War. Res. Vol. 25, No. 12, pp.

1509-1513, 1991
Printed in Great Britain. All rights reserved

0043-1354/91 $3.00+0.00
Copyright 1991 Pergamon Press plc

CONVERSION OF ACETATE, PROPIONATE A N D

BUTYRATE TO METHANE U N D E R THERMOPHILIC
CONDITIONS IN BATCH REACTORS
MUSTAFA (~ZTURK

Department of Environmental Engineering, Ylldlz University, 80750 Yfldtz, Istanbul, Turkey

(First received June 1990; accepted in revised form May 1991)

Abstract--These experiments were performed to determine the degradation of VFA (acetate, propionate
and butyrate) and the maximum methanogenic activity of granular sludge from the thermophilic anaerobic
digestion of pure molasses. The compositions of acetate, propionate and butyrate used as substrate were
25:35:40. The tests were performed at constant temperature (55C) and pH 7 on two duplicate batch
reactors (I and II) running in parallel and were repeated to show the effect of acclimatization. During the
first feeding, there was a significant lag phase and after about 23 h incubation the volumes of CH4 gases
produced from two reactors were only about 20 and 490 ml, respectively. In this experiment, propionate
was converted to acetate only after the initial concentrations of butyrate and acetate had completely
degraded. Acetate formed from propionate was immediately converted to methane and carbon dioxide.
The maximum methanogenic activities of the first feeding were not high because the natural populations
of the propionate-degrading bacteria were low and the sludge adapted itself to the VFAs' substrate very
slowly. In a second experiment with the same sludge, the maximum methanogenic activities of the second
feedings were about 1.60 times higher than those of the first feedings because of the adaptation of the
sludge and increase of populations of the propionate-degrading bacteria.
Key words--thermophilic, anaerobic, propionate, batch reactor, methanogenic activity, molasses

INTRODUCTION

The anaerobic digestion process is one of the major

biological waste treatment processes in use today.
This process has been popular in the waste treatment
field, because it has many advantages such as a high
treatment efficiency and methane-producing ability
(Lin et al., 1986).
Volatile acids formed as intermediate compounds
during anaerobic treatment are oxidized by floxidation to hydrogen, carbon dioxide and acetate,
and the processes are termed dehydrogenation and
acetogenesis, respectively. The last stage is methanogenesis (McCarty, 1964; McCarty and Smith,
1986). Methanogenesis involves the reduction of
carbon dioxide to methane using hydrogen by relatively fast growing pH-sensitive autrotrophic bacteria. Methanogens also catalyze the reduction of
acetate to methane and carbon dioxide (Denac et al.,
1988).
Thermophilic anaerobic digestion systems offer
attractive kinetic advantages in comparison with
mesophilic systems. The maximum specific growth
rates and activities of thermophilic bacteria are
higher than those of mesophilic bacteria, whereas
their properties are generally similar to those of
mesophilic homologs and the substrate saturation
constants are in the same range (Wiegant, 1986),
Hydrogen concentration during maximum growth
or activities of butyrate converting bacteria is 4.9
times higher than the concentration during maximum

propionate conversion. As a consequence, propionate

degradation will be highly inhibited during periods of
high activity of the butyrate converting bacteria.
Under thermophilic conditions butyrate degrading
bacteria are approx. 6 times less sensitive against
hydrogen gas than those of propionate. If the hydrogen partial pressure in the reactor is low, acetogens
convert propionate and butyrate to acetate, hydrogen
and carbon dioxide, but at high partial pressures
of hydrogen they are inhibited and propionate and
butyrate are not fermented (Table 1). Maximum
conversion of propionate and butyrate to methane
gas is possible only at low hydrogen partial pressure
(Samsoon et al., 1981).
Acetate has been described as the least toxic of the
volatile acids, while propionate has often been implicated as a major cause of digester failure (Stronach
et al., 1986). Propionate conversion appeared to be
strongly inhibited, probably because propionate oxidation is thermodynamically rather unfavorable in
anaerobic digestion. Methane production from propionate in batch experiments is believed to be slower
than that from butyrate or acetate (Gijzen et al.,
1988; Hanaki et al., 1987).
The methanogenic activity of granular sludges is
related to their source and previous feedstocks.
During the first feeding the sludge adapts itself to the
volatile fattty acid substrate. The activity in the
second feeding is higher than the first. This increase
may be assumed to be the result of adaption (Field
and Sierra, 1989).

