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7th International Conference ORBIT, 2010

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7th International Conference ORBIT 2010, Organic Resources in the Carbon Economy, 29th June to 3rd July 2010 Heraklion Crete, Greece. ...

7th International Conference ORBIT 2010, Organic Resources in the Carbon Economy, 29th June to 3rd July 2010 Heraklion Crete, Greece.
Partly published in: Koplimaa, M.; Menert, A.; Blonskaja, V.; Kurissoo, T.; Zub, S.; Saareleht, M.; Vaarmets, E.; Menert, T. (2010). Liquid and gas chromatographic studies of the anaerobic degradation of baker’s yeast wastewater. Procedia Chemistry, 2(S1), 120 - 129.

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    7th International Conference ORBIT, 2010 7th International Conference ORBIT, 2010 Presentation Transcript

    • Sulfate-dependent anaerobic ammonium oxidation in baker’s yeast wastewaterErgo Rikmann, Anne Menert, Viktoria Blonskaja, Tõnu Kurissoo,Sergei Zub, Toomas Tenno 1
    • Substrate conversion patterns associated with anaerobic digestion 1. Hydrolysis of organic polymers; 2. Fermentation of organic monomers; 3. Oxidation of propionic and butyric acids and alcohols by OHPA; 4. Acetogenic respiration of bicarbonate; 5. Oxidation of propionic and butyric acids and alcohols by SRB and NRB; 6. Oxidation of acetic acid by SRB and NRB; 7. Oxidation of hydrogen by SRB and NRB; 8. Aceticlastic methane fermentation; 9. Methanogenic respiration of bicarbonate OHPA – obligatory hydrogen producing anaerobes SRB – sulfate reducing bacteria NRB – nitrate reducing bacteria (Harper and Pohland, 1987) Anaerobic treatment of high S and high N content wastewaterSoil Micro-organisms as Indicator for the Biological Quality of Soils
    • Simultaneous removal of NH4+-N and SO42− in a methanogenic reactor Using “traditional” wastewater treatment methods, removal of sulphur and nitrogen compounds takes place separately. Anaerobic sulfate reduction is accompanied with formation of toxic and corrosive H2S that may inhibit bioprocesses and damage wastewater treatment apparatus. Biological nitrogen removal cannot be achieved entirely under anaerobic or entirely under aerobic conditions, it needs a combination of aerobic and anaerobic processes.Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 3 S. Zub, T. Tenno
    • Objectives of this study• mutual interactions between sulphur and nitrogen compounds in anaerobic wastewater treatment process;• methylotrophic methanogenesis and mechanism of anaerobic degradation of betaine;• a possible link between sulphate reduction and anaerobic ammonium oxidation (anammox-process) under anaerobic conditions. 4 Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece
    • “Classical” anammox-protsessNH4+ + NO2- → N2 ↑ + 2H2O G0 = -360 kJ/molParticipating bacteria: Brocadia anammoxidans; Kuenenia stuttgartiensis; Scalindua sorokinii; Scalindua brodae; Scalindua wagneri; KSU-1 (Anammoxoglobus propionicus) propionate consuming (Anammoxoglobus sulfate) sulfate consuming A multistep process, NH4+ is oxidized over intermediate products NH2OH and NH2-NH2 formation, by-products small amounts of N2O, NO, NO2. Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 5 T. Kurissoo, S. Zub, T. Tenno
    • Anammox- Anammoxosome microorganisms possess a specific organelle - anammoxosome Kartal, B. et. al., Candidatus ‘‘Anammoxoglobus propionicus’’ a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol. 30 (2007) 39–49Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 6 S. Zub, T. Tenno
    • Thermodynamics of some reactions of ammonia oxidation and sulfate reduction Yang, et. al, 2009Reaction ∆G0 Feasibility in (kJ/mol) man made environmentNH4+ + NO2− → N2↑+ 2H2O −360 feasibleCH4 + SO42− → HS− + HCO3− + H2O −16.6 feasible(anaerobic oxidation of methane)8NH4+ + SO42− →4N2↑+ 3H2S + 12H2O + 5H+ −22 sometimes(at higher ratio of NH4+/ SO42− ) feasible2NH4+ + SO42−→ N2↑ + S0 + 4H2O −45.35 sometimes(at lower ratio of NH4+/ SO42− ) feasible Soil Micro-organisms as Indicator for the Biological Quality of Soils
    • Sulfate-dependent anammox-processFdz.-Polanco 2001: previously unpublished interaction found between SO42− andNH4+ in anaerobic environment2 NH4+ + SO42− → S0 colloid + N2↑ + 4 H2O G0 = - 46 kJ/molTwo-stage process, with formation of nitrite as an intermediate, using sulfate asthe terminal electron acceptor:NH4+ + SO42− → S0 colloid + NO2− + 2 H2O G0 = + 314 kJ/molNH4+ + NO2− → N2↑ + 2 H2O G0 = - 360 kJ/mol The anammox-process was achieved for the first time in a methanogenic reactor simultaneously with methanogenesis at a good performance of the methanogenesis. COD removal was high, biogas contained a significant amount of N2 and formation of S0 (colloidal sulphur) was observed in the liquid phase of the reactor.
