Appl Microbiol Biotechnol (1995) 43:1001-1005

Springer-Verlag 1995

R. Lepist6 J. A. Rintala

Acetate treatment in 70C upflow anaerobic sludge-blanket

(UASB) reactors: start-up with thermophilic inocula
and the kinetics of the UASBsludges

Received: 29 September 1994/Received revision: 18 January 1995/Accepted: 23 January 1995

Abstraet This study focused on the use of thermophilic

anaerobic granulae in the start-up of 70C acetate-fed
upflow anaerobic sludge-blanket (UASB) reactors and
the kinetics of granulae grown at 70C. In the UASB
reactors, chemical oxygen demand removal commenced within 48 h of the start-up. The maximum
reduction in chemical oxygen demand was 84% with
the feed containing yeast and 71% without a yeast
supplement. In the bioassays, the yeast-grown sludge
converted 98% of the acetate consumed to methane as
compared to 92% for the sludge grown without yeast.
The highest initial specific methane production rate
(/~cH4) of the UASB sludges grown at 70C was
0.088 h - 1 at an acetate concentration of 4.6 mM. The
higher initial acetate concentration was found to prolong the lag-phase in methane production significantly
and to decrease the #cn4. The half-saturation constant
(Ks), the inhibition constant (Ki), the inhibition response coefficient (n), and the /XcH4.... calculated according to a modified Haldane equation, were
1.5 mM, 2.8 mM, 0.8, and 0.28 h - 1, respectively. The
prolonged starvation of the 70C sludge (15 days) decreased the/~cn~ from about 0.022 h - i to 0.011 h - 1 and
increased the lag phase in methane production from 6 h
to 24 h as compared to non-starved sludge.

Introduction

The temperature of many industrial process waters and

wastewaters exceeds 50C. Such waters are usually high
in organic content and hence potential candidates for
thermophilic anaerobic treatment. Several studies have
demonstrated the feasibility of thermophilic high-rate
R. Lepist6 (IE)- J. A. Rintala
Institute of Water and Environmental Engineering,
Tampere University of Technology, SC 108, P.O. Box 692,
FIN-33101 Tampere, Finland.
Fax: + 358-31-3162869

anaerobic reactors, such as upflow anaerobic sludgeblanket (UASB) reactors, for treating industrial process
waters and wastewaters (e.g. Wiegant et al. 1985;
Lepistt5 and Rintala 1994). However, few if any fullscale thermophilic high-rate anaerobic reactors are
used to treat industrial wastewaters.
We are developing and applying thermophilic anaerobic processes as an internal purification method for
closed water circuits in pulp and paper production and
also for the treatment of the organic fraction of municipal solid waste. Process temperatures of about 70C
have been applied because some pulp and paper industry process waters are of that temperature and because
such temperatures may be high enough to sanitize
waste. The feasibility of operating UASB reactors at
temperatures of 70C (Rintala and Lepisti5 1992; Rintala et al. 1993) and 75C (Van Lier et al. 1992a) has
been demonstrated. However, specific methanogenic
activities have been shown to be lower at 70C than at
55C (Rintala et al. 1993).
Thermophilic UASB reactors are generally inoculated with sludge from mesophilic processes as thermophilic sludge is not commonly available. The startup of a thermophilic process with mesophilic inoculum
is possible, but it requires a relatively extended period
of time, especially at 70C and 75C (Van Lier et al.
1992a, b; Rintala et al. 1993). On the other hand, using
thermophilic inocula to start up a thermophilic reactor
could reduce the start-up period and minimize the
possible inhibition effects from the die-off of mesophilic
organisms. Varel et al. (1980) reported a fast start-up of
a 60C anaerobic digester inoculated with 55C sludge.
Acetate is responsible for more than 65% of the
methane production in anaerobic thermophilic (Zinder
et al. 1984; Mackie and Bryant 1981) and mesophilic
digesters (Smith and Mah 1966; Mountfort and Asher
1978). In thermophilic anaerobic processes, the conversion of acetate is carried out by aceticlastic methanogens, Methanothrix and Methanosarcina (Zinder
1990) and/or by syntrophic cocultures, which consist of

