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Dark fermentation of biomass and organic waste for production of
renewables
T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
Dark fermentation
The efficiency of dark fermentation (yield of biogas, etc) depends on a number of process parameters (kind of substrate, loading rate, hydraulic retention time, etc) of which the hydrolysis rate of organic material has the precedence.
Pretreatment of material is applied for increasing it, resulting in converting the substrate more accessible to anaerobic
2
converting the substrate more accessible to anaerobic microorganisms, accompanied by acceleration of digestion process, increase of methane yield, decrease of the amount of digested sludge and improvement of the process energy balance.
Anaerobic digestion of organic material needs additional heat however this can be compensated by the methane evolved in the same process and its energetic value.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
The main goal of sludge treatment
so far has been caused by environmental issues (hygienization and stabilization) in order to produce biogas (methane) and to use residual sludge in agriculture.
Anaerobic digestion is most commonly used for sludge treatment whereas part of organic matter of sludge is decayed by bacteria into CH4 and CO2.
3
A novel challenge for use of residual sludge would be production of H2
in addition to CH4. Moreover, it is advantageous to steer the fermenting process towards production of H2 instead of methane because combustion of methane causes CO2-release. Methane itself is a dangerous greenhouse gas having a 21 times higher global warming potential as compared to CO2. Production of bioH2 from sludge would thus effect positively on climate change mitigation.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
The main aims and working hypothesis of the study
Thermal pre-treatment of organic material partly or entirely increases its biodegradability. Although this procedure needs additional heat this can be compensated by the energetic value of methane evolved.
Humic substances well known for their ability to facilitate thebioremediation of pollutants can be possibly applied in the
4
bioremediation of pollutants can be possibly applied in thetreatment of wastewaters.
Although members of biogas producing microbial community are engaged in metabolic symbiosis, it is possible to characterize this community and find out key species necessary for normaloperation of the digester.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Scheme of Tallinn Wastewater Treatmentplant
Outlet
Waste activated sludge
(residual sludge)
InletMethane
reactors
Storage of
digested sludge
Storage of
sludge
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Influence of various pretreatment techniques on thebiodegradability of residual sludge
Temperature(T)
Ratio (R)
Minimum +70 °C 1:1:8
Average +85 °C 1:4.5:4.5
Maximum +95 °C 1:8:1
A two-factor three-level full factorial design was usedfor planning the experiment and
calculating the data produced
Bottle # Temperature Level Ratio Level
1 +70 °C -1 1:1:8 -1
2 +70 °C -1 1:4.5:4.5 0
7
3 +70 °C -1 1:8:1 1
4 +85 °C 0 1:1:8 -1
5 +85 °C 0 1:4.5:4.5 0
6 +85 °C 0 1:8:1 1
7 +95 °C 1 1:1:8 -1
8 +95 °C 1 1:4.5:4.5 0
9 +95 °C 1 1:8:1 1
Experimental factors - pre-treatment
temperature and ratio of individual
components of the mixture (inoculum,
treated sludge, raw sludge), were varied
at three levels.
Response factors - cumulative biogas
production, cumulative VFAs production,
ratio of propionate and acetate
14-17 June 2009T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Monitoring of anaerobic digestion process
Gas sample
Liquid sample
8
Oxitop® Control AN 6, WTW Germany
Respirometric Oxitop® method, normally exploited for biodegradability tests and BOD5 measurement can be used for monitoring the digestion process