1509

MUSTAFAOZTORK

1510

Table 1. AG values of some relevant reactionsof 25 and 55C under the followingconditions;concentrationsof 0.01 M, pH = 7.2, 20% CO2
in the dry biogas and ionic strength I ~ 0.087 M
Reaction
Substrates

AG(kJ mol- i)
Products

25C

55C

Equation

Acetic+ H20
CH4+ HCO~-+ H +
-26.9
-32.8
4H2+ H + + H C O 3
C H 4 + 3HzO
- 127.74 - 22.84 log(pH2)
- 114.98 -- 25.15 log(pH2)
Acetic+ 4H20
2 H C O 3 + 4H2+ 2H
100.83 + 22.84 Iog(pH2)
82.17 + 25.15 log(pH2)
Propionic+ 3H20
Acetic+ HCOf + 3H2H+
62.55 + 17.11 Iog(pH:)
48.12 + 18.87log(pH2)
Butyric + 2H20
2 Acetic+ 2H~
32.55 + 11.42log(pH2)
22.84 + 12.55Iog(pH2)
Acetic+ propionate + H + + 2H2 Valerate+ 2H20
-48.1
pH2 is the partial pressure of H2. 1/aM of H 2 correspondswith 1270ppm at 25C and 1370ppm at 55C in the gas (Wiegant et

1
2
3
4
5
6
al., 1986).

Table 2. Compositionof stock solutions of nutrients and trace elements

Nutrient

(g/I)

NH4CI
KH2PO4
CaC12.2H20
MgSO4.4H20

170
37
8
9

Trace element (g/I)

FeCI3.4HzO
COC12-6H20
MnCI2"4H20
CuClz.2HzO
ZnCI2
H3BO3

Usually the m e t h a n e p r o d u c t i o n rate during the

second feeding is more than 30% higher than during
the first feeding (Manual Anaerobic Laboratory,
1989).
In this study, specific methanogenic activities o f
granular sludge from the thermophilic anaerobic
digestion o f pure molasses and conversion o f
butyrate, p r o p i o n a t e and acetate fed as substrate to
methane were investigated in two batch reactors
running in parallel under thermophilic conditions.
MATERIALS AND METHODS
The experiments were performed in Plexiglas cylindrical
reactors with a working volume of 5 dm 3. Reactors were
operated at thermophilic conditions (55 + IC). Two reactors were run in parallel. To each reactor, which contained
15 ml nutrient solution and 2 ml trace elements solution,
2.5g NaHCO 3 buffer was added and they were filled
with oxygen-free water. Table 2 shows the composition
of stock solutions of nutrients and trace elements. Nitrogen
gas was passed through the solutions for 5 min. Granular
sludge used in the experiments was originally obtained
from an UASB reactor treating pure molasses at thermophilic conditions. The sludge concentration used was
2.5 g organic solids (OS) per liter. The volatile fatty acid
(VFA) substrates used throughout the experiments were
obtained from a stock solution containing 100:100:100g
acetate:propionate:butyrate per kilogram with a pH of 7
neutralized with NaOH solution. The chemical oxygen
demand ratio of the VFA stock was 25:35:40 of the total
COD for C2, C 3 and C4, respectively. Exact concentrations
of VFA used in the first and second experiments were
2.97-3.007g COD per liter in the first reactor and
2.26-2.554 g COD per liter in the second reactor. The VFA
standards (C2-C4) were obtained from Merck. The reactors
were again flushed with nitrogen gas for another 3 min. The
reactor vessels were connected to a gas measurement system.
The solutions were continuously stirred for a minimum of
6 s every 3 min during the experiment (Fig. 1).
Methane production removed from CO 2 in the gas was
measured, daily. Volatile fatty acids (VFA) in the samples
taken from each batch reactor were analyzed in a gas
chromatograph equipped with a 2 m x 2 mm i.d. column
packed with 10% Fluorad FC 431 on supercoat
(100-120 mesh). The carrier gas (N2) was saturated with
formic acid and flow rate set at 35mlmin -~. The oven

