The document discusses sulfate-dependent anaerobic ammonium oxidation (anammox) in wastewater from a baker's yeast factory. It suggests that anammox bacteria can use sulfate as a terminal electron acceptor to oxidize ammonium to nitrite, followed by the standard anammox reaction of oxidizing ammonium and nitrite to nitrogen gas. This two-step anammox process was observed in a methanogenic reactor treating high-sulfate, high-nitrogen wastewater. The document also examines how the organic compound betaine, present in yeast wastewater, may influence sulfate reduction and methanogenesis during anaerobic treatment.

1. Sulfate-dependent anaerobic
ammonium oxidation in baker’s
yeast wastewater
Ergo Rikmann, Anne Menert, Viktoria Blonskaja, Tõnu Kurissoo,
Sergei Zub, Toomas Tenno
1

2. 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
wastewater
Soil Micro-organisms as Indicator for the Biological Quality of
Soils

3. 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

4. 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

5. “Classical” anammox-protsess
NH4+ + NO2- → N2 ↑ + 2H2O G0 = -360 kJ/mol
Participating 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

6. 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)
39–49
Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 6
S. Zub, T. Tenno

7. Thermodynamics of some reactions of
ammonia oxidation and sulfate reduction
Yang, et. al, 2009
Reaction ∆G0 Feasibility in
(kJ/mol) man made
environment
NH4+ + NO2− → N2↑+ 2H2O −360 feasible
CH4 + 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− ) feasible
2NH4+ + 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

8. Sulfate-dependent anammox-process
Fdz.-Polanco 2001: previously unpublished interaction found between SO42− and
NH4+ in anaerobic environment
2 NH4+ + SO42− → S0 colloid + N2↑ + 4 H2O G0 = - 46 kJ/mol
Two-stage process, with formation of nitrite as an intermediate, using sulfate as
the terminal electron acceptor:
NH4+ + SO42− → S0 colloid + NO2− + 2 H2O G0 = + 314 kJ/mol
NH4+ + 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.

9. 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

10. 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/mol
The mechanism of the first stage NH4+ + SO42− → S0 colloid + NO2− + 2 H2O involves several
possible 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 .

11. 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

12. 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

13. 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

14. Betaine (N,N,N-trimetylglycine)
Fdz-Polanco 2001, making discovery
of the sulfate-dependent anammox-
process, was investigating anaerobic
treatment of sugar beet vinasse H3C
wastewater. Sugar beet molasses O
has a characteristic high betaine
content (up to 6%). H3C +
N
Earlier studies on yeast separation OH
wastewater from Salutaguse yeast H3C
factory have given evidence that
betaine affects significantly the
perfomance of anaerobic wastewater
treatment process (Koplimaa et.al
2009).
Betaine degrades quickly in
anaerobic batch-cultures at pH values
exceeding 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

15. Betaine concentration decreased 20-95% as early as in the first
day in anaerobic batch-cultures treating yeast separation
wastewater (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 0
Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 15
S. Zub, T. Tenno

16. 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 H2O
trimethylglycine + 1.32 serine + H2O → trimethylamine + 2 acetate + 1.32 CO2 + 1.32 NH3
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)

17. Formation of NH4+ was accompanied by CH4
production
Nordic Archaeal Network Meeting 2010, May 20 – 22, 17
Södergarn/Lidingö

18. Acetate and trimethylamine are good carbon and energy sources for
acetotrophic methanogens (e.g. Methanobacterium soehngenii) and
methylotrophic methanogens (e.g. Methanosarcina barkeri):
CH3COOH → CH4 + CO2
4 (CH3)3N + 12 H2O → 9 CH4 + 3 CO2 + 6 H2O +4 NH3
At the expense of NH4 it is possible to reduce sulfate
2 NH4+ + SO42− → S0 colloid + N2↑ + 4 H2O
E. Rikmann, A. Menert, V. Blonskaja, T. Kurissoo, 18
Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece
S. Zub, T. Tenno

19. Effect of betaine on the sulfate-dependent anammox-process and
methanogenesis
At least some strains of sulfate-reducing bacteria (SRB) Desulfuromonas are capable to both
hydrolyse 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 processes
of 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

20. Selected substrates and
methane producing reactions
Reactions ∆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 + 2H2O
Aceticlastic reaction:
CH3COO- + H+ = CO2 + CH4 -31,0 25
Disproportionation 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., 1983
Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 20
T. Kurissoo, S. Zub, T. Tenno

21. Fermentative conversion of betaine may bind a
significant fraction of the sulfate-reducing microbial
community,
reducing the use of SO42− as an electon acceptor to oxydize organic substrates. This
would improve the position of the methanogenetic microflora in competition with the SRB
for available organic substrates.
Trimethylamine is an applicable substrate for methanogenic archea from genus
Methanosarcina:
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 wide
range of different substrates. They are able to perform methanogenesis, using
methylotrophic, acetoclastic and hydrogenotrophic pathways.
Occupation of the methylotrophic niche for methanogenesis may help them to achieve a
competitive advantage over sulfate reducing bacteria (SRB ) for substrates available..
The practical output - a more stable and effective perfomance of
methane tank.

22. 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, 22
Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece
S. Zub, T. Tenno

23. Genus Methanosarcina, coloured
archeal strains –
obtained from Tallinn WWTP
Soil Micro-organisms as Indicator for the Biological Quality of
Soils

24. Archae from the genus Methanosarcina
Genus Methanosarcina, sequences determined
closiest to species Methanosarcina mazei and
Methanosarcina 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

25. 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

26. Additional amounts of acetate released from betaine
degradation facilitate growth of acetoclastic methanogens
CH4 formation from intermediates of betaine degradation increses CH4 yield per unit of
COD utilized (in addition to other above-mentioned mechanisms)
CH3COOH → CH4 + CO2
NH4+ produced from methanogenic conversion of trimethylamine (TMA) provides
substrate for microorganisms participating in the chain of reactions of sulfate-dependent
anammox process. Production of colloidal sulphur instead of H2S reduces the general
inhibiting 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 H2O
Betaine may be a key compound to reach and maintain the dynamic equilibrium in an
anaerobic 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

27. RUSSIA
Salutaguse
RUSSIA
AS Salutaguse Yeast Factory
(Subsidary of Lallemand Inc)
27
Tallinn University of Technology

28. Production parameters of
Salutaguse Yeast Factory
From 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-1
dry 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

29. 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.

30. 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 fragments
ST + 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

31. 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 GeneRuler
ST 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.

32. Bacteria from Salutaguse yeast factory wastewater
sludge determined by DGGE (Planctomycetes-specific
forward praimer Pla46F and anammox-specific reverse praimer
Amx368r 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.

33. 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

34. 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 Vilu
Orbit 2010, 29 June – 3 July, Heraklion Crete, Greece E. Rikmann, A. Menert, V. Blonskaja, 34
T. Kurissoo, S. Zub, T. Tenno

35. Thank you for your attention!
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