Молекулярно-генетическая и морфологическая характеристика Micractinium thermotolerans и M. inermum (Trebouxiophyceae, Chlorophyta) из пирокластических отложений полуострова Камчатка (Россия)

В ходе исследования разнообразия водорослей пирокластических отложений вулканов Шивелуч и Горелый (полуостров Камчатка) были выделены Chlorella-подобные штаммы зеленых водорослей VCA-72 и VCA-93 из проб, отобранных вдоль русла р. Байдарная на вулкане Шивелуч и на выходе термальных паров по краю кальдеры на южном склоне вулкана Горелый в 2018 и 2020 гг. соответственно. Идентификация штаммов выполнялась в рамках комплексного подхода микроскопическими и молекулярно-генетическими методами, включающими предварительную идентификацию, получение нуклеотидных последовательностей малой субъединицы и внутреннего транскрибируемого спейсера рРНК, построение филогенетических деревьев и вторичных структур участков ITS1 и ITS2 рРНК. На филогенетическом древе штамм VCA-93 кластеризовался в видовой кладе Micractinium thermotolerans. При сравнении моделей спиральных доменов ITS1 и ITS2 у M. thermotolerans различий не выявлено. Штамм VCA-72 занимал базальное положение в кладе M. inermum. Модели вторичной структуры спиралей штамма VCA-72 в целом были сходны с моделями для M. inermum, однако отмечена внутривидовая вариабельность, обусловленная в основном заменами в верхушечных и боковых петлях. В спиральных участках исследуемых штаммов M. inermum обнаружено пять замен hCBC, тогда как замен CBC обнаружено не было. Детальный анализ морфологии и жизненного цикла позволил выявить в стареющих культурах клетки, которые по размеру значительно превышают вегетативные и имеют грушевидную, овальную и эллипсоидную формы с неглубоким широким сужением в центре. Также в стареющих культурах обоих видов были выявлены клетки с бесцветными липидными каплями. Способность синтезировать и накапливать липиды говорит о большом потенциале штаммов для производства биодизельного топлива. Обзор местообитаний предыдущих и новых находок позволяет сделать вывод об экологической пластичности исследуемых видов. Полученные результаты дополняют сведения о биогеографии видов: M. inermum обнаружен впервые на территории России, а M. thermotolerans – на полуострове Камчатка.

1. Abdullin Sh.R., Nikulin A.Yu., Bagmet V.B., Nikulin V.Yu., Goncharov A.A. New cyanobacterium Aliterella vladivostokensis sp. nov. (Aliterellaceae, Chroococcidiopsidales), isolated from temperate monsoon climate zone (Vladivostok, Russia). Phytotaxa. 2021;517: 221-233. https://doi.org/10.11646/phytotaxa.527.3.7

2. Abou-Shanab R.A.I., El-Dalatony M.M., El-Sheekh M.M. Cultivation of a new microalga, Micractinium reisseri, in municipal wastewater for nutrient removal, biomass, lipid, and fatty acid production. Biotechnol. Bioprocess Eng. 2014;19:510-518. https://doi.org/10.1007/s12257- 013-0485-z

3. Akaike H. A new look at the statistical model identification. IEEE Trans. Autom. Control. 1974;19:716-723. https://doi.org/10.1109/TAC.1974.1100705

4. Andersen R.A. Algal Culturing Techniques. New York: Elsevier Acad. Press, 2005

5. Andreyeva V.M. Soil and Aerophilic Green Algae (Chlorophyta: Tetrasporales, Chlorococcales, Chlorosarcinales). St. Petersburg: Nauka Publ., 1998 (in Russian)

6. Banskota A.H., Hui J.P.M., Jones A., McGinn P.J. Characterization of neutral lipids of the oleaginous alga Micractinum inermum. Molecules. 2024;29:359. https://doi.org/10.3390/molecules29020359

7. Bonfield J.K., Smith K.F., Staden R. A new DNA sequence assembly program. Nucleic Acids Res. 1995;23(24):4992-4999. https://doi.org/10.1093/nar/23.24.4992

8. Bouarab L., Dauta A., Loudiki M. Heterotrophic and mixotrophic growth of Micractinium pusillum Fresenius in the presence of acetate and glucose: effect of light and acetate gradient concentration. Water Res. 2004;38(11):2706-2712. https://doi.org/10.1016/j.watres.2004.03.021

9. Caisová L., Marin B., Melkonian M. A consensus secondary structure of ITS2 in the Chlorophyta identified by phylogenetic reconstruction. Protist. 2013;164(4):482-496. https://doi.org/10.1016/j.protis.2013.04.005

