October 7, 2021: I came to ELSI after 2 months. I met Shawn in August for a day for fun and discussion. I also talked with him online and exchanged emails during the time, and we have been continuing collaboration even out of ELSI. During the time, I presented our research at the annual meeting of the Botanical Society and submitted the titles and abstracts for the annual meetings of the Japanese Society of Microbial Ecology and the Japanese Society of Hot Spring Sciences.

During the last three weeks, Shigeru and I found two new important results, and I talked them to Shawn at lunchtime on the roof. A brief description of the findings is as follows.

(1) Shigeru was succeeded in amplifying Chloroflexus 16S rRNA genes by changing the conditions for the 1st PCR for amplicon analysis. His new conditions and the old conditions of the analysis company are as follows.

New 1st PCR conditions    temperature      time (second)
Initial denature                       98                       120
Denature                                98                       10       (xx cycles)
Aneal&extension                     68                       5
Final extension                       68                       60

Old 1st PCR conditions    temperature      time (second)
Initial denature                        94                       120
Denature                                 94                       30       (30 cycles)
Aneal                                      50                       30
extension                               72                       30
Final extension                       72                       300

Among 18 taxonomic groups present in more than 1% of total reads of either old or new analyses, three are significantly larger, and two are smaller in the read numbers.
Chloroflexus  7882 times more
A Thermus species   7.2 times more
Archaea  5.2 times more
Nitrospira  13 times less Chlorobi   20 times less
Other 13 taxonomic groups, including major Aquificae and Themodesulfobacteria, did not show a significant difference between the two conditions of the 1st PCR.

(2) I was in Nakabusa last week for three days with Etsuko to examine the concentrations of HS- and O2 before and after removing streamers and mats in the hot water current. The summary of the results and some discussion is as follows.
1. SH-,DO,Eh, and temperature were measured at 9 points, almost 1 m apart for each, along with the current before and after the removal of streamers and mats.
2.  The PowerPoint file includes four graphs, red lines are before and blue lines are after the removal.
3. HS- and O2 concentrations were increased by the removal, and the effect was more significant in O2 concentrations.
4. Changes along the current were very small from point 4 to 7, possibly because the current between them is along a large rock and narrow and rapid.
5. We tried to remove as many streamers as possible and additionally the surface sands of the bottom. We found significant microbial aggregates in the sands, and possibly invisible many microbes are also present in the sands.
6. As a result, we may have removed only a part of microbes in the stream and bottom sands.
7. I am thinking of additional in situ experiments using a plastic drain half-pipe or a sheet to avoid the effect of microbes in the bottom sands.

July 1, 2021: It was heavily raining this morning. I made a little conversation with Reiko Nagano about power harassment and sexual harassment and the changes in their meaning in the last two decades. It started with the appropriateness of mentioning the change of hairstyle of a lady colleague. Fatima kindly greeted me as she walked through the hallway. I started some practice to improve my English for paper writing and presentation.

Abstract due date of the presentation for the botanical society meeting with Shawn and Shigeru will be next Monday. I sent a draft of the abstract to them yesterday as follows. The presentation and abstract will be in Japanese.

Relationship between abundance of photosynthetic pigments and the synthesizing genes and redox conditions in microbial communities including thermophilic filamentous photosynthetic bacteria
◯Katsumi Matsuura1, 2), Shawn E. McGlynn2), Shigeru Kawai3)
  The green filamentous bacterium, Chloroflexus aggregans, grows up to 70°C, which is about the maximum temperature in all photosynthetic organisms. Since it is thought that the birth of life was as high as over 100°C, photosynthetic organisms that grow at high temperatures are important for understanding the emergence and early evolution of photosynthesis. In this study, C. aggregans in nature was investigated concerning the redox environmental conditions.
  Microbial communities, including C. aggregans, are well developed in hot springs of Nakabusa at 62°C to 70°C. Seven communities of various colors of olive green, orange, and pale brown were collected, and the absorption spectra were examined. The peak wavelengths of the near-infrared bacteriochlorophyll (BChl) c and a were all the same. On the other hand, there was a difference of 70 times in the amount of BChl c and 20 times in Bchl a. The abundance of BChl synthetic genes was 12 times different for bchX and 16 times for bchL, which was similar to Bchl a. The communities with low pigments and BChl synthetic genes were pale brown, under low hydrogen sulfide concentrations, high oxygen concentrations, and high redox potentials. Still, BChl synthetic genes had the same sequence in all communities. From these results, it was found that the same C. aggregans strain grows widely under various environmental conditions in Nakabusa hot springs.