    • Biochemical cycle of anorganic sulfur and nitrogen compounds (Zhang 2009)Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 9 T. Kurissoo, S. Zub, T. Tenno
    • Biochemical mechanism of the sulfate-dependant anammox-process:The second (final) stage of the sulfate-dependent anammox-process is the “classical”anammox-reaction: + − NH4 + NO2 → N2 ↑ + 2H2O G0 = - 360 kJ/molThe mechanism of the first stage NH4+ + SO42− → S0 colloid + NO2− + 2 H2O involves severalpossible options:1) An anammox-anammox symbiosis: Anammox-microflora (Planctomycetales) incorporates species that are capable to utilize other terminal electron acceptors than NO2- and NO3-. For example, SO42− (and theoretically Fe3+, Mn4+ etc.). Genetically closely related species may be involved as well In the first stage of the syntrophic chain, Planctomycetales species possessing specific metabolic pathways oxydize NH4+ to NO2−, applying an alternative electron acceptor subsequently, other Planctomycetales species carry out the “classical” anammox- reaction Liu 2008 discovered a new Planctomycete that was named Anammoxoglobus sulfate, able to oxydize NH4+ → NO2-, using SO42− as the terminal electron acceptor .
    • Biochemical mechanism involving anammox-anammox symbiosis: NH4+ + SO42− Anammoxoglobus sulphate, Other possible sulfate-reducing anammox- or anammox-related bacteria S0 colloid + NO2− + H2O “classical” anammox-microflora NH4+ N2↑ + 2H2O
    • Biochemical mechanism involving SRB – anammox symbiosis Anammox-microflora may form syntrophic chains with some species of sulfate- reducing microflora (SRB-bacteria), showing specific metabolic ability to oxydize NH4+ → NO2−. Theoretically, species reducing Fe3+ or Mn4+ may also be an option SRB – anammox symbiosis hypothesis was supported by Yang 2009 No individual species of SRB potentially involved have been specified or identified yet. Both stages take place in a single cell One can not exclude an option that there are Planctomycetes or related species capable to carry out transformations NH4+ → N2 in a single cell using alternative terminal electron acceptors like SO42−, without any assistance from other microorganisms.Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 12 S. Zub, T. Tenno
    • Biochemical mechanism involving SRB NH4+ + SO42- SRB (Hypotetically: Desulfosarcinales, other δ-proteobacterial SRB, other species, with no individual species identified yet) S0 colloid + NO2- + H2O “classical” anammox-microflora NH4+ N2↑ + 2H2O
    • Betaine (N,N,N-trimetylglycine)Fdz-Polanco 2001, making discoveryof the sulfate-dependent anammox-process, was investigating anaerobictreatment of sugar beet vinasse H3Cwastewater. Sugar beet molasses Ohas a characteristic high betainecontent (up to 6%). H3C + NEarlier studies on yeast separation OHwastewater from Salutaguse yeast H3Cfactory have given evidence thatbetaine affects significantly theperfomance of anaerobic wastewatertreatment process (Koplimaa et.al2009).Betaine degrades quickly inanaerobic batch-cultures at pH valuesexceeding 7 (optimal pH value is 7.4). E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 14 Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece S. Zub, T. Tenno
