Biological N and P removal in activated sludge processes

Biological N and P removal in activated sludge processes Sara Hallin Department of Microbiology, SLU Sara Hallin

Metabolism

Oxidation: KOLFÖRENING KOLDIOXID + ELEKTRONER + VÄTEJONER Reduktion: SYRE + ELEKTRONER + VÄTEJONER VATTEN Fullständig reaktion: KOLFÖRENING + SYRE KOLDIOXID + VATTEN BIOKEMISKT BUNDEN ENERGI

Fermentation: GLUKOS ETANOL + KOLDIOXID BIOKEMISKT BUNDEN ENERGI Fermentation av socker till etanol och koldioxid. En del av kolet i sockret har oxiderats till koldioxid medan en del har reducerats till etanol (vanlig sprit).

Traditionell ASP Organiskt material bryts ner av mikroorganismer i luftningsbassängen. Slammet (biomassa och organiskt material)avskiljs från det renade vattnet i sedimenteringsbassängen..

Microbiological reactions in the N cycle ATMOSPHERE Denitrification N 2 SOIL/WATER Nitrogen fixation N 2 O Organically bound nitrogen orgnh 2 NO Assimilation Mineralization/ Ammonification Assimilation NO 2 NH 4 + NO 2 NO 3 Nitrification Dissimilatory nitrate reduction to ammonium

Kväverening + NH 4 NH NO 2 OH NO NO 2 3 2 NO N 2 O N 2

Nitrification a two step oxidation process Ammonia oxidation Nitrite oxidation NH 3 NH NO 2 OH 2 NO 3

Regulation of nitrous oxide emissions NO 3 NO 2 NO N 2 O N 2 N removal

Nitrification Redox Denitrification Glucose Glykolysis 2 Pyruvate 2 ATP 2 NADH 8 NADH 2 FADH TCA cycle 2 GTP 6 CO 2 NH 4 + NO 2 ATP NADH FADH ATP ATP O 2 H 2 0 NO 3 NO 2 NO N 2 O N 2 +

Ammonia oxidation: NH 3 + 1,5O 2 NO 2 + H + + H 2 O

Nitrite oxidation: NO 2 + ½ O 2 NO 3 Periplams OH

Metabolism

Carbon fixation Cell constituents Growth Lots of ATP needed! NADPH required!

Energetic constraints 1. ATP and NADH (reducing power) requirements in for Cfixation in Calvin cycle 2. NADH formed by reverse e flow: 2e Cyt c e e Cyt c O 2 NAD +

Ammonia Denitrifier oxidizer Diversity diversity in soil Bacteria Eukarya Archaea Ammonia oxidizers

Ammonia oxidizing archaea and bacteria (AOA and AOB), nitrite oxidizing bacteria (NOB) The organisms NH 3 oxidizers Proteobacteria: Nitrosomonas Nitrosococcus Nitrosospira NO 2 oxidizers Bacteria: Nitrobacter Nitrospira Thaumarchaeota: Nitrosopumilis

Screening of a 1215 Mb soil metagenomic library amoa amoa 16S NH 3 NH 2 OH NO 2 Treusch et al. 2005 Env Microbiol 7, 19851995

Highaffinity ammonia oxidation by AOA AOA: Nitrosopumilis maritimus ( ) AOB: Nitrosomonas spp; Nitrosospira spp.( ) nitrification in ocean water ( ) nitrification in soils ( ) lowest K m for ammonium assimilation ( ). (MartensHabbena et al. Nature, 2009)

Kväverening + NH 4 NH NO 2 OH NO NO 2 3 2 NO N 2 O N 2

Microbiological reactions in the N cycle ATMOSPHERE Denitrification N 2 SOIL/WATER Nitrogen fixation N 2 O Organically bound nitrogen orgnh 2 NO Assimilation Mineralization/ Ammonification Assimilation NO 2 NH 4 + NO 2 NO 3 Nitrification Dissimilatory nitrate reduction to ammonium

Denitrification pathway Pathway NO / + 3 = O NO 2 2

Denitrification: anaerobic respiration ATP Organic compound CO 2 Carbon flow Electron flow Biosynthesis NO 3, (NO 2, N 2 O)

Denitrification Cytoplasma NO 2 NO 3 NADH 2 NAD + nar 2e 2e 2enor e NO 3 Periplasma H + Proton motive force nir NO 2 NO NO N 2 O nos N 2 O N 2

Denitrifier diversity Diversity Bacteria Eukarya Archaea Denitrifiers

Nitrifikation Redox Denitrifikation Glucose Glykolysis 2 Pyruvate 2 ATP 2 NADH 8 NADH 2 FADH TCA cycle 2 GTP 6 CO 2 NH 4 + NO 2 ATP NADH FADH ATP ATP O 2 H 2 0 NO 3 NO 2 NO N 2 O N 2 +

Nitrifierande bakterier Denitrifierande bakterier Nitrifierare finns i mark och vatten Denitrifierare finns nästan överallt Bara några få arter Nitrifikation är två energigivande processer som utförs av två olika grupper av bakterier Nitrifierare växer långsamt Många bakteriesläkten Denitrifikation är en alternativ andningsprocess i frånvaro av syre Denitrifierare är växer oftast snabbt

Nitrogen reduction (%) Denitrification rate (mg N 2 ON g 1 VSS h 1 ) Effekt av extern kolkälla på kvävereningen a 100 1. Kvävereningsgrad (%): 2. Denitrifikastionskapacitet: b 15 80 E 60 E 10 40 5 20 R R 0 0 10 20 30 40 50 60 Time (days) Tid (dagar) 0 0 10 20 30 40 50 60 Time (days) Tid (dagar) R = Fördenitrifikation utan extern kolkälla E = Fördenitrifikation med etanoltillsats

Intermittent dosering av etanol i en fördenitrifikationsprocess Hasselblad & Hallin. 1998. Wat. Sci.Technol.

