Investigation of Formation and Development of Anammox Biofilms by Light, Epifluorescence, and Electron Microscopy
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RIMENTAL ARTICLES
Investigation of Formation and Development of Anammox Biofilms by Light, Epifluorescence, and Electron Microscopy A. Yu. Kallistovaa, *, Yu. A. Nikolaeva, A. V. Mardanovb, Yu. Yu. Berestovskayaa, V. A. Gracheva, N. A. Kostrikinaa, A. V. Pelevinaa, N. V. Ravinb, and N. V. Pimenova a
Winogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, 119071 Russia b Institute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, 119071 Russia *e-mail: [email protected] Received June 15, 2020; revised July 6, 2020; accepted July 7, 2020
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. Keywords: anammox bacteria, Ca. “Brocadia,” Ca. “Jettenia,” microscopy methods, autofluorescence, acetate, formate DOI: 10.1134/S0026261720060077
Anammox bacteria, planctomycetes of the order Ca. “Brocadiales,” play an important role in the global nitrogen cycle and are used in biotechnology for ammonium removal from wastewater. These bacteria carry out anaerobic oxidation of ammonium with nitrite to dinitrogen gas, which is coupled to production of small amounts of nitrate (Strous et al., 1998). Anammox bacteria are also capable of anaerobic oxidation of such volatile fatty acids (VFA) as formate, acetate, and propionate, coupled to dissimilatory nitrate reduction. In the course of th
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