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Change of VFAs composition during anaerobic mesophilic digestion (t=37°°°°)
inoculum : treated sludge (70 Co) : raw sludge; 1 : 1 : 8
0
5
10
15
20
25
Acid
co
ncentr
atio
n (
mM
)
0
1
2
3
4
5
Gas p
rodu
ctio
n (
mM
d-1)
Gas production rateAceticPropionicButyricValeric
a inoculum : treated sludge (70 Co) : raw sludge; 1 : 1 : 8
0
5
10
15
20
25
Acid
co
ncentr
atio
n (
mM
)
0
1
2
3
4
5
Gas p
rodu
ctio
n (
mM
d-1)
Gas production rateAceticPropionicButyricValeric
a inoculum : treated sludge (95 Co) : raw sludge; 1 : 4.5 : 4.5
0
5
10
15
20
25
30
35
0 5 10 15 20 25 30 35 40
Acid
co
ncen
trati
on
(m
M)
0
1
2
3
4
5
Gas p
rod
ucti
on
(m
M d
-1)
Gas production rateAceticPropionicButyricValeric
c inoculum : treated sludge (95 Co) : raw sludge; 1 : 4.5 : 4.5
0
5
10
15
20
25
30
35
0 5 10 15 20 25 30 35 40
Acid
co
ncen
trati
on
(m
M)
0
1
2
3
4
5
Gas p
rod
ucti
on
(m
M d
-1)
Gas production rateAceticPropionicButyricValeric
c
0 5 10 15 20 25 30 35 40 45
Time (d)
0 5 10 15 20 25 30 35 40 45
Time (d)
inoculum : treated sludge (85 Co) : raw sludge; 1 : 4.5 : 4.5
0
5
10
15
20
25
0 5 10 15 20 25 30 35 40 45
Time (d)
Acid
concentr
ation
(m
M)
0
1
2
3
4
5
Gas p
roductio
n (
mM
d-1
)
Gas production rateAceticPropionicButyricValeric
b inoculum : treated sludge (85 Co) : raw sludge; 1 : 4.5 : 4.5
0
5
10
15
20
25
0 5 10 15 20 25 30 35 40 45
Time (d)
Acid
concentr
ation
(m
M)
0
1
2
3
4
5
Gas p
roductio
n (
mM
d-1
)
Gas production rateAceticPropionicButyricValeric
b0 5 10 15 20 25 30 35 40
Time (d)
0 5 10 15 20 25 30 35 40
Time (d)
Depending on treatment temperature and sludge make-up
(ratio of inoculum, treated and untreated sludge) three
different VFAs formation profiles were observed:
acetate was formed in two stages with maximums up to 15
mM on the day 10 and day 20, with abundant biogas and
methane generation (balanced growth);
moderate VFAs formation, poor consumption of VFAs and
biogas evolution (overproduction of propionate);
large amounts of acetate were formed only at the beginning of
digestion with moderate biogas and methane formation
(overproduction of acetate);
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Global optimisation with desirability function
0,50
0,60
0,70
0,80
0,90
1,00
global optimisation with desirability function
(gas, cum acids, max prp/ac)
0,90-1,00
0,80-0,90
0,70-0,80
0,60-0,70
0,50-0,601 : 5.4 : 2.6
1 : 6.3 : 1.7
1 : 7.1 : 0.9
1 : 8.0 : 1.0
Mix
ture
, ra
tio
Global optimisation with desirability function
(gas, cum acids, max prp/ac)
0,80-1,00
From the 32 design for each response, model of second-degree polynomial function was used. To find the optimum point for all the responses simultaneously, graphical study of the desirability function was performed. By plotting the overall desirability values against the two factors, temperature and ratio of components, it was possible to find optimum conditions for all responses together.
10
70 74 78 81 85 88 90 93 95
1 : 1
.0 : 8
.0
1 : 2
.3 : 5
.7
1 : 4
.5 : 4
.5
1 : 6
.3 : 1
.7
1 : 8
.0 : 1
.0
0,00
0,10
0,20
0,30
0,40
0,50
Tempmix
0,50-0,60
0,40-0,50
0,30-0,40
0,20-0,30
0,10-0,20
0,00-0,10
70 74 78 81 85 88 90 93 95
1 : 1.0 : 8.0
1 : 1.1 : 7.9
1 : 2.3 : 5.7
1 : 3.4 :4.6
1 : 4.5 : 4.5
Temperature, °C
Mix
ture
, ra
tio
0,80-1,00
0,60-0,80
0,40-0,60
0,20-0,40
0,00-0,20
Optimum conditions regarding biogas production, cumulative VFAs production
and propionate acetate ratio were t=70°C and ratio of inoculum : treated
sludge and raw sludge 1 : 8 : 1.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Biogaasi tootlikuse sõltuvus osalisest termilisest eeltöötlusest (70 oC)
40
60
80
100B
iog
aasi h
ulk
(m
mo
l)
10% inokulum + 80% toormuda + 10% 70C
10% inokulum + 90% toormuda
Rate of biogas production
11
0
20
0 5 10 15 20 25 30 35 40
Aeg (päeva)
Bio
gaasi h
ulk
(m
mo
l)
Later on it was found that even partial thermal pre-treatment (10% of raw sludge) increased the production of biogas up to 20%. Considering products of metabolism during anaerobic digestion the acetoclasticmetanogenesis dominated.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Production of VFAs in residual sludgewith low proportion of pre-treated sludge (1:1:8)