Kväverening + NH 4 NH NO 2 OH NO NO 2 3 2 NO N 2 O N 2

N 2 O producing processes and NO 3 leaching N 2 O N 2 O NO + NH 4 NH 2 OH NO 2 NO 3 NO 2 NO N 2 O N 2 NO 3

D I E T A G R I C U L T U R E E N V I R O N M E N T

World greenhouse gas emissions by sector

Regulation of N 2 O emissions NO 3 NO 2 NO N 2 O N 2

N 2 O/(N 2 O+ N 2 ) Microbes without nosz (N 2 O reductase gene) 1/3 of denitrifier genomes lack nosz (Jones et al. 2008 Molec Biol Evol) 0,8 Manipulation of soil denitrifier community showed direct causality link between the community composition and potential N 2 O emissions. (Philippot et al. 2011 Global Change Biol.) 0,6 0,4 0,2 0 Ratio of N 2 Oproducers

Gene copy ng 1 DNA Gene copy ng 1 DNA ng N Cattle Importance impact of on root the derived denitrifying carbon commmunity Potental denitrification N 2 O/(N 2 O+N 2 ) 500 1500 2500 10 30 50 70 g 1 dry soil h 1 Total bacteria (16S rrna) Proportion of denitrifiers genetically capable to reduce N 2 O (% nosz/16s rdna) Denitrifier genetically capable to reduce N 2 O (nosz) 1. 10 4 2.10 4 3.10 0.5 4 4.10 0.8 4 1.1 1.10 1.4 2 2.10 2 NO 3 NO 2 NO NNO 2 O nosz NO 2 3.10 2 N E Environmental Microbiology (2009) 11(6), 15181526 W S Mapping fieldscale spatial patterns of size and activity of the denitrifier community Low cattle impact 40 m High cattle impact Medium cattle impact Laurent Philippot, 1,2,* Jiri Ćuhel, 3 Nicolas P A Saby, 4 Dominique Chèneby, 1,2 Alicia Chroňáková, 3 David Bru, 1,2 Dominique Arrouays 4, Fabrice Martin Laurent 1,2 and Miloslav Śimek 3 (From L. Philippot, INRA)

Regulation of N 2 O emissions NO 3 NO 2 NO N 2 O N 2

Microbes that only have nosz (N 2 O reductase gene) Some organisms only have nosz and are potential N 2 O sinks. (Graf et al. in prep.)

Biological phosphorus removal ANAEROBIC AEROBIC Short chain fatty acids Energy Phosphate O 2 CO 2 +H 2 O Energy Phosphate Energy consumption for uptake of soluble organics. ATP and PO43 is released. Energy is conserved as polyphosphate granules. Uptake of PO 4 3. Consumption of stored products (PHB).

PHB synthesis & degradation Acetic acid AcetylCoA AcetoacetylCoA Acetoacetate ßhyroxybutyrylCoA ßhydroxybutyrate Polyßhyroxybutyrate (PHB)

Denitrification Biological phosphorus removal Recirculation of NO 3 ANAEROBIC REACTOR AEROBIC REACTOR SEDIMENTATION Organics Energy PO 4 3 O 2 CO 2 +H 2 O Energy PO 4 3

Nremoval in wetlands

Anaerobic zone Constructed wetlands Diffusion through aerenchyma CH 4 O 2 CO 2 CH 4 N 2 O N 2 O 2 Water CO 2 O 2 O 2 +NH 3 NO 3 O 2 NO 3 N 2 O N 2 Aerobic zone NO 3 Reduction zone NO 3 O 2 + NH 3 SO 2 4 Reduction zone CO 2 Reduction zone Acetate CH 4 Root exudates H 2 +CO 2

µg N/g DW/h Plants affect denitrification Ruiz et al., 2009, FEMS Microbiol. Ecol.

Wetland plants effects Rhizospehere Sediment DGGE of nosz Low rates High rates Typha and Fragmites select nosz communities Seasonal differences Ruiz et al., 2009, FEMS Microbiol. Ecol.

Conclusions and outlook Typha and Phragmites select nosz communities Typha and Phragmites increase denitrification activity Seasonal differences Is increase in rhizosphere enough for increased capacity of wetland? What about the abundance of denitrifiers?

Ekeby wetland in Eskilstuna Ekeby Constructed Wetland Total area: 36 ha Flow: ~45000m 3 /day

Waterflow paths Kjellin et al., 2007, Wat. Res.

Nremoval in mining impacted waters