June 11, 2021: Shawn was working at home today, and we exchanged emails. I explained the current situation of our collaboration, especially Shigeru’s trial for analyzing photosynthetic genes to find out the missing C. aggregans DNA in our 16S rRNA analyses. Shawn will be left for the US from June 17-July 17, and we will see next time in August. I hope Shigeru will get some significant results to proceed with our study. Shawn let me know about the annual meeting of the society of evolutionary studies, Japan, held in Tokyo Metropolitan University in August. The symposium program seems to be interesting to me, and I will probably attend it.

June 3, 2021: I was intended to come to ELSI last Thursday, but Shawn sent me an email that the university had a problem and people were not allowed to enter the campus until 13:00. Then we talked on zoom, with Shigeru, about the presentation at the online meeting of the Japanese Society of Photosynthetic Research, which was held the day after. I have got significant suggestions and slide corrections from Shawn, and the presentation was successful. The PowerPoint files were uploaded to this website in Japanese and English. Today, I have talked to three people in my office for the first time. Professor Tomoaki Matsuura, who shares the room but rarely uses it, come to the room, and we talked for minutes about our background, research interests, and the situation of ELSI. Reiko Nagano came with some stationary for me and chatted a while, and another person we have talked to several times, but I forgot her name, gave me a handmade cupcake. I have read something about high-throughput sequencing and seen some related videos on the internet. Now I understand some about the “pipeline.” Before leaving, I will talk with Shawn about a possible suggestion of a research topic (article collections) for an open-access journal on the roof.

May 13, 2021: Because of a field trip to Nakabusa and the golden week, I came to ELSI after 4 weeks. During that time, I contacted Shawn and Shigeru about presentations at annual meetings of photosynthesis society and botanical society. Titles are, originally in Japanese, “An autotrophic microbial community including Chloroflexus aggregans in Nakabusa hot springs as a hypothetical model of the emergence of photosynthesis from chemosynthesis” and “Photosynthetic pigments and pigment-synthesizing genes and redox conditions in microbial communities containing the thermophilic photosynthetic bacterium, Chloroflexus.” I talked to Shawn from 11:00 for 1 hour. After confirming the titles above, we talked good and bad points of Japanese society and the system, starting from COVID-19 measures. Peopled minds are so resistant to changes. I talked about “Deru kui-ha utaeru,” meaning “The stake that sticks out gets hammered in.” And I told the situation was different in the periods of Meiji Reformation and soon after world war II.

April 15, 2021: I have talked to Shawn from 15:00 to 16:00 at ELSI-1. Presentations at 4 national conferences from May to November were discussed using possible titles described below on April 8. Several suggestions were raised, e.g., emphasizing the monitoring of the internal redox state through bacterial population there for the JAME meeting and desirable microbe-free measurements of hydrogen sulfide for the JSHSS meeting. In terms of the first photosynthesis, comparisons with various models should be clarified. I have talked about Shigeru’s recent publication (Microorganisms 2021, 9, 650) on daily cycle changes of the mode of energy transduction in relation to the multifunction of it in Chloroflexus aggregans, and Shawn was very interested in it. Shawn was a little hesitant to be a co-author to these presentations, saying that his contributions have been minor. Still, I asked to be the co-author since the discussion between us is very important for the studies, and the acceptance of me in ELSI is also significant for the studies.