Inokulum: töödeldud sete (70oC): toorsete 1:1:8
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50 55Aeg, päev
Org
.üh
end
id,
mM
0
10
20
30
40
50
60
70
Met
aan
i,
mm
ol
Propionaat Atsetaat Võihape Laktaat etanool Metaan
70°°°°
Inokulum: töödeldud sete (95oC): toorsete 1:1:8
40
50
60
70
Org
. ü
hen
did
,
40
50
60
70
Meta
an
i,
mm
ol
95°°°°
Methane yields
Metaani saagised
0,44
0,30 0,280,30
0,40
0,50
0,60
m3 m
etaa
ni/
kg K
HT
Metaani erisaagis: m3 CH4/kg ärastatud KHT
Metaani saagis: m3 CH4/kg KHT
Specific yield of methane m3 CH4/kg COD removed
Methane yield m3 CH4/kg COD
12
Propionaat Atsetaat Võihape Laktaat etanool Metaan
Inokulum: töödeldud sete (85oC): toorsete 1:1:8
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25 30 35 40 45 50 55
Aeg, päev
Org
. ü
hen
did
,
mM
0
10
20
30
40
50
60
70
80
Meta
an
i,
mm
ol
Propionaat Atsetaat Võihape Laktaat etanool Metaan
85°°°°
0
10
20
30
0 5 10 15 20 25 30 35 40 45 50 55Aeg, päev
Org
. ü
hen
did
,
mM
0
10
20
30
Meta
an
i,
mm
ol
Propionaat Atsetaat Võihape Laktaat Etanool Metaan
0,18 0,16 0,14
0,00
0,10
0,20
0,30
inokulum: töödeldud
(+70C): toorsete; 1:1:8
inokulum: töödeldud
(+85C): toorsete; 1:1:8
inokulum: töödeldud
(+95C): toorsete; 1:1:8
Katsesegu koostis
m3 m
etaa
ni/
kg K
HT
14-17 June 2009T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Humic substances
Humic substances as potential electron
acceptors facilitate degradation of recalcitrant
compounds. One of the objects under study
was residual sludge from WWTP that can be
modified by thermal pre-treatment. Humic
substances were extracted from various
environments - ecologically clean environment
(peat) and polluted environment (wastewater
sediments) and fractionated to humic and fulvic
13T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
sediments) and fractionated to humic and fulvic
acids.
The fractions obtained were characterized by
size-exclusion chromatrography (HPLC-SEC),
calculating average molecular masses of humic
substances and determining molecular mass
distribution. By these parameters it was
possible to make conclusions on the behaviour
of humic substances in these environments.
14-17 June 200912th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Molecular masses of humic and fulvic acids and polydispersity in peat samples
Weight average
molecular mass, Mw
Numeral average
molecular mass, Mn
Polydispersity
Sample 1
Fulvic acid 8360 583 14
Humic acid 84300 332 254
Sample 2
Fulvic acid 10200 500 21
Humic acid 140300 171 610
Sample 3
Fulvic acid 52200 403 130
Fulvohape, turvas
0 10 20 30
Retentsiooni aeg, min
UV
, 254 n
m
Fulvic acid, peat
Retention time, minHumic acid 228300 579 394
Sample 4
Fulvic acid 43200 538 80
Humic acid 170800 318 537
ii
i
i
ii
nMh
h
N
MNM
/∑
∑=
∑
∑=
i
ii
ii
ii
wh
Mh
MN
MNM
∑
∑=
∑
∑=
2
wM nM
/
Retentsiooni aeg, minRetention time, min
Humiinhape, turvas
0 10 20 30
Retentsiooni aeg, min
UV
, 254 n
m
Humic acid, peat
Retention time, min
Co-digestion
Co-digestion - simultaneous digestion of homogeneous mixture oftwo or more substrates improves the processing qualities of differentwastes and increases the production of biogas. Usually a major amount of basic substrate (e.g. manure or sewage sludge) is mixedand digested together with minor amounts of a single or a variety ofadditional substrates (garbage waste, paper mill residues, slaughterhouse waste, animal manures, saw dust, energy crops, food industrywaste, pharmaceutical wastes). Co-digestion offers several
15
waste, pharmaceutical wastes). Co-digestion offers severalecological, technological and economical benefits:
� digester operational advantages, � improved overall process economics (higher biogas yield - 40-200%)� most of chemical energy of the substrate is turned into biogas, less
spent solids are to be processed.