1002

acetate-oxidizing organisms and hydrogen-utilizing

methanogens (Zinder and Koch 1984). Aceticlastic
methanogens convert acetate via decarboxylation
(Smith and Mah 1980; Zehnder et al. 1980), while the
coculture first oxidizes acetate to CO2 and H2, which is
followed by the reduction of CO2 to C H , (Lee and
Zinder 1988a; Petersen and Ahring 1991).
Little is known about high-rate anaerobic processes
at temperatures of 70C or above. The aim of this study
was to evaluate the start-up of acetate-fed, 70C UASB
reactors with thermophilic inocula and the characteristics of sludges grown at 70C. We also studied the
effects of yeast supplements on the start-up of the
UASB reactors.

Materials and methods

Reactor experiments
Two UASB glass reactors (R1 and R2) with a working volume of
0.201 were used in this study. The reactors were placed in a temperature-controlled water bath at 70C. The feed solutions were pumped into the reactors through tubes placed in the water bath to
ensure proper temperatures along the reactor height.
The reactors were inoculated with a mixture of thermophilic
granular sludges (1.9 g volatile solids each). The sludges were obtained from acetate-fed 70C (1.4 g volatile solids) UASB reactors
and from a 55C UASB (0.5 g volatile solids) reactor fed with
pulp-mill white water. The 70C and 55C sludges, prior to use in
this experiment, were refrigerated under reduced atmosphere at 4C
for about 2 weeks and 1 year, respectively.
R1 was fed with a 4 8 - 5 2 m M acetate (as sodium acetate;
2.9-3.2 g 1-1 chemical oxygen demand, COD) solution containing
0.1 g 1-1 of yeast extract while R2 was fed with a similar solution but
the yeast was omitted. Both feed solutions contained macro and
trace nutrients, as previously described (Rintala et al. 1992) and
modified as follows: N a H C O 3 was omitted, and l l 0 m g 1 - 1
CaC12'2H20 was added. The pH of the feed solutions was a d j u s t e d
to 6.8-7.0 with NaOH. F r o m experiment day 36 onwards the pH of
the feed was adjusted to 5.7-6.3. The feed solutions were prepared
one or two times a week, flushed with N 2 for 20 rain to remove
oxygen, and stored under N 2 at 4C.

Bioassays
At the end of the UASB reactor experiments, the sludges from R1
and R2 were used in bioassay studies either separately or mixed. The
bioassays were performed in either 50 ml vials with 29 ml medium or
in 120-ml vials with 51-ml medium. In all assays, the media contained macro and trace nutrients, as previously described (Rintala et
al. 1992). The pH of all the assays was adjusted to 6.9 _+ 0.1 with
NaOH. All the addition were done under 80% N2/20% CO 2. The
vials were subsequently sealed with rubber stoppers and aluminum
crimps and the head space was flushed with 80% N2/20% CO 2.
Finally, NazS.9H20 was added to the vials from an anaerobic sterile
stock solution to give a final concentration of 0.25 gl-1. The vials
were incubated in static culture at 70C.
In the assays for the conversion of acetate to methane, 4 ml sludge
from either R1 (0.11 g volatile solids) or R2 (0.07 g volatile solids)
was separately added into the 120-ml vials; 53-55 m M acetate was
added as substrate. The assays were performed in quadruplicate, and
two vials without acetate served as controls.

In the K s and the starvation assays, 1.7 m 1mixed sludge (Rl-to-R2

ratio 1: 1) was distributed to the 50-ml vials. In the K s assays, acetate
of 0.64, 0.90, 2.6,4.6, 8.3, 15, 41, 76, 114, and 218 m M were added. In
the starvation assays, 50 m M acetate was added into the vials after
1,2,3,6, and 15 days of incubation without any substrate. Each
assay was run in duplicate or triplicate, and three vials without
acetate served as controls.