April 8, 2021: Possibly because of the increase of corona infection, the number of students in the building seems to be small. I decided to the door open when I am in the office room otherwise I feel lonely in the room. I posted the following message on the door.

Katsumi Matsuura: A collaborator of Shawn McGlynn
Present in ELSI on Thursday or Friday
Professor Emeritus, Tokyo Metropolitan Univ.
Research project: Early evolution of energy transduction and electron transfer through H/C/N/S/Fe in thermophilic microbial communities
Field research site: Nakabusa hot springs in Nagano Prefecture
Previous studies: Mechanisms, ecology, and evolution of energy transduction and electron transfer in photosynthetic bacteria
Please come in and talk. Anyone is welcome.

I am thinking to make poster or oral presentations in the following national conferences.

Microbial Ecology Oct 31-Nov 2 Niigata
Diversity of redox environments of weakly-alkaline hydrogen-sulfide hot springs in Nakabusa and the development of chemosynthetic and anoxygenic-photosynthetic microbial communities. Matsuura, McGlynn, Kawai
Hot Spring Sciences Sept 13-15 Kusatsu onsen, Gumma
Development of microbial communities in the outdoor running water of Nakabusa hot springs and the reduction of hydrogen sulfide concentration in the water. Matsuura, Kawai, McGlynn
Plant Sciences Sept 18-19 Minami-Osawa
Development of diverse microbial communities including the thermophilic photosynthetic bacterium Chloroflexus in Nakabusa hot springs and the relative distribution of photosynthetic pigments and pigment-synthesizing genes. Matsuura, McGlynn, Kawai

April 1, 2021: New school year starts today. Before coming to ELSI, I tested a new scanning visible spectrophotometer at home. I am planning to use it at the field sites of Nakabusa. Last year, I used a tiny spectrophotometer, named GoSpectro, attached to an iPhone. It worked for the absorption spectra and the H2S calorimetric determination. However, the operation was a little difficult and time-consuming, and wavelength range and data dynamic range were insufficient. I found a full spec scanning spectrophotometer is available with a price of 260,000 yen with tax from AsOne. Then I bought one with an AC-100V lithium battery and a backpack carrier to bring down them to the riverside. The test was successful, and I will make the field test within this month at Nakabusa hot springs.

Then I came to ELSI. I enjoyed talking with Shawn having lunch together on the roof of the ELSI-1 building. It was warm and comfortable. We talked about the differences in tax return forms, pension system, security, buying houses, and others between the US and Japan. I also talked about the new graduate program of ELSI starting next year, commuting by car to Japanese universities, and other things. We will talk about science next week.

March 25, 2021: Cherry blossoms are in full bloom on the campus of Tokyo Tech. The spring has come. I walked up to Nakabusa hot springs on the weekend before last, and the last Friday was the day of the graduation ceremony at the Japanese language school where I am working as a principal for 3 days a week. So I came to ELSI after 3 weeks. My office desk was moved from Room 209 to 211, a small room with 3 desks. The room is for temporal usage for experienced people; quiet but lonely. I have got a key to the room.

After finishing preliminary analyses of data, I am reading textbooks on molecular evolution, reconsidering the stories of possible papers. Titles of the textbooks are “Fundamentals of Molecular Evolution” by Wen-Hsiung Li and Dan Graur and “An Introduction to Molecular Evolution and Phylogenetics” by Lindell Bromham. I am enjoying reading the textbooks remembering the evolutionary-genetic lecture by Osamu Kitagawa in my undergraduate days.

March 4, 2021: I got a new MacBook Air (M1 tip) last Monday and transferred files from the old MacBook Pro. An important new trial was to keep the data and analysis files in a more reliable computer and an online storage place for the security of the data. M1 mac seems to be good in data security, and OneDrive came with Microsoft 356 for Excel, Word, etc. is convenient to synchronize local and online storage. I have succeeded in setting up these things. MEGAX and SnapGene programs work very fast on the new machine. Further analyses of the data and writing the paper drafts will be smooth with this computer.