14-17 June 2009T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Dependence of biogas yield on thermal pre-treatment (+70°C) and addition of humic substances (peat)
Thermal pre-treatment ofresidual sludgeappeared to be aneffective pre-treatmenttechnique, the best was 20
30
40
50
60
70
Meth
an
e (
mm
ol)
16
technique, the best wascombination of thermalpre-treatment with thesimultaneous use ofhumic substances.
0
10
20
0 10 20 30 40 50 60 70
Time (days)10% inokulum + 80% raw sludge + 10% 70Cpre-treated sludge10% inoculum + 80% raw sludge + 10% 70C pre-treated sludge + 10 ml peat10% inoculum + 80% raw sludge + 10% 70C pre-treated sludge + 30ml peat10% inoculum + 80% raw sludge + 10% 70C pre-treated sludge + 1,6g AQDS10% inoculum + 90% raw sludge10% inoculum + 90% raw sludge + 10ml peat10% inoculum + 90% raw sludge + 30ml peat
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Buffering capacity ofhumic substances prevents accumulation of organic acids
Inoculum 10%, raw sludge 90%
20 60
Acetic acid Propionic acidButyric acid Lactic acidMethane
Inoculum 10%, raw sludge 90%
+ 10ml peat
10
15
20
Org
an
ic c
om
po
un
ds (
mm
ol/L
)
30
45
60
Meth
an
e (
mm
ol)
Lactic acid Acetic acidPropionic acid Butyric acidMethane
17
0
5
10
15
0 10 20 30 40 50 60
Time (days)
Org
an
ic c
om
po
un
ds
(m
mo
l/L
)
0
15
30
45
Me
tha
ne
(m
mo
l)
0
5
10
0 10 20 30 40 50 60
Time (days)O
rgan
ic c
om
po
un
ds (
mm
ol/L
)
0
15
30
Meth
an
e (
mm
ol)
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Some conclusions on use of humic substances
� Peat as asource of humic substances accelerates the process of anaerobic
digestion, increases the yield of biogas and makes the process more effective
and stabile.
� One reason might be the buffering capacity of humic substances that does
not allow organic acids to accumulate and leads the process towards more
oxidized products. Humic substances act as electron carriers and mediators
on decaying the organic substance.
� The combination of thermal pre-treatment with addition of humic substances
18
� The combination of thermal pre-treatment with addition of humic substances
(peat) accelerated the process most. Use of only 10% of pre-treated sludge in
the mixture is sufficient, 80% of pre-treated sludge in the mixture resulted in
less biogas yield.
� Antraquinone-2,6-disulphonate(AQDS) is not a good model for humic
substances. Quinone respiration was preferred as to methane respiration – in
the experiments with AQDS, methane yield was 2-4 times less as compared
to peat. AQDS performed as terminal electron acceptor.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Humic substances as co-digestion substrates
� Humic substances well known for their ability to facilitate the bioremediation of pollutants can be possibly applied in the treatment of wastewaters. Experiments have clarified by now the effect humic substances (one of co-substrates) on the production of biogas.
� Thermal pre-treatment of residual sludge appeared to be an effective pre-treatment alternative, the best was combination of thermal pre-treatment with the simultaneous use of humic substances.
