Appl Microbiol Biotechnol (1997) 47: 640±644
ORIGINAL PAPER
L. BjoÈrnsson á B. Mattiasson á T. Henrysson
Effects of support material on the pattern
of volatile fatty acid accumulation at overload
in anaerobic digestion of semi-solid waste
Received: 3 December 1996 / Received revision: 7 February 1997 / Accepted: 14 February 1997
Abstract Anaerobic degradation of a semi-solid waste
with a total solids content of 4% particulate matter,
much of it insoluble, was investigated in four laboratory-
scale reactors. Two of the reactors were equipped with
di€erent textile materials for immobilisation of micro-
organisms, while the other two were used as continu-
ously-stirred-tank reactor references. A constant organic
loading rate and hydraulic retention time were used in
the start-up period; the hydraulic retention time was
then decreased and the e€ects of this change were
monitored. Volatile fatty acid (VFA) concentration and
pH were chosen as indicators of the microbial status in
the reactors. The reactors with support material showed
a greater resistance to overload than did the continu-
ously-stirred-tank reactors. This is in agreement with
many studies undertaken on the anaerobic treatment of
wastewater. However, no problems with clogging oc-
curred, showing that a support material is also appli-
cable in systems treating waste containing large amounts
of insoluble, particulate matter. The pH was compara-
ble to VFA for indicating an approaching process fail-
ure. However, the pattern of VFA accumulation was
qualitatively di€erent between the reactors with and
without support material. Obviously the metabolic pat-
tern of mixed cultures changes when the microorganisms
are immobilised.
Introduction
Anaerobic digestion is recognised as a process that can
be used to treat a broad range of highly concentrated
organic wastes e€ectively and, simultaneously, recover
energy in the form of biogas (Ghosh et al. 1975).
L. BjoÈrnsson (&) á B. Mattiasson á T. Henrysson
Department of Biotechnology,
Centre for Chemistry and Chemical Engineering,
Lund University, P.O. Box 124, S-221 00
Lund, Sweden
Ó Springer-Verlag 1997
However, there are problems with instability at start-up
and in the operation of the anaerobic process (Gijzen
et al. 1988; Huysman et al. 1983). This depends mainly on
the low growth rates and the sensitivity to pH variations
of anaerobic bacteria (Rozzi 1991). Much research has
been carried out on increasing the stability of the process
through improved process design. It has been shown
that this can be done by using a support material to
retain the slow-growing microorganisms within the
process. This approach has been successfully applied to
the anaerobic treatment of waste water containing high
amounts of soluble organic matter (Anderson et al.
1994; Defour et al. 1994; van den Berg and Kennedy
1981; Yee et al. 1992). However, the use of support
material for anaerobic digestion of wastes with high
amounts of particulate matter has not previously been
studied extensively. The combination of support mate-
rial and particulate matter leads to a potential risk of
clogging, and, thus, the continuously-stirred-tank reac-
tor (CSTR) is still the most commonly used process
con®guration for treatment of this kind of waste.
Another important factor for ecient operation of
the anaerobic digestion processes, extensively studied in
recent years, is the identification of parameters that
could be used for monitoring the process (Cobb and Hill
1991). The parameter monitored should re¯ect the cur-
rent metabolic status of the active organisms in the
system. There are several suggestions in the literature
regarding the choice of parameter to measure to indicate
instability before the total failure of the process.
One frequently investigated indicator is the amount
of volatile fatty acids (VFA). VFA are intermediates in
the anaerobic degradation process. If the substrate is
easily hydrolysed, further acidogenic fermentation is a
rapid process (Bryant 1979). However, methanogens
grow more slowly than the acidogens upstream in the
food chain and, therefore, an organic overload will in-
duce a build-up of VFA (Rozzi 1991). It is commonly
agreed that VFA build-up is the result of unbalanced
digestion conditions. The concentration of individual
VFA, especially acetic, propionic and butyric acid, can

be considered the best control parameters in the liquid
phase. This is because they indicate the metabolic state
of the most delicate microbial groups in the anaerobic
system: the obligate hydrogen-producing acetogens and
the acetoclastic methanogens (Rozzi 1991). Earlier in-
vestigations have, however, given contradictory infor-
mation on the pattern of VFA build-up at overload. It
has been shown, by several researchers, that build-up of
propionic acid is the ®rst sign of reactor overload in the
CSTR process (Renard 1991; Marchaim and Krause
1993; Hill et al. 1987), whereas others report that acetic
acid accumulates ®rst at the overload of both CSTR and
reactors with attached growth (Cobb and Hill 1991).
Another important indicator is alkalinity (Jenkins
et al. 1991; Ripley et al. 1986). If the amounts of VFA
are low, the bu€er capacity in the pH range optimal for
anaerobic digestion is almost totally dependent on the
carbonate weak acid/base bu€ering subsystem (Pre-
torius 1994). Wastewater containing carbohydrates,
VFA, and lipids would not generate bicarbonate alka-
linity at all when anaerobically degraded (Anderson and
Yang 1992). If the substrate is dominated by these
compounds, the alkalinity needed to maintain the pH is
dependent on the prevailing partial pressure of carbon
dioxide in the reactor (Pretorius 1994). At low initial
alkalinity in the process, the decrease in pH will be
larger at the increased VFA concentrations caused by
overload. Therefore, pH is more suited to monitoring
digesters fed on wastes which produce low bicarbonate
alkalinities during treatment (Rozzi 1991).