    • Betaine concentration decreased 20-95% as early as in the firstday in anaerobic batch-cultures treating yeast separationwastewater (Koplimaa et.al 2009). -1 Sample Ratio Proportion of Substrate Inoculum Buffer Concentration of betaine, g L inoculum : inoculum in (separation (from anoxic (pH substrate mixture, % water), mL reactor), mL 5,7) Day 0 Day 4 Day Day Day 12 57 120 ST1 1:5 20 240 60 X 3.787 1.907 0.565 0 0 ST2 3:10 30 210 90 X 3.574 1.713 0 0 0 ST3 2:5 40 180 120 X 2.979 0.489 0 0 0 ST4 1:5 20 240 60 0.501 0.197 0 0 0 ST5 3:10 30 210 90 0.229 0 0 0 0 ST6 2:5 40 180 120 0.549 0 0 0 0Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 15 S. Zub, T. Tenno
    • A sign indicating betaine degradation was formation of NH4+ that occurred if pH>6.Halophilic fermentative bacteria (Haloanaerobacter SG 3903T, Moune, 1999)2.5 trimethylglycine + 4.04 H2 → 2-propanol + 2.5 trimethylamine + 0.95 acetate + 0.1 CO2 +1.9 H2Otrimethylglycine + 1.32 serine + H2O → trimethylamine + 2 acetate + 1.32 CO2 + 1.32 NH3Fermentation mediated by Clostridia (Clostridium sporogenes, Naumann, 1983)R- CH(NH2)-COOH + 2 betaine + 2 H2O → R-COOH + CO2 + NH3 + 2 trimethylamine + 2 acetate(R- CH(NH2)-COOH – alanine, valine, leucine or isoleucine)
    • Formation of NH4+ was accompanied by CH4 production Nordic Archaeal Network Meeting 2010, May 20 – 22, 17 Södergarn/Lidingö
    • Acetate and trimethylamine are good carbon and energy sources foracetotrophic methanogens (e.g. Methanobacterium soehngenii) andmethylotrophic methanogens (e.g. Methanosarcina barkeri):CH3COOH → CH4 + CO24 (CH3)3N + 12 H2O → 9 CH4 + 3 CO2 + 6 H2O +4 NH3At the expense of NH4 it is possible to reduce sulfate2 NH4+ + SO42− → S0 colloid + N2↑ + 4 H2O E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 18Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece S. Zub, T. Tenno
    • Effect of betaine on the sulfate-dependent anammox-process and methanogenesisAt least some strains of sulfate-reducing bacteria (SRB) Desulfuromonas are capable to bothhydrolyse and oxydize betaine (Heijthuijsen ,1989) : betaine + 0,5 H2O → 4 (CH3)3N (trimethylamine) + 0,75 CH3COO− + 0,5 CO2 betaine + 2 H+ → TMA + CH3COO− 0,25 CH3COO− + 0,5 H2O → 0,5 CO2 + 2 H+Betaine hydrolysis and fermentative anaerobic oxydation are likely the predominant processesof anaerobic betaine conversion in yeast separation wastewater.Alternative processes and pathways: Fermentation mediated by Clostridia (Clostridium sporogenes Naumann, 1983) R- CH(NH2)-COOH + 2 betaine + 2 H2O → R-COOH + CO2 + NH3 + 2 trimethylamine + 2 acetate, R- CH(NH2)-COOH – alanine, valine, leucine or isoleucine, ) Trimethylamine formation by halophilic fermentative bacteria (Haloanaerobacter SG 3903T, Moune, 1999) 2.5 trimethylglycine + 4.04 H2 → 2-propanol + 2.5 trimethylamine + 0.95 acetate + 0.1 CO2 +1.9 H2O trimethylglycine + 1.32 serine + H2O →trimethylamine + 2 acetate + 1.32 CO2 + 1.32 NH3 Formation of N,N-dimethylglycine accompanied with sulfate reduction mediated by Desulfobacteria (Heijthuijsen ,1989) 4 betaine + 3 SO42- → 4 N,N-dimethylglycine + 4 CO2 + 3 HS- + H+ + 4 H2O
