Abstract
Two activated sludge cultures, seeded with activated sludge from the same source, were cultivated for 370 days in synthetic wastewater. Both cultures were transferred weekly to fresh medium; one culture was operated at high dissolved oxygen (DO) (near saturation) and the other at low DO (0.25 mg O2/L). There were significant changes in the abundances of bacterial species and phyla present in each culture throughout the 370-day operational period. In the low DO culture, over time, there was a continuously increasing proportion of cells of species known to encode truncated hemoglobins (Hbs). These are the types of Hbs which may enhance delivery of oxygen to the respiratory chain, to enhance ATP production, especially under low aeration conditions. The levels of heme b, the heme found in Vitreoscilla hemoglobin, increased in parallel to the increase in Hb-encoding species, to much higher levels in the low DO culture than in the high DO culture. Specific oxygen uptake rates increased by 3 % for the high DO culture near the end of the 370-day period, while those for the low DO culture increased steadily to a level 28 % higher than that of the starting culture. Thus, imposition of low DO conditions may, due to selection for Hb-expressing species, be useful in developing bacterial communities with enhanced ability to function efficiently in aerobic wastewater treatment, especially under low aeration conditions.
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Arnaldos M, Kunkel SA, Stark BC, Pagilla KR (2013) Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions. Appl Microbiol Biotechnol 97:10211–10221
Arnaldos M, Kunkel SA, Stark BC, Pagilla KR (2014) Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions. Appl Microbiol Biotechnol 98:3231–3239
Arnaldos M, Pagilla KR (2014) Implementation of a demand-side approach to reduce aeration requirements of activated sludge systems: directed acclimation of biomass and its effect at the process level. Water Res 62:147–155
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2003) Introduction of plasmid DNA into cells. Curr Protoc Mol Biol 37:1.8.1–1.8.2. John Wiley & Sons, Inc., New York
Fish PA, Webster DA, Stark BC (2000) Vitreoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo. J Molec Catalysis B: Enzymatic 9:75–82
Forrester MT, Eyler CE, Rich JN (2011) Bacterial flavohemoglobin: a molecular tool to probe mammalian nitric oxide biology. BioTechniques 50:41–45
Frey AD, Kallio PT (2003) Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology. FEMS Microbiol Rev 27:525–545
Jenkins D, Richard MG, Daigger GT (2004) Manual on the causes and control of activated sludge bulking, foaming, and other solids separation problems. IWA Publishing, London
Kumar A, Nag M, Basak S (2013) Truncated or 2/2 hemoglobins: a new class of globins with novel structure and function. J Proteins Proteomics 4:45–64
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 82:6955–6959
Law Y, Ye L, Pan Y, Yuan Z (2012) Nitrous oxide emissions from wastewater treatment processes. Philo Transact Roy Soc Biolog 367:1265–1277
Lin JM, Stark BC, Webster DA (2003) Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (DNT) dioxygenase activity of Burkholderia and on DNT degradation in two-phase bioreactors. J Indust Microbiol Biotechnol 30:362–368
Muyzer G, Dewaal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S ribosomal RNA. Appl Environ Microbiol 59:695–700
Pagilla KR, Sattayatewa C, Urgun-Demirtas M, Baek S (2011) Effect of influent N speciation on organic nitrogen occurrence in activated sludge process effluents. Water Env Res 83:761–766
Park KW, Kim KJ, Howard AJ, Stark BC, Webster DA (2002) Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases. J Biol Chem 277:33334–33337
Pathania R, Navani NK, Rajamohan G, Dikshit KL (2002) Mycobacterium tuberculosis hemoglobin HbO associates with membranes and stimulates cellular respiration of recombinant Escherichia coli. J Biol Chem 277:15293–15302
Ramandeep HK-W, Raje M, Kim KJ, Stark BC, Dikshit KL, Webster DA (2001) Vitreoscilla hemoglobin: intracellular localization and binding to membranes. J Biol Chem 276:24781–24789
Reedy CJ, Elvekrog MM, Gibney BR (2008) Development of a heme protein structure-electrochemical function database. Nucl Acids Res 36:D307–D313
Rittmann BE, McCarty PL (2001) Environmental biotechnology: principles and applications. McGraw-Hill, New York
Rosso D, Stenstrom MK, Larson LE (2008) Aeration of large-scale municipal wastewater treatment plants: state of the art. Water Sci Technol 57:973–978
Stark B, Pagilla KR, Dikshit KL (2015) Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation. Appl Microbiol Biotechnol 99:1627–1636
Vinogradov SN, Hoogewijs D, Bailey X, Arredondo-Peter R, Gough J, Dewilde S, Moens L, Vanfleteren JR (2006) A phylogenomic profile of globins. BMC Evol Biol 6:31
Wainwright LM, Elvers KT, Park SF, Poole RK (2005) A truncated haemoglobin implicated in oxygen metabolism by the microaerophilic food-borne pathogen Campylobacter jejuni. Microbiology 151:4079–4091
Wainwright LM, Wang Y, Park SF, Yeh S-R, Poole RK (2006) Purification and spectroscopic characterization of Ctb, a group III truncated hemoglobin implicated in oxygen metabolism in the food-borne pathogen Campylobacter jejuni. Biochemistry 45:6003–6011
Webster DA (1987) Structure and function of bacterial hemoglobin and related proteins. In: Eichorn GC, Marzilli LG (eds) Advances in inorganic biochemistry, Vol. 7. Elsevier, New York, pp. 245–265
Winkler MKH, Kleerebezem R, de Bruin L, Verheijen PJT, Abbas BA, Habermacher JM (2013) Microbial diversity differences within aerobic granular sludge and activated sludge flocs. Appl Microbiol Biotechnol 97:7447–7458
Wittenberg JB, Bolognesi M, Wittenberg BA, Guertin M (2002) Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes, and plants. J Biol Chem 277:871–874
Yamanaka T (1992) The biochemistry of bacterial cytochromes. Japan Scientific Societies-Springer, Tokyo
Yeh DC, Thorsteinsson MV, Bevan DR, Potts M, Ja Mar GN (2000) Solution 1H NMR study of the heme cavity and folding topology of the abbreviated chain 118-residue globin from the Cyanobacterium Nostoc commune. Biochemistry 39:1389–1399.
Zhang L, Li Y, Wang Z, Xia Y, Chen W, Tang K (2007) Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnol Adv 25:123–136
Acknowledgments
We would like to thank the Stickney WWTP for providing access to their aeration tanks for sampling of activated sludge and Dr. Marina Arnaldos for her expertise, which greatly aided the starting of this project.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
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The authors declare that they have no competing interests.
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Kunkel, S.A., Pagilla, K.R. & Stark, B.C. Directed evolution to produce sludge communities with improved oxygen uptake abilities. Appl Microbiol Biotechnol 99, 10725–10734 (2015). https://doi.org/10.1007/s00253-015-6891-8
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DOI: https://doi.org/10.1007/s00253-015-6891-8