In this investigation, VFA concentration and
pH were chosen as indicators of reactor status. The
purpose was to compare the response of these parame-
ters in systems with and without support material to the
e€ect of a sudden change in hydraulic retention time.
Materials and methods
Substrate and inoculum
A semi-solid waste from a potato-processing plant was used as a
substrate. The waste had a total solids content of about 4%, of
which 85% were volatile solids (APHA, AWWA, WPCF 1985).
The substrate contained a large amount of insoluble, particulate
matter. Approximately half of the volatile solids was starch. In
addition, the substrate contained on average 5 g/l lactic acid, 1 g/l
acetic acid, 0.5 g/l propionic acid, 0.5 g/l n-butyric acid and 2 g/l
ethanol. Long-term storage was at )20 °C and the substrate was
kept at 4 °C just before use. The inoculum used was taken from a
large-scale biogas plant operating on this potato waste.
Support material
The most common process for anaerobic treatment of semi-solid
waste is the CSTR. The support material used in this investigation
was chosen to be suitable for upgrading to such a process, that is,
the material had to have an open structure to prevent clogging. The
material also needed to be both available at low cost and suitable
for the immobilisation of slow-growing microorganisms. Two
synthetic-®bre materials were chosen: a band with a width of
15 mm and a 5-mm-thick surface of small loops, and a 10-mm-
wide band with a bushy fringe; this was originally 200 mm long
641
but was cut to 100 mm for this application. Both materials are
commercially available and were supplied by Anox AB (Ideon
Science Park, Lund, Sweden).
Reactor design
Two of the four reactors were used as CSTR references with vol-
umes of 550 ml and 600 ml respectively. The third had 85 cm of
the band support material and a volume of 600 ml. The fourth had
a volume of 500 ml and 35 cm of the fringed support material.
The experimental set-up (Fig. 1) consisted of glass reactors
maintained at a temperature of 35 ‹ 2 °C and sealed with rubber
stoppers, each with in- and outlets for liquid and an outlet for gas.
The gas outlet of each reactor was connected to a gas meter, where
the biogas was collected using the water-displacement principle,
and was where gas samples were taken. The liquid in- and outlets
had two ports each, one connected to a peristaltic pump with a
timer for automatic feeding or withdrawal, and the other used for
manual sampling and feeding.
Experimental procedure
The potato waste was dispersed in order to get a pumpable slurry,
and then diluted to obtain a constant organic loading rate at de-
creased hydraulic retention time (tHR). Volatile solids were used to
provide a rough approximation of the amount of organic material
in the substrate, and the organic loading rate was reported as g
volatile solids l)1 day)1. Substrate was added in a semi-continuous
way once a day, either manually or automatically. Over a start-up
period of 120 days, an organic loading rate of 1.7 ‹ 0.3 g volatile
solids l)1 day)1 and a tHR of 20 ‹ 2 days was applied to the sys-
tem. This is the normal operating conditions for the CSTR process
(Ghosh 1984). At day 120, tHR was decreased from 20 to 15 days
with the organic loading rate remaining constant.
Analytical methods
Volatile fatty acids were monitored with HPLC, with a BioRad
125-0115 column for fermentation monitoring. The column tem-
perature was 65 °C. Sulphuric acid (5 mM) was used as a mobile
phase and the liquid ¯ow was 0.8 ml/min. The UV absorbance at
208 nm was used for peak detection.
Total solids and volatile solids were determined according to
standard methods (APHA, AWWA, WPCF 1985).
Measurement of pH was made o€-line with a pH electrode
(Schott-GeraÈte). Analysis of the substrate starch content was car-
ried out using a modi®ed version of an analysis developed by
Lyckeby StaÈrkelsen (Kristianstad, Sweden). The method includes
the enzymatic degradation of starch to glucose with thermostable
a-amylase and amyloglucosidase. The amount of glucose was then
analysed on HPLC, with the same conditions as above, but with a
refractive-index detector.
Fig. 1 Schematic outline of the experimental set-up
642
Results
During the start up-period, that is, days 1±120, the pH in
the CSTR references was 7.1 ‹ 0.3. In the reactors with
support material, the pH was higher: 7.2 ‹ 0.2 for the
reactor with the band support and 7.3 ‹ 0.2 for the
reactor with the fringe. At day 120, the tHR was decreased
from 20 days to 15 days with the organic loading rate
kept constant. For the CSTR references, this resulted in
an immediate drop in pH. In the reactors with the sup-
port materials, the pH drop was delayed about 15 days
after the tHR change (Fig. 2).
The two CSTR references showed similar behaviour
in the accumulation of VFA after the tHR was decreased
(Fig. 3). The ®rst response to overload was an increased
concentration of propionic acid.