    • Selected substrates and methane producing reactionsReactions ∆G’o (kJ/mol) T (°C)Hydrogenotrophic reactions: CO2 + 4H2 = CH4 + 2H2O -131 35 4CHOO- + 4H+ = CH4 + 3CO2 + 2H2O -144,5 4 (2-propanol) + CO2 = CH4 + 4 acetone + 2H2OAceticlastic reaction: CH3COO- + H+ = CO2 + CH4 -31,0 25Disproportionation reactions: 4CH3OH + 2H2O = 3CH4 + CO2 + 4H2O -319,5 35 4CH3OH + CH3COO- = 4 CH4 + 2 HCO3- + H+ -346 CH3OH + H2 = CH4 + H2O -113 4CH3NH3+ + 3H2O = 3CH4 + HCO3- + 4NH4 + + H+ -225 2 (CH3)2NH2+ + 3H2O = 3CH4 + HCO3- + 2NH4+ + H+ -220 4(CH3)3NH+ + 9 H2O = 9 CH4 + 3HCO3- + 4NH4+ + 3H+ -670 2Dimethyl sulfide + 2H2 = 3CH4 + CO2 + H2S Jones, 1991; Thauer, 1977; Zinder, 1993; Lovley et al., 1983Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 20 T. Kurissoo, S. Zub, T. Tenno
    • Fermentative conversion of betaine may bind asignificant fraction of the sulfate-reducing microbialcommunity,reducing the use of SO42− as an electon acceptor to oxydize organic substrates. Thiswould improve the position of the methanogenetic microflora in competition with the SRBfor available organic substrates.Trimethylamine is an applicable substrate for methanogenic archea from genusMethanosarcina: 4 (CH3)3N + 12 H2O → 9 CH4 + 3 CO2 + 6 H2O + 4 NH3 ↑Methanosarcina has a very versatile metabolism, possessing an ability to utilize a widerange of different substrates. They are able to perform methanogenesis, usingmethylotrophic, acetoclastic and hydrogenotrophic pathways.Occupation of the methylotrophic niche for methanogenesis may help them to achieve acompetitive advantage over sulfate reducing bacteria (SRB ) for substrates available.. The practical output - a more stable and effective perfomance of methane tank.
    • Archea from Tallinn WWTP determined with DGGE 70ºC 85ºC 95ºC 70ºC 85ºC 95ºC 3 14 1 11 4 8 10 15 16 5 17 2 18 6 2 0 9 19 7 12 21 13 22 23 E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 22Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece S. Zub, T. Tenno
    • Genus Methanosarcina, coloured archeal strains – obtained from Tallinn WWTPSoil Micro-organisms as Indicator for the Biological Quality of Soils
    • Archae from the genus MethanosarcinaGenus Methanosarcina, sequences determinedclosiest to species Methanosarcina mazei andMethanosarcina barkeri.Genus Methanosarcina, family Methanosarcinaceae,order Methanosarcinales,class Methanomicrobia,phylum Euryarchaeota. Multicell form of Methanosarcina acetivorans (http://www- 22nd amino acid – genome.wi.mit.edu/annotation/microbes/methanosarcin pyrrolysine from a/background.html) Methanosarcina barkeri Methanosarcinae have the largest genome among archea – the genome of M. acetivorans has 5,751,492 nucleotides (Galagan et al., 2002). Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 24 T. Kurissoo, S. Zub, T. Tenno
    • Methanosarcinae – anaerobic methanogens Methanosarcinae have specific pathway for methane production – methylotrophic methanogenesis using methanol, methylamines and methyltiols for methane production (Galagan et al., 2002). Three pathways of methanogenesis Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 25 T. Kurissoo, S. Zub, T. Tenno
    • Additional amounts of acetate released from betainedegradation facilitate growth of acetoclastic methanogensCH4 formation from intermediates of betaine degradation increses CH4 yield per unit ofCOD utilized (in addition to other above-mentioned mechanisms) CH3COOH → CH4 + CO2NH4+ produced from methanogenic conversion of trimethylamine (TMA) providessubstrate for microorganisms participating in the chain of reactions of sulfate-dependentanammox process. Production of colloidal sulphur instead of H2S reduces the generalinhibiting effect from H2S to the full microbial consortium of the methane reactor. 4(CH3)3NH+ + 9 H2O = 9 CH4 + 3HCO3- + 4NH4+ + 3H+ 2 NH4+ + SO42− → S0 colloid + N2↑ + 4 H2OBetaine may be a key compound to reach and maintain the dynamic equilibrium in ananaerobic reactor in a way that simultaneous progression of methanogenesis and(sulfate-dependent) anammox-process become feasible in the same reactor. Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 26 S. Zub, T. Tenno