19
� Humic substances can increase the yield of biogas as electron donors and additional carbon sources. 50 or 150 mmol of extra carbon was added as humic substances, however, in the experiments only 10-30 or 40-60 mmol methane was formed and twice less CO2. Evidently, the rest of carbon was bound into refractory inert polymeric compounds.The resultant digestate is more stabile and has higher dry solids content.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Substrate conversion patterns associated with anaerobic treatment of
wastewaters
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)
Some redox half-reactions responsible for anaerobic microbial conversion of selected substrates
Reactions
Oxidations (electron donating reactions) ∆G0, KJ
Propionate→Acetate: CH3CH2COO- + 3H2O → CH3COO- + H+ + HCO3- + 3H2 +76.1
Butyrate →Acetate: CH3CH2CH2COO- + 2H2O → 2CH3COO- + H+ + 2H2 +48.1
Ethanol →Acetate: CH3CH2OH + H2O → CH3COO- + H+ + 2H2 +9.6
Lactate →Acetate: CH3CHOHCOO- + 2H2O → CH3COO- + HCO3- + H+ + 2H2 -4.2
Acetate →Methane: CH3COO- + H2O → HCO3- + CH4 -31.0
21
Acetate →Methane: CH3COO- + H2O → HCO3- + CH4 -31.0
Respirative (electron accepting reactions)
HCO3- →Acetate: 2HCO3
- + 4H2 + H+ → CH3COO- + 4H2O -104.6
HCO3- →Methane: HCO3
- + 4H2 + H+ → CH4 + 3H2O -135.6
Sulfate→ Sulfide: SO42- + 4H2 + H+ → HS- + 4H2O - 151.9
CH3COO- + SO42- + H+ → 2HCO3
- + H2S -59.9
Nitrate →Ammonia: NO3- + 4H2 + 2H+ → NH4
+ + 3H2O -599.6
CH3COO- + NO3- + H+ + H2O → 2HCO3
- + NH4+ -511.4
Nitrate → Nitrogen gas: 2NO3- + 5H2 + 2H+ → N2 + 6H2O -1120.5
Adapted from Harper and Pohland, 1986)
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Selected substrates and methaneproducing 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
22
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
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Conversion of acetate to methane
23
Corr – corrinoid containing protein;
CODH – carbon monooxide dehydrogenase
CH3COO- + H+ = CO2 + CH4 ∆G°’ = - 31 kJ
Madigan, M. T. & Martinko, J. M. 2006. Brock Biology of
Microorganisms“ 11th Edition, Southern Illinois University,
Carbondale
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
The methanogenic pathway of CO2 reduction with H2
MF – metanofurane, coenzyme participatingin C1 transfer from CO2 to CH4;
MP – metanopterine, conenzyme, C1 carrier in intermediate stages;
CoM – coenzyme M, participates inconversion of methyl group (CH3) to CH4
F – reduced coenzyme F ;
24
CO2 + 4H2 → CH4 + 2H2O ∆G°’ = - 135,6 kJ
F420red – reduced coenzyme F420;
F430 – coenzyme F430
CoB – coenzyme B
Yellow – C-atom to be reduced;
Brown – electron donor
Madigan, M. T. & Martinko, J. M. 2006. Brock Biology of
Microorganisms“ 11th Edition, Southern Illinois University,
Carbondale
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Methanogenic archea
Genus Suitable substrates for
methanogenesis
Methanobacteriale
Methanobacterium
Methanobrevibacter
Methanosphaera
Methanothermus
Methanothermobacter
H2 + CO2, formic acid
H2 + CO2, formic acid
methanol + H2 (both necessary)
H2 + CO2, (S0); hyper thermophilic
H2 + CO2, formic acid; thermophilic
Methanococcale
Methanococcus
Methanothermococcus
H2 + CO2, CO2 + pyruvate, formic acid
H2 + CO2, formic acid
Methanosarcinale
Methanosarcina
Methanolobus
Methanohalobium
Methanococcoides
H2 + CO2, methanol, methylamines, acetate
methanol, methylamines
methanol, methylamines; halophilic
methanol, methylamines
25
Methanocaldococcus
Methanotorris
H2 + CO2
H2 + CO2
Methanomicrobiale
Methanomicrobium
Methanogenium
Methanospirillum
Methanoplanus
Methnocorposculum
Methanoculleus
Methanofollis
Methanolacinia
H2 + CO2, formic acid
H2 + CO2, formic acid
H2 + CO2, formic acid
H2 + CO2, formic acid
H2 + CO2, formic acid, alcohols
H2 + CO2, alcohols, formic acid
H2 + CO2, formic acid
H2 + CO2, alcohols
Methanohalophilus
Methanosaeta
Methanosalsum
methanol, methylamines, methylsulfides; halophilic
acetate
methanol, methylamines, dimethylsulfide
Methanopyrales
Methanopyrus H2 + CO2; hyper thermophilic (110°C)
Madigan, M. T. & Martinko, J. M. 2006. Brock Biology of
Microorganisms“ 11th Edition, Southern Illinois University,
Carbondale
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
DNA-based methods for direct process monitoring of the consortia used
In industrial applications the use of mixed cultures from organic wastes for CH4 and H2 production is more advantageous as pure cultures can easily become contaminated with H2 consumers. Many members of these microbial communities are non-culturable bacteria. Thus for characterization of these potent CH4 and H2 producing