Fig. 2 Changes in pH in the four reactors after decreased hydraulic
retention time
Fig. 3 Accumulation of volatile fatty acids for the continuously-
stirred-tank reactor references 1 (A) and 2 (B)
Fig. 4 Accumulation of volatile fatty acids for the reactors with the
band support material (A) and the fringe support material (B)
The two reactors with support material had higher
concentrations of acetic acid and propionic acid during
start-up. The pH in the reactor with the band carrier
dropped earlier than in the one with the fringed material,
and the accumulation of VFA also started more rapidly.
The ®rst indication of overload was an increase of acetic
acid concentration in both the reactors with support
material (Fig. 4).
Discussion
The reactors with support material showed a greater
resistance to overload than did the CSTR. This was
expected and is consistent with many studies on the
anaerobic treatment of waste water (Hickey et al. 1991).
The new ®nding in this study is that a solid support can
be applicable in a process for anaerobic digestion of
wastes with a high content of particulate matter without
causing clogging problems. The reason behind the in-
creased stability is the improved retention of slow-
growing microorganisms in the process.
There was a small di€erence in the resistance to
failure between the two reactors with carrier materials,
which can be explained by their di€ering ability to retain
the microorganisms in the system. Observation under a
microscope showed that the biomass was not growing
attached to the surface, but was entrapped in voids of
the material. The fringed material, which gave the best
resistance to failure, had long porous ®bres to enclose
cell colonies, whereas the band had a smaller surface
area for cell immobilisation.
The reactors with support material had higher
VFA concentrations during the start-up period. High

concentrations of propionic acid at low organic load and
long retention time have been reported previously
(Mosey and Fernandes 1989). However, after a start-up
period of 120 days, the VFA level would have been ex-
pected to stabilise at a lower level. In spite of the higher
concentration of VFA at the start-up level, the pH was
higher in the reactors with growth-support material than
in the CSTR. This indicates that, apart from a higher
concentration of microorganisms, the bu€ering capacity
is greater in the reactors with support material.
The rise in the VFA concentration at overload gave a
corresponding drop in pH because the system had a low
bu€ering capacity. In this system, pH is as good as VFA
for indicating an approaching process failure, but the
use of pH for monitoring can only be recommended in
systems where the bu€ering capacity is low. However, it
is interesting to note that the pattern of VFA accumu-
lation is qualitatively di€erent in the reactors with and
without support material.
In the CSTR the accumulation of propionic acid was
the ®rst indication of overload, while an increased con-
centration of acetic acid was the ®rst sign in the reactors
with support material. Increased amounts of propionic
acid have been reported as a consequence of reactor
overload (Archer 1983; Hill et al. 1987; Marchaim and
Krause 1993; Renard 1991), which is in agreement with
the results from the CSTR. However, the reactors with
support material show increased acetic acid concentra-
tions as a ®rst response to overload. These results show
the importance of careful investigations before choosing
an indicator parameter for monitoring reactor status,
since the metabolic pattern of mixed cultures obviously
changes when the microorganisms are immobilised.
The most slow-growing and delicate microbial groups
in the anaerobic system are the propionate-assimilating,
obligate hydrogen-producing acetogens and the aceto-
clastic methanogens (Mosey and Fernandes 1989; Rozzi
1991). It is known that the growth rate of the propionate-
assimilating organisms is very slow since the free-energy
gain, and therefore the yield, from the conversion of
propionate to acetate is low (Barthakur et al. 1991;
Bryant 1979). There is also a critical e€ect of the partial
pressure of hydrogen (pH2) on the formation and degra-
dation of propionate. Glucose is fermented ®rst to
pyruvic acid, via the Embden-Meyerhof pathway, and it
has been suggested that thereafter the pathways depend
on the pH2 conditions (Moosbrugger et al. 1993; Mosey
and Fernandes 1989). Under low-pH2 conditions pyruvic
acid is suggested to be converted to acetic acid only,
whereas under high-pH2 conditions both acetic acid and
propionic acid are formed (Moosbrugger et al. 1993).
Further, high pH2 in the reactor inhibits the propionate-
assimilating organisms, giving an accumulation of
propionic acid. This could explain why propionic acid
accumulates in the CSTR, suggesting that, in these sys-
tems, the stress of overload causes increased pH2. In the
systems with support material, there is probably a relative
advantage for the hydrogen-assimilating methanogens,
and a low pH2 is maintained. This would direct the fer-
643
mentation towards the formation of acetic acid. The
limiting step instead becomes the acetoclastic meth-
anogens causing a build up of acetic acid at overload.
It should be stressed that these results are only valid
for digestion of easily hydrolysable semi-solid wastes
since the digestion of poorly hydrolysable wastes is
limited by the hydrolysis and not by methanogenesis
(Kaspar and Wuhrmann 1978).
Acknowledgements The support from Swedish International
Development Corporation Agency (Sida) and valuable technical
advice from Lyckeby StaÈrkelsen is gratefully acknowledged.
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