Analyses
COD, volatile solids and total solids were measured according to the
Standard Methods for the Examination of Water and Wastewater
(APHA 1992). C O D measurements were determined for samples
obtained through filtration by G F / A (Whatman). The pH was measured with a Radiometer combined electrode, model G K 2401C. The
methane content in the gas was determined using a Perkin-Elmer
Sigma 300 gas chromatograph, equipped with a hot-wire detector
and fitted with a Porapak Q stainless-steel column (mesh 80/100;
2 m x 0.32 cm). Helium at 30 ml/min was used as a carrier gas.
Acetate was measured with a Hewlett Packard 5890 series II gas
chromatograph, equipped with a flame ionization detector and an
automatic sampler. The gas chromatograph was fitted with an
H P - F F A P (Hewlett-Packard cross-linked polyethylene glycol-TAP)
column (15 m x 0.53 mm x 1.0 gin). Helium was used as a carrier gas
and nitrogen as an auxiliary gas. Prior to injection into the gas
chromatograph, the samples were centrifuged at 10 000 g for 15 min
and acidified with formic acid to a pH of less than 2.

Calculations
The specific methane production rate (/~cH4)was used to describe the
growth rate of the culture. In this study, the #cm was determined by
taking the slope of the best-line fit using the linear regression of the
natural logarithm of the initial cumulative methane production
versus time. The lag phase was defined as the time to the first
observation of methane production exceeding 1% in the head space
of the vials, which amounted to about 9 gM in the 50-ml bottles and
about 29 g M in the 120-ml bottles. The kinetic values were calculated according to a modified Haldane equation (e.g. Wu et al.
1988) as follows:

~cm =

#CH4max

1 + Ks/S + (S/KI)"

(1)

Where/~cmm,x is the maximum #cH4, S is the substrate concentration, K s is the half-saturation constant, K i is the inhibition constant,
and n represents the inhibition response coefficient. The #cH, values
reported in the abstract and the text represent the average of the
duplicate or the triplicate of each of the acetate concentrations (see
bioassays above).

Results
UASB reactors
The two UASB reactors were run at 70C for 3 months,
with a constant hydraulic retention time (calculated per
total reactor volume) of 1 0 _ 0.5 h and a load of
6.6 _+ 0.4 g C O D 1- 1 d a y - 1. The effluent COD, except
for the first 14 days, was consistently lower for R1 than
for R2 (Fig. 1). C O D reduction and methane production (data not shown) commenced in both reactors
shortly after start-up (less than 48 h), with a C O D

1003
3500
mgl~
3000

Table 1 Methane produced, residual acetate, and specific methane

production rate (gcnj of sludges from reactors R1 and R2 in bioassays at 70C

2500

Sludge Acetate
CH4
source consumed produced
(mM) a
(mM) ~

DO

2000

o 1500
1000

R1
R2

CH4
Residual
produced acetate ~
(% of
(raM) ~
acetate
removed) ~

#cn,
(h -~)

54.2 +_ 1.1 53.5 0.2 98.7 _+ 1.7 0.3 + 0.01 0.025 _+ 0.002
49.4 _+ 2.2 45.2 _+ 0.8 91.7 +_ 2.3 5.2 _+ 1.3 0.024 _+ 0.002

500

a After 239 h of incubation

20

40

60

80

100

Time (days)
0.12

Fig. 1 Chemical oxygen demand (COD) effluent concentrations

from reactors R1 and R2

reduction of 25%-30%. The highest C O D removal

efficiency was 84% for R1 and 71% for R2, corresponding to C O D removal rates of 5.4 and 4.7g
COD1-1 day -1 for R1 and R2, respectively. The final
sludge volatile to total solids ratio was 0.27 for R1 and
0.34 for R2 as compared to an initial ratio of 0.32 for
both reactors. No excessive wash-out of the granulae
was observed.

h!
0.10

-- calculated values
z measured values

0.08

:L I
~EE
0.06

0.04

0.02

0
20

40

60

80

100 120 140 160 180 200 220 240 260

Acetate (raM)

Acetate conversion to methane

The methane recovery of the added acetate and the
#cH~ of the different sludges obtained from the reactors
at the end of the experimental periods were studied.
The lag phases in the methane production of both
sludges were less than 6 h. More acetate was converted
to methane by R1 sludge than by R2 sludge (Table 1).
The residual acetate concentration was higher for R2
sludge than for R1 sludge (Table 1). The #cn~ values
were similar for both sludges (Table 1). The final pH
was 7.5 for both sludges.