February 26, 2021: I made a two-hour review discussion with Shawn on our collaborative studies on ecology and evolution of electron and energy flow in Nakabusa microbial communities since October 2020. The following 7 points are the summary of the discussion.

1. Numerical estimation of the redox states of 16 microbial communities and 100 major OTUs n Nakabusa hot springs.

Taking advantage of the large differences in hydrogen sulfide concentration/oxygen concentration/redox potential in the water around the 16 microbial communities and that the different adaptability of each microbial species to different redox environments, the redox index of each community and each OTU were estimated. Initially, the calculation was carried out using only microbial species with somewhat known redox properties, and the temporal redox indexes of the communities were obtained. The redox index of the microbial species was recalculated using the obtained redox indexes of the communities and the relative abundance of each species. By repeating such calculations, the redox indexes of the communities and the redox indexes of the microbial species converged to certain values. By this method, the redox indexes of communities and microbial species were estimated. Consequently, the redox environments of microbial species, only sequences of which could be known, were estimated. By combining this method with in situ hybridization in the future, it may be possible to use it as a micro biosensor for redox states in microbial communities.

2. A simple high-temperature microbial community dominated by only two chemosynthetic bacteria (additional experiments in preparation)

A black microbial streamer was developed in an alkaline spring containing hydrogen sulfide at 86 ° C in Nakabusa Onsen. Amplicon analysis of 16S ribosomal RNA revealed that 68% was Aquificae, which is closely related to Hydrogenobacter, and 29% was Thermodesulfobacteria, which is closely related to Caldimicrobium. These two species accounted for 97% of the 16S ribosomal RNA in the community, making it a very simple community for an open outdoor environment. The reason for the simple species composition is considered that it is extremely hot, the relationship between the two species is particularly strong, the secreting soluble components are easy to flow in vigorous running water, and the old streamer is easy to tear off. It is considered that Hydrogenobacter obtains energy by oxidizing hydrogen sulfide with low-concentration oxygen to form elemental sulfur, and Caldimicrobium obtains energy by disproportionation of elemental sulfur. It was considered that the high concentration of hydrogen sulfide, which was generated in the microbial streamer by the disproportionation of elemental sulfur, combined with the divalent iron ions in the running water and precipitated and turned black.

3. Franboydal pyrite in high-temperature chemosynthetic microbial communities oxidizing hydrogen sulfide (experiments in preparation)

In alkaline hot springs containing hydrogen sulfide from 70°C to 80°C, micro mineral crystals thought to be framboidal pyrite were present on the surface of microorganisms in gray, white, and transparent pale-tan streamers and mats. Similar framboidal pyrite was also observed in the culture of isolated Hydrogenobacter sp. under certain conditions. Pyrite is known to be abundant in Archean sediments, and the involvement of microbial activity in the early stages of the Earth has been postulated. These results are important in considering what kinds of microorganisms may have been involved in pyrite formation in Archean environments.

4. Sulfur metabolism and energy conversion in microbial communities including Chloroflexus in Nakabusa hot springs (16S rRNA experiments, in preparation)

Kawai et al. have previously shown that the coexistence of Chloroflexus and elemental-sulfur-disproportionating bacteria enables the growth of both strains in the anaerobic photo-dependent conditions in the presence of hydrogen sulfide in vitro. We investigated whether such a relationship also exists in natural environments. From the results of amplicon analysis of BchL and BchX, which are nifH homologs, the communities with high hydrogen sulfide concentration and low oxygen concentration contained almost 100% Chloroflexus aggregans and no C. aurantiacus. On the other hand, in the communities with low hydrogen sulfide concentration and high oxygen concentration, the proportion of C. aurantiacus increased to 2-20%. In such environments, the number of reads of C. aggregans was reduced to 1/3-1/10. OTU closely related to Calcimicrobiu, sulfur disproportionating bacterium, was present at a high rate of 3% to 14% only in the communities with high hydrogen sulfide concentration and low oxygen concentration. The symbiotic relationship between Chloroflexus and elemental-sulfur-disproportionating bacteria was considered to be established in highly anaerobic Chloroflexus communities in natural environments.