10. Chae H., Lim S., Kim H.S., Choi H.G., Kim J.H. Morphology and phylogenetic relationships of Micractinium (Chlorellaceae, Trebouxiophyceae) taxa, including three new species from Antarctica. Algae. 2019;34(4):267-275. https://doi.org/10.4490/algae.2019.34.10.15

11. Coleman A.W. The significance of a coincidence between evolutionary landmarks found in mating affinity and a DNA sequence. Protist. 2000;151(1):1-9. https://doi.org/10.1078/1434-4610-00002

12. Coleman A.W. Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure. Trends Genet. 2015;31(3):157- 163. https://doi.org/10.1016/j.tig.2015.01.002

13. Darienko T., Pröschold T. Reevaluation and discovery of new species of the rare genus Watanabea and establishment of Massjukichlorella gen. nov. (Trebouxiophyceae, Chlorophyta) using an integrative approach. J. Phycol. 2019;55:493-499. https://doi.org/10.1111/jpy.12830

14. Darriba D., Taboada G., Doallo R., Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods. 2012;9: 772. https://doi.org/10.1038/nmeth.2109

15. Darty K., Denise A., Ponty Y. VARNA: interactive drawing and editing of the RNA secondary structure. Bioinformatics. 2009;25:1974- 1975. https://doi.org/10.1093/bioinformatics/btp250

16. Dickinson K.E., Lalonde C.G., McGinn P.J. Effects of spectral light quality and carbon dioxide on the physiology of Micractinium inermum: growth, photosynthesis, and biochemical composition. J. Appl. Phycol. 2019;31:3385-3396. https://doi.org/10.1007/s10811-019-01880-z

17. Echt C.S., Erdahl L.A., McCoy T.J. Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa. Genome. 1992;35(1):84-87. https://doi.org/10.1139/g92-014

18. Fernandes B., Teixeira J., Dragone G., Vicente A.A., Kawano S., Bišová K., Vítová M. Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri. Bioresour. Technol. 2013;144:268-274. https://doi.org/10.1016/j.biortech.2013.06.096

19. Fresenius G. Beiträge zur Kenntniss mikrokopischer Organismen. Abh. Senckenberg. Naturforsch. Ges. 1858;2(2):211-242. https://doi.org/10.5962/bhl.title.2137

20. Galtier N., Gouy M., Gautier C. Seaview and phylo-win: two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci. 1996;12:543-548. https://doi.org/10.1093/bioinformatics/12.6.543

21. Ganuza E., Sellers C.E., Bennett B.W., Carney L.T. A novel treatment protects Chlorella at commercial scale from the predatory bacterium Vampirovibrio chlorellavorus. Front. Microbiol. 2016;7:188348. https://doi.org/10.3389/fmicb.2016.00848

22. Goka K., Yokoyama J., Une Y., Kuroki T., Suzuki K., Nakahara M., Kobayashi A., Inaba S., Mizutani T., Hyatt A.D. Amphibian chytridiomycosis in Japan: distribution, haplotypes and possible route of entry into Japan. Mol. Ecol. 2009;18(23):4757-4774. https://doi.org/10.1111/j.1365-294X.2009.04384.x

23. Gollerbah M.M., Shtina E.A. Soil Algae. St. Petersburg: Nauka Publ., 1969 (in Russian)

24. Guiry M.D., Guiry G.M. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. (Cited on April 15, 2024). Available from: http://www.algaebase.org

25. Ho S.H., Chen C.Y., Chang J.S. Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N. Bioresour. Technol. 2012;113:244-252. https://doi.org/10.1016/j.biortech.2011.11.133

26. Hoef-Emden K., Melkonian M. Revision of the genus Cryptomonas (Cryptophyceae): a combination of molecular phylogeny and mor- phology provides insights into a long-hidden dimorphism. Protist. 2003;154:371-409. https://doi.org/10.1078/143446103322454130

27. Hong J.W., Jo S.-W., Cho H.-W., Nam S.W., Shin W., Park K.M., Lee K.I., Yoon H.-S. Phylogeny, morphology, and physiology of Micractinium strains isolated from shallow ephemeral freshwater in Antarctica. Phycol. Res. 2015;63:212-218. https://doi.org/10.1111/pre.12097

28. Hoshina R., Fujiwara Y. Molecular characterization of Chlorella cultures of the National Institute for Environmental Studies culture collection with description of Micractinium inermum sp. nov., Didymogenes sphaerica sp. nov., and Didymogenes soliella sp. nov. (Chlorellaceae, Trebouxiophyceae). Phycol. Res. 2013;31:124-132. https://doi.org/10.1111/pre.12010