2
2
0.5
0.03
0.05
0.05

(NH4)rMoTO2.4H20
Na2SeO3.5H20
NiCI2"6H20
EDTA
HCI 36%
Resazurin

0.09
0.1
0.05
1
0.001
0.5

temperature was set at 130C. The FID detector signal was

processed with a SP41 Spectra Physics Integrator.
About 163 h later, all the VFA added in the first feeding
degraded and the second feedings were made and again
methane production was measured. The concentrations of
VFA were also analyzed by gas chromatography.
RESULTS
During the experiments two feedings were made
and the temperature and p H o f solutions in the batch
reactors remained fai,'y constant at 55C and 7,
respectively. By the end o f 22.66 h methane gas
volumes from reactors I and II were 20 ml ( 1 0 m g
C H 4 C O D l -l) and 4 9 0 m l (257mg CH4 C O D l-l),
respectively. But the methane gas production began
to increase sharply after this time [Fig. 2(a)].
During the first experiment, butyrate degraded
rapidly, as shown in Figs 3 and 4, but the acetate
produced did not immediately convert to m e t h a n e
and concentrations o f acetate and valerate continuously increased. After the degradation o f butyrate
was completed, acetate started to be converted to
methane and carbon dioxide. A b o u t 90.84h later,
Stirred
SampLe
Points

Soda Lime pe~ets

KOH(5%)

TefLon
bag
Digester

Fig. 1. The stirred batch anaerobic digestion assay set-ups

with a Teflon bag for measuring the methane gas
production.

Degradation of thermophilic VFAs

1511

(a)

but.yr ate

2--

j/o

'Reactor Z-first feeding

/ , ~ "

;1
E
3

/~L /

O> 0

20

E3
0

40

60

80

100

120

140

160
>

(b)

40

yo

I
20

/ f J

120

160

Fig. 4. The anaerobic degradation of volatile fatty acids

during the first feeding at 55C in batch reactor II.

~-Reoctor I-second feeding

I
40

I
60

I
80

I
100

I
120

I
140

I
160

Time ( h )

Fig. 2(a and b). The methanogenic activity of pure molasses

granular sludge at 55C during batch anaerobic digestion
assays in batch reactors I and II.

degradation of all the acetate was completed and

subsequently propionate was converted to acetate.
Acetate formed from propionate was immediately
converted to methane and carbon dioxide (Figs 3
and 4).
The degradation times of acetate, propionate and
butyrate for the first feedings are 90.84, 162.92 and
42.5 h, respectively.
The maximum methanogenic activity is expressed
in milliliters (or g C H 4 C O D ) methane gas production
per gVSS of granular sludge in unit time. The
methanogenic activity of granular sludge is related to
their source and previous feedstocks. The maximum
methanogenic activities of the first feeding in reactors
I and II were as low as 0.196, 0.201 g CH4 COD/gVSS
day, respectively, because the adaptation of sludge
itself to VFA substrate and conversion of propionate
to acetate took a long time.

During the first feeding the methane obtained was

77 and 79% from VFA based on COD, respectively.
The activity period is the time period when the
methane gas production rate is at its highest during
the feeding. The time period should at least cover
about 50% VFA used. The activity period of the
first feeding in reactors I and II was calculated to be
about 80 h.
The methane gas volumes were high for the first
days of the second experiment and about 46.6 h later
methane production rate continuously decreased as
shown in Fig. 2(b).
Butyrate and propionate degraded together
throughout the second tests and acetate formed from
these substrates (C3, C4) was immediately converted
to methane and carbon dioxide. Afterwards acetate
substrate which was added in the second feeding was
converted to methane and carbon dioxide (Figs 5
and 6).
The degradation times of acetate, propionate and
butyrate for the second feeding were 71, 75 and 27 h,
respectively.
The maximum methanogenic activities of the second feeding in reactors I and II were 0.285, 0.352 g
CH4 COD/gVSS day, respectively. Increase in the

2I

ta

/otyrote

butyrate

propionote

80

Time ( h )
cond feeding

I"
0

40

80

120

160

T i m e (h)

Fig. 3. The anaerobic degradation of volatile fatty acids

during the first feeding at 55C in batch reactor I.