5. Diverse abundance and estimated energy metabolism of Archaea in the various microbial communities of Nakabusa hot springs

The presence of Archaea was analyzed in 16 communities from Nakabusa hot springs with various environmental conditions such as temperature, hydrogen sulfide concentration, and oxygen concentration. The ratio of Archaea to all Prokaryotes varied significantly from 0% to 17%. 0% were found in 2 communities, 0.005% to 0.1% in 3 communities, 0.1% to 1% in 7 communities, and 1% to 17% in 4 communities. The Archaea-rich communities were considered to be under ambient reducing conditions. The communities seemed to be older, inside of which were highly reducing conditions. It was considered that there are few Archaea adapted to oxygen respiration in Nakabusa hot springs. The Archaea related to mainly detected OTUs were as follows; Desulfrococcus doing sulfur respiration (H2⇒S0); Sulfophobococcus, whose growth is inhibited by elemental sulfur; Nitrososphaera, which oxidizes ammonia, Archaeoglobus, which reduces sulfate, an autotrophic methanogen related to Methanothermobacter, a heterotrophic methanogen related to Methanosaeta. Of these, Methanothermobacter and Archaeoglobus were also detected in nifH analysis, and Archaeoglobus was also detected in aprA analysis. The finding that the abundance and estimated species of Archaea differed significantly among the communities is important for future studies of the ecology and evolution of Archaea in terrestrial weakly-alkaline hot springs.

6. Distribution of nitrogen-fixing gene nifH in various chemosynthetic and anoxygenic photosynthetic microbial communities in Nakabusa hot springs

Nishihara et al. have previously shown that (1) nitrogen-fixing activity, (2) presence of nitrogen-fixing gene nifH, and (3) isolation of Hydrogenobacter with nitrogen-fixing activity in the chemosynthetic microbial community in the 75°C regions of Nakabusa hot springs. In this study, we analyzed the nitrogen-fixing gene nifH in 16 various chemosynthetic and anoxygenic photosynthetic microbial communities from 62°C to 88°C. Almost no nifH was detected in the community above 80°C. The reason for the no detection may be that the nitrogen-fixing microorganisms are not existing under the temperature in any place, or that the minute amount of ammonia in the source water is enough for the nitrogen requirement. As Nishihara showed, nifH of Hydrogenobacter and Caldicellulosiruptor was detected in the chemosynthetic communities at 75°C to 79°C. In contrast, in highly anaerobic communities containing Chloroflexus between 62°C to 70°C, nifH was mostly that of NItrospira. However, Hydrogenobacter nifH was mainly detected when the surrounding oxygen concentration was high in the same temperature range containing Chloroflexus. From these results, nitrogen fixation in the high-temperature range of Nakabusa hot springs is mainly carried out by Hydrogenobacter under micro to semi aerobic conditions, NItrospira under strongly anaerobic conditions at a temperature less than 70°C, and Caldicellulosiruptor under anaerobic conditions close to microaerobic conditions. Since the abundance of Caldicellulosiruptor was very small in the results of 16S rRNA gene analysis, it is possible that the nifH gene is present in another species due to horizontal gene transfer.

7. Genetic diversity of the gene aprA involved in sulfur disproportionation in Nakabusa hot springs and its relation to temperature adaptation

Sulfur disproportionating bacteria and sulfate-reducing bacteria play important ecological roles in chemosynthetic and anoxygenic photosynthetic microbial communities in Nakabusa hot springs. The amplicon analysis of the gene aprA involved was performed in both desproportionating and reducing reactions. The major aprA genes were estimated mostly to belong to two species closely related to Caldimicrobium, and in addition to species closely related to Thermodesulfobacteria geofontis and a species related to Themodesulfovibrio. (In addition, Arcaeglobus of delta proteobacteria and Archaea was detected.) It was speculated that Caldimicrobium was involved in the sulfur disproportionation reaction, and T. geofontisto and Themodesulfovibrio were involved in sulfate reduction. High genetic diversity was observed in Caldimicrobium, suggesting that both synonymous and non-synonymous substitutions have long been conserved in the communities in Nakabusa. This suggests that sulfur disproportionating bacteria of the genus Caldimicrobium are indispensable members in the weakly alkaline hydrogen-sulfide springs in this temperature range, and the bacterial population has been present in a significant size for many years (hundreds of millions of years?). It was also suggested that non-synonymous permutations might be related to temperature adaptation.