    • RUSSIA Salutaguse RUSSIAAS Salutaguse Yeast Factory(Subsidary of Lallemand Inc) 27 Tallinn University of Technology
    • Production parameters of Salutaguse Yeast FactoryFrom 8000 m3 100% beet molasses per year 5000 tons of compressed yeast, that produce 99 000 m3 wastewaters per year.average 270 m3 wastewaters day-1dry matter 152 - 408 g L-1 COD 30000 – 80000 mg O2 L-1 BOD 16500 – 44500 mg O2 L-1 (COD/BOD – 1.5-1.8) Ntotal 3000 - 4000 mg L-1 Ptotal 30 - 90 mg L-1 SO42- 4000 - 12000 mg L-1 (COD/SO42- – 4-8)Incoming loading is comparable to ~50 000 population equivalents Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 28 S. Zub, T. Tenno
    • The wastewater treatment system of AS Salutaguse Yeast Factory Zub, S. Combined treatment of sulfate-rich molasses wastewater from yeast industry. Technology optimization. TUT Press, Tallinn 2007, 136 pp.
    • Bacteria from Salutaguse yeast factory wastewater sludge determined by PCR-DGGE. General eubacterial praimers BacV3f-GC and 907r were used to amplify bacterial 16S rRNA fragmentsST + MQ 3SK 3SK 2SK 2SK 1SK 1SK 9 9 7 7 6 6 5 5 4 4 3 3 2 2 1 1 ST Leuconostoc sp. Leuconostoc garlicum Lactococcus sp. Citrobacter gillenii Pantoea sp. Citrobacter sp. Kluyvera ascorbata
    • Bacteria from Salutaguse yeast factory Aerotank Anaerobic reactor wastewater sludge determined by DGGE Anoxic General eubacterial praimers BacV3f-GC and 907r were reactor 100 bp DNA Ladder used to amplify bacterial 16S rRNA fragments GeneRulerST 3sk 4sk 5sk 1 3 4 5 6 7 8 An R1 3sk 4sk 5sk 1 3 4 5 6 7 8 An R1 ST Porphyromonadaceae sp. Bacteroidetes sp. Cryomorphaceae sp. Planctomycetaceae sp. Thauera sp. Bacilli sp.
    • Bacteria from Salutaguse yeast factory wastewatersludge determined by DGGE (Planctomycetes-specificforward praimer Pla46F and anammox-specific reverse praimerAmx368r were used)ST MQ1 MQ2 1 3 4 5 6 7 8 9 An R1 R2 51 81 R21 ST Nested PCR was used. All 16S rDNA sequences were amplified in the first round with the widerange praimer set 27f and 1492r. The second round of PCR was performed using specific praimers for anammox bacteria: Praimers Pla46f GC and Amx368r Carnobacterium sp. Spirochaetes sp. Verrucomicrobia sp.
    • Position of the anammox-bacteria in the phylogenetic tree Wagner, M, & Horn, M. The Planctomycetes, Verrucomicrobia, Chlamydiae and sister phyla comprise a superphylum with biotechnological and medical relevance. Curr. Op. in Biotechnol. 2006, 17:241–249
    • Acknowledgement The financial support from Estonian Science Foundation (Grant No 5889), Nordic Energy Research (Grant No 06-Hydr-C13) Enterprise Estonia (Grant No EU27358) are gratefully acknowledged. Special thanks to team members: Liis Loorits Jaanus Suurväli Ergo Rikmann Peep Pitk Raivo ViluOrbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 34 T. Kurissoo, S. Zub, T. Tenno
    • Thank you for your attention! 35