29. Huelsenbeck J.P., Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001;17:754-755. https://doi.org/10.1093/bioinformatics/17.8.754

30. Kim I., Yang H.-M., Park C.W., Yoon I.-H., Seo B.-K., Kim E.-K., Ryu B.-G. Removal of radioactive cesium from an aqueous solution via bioaccumulation by microalgae and magnetic separation. Sci. Rep. 2019;9:10149. https://doi.org/10.1038/s41598-019-46586-x

31. Knothe G. Improving biodiesel fuel properties by modifying fatty ester composition. Energy Environ. Sci. 2009;2:2759-2766. https://doi.org/10.1039/B903941D

32. Komárek J., Fott B. Chlorophyceae (Grünalgen). Ordnung Chlorococcales. In: Huber-Pestalozzi G. (Ed.) Das Phytoplankton des Süsswassers. 7. Teil. 1. Stuttgart: Schweizerbart‘sche Verlagsbuchhandlung, 1983;1-1044

33. Komárek J., Kaštovský J., Mareš J., Johansen J.R. Taxonomic classification of cyanoprokaryotes (cyanobacterial genera), using a polyphasic approach. Preslia. 2014;86(4):295-335

34. Kozlov A.M., Darriba D., Flouri T., Morel B., Stamatakis A. RAxMLNG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35:4453-4455. https://doi.org/10.1093/bioinformatics/btz305

35. Krivina E., Sinetova M., Savchenko T., Degtyaryov E., Tebina E., Temraleeva A. Micractinium lacustre and M. thermotolerans spp. nov. (Trebouxiophyceae, Chlorophyta): taxonomy, temperature-dependent growth, photosynthetic characteristics and fatty acid composition. Algal Res. 2023;71:103042. https://doi.org/10.1016/j.algal.2023.103042

36. López-García P., Philippe H., Gail F., Moreira D. Autochthonous eukaryotic diversity in hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic Ridge. Proc. Natl. Acad. Sci. USA. 2003;100:697-702. https://doi.org/10.1073/pnas.0235779100

37. Luo W., Krienitz L., Pflugmacher S., Walz N. Genus and species concept in Chlorella and Micractinium (Chlorophyta, Chlorellaceae): genotype versus phenotypical variability under ecosystem conditions. SIL Proceedings. 2005;29(1):170-173. https://doi.org/10.1080/03680770.2005.11901988

38. Luo W., Pflugmacher S., Pröschold T., Walz N., Krienitz L. Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae). Protist. 2006;157:315-333. https://doi.org/10.1016/j.protis.2006.05.006

39. Marin B., Klingberg M., Melkonian M. Phylogenetic relationships among the Cryptophyta: analyses of nuclear-encoded SSU rRNA sequences support the monophyly of extant plastid-containing lineages. Protist. 1998;149:265-276. https://doi.org/10.1016/S1434-4610(98)70033-1

40. Marin B., Palm A., Klingberg M., Melkonian M. Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure. Protist. 2003;154:99-145. https://doi.org/10.1078/143446103764928521

41. McFadden G.I., Melkonian M. Use of Hepes buffer for microalgal culture media and fixation for electron microscopy. Phycologia. 1986; 25:551-557. https://doi.org/10.2216/i0031-8884-25-4-551.1

42. Mikhailyuk T., Lukešová A., Glaser K., Holzinger A., Obwegeser S., Nyporko S., Friedl T., Karsten U. New taxa of streptophyte algae (Streptophyta) from terrestrial habitats revealed using an integrative approach. Protist. 2018;169:406-431. https://doi.org/10.1016/j.protis.2018.03.002

43. Nikulin V.Yu., Nikulin A.Yu., Gontcharov A.A., Bagmet V.B., Abdullin Sh.R. Oogamochlamys kurilensis sp. nov. (Chlorophyta, Volvocales) from the soils of Iturup Island (Sakhalin Region, Russia). Plants. 2023;12:3350. https://doi.org/10.3390/plants12193350

44. Onay M., Sonmez C.A., Oktem H., Yücel M. Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of Central Anatolia. Bioresour. Technol. 2014;169:62-71. https://doi.org/10.1016/j.biortech.2014.06.078

45. Ota S., Morita A., Ohnuki S., Hirata A., Sekida S., Okuda K., Ohya Y., Kawano S. Carotenoid dynamics and lipid droplet containing astaxanthin in response to light in the green alga Haematococcus pluvialis. Sci. Rep. 2018;8:5617. https://doi.org/10.1038/s41598-018-23854-w