Fig. 2 Specific methane production rate (#cH4) of the mixed sludge

at various initial acetate concentrations. The calculated values were
obtained using a modified Haldane equation (Eq. 1). The data fit was
determined according to non-linear least-squares regression using
the Excel (5.0) add-in program solver. The measured #~, values
represent the duplicate or the triplicate values of each of tl~e~acetate
concentrations

especially at acetate concentrations of less than 50 mM.

From Eq. 1 the Ks, Ki, and #Cn4max values were calculated to be about 1.5 mM, 2.8 raM, and 0.28 h -1
respectively, while n was 0.8. The absolute average
error was 0.0112 h -1. Various fits for the data were
performed resulting in negligible change in the
Ks, Ki, #cn~.... and n values (data not shown).

K~
The effects of the initial acetate concentrations on
#cH4 were studied. The lag phase values in methane
production were similar (4-5 h) when the initial acetate
concentrations ranged from 0.64 m M to 8.3 mM and
then increased with the increase of the acetate concentration, ranging from less'than 16 h to less than 235 h
with acetate concentrations of 15-218 raM. The highest
#cn~ (0.088 h -1) was determined at 4.6 mM acetate
(Fig. 2). The #cn4 value decreased with the increase of
acetate concentrations. At 15 mM acetate concentration #ci~ value had decreased to 0.048 h - 1.
O u r data did not fit the classical Monod or
Michaelis-Menten equations. However, when a modified Haldane equation (Eq. 1), which accounted for
substrate inhibition, was used, a clear fit was observed,

Starvation
The effects of the starvation of the sludge at 70C on the
#cn4 were evaluated. The lag phase in methane production was similar for starvation of 1-3 days and increased with increasing starvation time (Table 2). The
#cH4 values were similar for the 1- to 3-day starvation
time, while the values decreased as the starvation time
increased (Table 2).

Discussion
The results of this study show that anaerobic treatment
of acetate is feasible and reliable in UASB reactors at

1004

70C. The COD removal and loading rates compared

favourably with those reported for UASB reactors at
70-75C (Table 3).
Ours was apparently the first start-up of a 70C
UASB reactor with thermophilic (55C and 70C)
sludge reported so far. The utilization of the substrate
commenced rapidly (within 2 days) even at an initial
loading rate of about 7 g COD 1- 1 d a y - 1. The start-up
of a 70C UASB reactor with mesophilic granular
sludge and half the loading rate of this study (Table 3)
(Rintala et al. 1993) required about 2 months to reach
an effluent COD concentration of 500-700 mg 1-1, as
compared to about 1 month in this study. Van Lier et
al. (1992a) reported a stoppage in methane production
during the first 12 days of the start-up of a 75C UASB
reactor with mesophilic granular sludge at a loading rate
of 7-8 g COD1-1 day -a. They also reported a rapid
decrease of the sludge bed volume due to a severe
wash-out of the fine biomass. Thermophilic granulae
(55C) were reported to be less dense and more likely to
wash-out than mesophilic sludges (Van Lier et al. 1992a).
In the present study, the granulae were not specifically
less dense and no excessive wash-out was noticed.
The addition of yeast extract enhanced COD removal efficiency in the UASB reactors and also resulted
in higher #cm values and a higher percentage methane
recovery in the bioassays with yeast-cultivated sludge
than in the bioassays with sludge cultivated without
yeast. The effect of yeast may be due to its mineral and
vitamin content and to its being a source of some
amino acids for biosynthesis (Bryant 1971; Smith and
Mah 1980; Brunetti et al. 1983). Shen et al. (1993)
demonstrated that adding yeast to 55C UASB reactors enhanced the COD removal rate with no effect on
the specific methanogenic activity.