February 18, 2021: Preliminary analyses of nifH and aprA amplicons were almost finished as follows.
A tentative title of nifH analysis is “Temperature and oxygen-concentration dependent distribution of dinitrogenase reductase gene (nifH) in thermophilic chemosynthetic and anoxygenic photosynthetic microbial communities in Nakabusa hot springs.”
1. When the OTU of nifH (partial, 343bp, 114aa) was analyzed using 14 different communities, the main taxa were Hydrogenobacter (29%), Caldicellulosiruptor (29%), and Nitrospira. It was (42%). Almost no nifH was detected in the 86°C communities.
2. Hydrogenobacter and Caldicellulosiruptor were abundantly detected in the chemosynthetic communities at 79°C (site A) and 76°C (site B). Hydrogenobacter seemed microaerobic chemoautotroph, and Caldicellulosiruptor seemed fermentative and grown under anaerobic conditions where the redox potential was not so low.
3. In the mats with well-developed Chloroflexus at 66°C, most of the nitrogen-fixing genes detected were those of Nitrospira. Nitrospira grows in highly anaerobic environments. Nitrospira was not detected above 70°C at all except for the contaminated level.
4. The nifH gene, which is closely related to each of the nitrogen-fixing bacteria Hydrogenobacter sp. 1-6 and Hydrogenobacter sp. 2-18 isolated by Arisa Nishihara, was detected in this study.
5. From these results, it became clear that bacteria containing nitrogen-fixing genes were present in an almost comparable amount in communities at temperatures from 62 ° C to 79 ° C in Nakabusa hot springs under different temperatures, hydrogen-sulfide concentrations, and oxygen concentrations of the microbial communities.
A tentative title of aprA analysis is “High genetic diversity of adenylphosphosulfate reductase in thermophilic chemosynthetic and anoxygenic photosynthetic microbial communities in Nakabusa hot springs.”
1. Using 14 different microbial communities, 160 OTUs of aprA (partial, 367bp, 122aa) were detected, and 65 of them, with more than 0.1% of the total number of reads, were analyzed. Among the 65 OTUs, 5 were Thermomodesulfovibrio, 4 were Thermomodesulfobacterium geofontis, and 56 were 2 groups of Caldimicrobium.
2. Caldimicrobium OTUs have a very high genetic diversity, and its relation with temperature adaptation was speculated. The presence of many synonymous substitution mutations in the same place suggests that the Caldimicrobium population at Nakabusa hot springs has been stable and abundant.
3. There were 14 OTUs considered to be the same species as Caldimicrobium thiodismutans, and the total number of reads accounted for about 35%, and the protein sequence was divided into 5 groups. The same protein sequence clade containing 7 OTUs was obtained mainly from the community at 66 ° C. Another identical protein sequence clade containing four OTUs was obtained from the 76 ° C and 66 ° C communities.
4. There were 42 OTUs, which are considered another species of Caldimicrobium, and the total number of reads accounted for about 50%, and the protein sequence was divided into 9 groups. A same-protein-sequence clade containing 13 OTUs was obtained mainly from the 79°C and 66°C communities. Another same-protein-sequence clade containing 8 OTUs was obtained mostly from the 79°C community. The other same-protein-sequence clade containing 1 OTUs was obtained from the 86°C and 79°C communities. These groups seem adapted to higher temperatures than the group closer to Caldimicrobium thiodismutans.
5. The finding that each clade containing many synonymous substitutions adapts to different temperatures suggests that the intraspecific diversity detected in this study may be related to the adaptation to different temperatures.