46. Park S., Kim J., Yoon Y., Park Y., Lee T. Blending water-and nutrientsource wastewaters for cost-effective cultivation of high lipid content microalgal species Micractinium inermum NLP-F014. Bioresourc. Technol. 2015;198:388-394. https://doi.org/10.1016/j.biortech.2015.09.038

47. Piligaev A.V., Sorokina K.N., Shashkov M.V., Parmon V.N. Screening and comparative metabolic profiling of high lipid content microalgae strains for application in wastewater treatment. Bioresour. Technol. 2018;250:538-547. https://doi.org/10.1016/j.biortech.2017.11.063

48. Pröschold T., Darienko T., Silva P.C., Reisser W., Krienitz L. The systematics of “Zoochlorella” revisited employing an integrative approach. Environ. Microbiol. 2011;13:350-364. https://doi.org/10.1111/j.1462-2920.2010.02333.x

49. Quintas-Nunes F., Brandão P.R., Barreto Crespo M.T., Glick B.R., Nascimento F.X. Plant growth promotion, phytohormone production and genomics of the rhizosphere-associated microalga, Micractinium rhizosphaerae sp. nov. Plants. 2023;12:651. https://doi.org/10.3390/ plants12030651

50. Rambaut A. FigTree v.1.4.4. 2018. http://tree.bio.ed.ac.uk/software/figtree (Access date: 01.03.2024)

51. Rambaut A., Drummond A.J., Xie D., Baele G., Suchard M.A. Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 2018;67:901-904. https://doi.org/10.1093/sysbio/syy032

52. Roleda M.Y., Slocombe S.P., Leakey R.J., Day J.G., Bell E.M., Stanley M.S. Effects of temperature and nutrient regimes on biomass and lipid production by six oleaginous microalgae in batch culture employing a two-phase cultivation strategy. Bioresour. Technol. 2013; 129:439-449. https://doi.org/10.1016/j.biortech.2012.11.043

53. Schlӧsser U.G. Additions to the Culture collection of algae since 1994. Bot. Acta. 1997;110:424-429. https://doi.org/10.1111/j.1438-8677.1997.tb00659.x

54. Shi M., Wei H., Chen Q., Wang X., Zhou W., Liu J. Exploring an isolate of the oleaginous alga Micractinium inermum for lipid production: molecular characterization and physiochemical analysis under multiple growth conditions. J. Appl. Phycol. 2019;31:1035-1046. https://doi.org/10.1007/s10811-018-1653-5

55. Smith R.T., Bangert K., Wilkinson S.J., Gilmour D.J. Synergistic carbon metabolism in a fast growing mixotrophic freshwater microalgal species Micractinium inermum. Biomass Bioenergy. 2015;82:73-86. https://doi.org/10.1016/j.biombioe.2015.04.023

56. Stamatakis A., Hoover P., Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst. Biol. 2008;57:758-771. https://doi.org/10.1080/10635150802429642

57. Starr R.C., Zeikus J.A. UTEX - the culture collection of algae at the University of Texas at Austin. 1993 list of cultures. J. Phycol. 1993;29:1-106. https://doi.org/10.1111/j.0022-3646.1993.00001.x

58. Sydney T., Marshall-Thompson J.-A., Kapoore R.V., Vaidyanathan S., Pandhal J., Fairclough J.P.A. The effect of high-intensity ultraviolet light to elicit microalgal cell lysis and enhance lipid extraction. Metabolites. 2018;8:65. https://doi.org/10.3390/metabo8040065

59. Weiss R.L. Fine structure of the snow alga (Chlamydomonas nivalis) and associated bacteria. J. Phycol. 1983;19:200-204. https://doi.org/10.1111/j.0022-3646.1983.00200.x

60. White T.J., Bruns T.D., Lee S.B., Taylor J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols - A Guide to Methods and Application. San Diego, CA, USA: Acad. Press, 1990;315-322

61. Wijffels R.H., Barbosa M.J. An outlook on microalgal biofuels. Science. 2010;329:796-799. https://doi.org/10.1126/science.1189003

62. Wolf M., Chen S., Song J., Ankenbrand M., Müller T. Compensatory base changes in ITS2 secondary structures correlate with the biological species concept despite intragenomic variability in ITS2 sequences - a proof of concept. PLoS One. 2013;8(6):e66726. https://doi.org/10.1371/journal.pone.0066726

63. Zhan J., Hong Y., Hu H. Effects of nitrogen sources and C/N ratios on the lipid producing potential of Chlorella sp. HQ. J. Microbiol. Biotechnol. 2016;26:1290-1302. https://doi.org/10.4014/jmb.1512.12074

64. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31(13):3406-3415. https://doi.org/10.1093/nar/gkg595.