40
Time ( h )

..+

80

Fig. 5. The anaerobic degradation of volatile fatty acids

during the second feeding at 55C in batch reactor I.

1512

MUSTAFAOZTURK

ta

butyrate

Q
0
c.)

>

40

80

Time (h)

Fig. 6. The anaerobic degradation of volatile fatty acids

during the second feeding at 55C in batch reactor II.
activity in reactors I and II was found to be 45 and
75%, respectively.
VFA conversion to methane was found to be 73
and 84% throughout the second feeding.
Because sludge adapted itself to VFA substrate and
all the VFA degraded within a short time, the activity
periods of the second feeding in reactors I and II were
as short as about 35 h.
DISCUSSION
The experimental results of the first feeding indicated that the granular sludge initially adapts to the
butyrate substrate. Butyrate is degraded to acetate
and hydrogen (Boone, 1989), but its conversion is
slow.
Acetate formed from butyrate is not immediately
converted to methane and carbon dioxide. As shown
in Figs 3 and 4, during the first 23 h incubation there
is a significant lag phase. After the first 23 h acetate
was fermented and hydrogen gas was also consumed
by carbon dioxide reducing methanogenic bacteria
and as a result methane gas production rate suddenly
rose. As in mesophilic digestors, acetate substrate
is quantitatively the most important precurser of
methane and is the source for about two thirds of
methane production in thermophilic digestors (Smith,
1966; Zinder, 1988).
After the initial concentrations of butyrate and
acetate had completely degraded, propionate substrate was converted by propionate degrading bacteria to acetate only. Acetate formed from propionate
was immediately converted to methane and carbon
dioxide (Figs 3 and 4).
When Figs 5 and 6 are examined during the second
feeding, it can be seen that there is no inhibition of
propionate degradation. It was observed that propionate and butyrate were simultaneously converted to
acetate, hydrogen and carbon dioxide, because of
adaptation of the propionate-degrading bacteria.
Acetate produced from butyrate and propionate
degraded rapidly to methane and carbon dioxide.

The degradation time of propionate in the second

feeding is 2.5 times shorter than that of the first
feeding. In this case, propionate-degrading bacteria
are the most sensitive. Again, according to the results
of the first and second feedings, the degradation time
of all the VFA in the second feeding is shorter than
that of the first feeding.
As can be seen from Fig, 2(b), during the second
feeding methane gas production rate is fairly high. It
is believed that the methanogenic consumption of
hydrogen serves to decrease the hydrogen concentrations below the inhibition level. Measurement in
continuous cultures have apparently confirmed this
phenomena (Kaspar, 1977).
Maximum methanogenic activities of the second
feeding are 1.45-1.75 times higher than those of the
first feeding. This is due to the fact that all the VFA
compounds during the second feeding degrade in
63% of the time of those of the first feeding, according to the activity period. This decrease may be
considered to be the result of adaptation of the sludge
and its propionate-degrading bacteria. As a result of
this situation the maximum methanogenic activity
should be determined after the second and/or third
feeding.
The percentage methanogenizations of the volatile
fatty acids in the first and second experiments are
77-79 and 73-84%, respectively. This suggests that
15-20% of the VFAs were converted to cells during
the first and second feeding.
In addition, acetate produced from propionate gas
was rapidly converted by methanogens to methane
and carbon dioxide, but acetate from butyrate is
converted to gases slowly (Figs 3 and 4).
This study suggests that natural populations of
propionate-degrading bacteria in many methanogenic sludges may be low and that it would
be worthwhile to routinely test for their ability to
degrade propionate when adapting them to new
feedstocks.
Acknowledgement--The author wishes to express his sincere
gratitude and thanks to the staff of Department of Water
Pollution Control, Agricultural University of Wageningen
and especially to Professor Dr G. Lettinga for their kind
efforts and contribution for the continuation and finalization of this work.
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Degradation of thermophilic VFAs

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1513

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