February 12, 2021: Some progress in phylogenetic and community analyses of nifH, aprA, and SSU rRNA amplicons.

February 5, 2021: The analysis and reference study of Archaea are continuing. Methanogens seem to be present in some Nakabusa communities. Several OTUs of hyperthermophilic sulfur metabolizing microbes are also present. NifH-like sequence related to a Methanogen was detected. From so far obtained results, Archaea members play important roles in the communities of Nakabusa hot springs in the temperature range of 62C-86C. Relative abundance of Archaea among microbes are diverse among examined 16 communities as follows; 16.646%, 0.669%, 9.390%, 0.410%, 4.103%, 0.116%, 0.005%, 0.000%, 0.491%, 0.840%, 0.000%, 0.309%, 0.388%, 0.326%, 0.042%, 5.334%. Old and complex communities may contain more Archaea populations. In the chemosynthetic communities at Site B at 86 and 79 ℃, microbes of Desulfurococcales are most abundant. In the chemosynthetic communities at Site B at the temperature of 76 ℃, Archaea was very rare, but some were related to Nitrososphaera.

January 29, 2021: DNA amplicon analysis data of nifH gene, aps reductase gene, and SSU gene with Archaea specific primers have arrived last Monday, and I made phylogenetic analyses of those data. I have discussed with Shawn about these analyses for 90 minutes. All three phylogenetic trees show OTU phylogenetic distribution is correlated with the temperature and redox conditions of the communities; 86C, 79C, 76C, and 66C, and 300 to 0 micromol/L of hydrogen sulfide. I compared protein trees of nifH and apsr with DNA trees of the enzymes and found diverse microevolution of the genes have occurred in some clade. We agree the finding is interesting so that some clades are highly adaptive and keep significant populations in Nakabusa hot springs for many years. We also agreed that several papers are worth publishing focusing on specific messages for readers with the data so far obtained from last September. We also talked about a summarized review paper after the several specific papers discussing the whole electron transfers in each community for the understanding various thermophilic microbial communities and the evolution of them.

January 15, 2021: I have talked to Shawn on line about possible papers on Nakabusa microbial communities. A black streamer paper could be possible as a separate paper describing the very simple community structure and the presence of iron-sulfur compounds. Iron-sulfur compounds in other communities may become another paper with data and findings by Shigeru and Mikako with a discussion of ancient pyrite formation. Estimation of oxidation-reduction states of communities and OTUs would become the first paper because the method of the estimation is new and valuable for further studies not only at Nakabusa hot springs but also at various dense microbial communities. The distribution of diverse Archaea in various communities in Nakabusa may become another paper.

January 7, 2021: I have written another research report aimed for a paper tentatively entitled “Iron depositing and sulfide metabolizing black microbial streamers with simple species composition in Nakabusa hot springs.” I talk to Shawn about it, and we agreed to proceed with the study. We also talked about the previous report on the redox of the communities. We may write several papers with data so far obtained and the coming amplicon data. I talked to Shawn that the comparative study of 16 different microbial communities with different redox states has been becoming very informative, something 20 times more than I expected.

December 24, 2020: Shawn was out of the institute. I wrote and sent Shawn a report paper about reduction-oxidation analysis of the communities and species (OTU’s) of Nakabusa hot springs, which I have done in the last few days. The report’s title is “Numerical estimation of reduction-oxidation conditions within diverse and related microbial communities and those of known and unknown species in the communities,” which may become the first outcome of the collaboration with Shawn and Shigeru.

December 17, 2020: I have continued to draw phylogenetic trees of major 7 bacterial phyla and another deeply blanching group of Nakabusa communities. I showed them to Shawn discussed them.