26
characterization of these potent CH4 and H2 producing microorganisms analyzing microbial communities, using culture independent techniques is inevitable.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Identifying on nonculturable bacteria by denaturating gradient gel electrophoresis (DGGE)
DNA extraction
Community DNA
Amplification of a fragment of 16S
rRNA gene (rDNA) using PCR
DGGE-gel. Different
bands in the gel
represent different
microbial speciesDenaturating
Checking PCR-
products using
agarose gel
elektrophoresis
Cutting out bands from
DGGE gel and
reamplifying DNA from
the bands
microbial species
CTGAATCGTA
Sequencing of
purified DNA
originated from
DGGE bands
Identifying
microorganisms by
comparing 16S rDNA
sequences to DNA
databases and
constructing a
phylogenetic tree
Denaturating
gradient gel
elecrrophoresis
(DGGE)
Phylogenetic tree
elektrophoresis
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Denaturating gradient gel electrophoresis (DGGE) of bacterial16S rRNA gene fragments
8
9
10
21
23
24
43
44
45
46
47
51
53
54 55
35
36
37
38 39
40
5261
60
62
6364
69
70
717280
77
78
79
81
84
85
8687
90
91
9293
97
98
99100
104
105
106
110
111
112
116
117
121
122
3
1
2
4
15
16
17
31
28
29
32
30 2211
1B 1B 4 4 4B 7A 7B
T=70°C T=85°C T=95°C
12
13
14
25
26
27
48
49
50
56
57
58
59
41
42
65
66
67
68
73
74
75
76
82
83
88
89
94
95
96
101
102
103
107
108
109
113
114
115
118
119
120
123
124
5
6
7
18
19
20
33
34
from samples ofthermally pre-treated residualsludge of WWTP
DGGEwas used in order to determine the impact of different pretreatment temperatures on the microbial community structure.
Characterization of microbial concortia
Most of the bacteria identified were representatives of the phyla Chloroflexi and Bacteroidetes;
All the archaeal strains identified were shown to represent the genus Methanosarcina – anaerobic methanogens, the main biogas
29
producers occurring in landfills, WWTPs, in sea sediments and mammal guts. These archaea are able to produce methane by all three known methanogenic pathways – the hydrogenothrophic, acetoclastic and methylotrophic pathway.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Phylogenetic analyses of bacteria, coloured bacterial strains –obtained from Tallinn WWTP
Most of the bacteria
identified were
representatives of the
phyla Chloroflexi and
Bacteroidetes
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
The most numerous bacteria were representatives from the phylum Chloroflexi.
The closiest species to the sequences determined was Levilinea saccharolytica
(B). Levilinea saccharolytica has been previously isolated as pure culture from
sugar industry wastewater (containing sucrose and easily degradable VFAs)
sludge granules (Yamada et al., 2005)
A – Anaerolinea thermolimosa; B – Levilinea saccharolytica; C – Leptolinea tardivitalis. (Yamada et al., 2005)
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Archea from Tallinn WWTP determined with DGGE
1
3
4 8 10
11
14
70ºC 85ºC 95ºC 70ºC 85ºC 95ºC
26
5
7
9
13
12
1516
1718
19
2
0
21
2223
Genus Methanosarcina, coloured archeal strains –coloured archeal strains –obtained from Tallinn WWTP
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
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.
34
phylum Euryarchaeota. Multicell form of Methanosarcina acetivorans
(http://www-
genome.wi.mit.edu/annotation/microbes/methano
sarcina/background.html)
Methanosarcinae have the largest genome among
archea – the genome of M. acetivorans has
5,751,492 nucleotides (Galagan et al., 2002).
22nd amino acid
aminohape –
pyrrolysine from
Methanosarcina
barkeri
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Methanosarcinae – anaerobic methanogens
Methanosarcinae have specific pathway for methane production – methylotrophic methanogenesis using methanol, methylamines and methyltiols for methane production (Galagan et al., 2002).
35
Three pathways ofmethanogenesis)
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology
Acknowledgement
The financial support from
the Estonian Science Foundation (Grant No 5889), from Nordic Energy Research (Grant No 06-Hydr-C13)and from Enterprise Estonia (Grant No EU27358) aregratefully acknowledged.
36
gratefully acknowledged.
14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,
M. Kaljurand, J. Suurväli, A. Menert
12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology