Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity
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a r t i c l e i n f o
around 70% of the methane produced in the digestion of
and carbon dioxide. When methanogenesis is not rapid
enough, volatile fatty acids (VFA) accumulate, whichmay lead
to lower pH and digester upsets.
The research presented in this paper focuses on the
production of methane from acetate by acetotrophic
Zahller (2004) and Bucher (2003) found that after batch feeding
their maximum capacity in anaerobic digestion and have
limited ability to handle high production of acetate. Noike et al.
(1985) found that aceticlastic methanogenesis proceededmore
slowly than hydrolysis of starches but more rapidly than
hydrolysis of cellulose. In the reactors of Bucher (2003) and
* Corresponding author. Tel.: 1 206 684 6532; fax: 1 206 903 0419.
Avai lab le a t www.sc iencedi rec t .com
wat e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 4E-mail address: firstname.lastname@example.org (A.S. Conklin).domestic sludge comes from the transformation of acetate to
methane usually by the aceticlastic methanogens (Jeris and
McCarty, 1965; Smith and Mah, 1966). The role of these
methanogens is important in maintaining the carbon flow
through the system via conversion of acetic acid to methane
of wastewater sludges that the gas production rate increased
and acetate accumulated, indicating that hydrolysis and
acidogenesis proceeded faster than methanogenesis. These
findings support those of Kaspar and Wuhrmann (1978) who
showed that aceticlasticmethanogens operate at nearly 50% of 2008 Elsevier Ltd. All rights reserved.
The degradation of organic matter in anaerobic digesters
occurs through three basic steps: hydrolysis, fermentation
and methanogenesis. Previous research has found that
methanogenesis and on the hypothesis that aceticlastic
methanogenesis is a key step in digestion of municipal
wastewater sludge which may determine the capacity of the
system to convert organicmatter tomethane and avoid system
upsets caused by VFA accumulation. In studies in our group,Article history:
Received 9 February 2008
Received in revised form
31 August 2008
Accepted 1 September 2008
Published online 1 October 2008
Anaerobic digester stability
Vmax,acDigester capacity0043-1354/$ see front matter 2008 Elsevidoi:10.1016/j.watres.2008.09.024a b s t r a c t
Aceticlastic methanogens are seen as a key to digester capacity and stability. This paper
develops and applies an assay to measure digester stability by measuring the maximum
aceticlastic methane production rate (Vmax,ac). The Vmax,ac in combination with acetate
concentrations was found to be an effective digestion monitoring tool to indicate process
upsets. At steady state, thermophilic, first stage and short SRT digesters generally had
a greater Vmax,ac than mesophilic, second stage or long SRT digesters. The ratio of the
Vmax,ac to the plant aceticlastic methane production rate, termed the Acetate Capacity
Number (ACN), is a measure of the excess capacity of the digester. Either Vmax,ac or ACN
can be used to estimate the capability to handle higher organic loading rates. Monod
modeling was used to predict Vmax,ac, ACN and maximum VS loading rates for mesophilic
and thermophilic digestion and for staged digesters to better understand expected diges-
tion capacity and stability.Box 352700, Seattle, WA 98195, USADepartment of Civil and Environmental Engineering, University ofHDR Inc., 500 108th Avenue NE, Suite 1200, Bellevue, WA 98004, USAd Washington, 201 More Hall,Monitoring the role of aceticlaActivity and capacity
A.S. Conklina,*, T. Chapmanb, J.D. Zahllerc,aCarollo Engineers, 1218 Third Avenue, Suite 1600, Seattle, WA 981bBrown and Caldwell, 701 Pike Street, Suite 1200, Seattle, WA 9810c
journa l homepage : www.er Ltd. All rights reserveds in anaerobic digestion:
D. Stenseld, J.F. Fergusond
ev ier . com/ loca te /wat res.
important because digester failure can be costly. There is
wa t e r r e s e a r c h 4 24896considerable debate in the literature as to the best way to
monitor digester stability. The parameters most commonly
discussed are: methane and carbon dioxide concentrations in
biogas (Callaghan et al., 1997), gas production or methane
production rates (Chynoweth et al., 1994), pH (Killilea et al.,
2000), alkalinity (Denac et al., 1988; Hawkes et al., 1992), gas
phase hydrogen concentration (Cord-Ruwisch et al., 1997) and
VFAs (Ahring et al., 1995). None of these methods can deter-
mine how close a digester is to failure. Methods that monitor
the activity of the aceticlasts, however, may measure the
capacity of the microbial community to use a key interme-
diate and thus indicate digester capacity and stability.
This research developed a batch bottle test aimed at
determining the activity of the aceticlastic methanogens
(Vmax,ac). The batch bottle test method used in this research is
a modification of batch bottle test assays developed and used
previously (James et al., 1990; Owen et al., 1979; Shelton and
Tiedje, 1984). This method uses replicate unfed and acetate-
fed bottles, monitoring methane produced over time and
determining Vmax,ac by subtracting 30% of the methane
production rate of the unfed bottles from the methane
production rate of the acetate-fed bottles. This parameter
directly indicates the maximum activity of the aceticlastic
methanogens and thus is a useful indicator of digester
capacity. The Vmax,ac parameter used in this research differs
from the aceticlastic capacity parameters developed previ-
ously both in how the parameter is calculated and in how the
parameter is measured. The excess digestion capacity can be
determined by comparing the Vmax,ac of a digesting sludge to
the plant aceticlastic methane production rate (Vplant,ac).
The objectives of this study were to develop a method to
measure digester capacity and aceticlastic stability in anaerobic
digestion, to apply the method to different anaerobic digesters,
and to investigate if variations can be related to different
digester operating conditions. This paper describes the devel-
opment of the Vmax,ac method. It then validates the use of the
Vmax,ac method by comparing it to traditional stability param-
eters during a forced upset of a bench-scale digester and an
unplanned upset of a full-scale digester. Finally the Vmax,ac and
Vplant,ac parameters were determined for full- and pilot-scale
digesters to gain understanding of digester capacity and
stability. Monod and Herbert (Roels, 1983) based kinetic equa-
tions were adapted to predict the acetate utilization capacity of
anaerobic digesters at different operating conditions.
2.1. Acetate utilization capacity (Vmax,ac) activity test
From the Monod adaptation of the MichaelisMenten equa-Zahller (2004) the initial burst of activity was likely from the
rapid hydrolysis of easily degradable substrates such as
starches and the slow degradation rates at the end of the
feeding cycle were from the hydrolysis of materials such as
Finding a method to monitor for digester capacity istion, the acetate utilization rate is a function of substrate
concentration (S ) and active biomass (Xa). When S[ the halfsaturation coefficient (KS) the MichaelisMenten equation
reduces to Eq. (1) and the initial substrate utilization rate
kXa Vmax (1)
where k equals the specific acetate growth rate.
The Vmax,ac test was conducted by adding 20 mL of digester
sludge to 55-mL serum bottles purged with an 80/20%mixture
of N2/CO2, bubbled through a 0.2% solution of titanium citrate.
One set of triplicate serum bottles was fed between 70 and
100 mM sodium acetate, while the other set of triplicate
bottles received an equal volume of water. Vmax,ac tests con-
ducted on mesophilic and thermophilic sludges were insen-
sitive to acetate concentrations above 50 mM sodium acetate.
Both sets were capped and shaken at 150 rpm in a 35 C waterbath. The methane production rate was determined by the
change in headspace methane concentration versus time,
typically for several hours.
In digesting sludge approximately 70% of the methane is
often produced by aceticlastic methanogens (Vac), and the
remaining 30% of the methane is produced by hydro-
genotrophic methanogens VH2 (Jeris and McCarty, 1965;Smith and Mah, 1966). Using this percentage, 30% of the
methane produced in the unfed bottles is assumed to be
produced by hydrogenotrophic methanogens VH2 ;u. Themethane produced from the aceticlastic methanogens in the
fed bottles (Vmax,ac) was then calculated as the gross methane
production rate of the fed bottles (Vtotal,f) less 30% of the unfed
methane production rate VH2 ;u. The total aceticlasticmethanogen biomass can be calculated to increase slightly
during the test as the acetate was consumed; however, this
biomass increase was not significant as indicated by linear
slopes of methane production versus time.
The error associated with the Vmax,ac values was deter-
mined using the linear mixed effects model (Pinheiro and
Bates, 2000) in order to account for multiple measurements
from three replicate bottles. Themixed effectmodel combines
both fixed effects (parameters associated with an entire pop-
ulation) with random effects (parameters associated with the
individual units). The linear mixed effect model is a function
in the data analysis programR version 1.9.0 (The R Foundation
for Statistical Computing, ISBN 3-900051-00-3). The program
uses the restricted maximum likelihood (REML) method to
estimate the errors associated with the linear fits. The stan-
dard error of the Vmax,ac value was then determined by the
linear combination of the standard errors for the slopes of the
unfed and fed bottles.
2.2. Vplant,ac determination and acetate capacity number
Since Vmax,ac depends on the acetate using biomass, Xa, which
in turn depends on digester loading, it is important to
compare the maximum rate of acetate use to the actual rate
occurring in the digester. The plant methane production rate
from acetate was calculated from volatile solids (VS) removal
data as shown in Eq. (2).
( 2 0 0 8 ) 4 8 9 5 4 9 0 4QV
digesters in this study were completely mixed, the effective
Plant (SP) in Renton, WA; Chambers Creek Wastewater
into a Carle Series 100 AGC Gas Chromatograph (Chandler
Engineering, Tulsa, OK) with a reduction gas detector (RGD)
wat e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 4 4897Treatment Plant (CC) in University Place, WA; the Annacis
Island Treatment Plant (AI) in Vancouver, BC; and the Central
Treatment Plant in Tacoma, WA (Tac). All digesters treated
mixed primary and secondary waste sludges with VS
concentrations that ranged from 3.0 to 5.6%. Temperature-
phased anaerobic digestion pilot plants were sampled at WP
and SP. Three temperature-phased digesters are used at Tac,
which has an initial autothermal aerobic digester. Annacis
Island digesters are a two-stage thermophilic system.
Samples were collected and immediately transported to labs
at the University of Washington where Vmax,ac tests were
2.4. Lab-scale digester operation
One bench-scale digesters was operated for this study and
was used tomeasure the effects of digester upset. The digester
was completely mixed, maintained in a 35 C constanttemperature chamber, and seeded with 4 L of digesting sludge
from the WP. The digester had an operating volume of 4-L,
a total volume of around 6-L and was manually fed thickened
raw sludge (50% primary sludge and 50% waste activated
sludge) daily tomaintain a 10 day SRT. An upset conditionwas
induced by daily dosing with CuCl2. The digester received
40 mg/Lreactor CuCl2 on day 0, followed by 20 mg/L on days 14,Vplant,ac depended to some extent on loading for the previous
days. To account for this, the Vplant,ac for a given day was
determined by averaging values for the 510 days prior to
The ratio of Vmax,ac to Vplant,ac, termed the acetate capacity
number (ACN) or the excess capacity of the digester for acetate
use, indicates the capacity of aceticlastic methanogens to
handle shock loads. A value close to one indicates that the
aceticlastic methanogens are operating near their maximum
rate and any additional feed will result in an upset condition,
and a value below one indicates acetate accumulation and
a condition approaching failure (Zahller et al., 2007).
2.3. Wastewater treatment plant samples
Anaerobic digesting sludge samples were obtained from five
municipal wastewater treatment plants for this study: West
Point Treatment Plant (WP) in Seattle, WA; South Treatmentwhere Q is digester feed rate (L/d), V is digester volume (L),
Rfeed and Rdigester are chemical oxygen demand (COD) to VS
ratios of the feed and digesting sludge, respectively, VSfeed and
VSdigester are the VS concentrations (g/L) in the feed and
digester, respectively, 0:395 LCH4=gCOD at 35C represents the
stoichiometry of COD conversion to methane, and 0.7 is the
fraction of COD degraded via acetate.
The Vplant,ac rate can also be determined based on the
product of the gas production rate and the digester gas
methane content. However, gas production data were not
thought to be accurate at several plants, and we chose to use
the VS-based calculation for all plants surveyed. Since the30 mg/L on day 5, 40 mg/L on day 6, 30 mg/L on day 7, 60 mg/L
on day 8 and 50 mg/L on days 914.(SRI Instruments, Torrance, CA). The carrier gas was N2, and
the oven temperature was maintained at 110 C.
2.6. Acetotroph modeling
Amodel that combines Monod kinetics for aceticlastic growth
and overall first order conversion of degradable substrates
allows prediction of the effects of SRT, temperature and
staging in completelymixed digesters andwith comparison to
Vmax,ac and ACN measurements, helps to interpret the
differences that we observe. The aceticlast growth equation
relates the biomass growth rate of the aceticlasts (rxa) to
biomass yield (Y ), kd, substrate utilization rate (rS) and Xa. The
aceticlastic biomass in digesters can be estimated fromamass
balance equation for a completely mixed reactor with no
influent biomass (Eq. (3)), incorporating biomass growth and
QXa YrS kdXaV (3)
where V volume of the digester (L) and Q volumetric flowrate (L/d).
Assuming a completely mixed, continuous flow reactor at
steady state, dX=dt equals 0, rS equals DS divided by the SRT,
and Eq. (3) can be solved for Xa, which in turn can be
substituted into Eq. (1) to determine Vmax,ac, assuming that
acetate consumed equals 70% of the COD reduction.
Vmax;ac k Y0:7DS1 kdSRT (4)2.5. Analytical methods
VS were measured according to Standard Methods (APHA
et al., 1995). The COD was determined according to a modifi-
cation of Standard Methods 5220 D by adding 2 mL of diluted
sludge to a HACH high range (01500 mgCOD/L) COD vial and
heating the vial for 2 h at 150 C. The CODwas measured witha HACH DR/4000U spectrophotometer (Loveland, CO). The pH
was determined with a Corning general purpose combination
probe and a Beckman B11 pH meter (Beckman Instruments,
Inc., Fullerton, CA). The alkalinity was determined by titrating
a 1:10 dilution of the sludge to pH 4.5 by the addition of
0.2 NH2SO4. This method followed Standard Methods 2320B.
VFAs and gas composition were measured by gas chro-
matography. The VFA concentrations were determined using
a HewlettPackard free fatty acid phase (FFAP) capillary
column (Agilent Technologies, Wilmington, DE) attached to
a flame ionization detector (FID) with helium as the carrier
gas. The oven temperature started at 80 C and after 1 minincreased to 120 C at 20 /min and then ramped to 205 C at10 /min. The methane and carbon dioxide were determinedby injecting 0.1 mL of reactor headspace onto a Hayesep
packed column (Supelco, Bellefonte, PA) attached to a thermal
conductivity detector (TCD) with helium as the carrier gas.
The oven temperature for the TCD was 110 C. Gas phasehydrogen sampleswere taken by injecting headspace samplesThe DS (or COD destruction) of a first stage (or single stage)
digester can be modeled by a first order relation (Batstone
4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 4et al., 2002). The mass balance for conversion of degradable
substrate in a completely mixed reactor at steady state is:
QS0 QS K1SV (5)which can be simplified to solve for the amount of substrate
that is degraded in terms of the influent substrate (S0):
where S0 influent degradable COD (mgCOD/L), DS de-gradable COD (mgCOD/L) removed in a first or single stage
digester and K1 first order degradation coefficient (d1).The assumption that sludge degradeswith a first order rate
equal to K1 is an approximation to describe the overall VS
reduction (Batstone et al., 2002) and simulates the rate-
limiting conversion of particulate degradable COD to soluble
intermediates, which are further stoichiometrically converted
to acetate. A single first order rate probably underestimates
degradation in short SRT systems (Conklin et al., 2004; Straub
et al., 2006) and overestimates degradation in second stage
digesters. An equation for Vmax,ac in a first stage digester can
be developed by substituting Eq. (4) into Eq. (6) (Eq. (7)).
The estimate of DS from Eq. (6) assumes that all acetate
created by degradation of particulate organics will be further
converted to methane by aceticlastic methanogens. In fact,
the methanogens grow relatively slowly and are washed out
at low SRT values. The minimum SRT can be estimated from
Yk kd (8)
In the following modeling of 1st stage digesters, curves for
Vmax,ac and ACN are truncated at calculated SRTmin values for
the aceticlasts. The ACN of first stage digesters can be calcu-
lated by assuming that the Vplant,ac equals 0.7DS/SRT. With
this assumption the ACN equals (Eq. (9)):
ACN kYSRT1 kdSRT (9)
In staged digestion, the majority of the biodegradable
substrate available for digestion is consumed in the first stage,
resulting in decreased biomass in the second stage due to little
growth and significant endogenous decay. The decrease in
Vmax,ac from the first to the second stage can be estimated
frommass balance equations on both digesters. The Vmax,ac of
the second stage is estimated by conducting a mass balance
for second stage digestion, which includes aceticlastic
biomass in the influent Xa1 (Eq. (10))
Xa1 Y0:7 S0
i1 kdSRT1 (10)
and assumes further first order degradation of the degradable
COD. The substrate utilization rate of DS2 can be represented
by Eq. (11).
wa t e r r e s e a r c h4898DS2 S01 KISRT1 K1SRT2
1 K1SRT2 (11)75 rpm to 200 rpm. A shaking intensity of 150 rpmwas chosen
for further tests.
The effects of refrigerated storage were determined by
performing aVmax,ac test on sludge samples fromWPdigesters
and then refrigerating the sample for a day and repeating the
test. Refrigerated storage decreased the Vmax,ac by 11 to 17%
(differences significant with 95% confidence). From this data,
it was concluded that refrigerated sludge storage should beBy substituting Eqs. (4), (10) and (11), the Vmax,ac of the second
stage digesters can be estimated as described in Eq. (12).
3.1. Developing the Vmax,ac test
In order to develop a method to measure the Vmax,ac, it was
important to verify that themethodwas in fact measuring the
maximum acetate utilization rate and that test conditions
were not inhibiting methanogenesis. The effects of three
parameters were tested: Na toxicity, shaking during incu-bation and refrigerated storage.
Previous research found that high concentrations of
cations were toxic to aceticlastic methanogens and that the
toxicity of the specific cations varied (Kugleman and McCarty,
1965; McCarty and McKinney, 1961). The effects of Na andCa2 cation toxicity on digester sludge from WP were deter-mined by adding digested sludge to serum bottles along with
either 75 mM of sodium acetate (NaCH3COO) or 37.5 mM
calcium acetate (Ca(CH3COO)2). This test found that sodium
acetate was degraded more rapidly 1:05 LCH4=Lsludge d thancalcium acetate 0:76 LCH4=Lsludge d. The inhibitory concen-tration of Na for aceticlastic methanogens in digester sludgefrom WP was determined by adding digester sludge and
75 mM sodium acetate to serum bottles along with various
concentrations of NaCl (0304 mM). The results indicated
that 100 mM Na was only slightly inhibitory and causeda 4% decrease in the acetate utilization rate, while Na
concentrations in excess of 200 mM resulted in greater than
90% decrease in acetate utilization rates. The results of these
two experiments lead to the conclusion that sodium acetate
was suitable for the test method at concentrations below
The degree of shaking during incubation may affect the
ability of aceticlastic methanogens to use the added acetate
and thus affect the Vmax,ac. This effect was determined by
measuring the Vmax,ac for sludge from WP at three different
shaking rates. This test found that sludge incubated with
shaking intensities of 0, 75 and 200 rpm had Vmax,ac values of
1.18 0.05, 1.29 0.05 and 1:34 0:03 LCH4=Lsludge d, respec-tively. These results showed that shaking did increase the
Vmax,ac of the digester sludge, but that an insignificant
increase was gained by increasing the shaking intensity fromavoided, and when possible, the Vmax,ac test should be
conducted immediately after sampling.
3.2. Application of Vmax,ac test to monitoringdigester upsets
3.2.1. Induced failure of a lab-scale digesterThe effectiveness of the Vmax,ac test in monitoring digester
capacity during failure was tested by inducing a digester upset
condition in a 10-day SRT, bench-scale digester. During the
course of the upset, the Vmax,ac along with other stability
parameters were monitored (acetate, headspace methane
fraction, headspace hydrogen concentration, biogas flow rate,
alkalinity and pH).
In Fig. 1 the progression of each measured parameter over
the 14 day CuCl2 addition is shown. The first noticeable effect
of inhibitionwas seen after 9 days of CuCl2 additionswhen the
Vmax,ac began to decline and the acetate concentration rose
above 100 mg/L. During the first 8 days, the Vmax,ac averaged
2:20 LCH4=Lsludge d and ranged from 2:15 LCH4=Lsludge d to
2:29 LCH4=Lsludge d. On day 9, the Vmax,ac dropped by
10% to 2:0 LCH4=Lsludge d (a difference that was significant with
95% confidence), followed by failure on day 12 with Vmax,ac of
1:3 LCH4=Lsludge d and ACNs less than 1. The steady state
Vplant,ac was determined from steady state COD destruction
data and equaled 1:4 LCH4=Lsludge d. The ACN equaled 1.5 for
the first 8 days of the CuCl2 addition and began to decrease (as
the Vmax,ac decreased) on day 9. The alkalinity, headspace
methane content, biogas production rate and pH were within
normal operating ranges after 9 days of CuCl2 addition and
began to decrease after 11 days of CuCl2 addition. Headspace
hydrogen concentrations showed no pattern that would be
useful with regard to process control. The most useful indi-
cators of the initial stages of inhibition were increased acetate
concentrations coupled with decreased Vmax,ac values.
3.2.2. Full-scale digester upsetThe Vmax,ac test was also used to monitor the recovery of
a full-scale digester from an upset condition. After March 6,
2002, the volatile acids in WP Digester 4 increased from stable
values around 60 mg/L to a peak concentration of 1200 mg/L
(20 mM as acetic acid). At this point the digester operators
decreased feeding, and the volatile acids gradually decreased
(Fig. 2a). Once the volatile acids were back to steady state
levels, feedingwas gradually increased to the steady state rate
of around 60 kgal/d, corresponding to a 30 day SRT. However,
after 20 days of normal operation, the volatile acids again
increased and peaked at 1800 mg/L (30 mM/L as acetic acid).
Again the digester operators decreased the feeding rate and
the volatile acids gradually dropped (Fig. 2a).
0 2 4 6 8 10 12 14Time(days)
wat e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 4 489901020
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14Time(days)
Fig. 1 Response of traditional stability measurements to
an induced pilot-scale digester failure. Note: X-axis shows
time from the start of the induced CuCl2 digester failure
test. (a) Shows the response of Vmax,ac, ACN and acetate
concentrations. (b) Shows the response of Gas Production,
headspace methane and hydrogen concentrations. (c)Shows the response of pH and alkalinity (Conklin et al.,
0 50 100 150Time from 3/1/02 (days)
s as A
30 130 230 330 430
Time from 3/1/02 (days)
Control Digester Vmax,ac Dig 4 Vmax,acControl Digester Vplant,ac Dig 4 Vplant,ac
Fig. 2 Vmax,ac monitoring of an unplanned full-scale
digester upset. Note: (a) shows the Digester 4 feeding rate,
volatile acids and Vmax,ac for 150 days after the first
digester upset. (b) Shows the continued response of Vmax,ac
and Vplant,ac for Digester 4 and the control digesters for 430
days after the upset in Digester 4 (Conklin et al., 2005).
At the peak in the second upset (day 54), the Vmax,acmonitoring began (Fig. 2a). Based on the acetate concentra-
tion, by day 70 (10 days after the second acetate peak) the
digester might be assumed to be have recovered from the
upset. However, Vmax,ac values were very low, indicating that
the digester would be able to use only a small portion of the
normal feed load. The increase in Vmax,ac rates followed the
same trend as the increase in feeding rate, which was
controlled by the operators (Fig. 2a). The possibility of another
digester failure was minimized by slowly increasing the
In order to compare the effects of the upset in Digester 4
with the measured Vmax,ac values, Vmax,ac of comparable
digesters wasmonitored at the same time (Digesters 2 and 5 in
Fig. 2b). The results of the testing show that the Vmax,ac of
Digester 4 was consistently below the Vmax,ac of the reference
digesters, even a year after the upset.
The Vplant,ac for reference digesters and Digester 4 was
similar after 120 days (Fig. 2b). However, the Vmax,ac for the
reference digesters was always higher than for Digester 4. The
ACN of Digester 4 averaged 1.3 0.14, while the ACN of thereference digesters (Digesters 2 and 5) averaged 1.6 0.16.
(Tac 1 (thermophilic), Tac 2 (unheated, 38 C) and Tac 3(mesophilic)), the first and second thermophilic stages fromAI
(AI 1 and AI 2, respectively) and CC. The operating conditions
of the digesters at the time the samples were collected are
summarized in Table 1.
Of the digestion systems tested, the majority of the Vmax,actests were performed on the WP digester system. A total of 46
Vmax,ac tests were performed on these five digesters over
a period of 3 years, 20032005. The average Vmax,ac was
0:93 LCH4=Lsludge d, and the average standard error of each
Vmax,ac test was 0:03 LCH4=Lsludge d. This low standard error
indicates that the test is reproducible. Additionally over the 3
years of testing the standard deviation of all the tests was
0:13 LCH4=Lsludge d, indicating that the digestion system was
stable over this time period. A similar conclusion can be based
on ACN values, which averaged 1.7 0.3 for all WP digestersover the 3 year period, indicating stability of the digestion
system. The ACN values indicate the aceticlastic metha-
nogens were normally functioning at about 59% of their
maximum capacity, a value closer to capacity than measured
by Kaspar and Wuhrmann (1978).
The results from the Vmax,ac tests (Table 1) suggest that the
digester Vmax,ac was affected by three parameters: digester
wa t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 449003.3. Application of Vmax,ac test to full- and pilot-scaledigesters
The Vmax,ac test method was used for various pilot and full-
scale digesters, including the WP digesters, both stages of the
WP pilot plant temperature-phased anaerobic digesters
(TPAD, thermophilic phase followed by a mesophilic phase)
(WP TPAD1 and WP TPAD2, respectively), the SP digesters (in
Renton, Washington), both stages of the SP TPAD pilot plant
(SP TPAD1 and SP TPAD2, respectively), both stages of the SP
mesophilic staged digestion test (SP 1 and SP 2), the three
staged digesters at the Central Wastewater Treatment Plant
Table 1 Vplant,ac, Vmax,ac and ACN values for anaerobic dig
Stage N Vmax,acLCH4=Lsludge d
SP 1 35 1st 3 1.17 (0.1) 1.0 (0
CC 37 1st 1 1.09 (NA) 0.8 (N
WP 35 1st 46 0.93 (0.1) 0.6 (0
CC 35 1st 2 0.88 (0.0) 0.4 (0
SP 35 1st 4 0.68 (0.0) 0.4 (0
WP TPAD1 55 1st 1 2.17 (NA) 2.8 (N
Tac 1 55 1st 4 1.49 (0.8) 1.1 (0
SP TPAD1 55 1st 2 1.32 (0.1) 1.2 (0
AI 1 55 1st 2 1.79 (0.2) 0.6 (0
Tac 2 38 2nd 1 0.16 (NA) 0.1 (N
WP TPAD2 35 2nd 1 0.11 (NA) 0.2 (N
SP TPAD2 35 2nd 3 0.29 (0.1) 0.1 (0
Tac 3 35 3rd 2 0.09 (0.0) 0.1 (0
SP 2 35 2nd 4 0.38 (0.0) 0.0 (0
AI 2 55 2nd 2 0.83 (0.5) 0.2 (0
Notes: Nnumber of samples analyzed, SP South Treatment Plant,Treatment Plant, AIAnnacis Island Treatment Plant, TPAD temSRT solids retention time.
Values provided in parenthesis are standard deviations of the N samplestemperature, digester SRT and staging in addition to degrad-
able COD of the feed sludge (S0). For 1st stage or single stage
digesters, the Vmax,ac for thermophilic digesters 1:6 0:6LCH4=Lsludge d was greater than the Vmax,ac for mesophilicdigesters 0:9 0:2 LCH4=Lsludge d. The Vmax,ac for second stagemesophilic digesters 0:3 0:3 LCH4=Lsludge d was much lowerthan for first stage digesters 1:0 0:3 LCH4=Lsludge d. Alldifferences were significant with 95% confidence. There also
was an apparent decrease in Vmax,ac with SRT for both meso-
philic and thermophilic digesters that will be discussed later.
These observations are qualitative since Vmax,ac depends on S0,
which varied between plants and was not directly measured.
er measured in this study
(days)VS Load(kg/m3 d)
VSR (%) Max VS Load(kg/m3 d)
1.2 (0.1) 17 (1) 3.5 (0.3) 64 (1) 4.2 (0.2)
1.3 (NA) 18 (NA) 2.7 (NA) 68 (NA) 3.6 (NA)
1.7 (0.3) 26 (3) 2.0 (0.3) 64 (3) 3.3 (0.5)
2.0 (0.1) 31 (2) 1.4 (0.1) 66 (2) 2.9 (0.1)
1.6 (0.2) 39 (2) 1.5 (0.2) 65 (3) 2.3 (0.1)
0.8 (NA) 4 (NA) 10.9 (NA) 57 (NA) 8.4 (NA)
1.3 (0.3) 8 (1) 4.1 (1.0) 58 (2) 5.7 (2.8)
1.1 (0.1) 13 (3) 3.9 (0.8) 62 (3) 4.4 (0.3)
3.0 (0.0) 18 (1) 2.1 (0.1) 58 (0) 6.4 (0.2)
1.5 (NA) 8 (NA) 1.5 (NA) 13 (NA) NA
0.5 (NA) 8 (NA) 2.3 (NA) 30 (NA) NA
2.6 (1.9) 16 (0) 1.2 (0.2) 21 (14) NA
1.1 (0.5) 7 (2) 1.5 (0.5) 12 (1) NA
8.3 (2.1) 18 (1) 1.2 (0.1) 6 (3) NA
4.4 (1.5) 6 (1) 2.9 (0.0) 10 (3) NA
Chambers Creek, WPWest Point Treatment Plant, Tac Tacomarature-phased anaerobic digestion, VSR volatile solids reduction,.
with gene copy numbers ranging from 4 107 to8 108 copies/mL, indicating likely aceticlastic activity in bothmesophilic and thermophilic digesters. Methanosaeta was
present at 25 to 1000 times the concentration of Meth-
anosarcina in the mesophilic systems, while the ratios ranged
from 16 to 1.6 in the thermophilic digesters. The overall
predominance of methane production through acetate seems
clear, and in our system very likely is via aceticlastic
This research found that the Vmax,ac of a digester was
affected by three parameters: SRT, temperature and staging,
and the ACN was affected by SRT and staging. The model for
aceticlastic activity (more properly, acetotrophic activity) was
used to explore these relations, using kinetic coefficients lis-
ted in Table 2.
Using Eqs. (7) and (9) and the kinetic coefficients listed in
wat e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 4 4901There has long been some controversy about the fate of
acetate in methanogenic systems. It is widely recognized that
6070% of the energy flow from complex substrates to
methane is via acetate as an intermediate. There are only two
genera of methanogens that transform acetate to methane,
Methanosaeta andMethanosarcina, and often one or two species
will predominate in an anaerobic system. With the high
maximum specific substrate utilization rate (k), half satura-
tion coefficient (KS) and decay coefficient (kd), Methanosarcina
will dominate when acetate concentrations are high (above
approximately 100 mg/L). However,Methanosaetawith a low k,
KS and kd values is expected to dominate when acetate
concentrations are at levels typically found in mesophilic
digesters. Acetate can also be oxidized by syntrophic bacteria,
associated with hydrogenotrophic methane production. This
pathway has been shown to be more important in thermo-
philic than inmesophilic digestion. Petersen andAhring (1991)
and Griffin et al. (1998) have shown that the aceticlastic genera
were relatively less common in a thermophilic digester than
in amesophilic digester. However, Zinder et al. (1984) found in
a thermophilic digester that about 2/3 of methane produced
from a municipal solid waste feed was from aceticlasts, and
Karakashev et al. (2006) found that when Methanosaetaceae
were present in thermophilic and mesophilic anaerobic
reactors, aceticlastic methanogenesis predominated. In work
related to this study most digester samples were analyzed by
quantitative polymerase chain reaction methods for theACN values do not depend on S0, but are less precise than
Vmax,ac, because of dependence on plant data and seldom-
measured conversion factors for VS to COD.
The digester ACN values are also displayed in Table 1.
For first stage or single stage digesters, the ACN for the 18-
day thermophilic digester (AI 1) was 3.0, which is signifi-
cantly higher than values of 1.2 and 1.3 (SP1 and CC) for 17
and 18 day SRT mesophilic digesters. ACNs for the ther-
mophilic digesters with SRTs less than 20 days were much
lower than for the lower SRT mesophilic digesters (WP
TPAD1 at 0.8, Tac 1 at 1.3, SP TPAD1 at 1.1). Values for low
SRT thermophilic digesters (less than 13 days) were 0.8, 1.1
and 1.3 (WP TPAD1, SP TAPD 1 and Tac 1, respectively).
Thus, mesophilic digesters with SRTs less than 20 days and
thermophilic digesters with SRTs less than 13 days were
found to have very little excess capacity.
ACN values for second stage digesters were often high,
indicating considerable excess capacity, but two values were
low (less than 1), likely because of inaccurate estimates of
The ACN value can be used to estimate the maximum VS
load that first stage anaerobic digesters can process. The max
VS load is simply the ACN multiplied by the average VS load
(Eq. (13)). The VSmax for the sampled digesters are shown in
kg VSm3 d
kg VSm3 d
ACN (13)number of 16S rRNA gene copies for Methanosaeta and Meth-
anosarcina. Abundant aceticlasts were found in all reactorsTable 2, a relationship between Vmax,ac, ACN and SRT can be
developed and is shown in Fig. 3 for mesophilic digestion with
a typical S0 of 78,000 mgCOD/L. The measured and calculated
Vmax,ac values for themesophilic digesters are similar (Fig. 3a),
and both calculated and measured values decrease with SRT.
