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Investigation of Formation and Development of Anammox Biofilms by Light, Epifluorescence, and Electron Microscopy

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Abstract—

The stages of formation and development of activated sludge biofilm carrying out the anammox process in a laboratory sequencing batch reactor (SBR) with complete biomass retention on the carrier were investigated using light, epifluorescence, and electron microscopy. Light microscopy revealed biofilm formation on the carrier to occur within one week. Rod-shaped and filamentous microorganisms were the first to attach on the carrier and acted as a skeleton for biofilm formation, thus playing an important role in colonization of the carrier. Epifluorescence microscopy revealed physiologically active anammox bacteria of the genera Candidatus “Brocadia” and Ca. “Jettenia” in the biofilms. Selective autofluorescence of the colonies of anammox bacteria Ca. “Jettenia” was observed. This autofluorescence was probably caused by specific proteins in the extracellular polymeric substances (EPS), their synthesis and/or amount depending on the colony age and the physiological state of the cells. Structural organization of the colonies of anammox bacteria was investigated by electron microscopy. High-throughput sequencing of the 16S rRNA gene fragments revealed the presence of sequences affiliated with members of the phyla Chloroflexi, Bacteroidetes, Planctomycetes, and Proteobacteria in the biofilms. Apart from anammox bacteria, anaerobic and facultatively anaerobic organotrophs, stage I nitrifiers, denitrifiers, and sulfate reducers were detected. Members of the genus Ca. “Brocadia” were predominant among the anammox bacteria, probably due to better adhesion of their cells to the carrier or to their competitive advantage over Ca. “Jettenia” in the presence of organic acids (acetate and formate) in the medium.

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Funding

The work was supported by the Russian Foundation for Basic Research, project no. 18-29-08008 mk) and by the Russian Federation Ministry of Science and Higher Education (State Assignment).

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Authors and Affiliations

  1. Winogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, 119071, Moscow, Russia

    A. Yu. Kallistova, Yu. A. Nikolaev, Yu. Yu. Berestovskaya, V. A. Grachev, N. A. Kostrikina, A. V. Pelevina & N. V. Pimenov

  2. Institute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences, 119071, Moscow, Russia

    A. V. Mardanov & N. V. Ravin

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  1. A. Yu. Kallistova
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  2. Yu. A. Nikolaev
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  3. A. V. Mardanov
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  4. Yu. Yu. Berestovskaya
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  5. V. A. Grachev
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  6. N. A. Kostrikina
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  8. N. V. Ravin
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  9. N. V. Pimenov
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Correspondence to A. Yu. Kallistova.

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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

AUTHORS CONTRIBUTION

AYK, YAN, AVM, NVR, and NVP conceived and designed the experiments and analyzed the results. VAG carried out reactor assembly and startup, maintained the reactor, and analyzed the physicochemical parameters. YYB and AVP carried out light microscopy. NAK performed electron microscopy. AYK conducted epifluorescence microscopy and wrote the paper. AVM and NVR performed molecular genetic analysis. NVP carried out general management. All authors discussed the results and commented on the manuscript.

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Translated by P. Sigalevich

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Kallistova, A.Y., Nikolaev, Y.A., Mardanov, A.V. et al. Investigation of Formation and Development of Anammox Biofilms by Light, Epifluorescence, and Electron Microscopy. Microbiology 89, 708–719 (2020). https://doi.org/10.1134/S0026261720060077

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