Table 2 The effects of starvation of the sludge (mixture from R1 and

R2) on the lag phase in methane production and on #cm at 70C
Starvation time

Lag phase (h)

#cH4 (h-1)

1
2
3
6
15

6
6
6
20
24

0.022
0.023
0.020
0.015
0.010

+_ 0.003
_+ 0.003
_+ 0.004
_+ 0.001
__ 0.001

The #ci~4 of 0.025-0.019 h - 1, found in this study at

41-76 m M acetate, was of the same magnitude as that
found by Rintala et al. (1993), who reported a #cH4 of
0.023-0.029 h-1 at 50 m M acetate at 70C. This suggests that similar bacteria, apparently a syntrophic
coculture consisting of acetate-oxidizing bacteria and
hydrogen-utilizing methanogens (Ahring and Rintala,
manuscript in preparation), were dominant in the
UASB reactors of this study and in the study by Rintala
et al. (1993).
Our results showed that methanogenic activity at
70C is highly substrate-dependent and that the measured maximum activity-contrary to current assumptions (e.g. by Rintala et al. 1993)-is comparable to
activities at 50-65C. The measured highest #c~4 was
0.088h-1
comparable to values (#Cn,ma~ of
0.058--0.086 h-1) obtained with acetate-utilizing thermophilic (55-65C) methanogens (Touzel and Albagnac 1984; Zinder et al. 1984; Touzel et al. 1985; Clarens
and Moletta 1990). The calculated low Ks and
Ki values indicate a high affinity for substrate with high
sensitivity to increase in the substrate concentration.
The inhibition at increasing acetate concentrations
could be due to undissociated acetate and/or to hydrogen partial pressure. The degree of inhibition in anaerobic systems is strongly dependent on the concentration of the undissociated forms of fatty acids (Fukuzaki
et al. 1990), which is a function of both total acids
concentration and pH (as reviewed in Buhr and Andrews 1977). Clarens and Moletta (1990) observed, at
55C and at increasing acetate concentrations, a decrease in the acetate utilization rate by Methanosarcina
sp. MSTA-1. Sodium ion (from sodium acetate) at
concentrations above 100-150 mM was shown to inhibit methanogens (Yang and Okos 1987; Ahring et al.
1991). In this study, the inhibition of the biomass at
a sodium acetate concentration of less than 5 0 m M
suggests that the observed effects were mainly due to
the acetate concentration.
An optimum hydrogen partial pressure is essential to
maintaining a favourable thermodynamic reaction of
the acetate oxidizers and the hydrogenotrophic methanogens in a thermophilic syntrophic coculture (Lee
and Zinder 1988b; Zinder 1990). Also hydrogen partial
pressure increases with an increase in temperature (Zinder 1990). In this study, the hydrogen partial pressure

Table 3 Load, hydraulic retention time (tn~), and chemical oxygen demand (COD) removaI characteristics of 70-75 C upflow anaerobic
sludge-blanket reactors (TMP thermomechanical pulping process water, VFA volatile fatty acids)
Feed

T (C)

Load
(g C O D 1- 1 day - 1)

COD
removaI (%)

tHR
(h)

Reference

Acetate
Acetate
TMP
VFA

70
70
70
75

6-7
3-4
4 5
7-8 a

84
90
55 60
60 a

9.5-10.5
24
15-16
18

This study
Rintala et al. 1993
Rintala and Lepist6 1992
van Lier et al. 1992

a Estimated from publihsed data

1005
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Acknowledgements This work was financially supported by the
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m a y h a v e i n c r e a s e d as a r e s u l t o f a c e t a t e o x i d a t i o n b y
a c e t a t e - o x i d i z i n g b a c t e r i a to a level t h a t i n h i b i t e d t h e i r
performance. This would mean that hydrogen oxidation by the methanogens was more limiting than
acetate oxidation.
S h o r t - t e r m s t a r v a t i o n ( 1 - 6 d a y s ) o f t h e 70C s l u d g e
h a d little o r n o effect o n t h e #cn4. H o w e v e r , p r o l o n g e d
s t a r v a t i o n c a u s e d a n i n c r e a s e in t h e l a g p h a s e a n d
a d e c r e a s e in t h e #CH4, i n d i c a t i n g a p o s s i b l e p a r t i a l
d e c a y of t h e b i o m a s s . O h t s u k i et al. (1992) s h o w e d t h a t
m e t h a n o g e n i c a c t i v i t y at 55C a n d u n d e r n o - l o a d c o n d i t i o n s d e c r e a s e d b y 3 0 % after 7 d a y s a n d b y 9 0 % after
68 d a y s of s t a r v a t i o n , w h i l e n o c h a n g e o c c u r r e d a t
30C.