December 10, 2020: I started to draw phylogenetic trees with MEGA. I found some difficulties in using the program and sent questions by email to Dr. Koichiro Tamura, a program developer. He is kind enough to answer the questions, and I started to use the program properly. Interesting results are coming out, and I used most of the time at ELSI. I took lunch with Shawn discussing OTU analysis of amplicon data.

December 3, 2020: I arrived at 12:10 and took lunch with Shawn talking about Fe-sulfur solid compounds associated with microbial streamers in Nakabusa hot springs. I have sent him 10 different streamer samples on November 20 for the analyses of iron compounds. Shigeru Kawai already had preliminary results about the compounds and sent me almost all the information he already had and suggested that we continue the study using the information. I made an English translation of his information, and Shawn has read it and was very interested in it. We discussed possible experiments about iron-sulfur compounds in various streamers, and what kind of papers are possible using expected results. For the currently going community-diversity paper, I would like to add fundamental information about the blackness of the streamer at 86℃, expected to be FeS micro solid. In the next paper, an interesting story from Shigeru may be realized. I asked Shawn to think about the next possible steps of iron stories using Nakabusa microbial communities. Then we moved to ELSI2 building and found the samples I have sent in a freezer. They were in good shape, and Fátima joined to check them. I asked Shawn to start the iron analysis of the samples since I would like to focus on the sequence and phylogenetic analyses for the first paper for a while.

November 13, 2020: I stayed at ELSI from 9:00 to 17:30, but I forgot to write this diary and wrote 2 weeks later. I did mostly desk work reading papers and planning research. I tried to use a spectrophotometer to take absorption spectra of microbial mats with cyanobacteria for a reference of peak wavelengths. The aim is to show the absence of them in Chloroflexus mats and chemosynthetic mats, the absorption spectra of those were taken in September at TMU. However, the machine was used by someone else, and I postponed the measurements for the next opportunity. I took lunch with Shawn on the roof under the sunny sky, talking about amplicon analysis, Fe SEM and X-ray analyses, immigration issues, and entrance examination for graduate school. In the afternoon, Fátima prepared sampling tubes for SEM and X-ray analyses of iron compounds in microbial streamers as well as iron concentration determination for me. Her preparation and explanation were perfect for easy sampling and packaging. I planned to collect the samples at Nakabusa hot springs on Nov. 19-20.

November 5, 2020: Commuting Ikegami line was stopped by accident, and I came through Tamagawa line and Meguro line and was late for 20 minutes. Shawn was waiting outside of the building. He cleaned up the laboratory bench for me and introduced me to laboratory managers. Then we met Fátima in the open space on the 1st floor. She showed me the chemical preparatory space, gas equipment, aseptic bench, and so on. I have got a full image of the preparation of anaerobic growth media. Shawe n came back to talk about iron-sulfur precipitate in chemosynthetic streamers in Nakabusa hot springs. We enjoyed Shigeru and MIkako’s data and planned the next experiments. I will come to Nakabusa 19 and 20 and pick up some samples for iron-sulfur and possible iron-sulfur particle analysis. We will make preparation for the sampling on 13th, Friday, next week. In the afternoon, Sebastian kindly showed me how to use the spectrophotometer, a computer attached to it, and a printer from my computer. Then I measured and printed the spectrum of a leaf from in-front-of the building successfully.

October 29, 2020: This is the first day of my research at ELSI. I met Shawn at the entrance of ELSI at 9:00 and went to the office to get the permission card to use experimental facilities. At first, they said I was not able to get a key card to get into the buildings, but after Shawn ask to issue it we waited a few minutes, and then they came out with a temporary key card and an application form for a personal card which I can get a week later. What a flexible institute! Then Shawn and I discussed my and Shigeru’s data of 16 different microbial communities from Nakabusa hot springs in terms of environmental conditions, absorption spectra, and 16S amplicon sequences for 3 hours in the open space in front of the office. We agreed to start collaboration on this project. Shawn brought two lunch boxes, one for me, and we enjoyed lunch on the roof under the comfortable warm autumn sunlight. I left ELSI at 13:00. My next visit will be on November 5, Thursday, at 9:00.