For AFM, why did the authors use hair dryer for drying? The blow of the air doesn't affect the sample? Why oven drying or freeze drying was not used which preserve the morphology better?
5- Why AFM image is placed in supplementary material while it is used in discussion? In conclusion also authors claim that AFM shows partly degradation of the cells during long term storage. The mentioned areas should be identified on the image. Also, AFM of the fresh new sample also must be added as reference for comparison of the two. Also, the authors must explain the purpose of doing AFM. Was it morphology or roughness? If morphology SEM wasn't conducted?
6- For TEM also images for new and preserved samples should be added for comparison. Also, for AFM images it should be clarified whether it is the old preserved sample or fresh one.
Author Response
We would like to thank the reviewer for the provided comments. We have substantially revised the manuscript and have provided the answers to the raised comments below.
COMMENT 1: Sample preparation and process for TEM is not explained. authors should add to their materials and characterization methods.
ANSWER 1: The description of the sample preparation is extended in Sec. 2.1 and the process for TEM is added in Sec. 2.2.
COMMENT 2: Second paragraph at part 2.2 doesn't make any sense. Authors must clarify that the paragraph is a mistake or they are trying to explain sth.
ANSWER 2: This paragraph is a mistake and removed.
COMMENT 3: Figure legends for fig. 2 are not unified. For all the charts / figure legends same font and same location should be used with proper size and readability to the reader.
ANSWER 3: This was corrected.
COMMENT 4: For AFM, why did the authors use hair dryer for drying? The blow of the air doesn't affect the sample? Why oven drying or freeze drying was not used which preserve the morphology better?
ANSWER 4: The word “hair dryer” is really confusing. In fact, to simplify the procedure, the sample to be dried was placed in an isolated "clean" volume and a special, non-domestic device was used to ensure its soft, gentle drying. This was corrected in text.
COMMENT 5: Why AFM image is placed in supplementary material while it is used in discussion? In conclusion also authors claim that AFM shows partly degradation of the cells during long term storage. The mentioned areas should be identified on the image. Also, AFM of the fresh new sample also must be added as reference for comparison of the two. Also, the authors must explain the purpose of doing AFM. Was it morphology or roughness? If morphology SEM wasn't conducted?
ANSWER 5: AFM was used for characterization of bacteria after long-term storage only. We were unable to obtain a high-quality image that would include both magnetosomes inside the intact and destroyed cells and chose a part of the image with the fragments of magnetosome chains and individual magnetosomes. Therefore, this Figure was placed in the Supplementary. The fragments of the magnetosome chain probably coupled by dipolar forced as well as separate magnetosomes can be seen on the image. Magnetosomes in the intact cell are presented in TEM image of Fig. 1. The AFM image of fresh new sample was not obtained on technical reasons.
COMMENT 6: For TEM also images for new and preserved samples should be added for comparison. Also, for AFM images it should be clarified whether it is the old preserved sample or fresh one.
ANSWER 6: The TEM images for the fresh and old samples are much alike. The fresh sample image would not yield more information.
Round 2
Reviewer 2 Report
The authors responded sufficiently to the points brought up.
