title: Floc Formation Reduces the pH Stress Experienced by Microorganisms Living in Alkaline Environments.
abstract: The survival of microorganisms within a cementitious geological disposal facility for radioactive wastes heavily depends on their ability to survive the calcium-dominated, hyperalkaline conditions resulting from the dissolution of the cementitious materials. The results from this study show that the formation of flocs, composed of a complex mixture of extracellular polymeric substances (EPS), provides protection against alkaline pH values up to 13.0. The flocs were dominated byAlishewanellaandDietziaspp., producing a mannose-rich carbohydrate fraction incorporating extracellular DNA, resulting in Ca2+sequestration. EPS provided a ∼10-μm thick layer around the cells within the center of the flocs, which were capable of growth at pH values of 11.0 and 11.5, maintaining internal pH values of 10.4 and 10.7, respectively. Microorganisms survived at a pH of 12.0, where an internal floc pH of 11.6 was observed, as was a reduced associated biomass. We observed limited floc survival (<2 weeks) at a pH of 13.0. This study demonstrates that flocs maintain lower internal pHs in response to the hyperalkaline conditions expected to occur within a cementitious geological disposal facility for radioactive wastes and indicates that floc communities within such a facility can survive at pHs up to 12.0.IMPORTANCEThe role of extracellular polymeric substances (EPS) in the survival of microorganisms in hyperalkaline conditions is poorly understood. Here, we present the taxonomy, morphology, and chemical characteristics of an EPS-based microbial floc, formed by a consortium isolated from an anthropogenic hyperalkaline site. Short-term (<2 weeks) survival of the flocs at a pH of 13 was observed, with indefinite survival observed at a pH of 12.0. Measurements from micro-pH electrodes (10-μm-diameter tip) demonstrated that flocs maintain lower internal pHs in response to hyperalkaline conditions (pH 11.0, 11.5, and 12.0), demonstrating that floc formation and EPS production are survival strategies under hyperalkaline conditions. The results indicate how microbial communities may survive and propagate within the hyperalkaline environment that is expected to prevail in a cementitious geological disposal facility for radioactive wastes; the results are also relevant to the wider extremophile community.
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom.
authors.forename: C J
authors.initials: CJ
authors.lastname: Charles
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom.
authors.forename: S P
authors.initials: SP
authors.lastname: Rout
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom.
authors.forename: K A
authors.initials: KA
authors.lastname: Patel
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom.
authors.forename: S
authors.initials: S
authors.lastname: Akbar
authors.affiliation: Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom.
authors.forename: A P
authors.initials: AP
authors.lastname: Laws
authors.affiliation: Bio-imaging Facility, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: B R
authors.initials: BR
authors.lastname: Jackson
authors.affiliation: Bio-imaging Facility, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: S A
authors.initials: SA
authors.lastname: Boxall
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom p.n.humphreys@hud.ac.uk.
authors.forename: P N
authors.initials: PN
authors.lastname: Humphreys
mesh_terms: D039903:Actinobacteria / Q000302:isolation & purification / Q000378:metabolism*; D034241:Alteromonadaceae / Q000302:isolation & purification / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development*; D006863:Hydrogen-Ion Concentration; D011108:Polymers / Q000378:metabolism; D011850:Radioactive Waste; D012722:Sewage / Q000737:chemistry*; D014865:Waste Disposal, Fluid
chemical_list: D011108:Polymers; D011850:Radioactive Waste; D012722:Sewage
keywords: Alishewanella; Dietzia; GDF; ILW; alkaliphile; alkaliphilic; biofilms; floc; flocculate; radioactive