The washout SRT for methanogens is around 7 days, and thus
the low SRT portions of the curves are not shown. For SRTs
above approximately 7 days the extent of COD hydrolysis
begins to plateau, the decay terms tends to reduce Xa, and
Vmax,ac decreases with increasing SRT. Calculated and
measured ACN values, on the other hand, increase with
increasing SRT (Fig. 3b), since Vplant,ac decreases more rapidly
than Vmax,ac with SRT.
It was seen that second stage digesters had very lowVmax,acrates and high ACN values for mesophilic and thermophilic
digestion systems. Fig. 4a displays the Vmax,ac predictions for
mesophilic series configuration with equal SRT in both stages,
along with measured Vmax,ac values from four sets of samples
from series operation at South Plant. Both 1st and 2nd stage
results fit model predictions when kd values are between 0.08
and 0.1 d1. These high kd values are consistent with ourmeasurements (Conklin, 2004), using a modified Vmax,ac test
method, but they are higher than values commonly used in
digestion models, which may range from 0.02 to 0.05 d1.Because aceticlasts grown in the first stage enter the
second stage digester, the Xa values for the second stage
Table 2 Monod and hydrolysis parameters used topredict Vmax,ac and ACN values
k (mgCOD/mgVSS d) 6a 18a
kd (d1) 0.082b
Y (gVSS/gCOD) 0.032c
KI (d1) 0.33d 0.34d
Degradable fraction 0.75e
a Batstone et al. (2002).
b Average value measured for digester sludge.
c Measured for a mesophilic culture of Methanosaeta sp. (Conklin,
d Moen et al. (2003).e Newton (1999).
0 5 10 15 20 25 30 35 40
wa t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 449020.0
0 5 10 15 20 25 30 35 40 45 50
bdigester (and Vmax,ac values) might be expected to be higher
than the first stage. However, in the second stage, both the
Vmax,ac and Vplant,ac values are depressed. The Vplant,ac rates
are considerably more affected due to the low VS destruction
rates in the second stage, so ACN values are high. This trend is
shown with the measured values listed in Table 1 and the
measured and modeled data presented in Fig. 4b.
The Vmax,ac values for mesophilic second stages of TPAD
systems are influenced by the reduced temperature, as well as
factors incorporated in Eq. (11). Effects of temperature on rates
of microbial processes are often modeled with the Arrhenius
equation. We found that the Vmax,ac for a thermophilic sludge
decreased by 73% when incubated at 35 C (Conklin, 2004).This decrease corresponds to a temperature coefficient (q) of
1.067, which is similar to values presented in the literature
(Wu et al., 1995). Using q equal to 1.067, and accounting for the
effects of staging (Eq. (11)) for the three temperature-phased
systems, the calculated ratios of Vmax,ac values for second
stage digester to the first stage digesters were 0.28, 0.17 and
0.25 for the WP TPAD, SP TPAD and the Tacoma digesters,
respectively. These ratios are significantly higher than the
0 5 10 15 20 25 30 35 40 45 50
Fig. 3 Full- and pilot-scale Vmax,ac and ACN
Measurements. Note: data points represent measured fist
stage Vmax,ac (a) and ACN (b) values from nine full- and
pilot-scale digesters from around the North West. Solid
line shows the model results, using the kinetic coefficients
in Table 2 and a typical initial substrate concentration of
78,000 mg COD/L.6
1st Stage2nd StageSP 1SP 2
1st Stage2nd StageSP 1SP 2
ameasured ratios of 0.05, 0.12, 0.03 and 0.1 for theWP TPAD, SP
TPAD and Tacoma digesters (first and secondmeasurements),
respectively. This difference could be due to a lack of
understanding of the hydrolysis rates (KI) or decay rates for
thermophilic sludge. As KI and kd values increase, the ratio of
the Vmax,ac values for the second stage to the first stage
This paper found that theVmax,ac gives a good indication of the
maximum acetate utilization rate for the acetotrophic
activity, which is likely dominated by aceticlastic metha-
nogens. For the best-characterized West Point digesters, the
average method error was approximately 3% of the average
Vmax,ac. Additionally over 3 years of testing (after the 2002
upset) the standard deviation of all the tests was
0:13 LCH4=Lsludge d, indicating that the digestion system was
stable over this time period.
Vmax,ac provides an additional tool to assess digester
conditions and changes in operating conditions. It does not
0 5 10 15 20 25 30 35 40SRT (days)
Fig. 4 Measured and predicted Vmax,ac and ACN for 1st and
2nd stage mesophilic digesters. Note: data points represent
measured first and second stage Vmax,ac (a) and ACN (b)
values from the SP series operation pilot-scale digesters.
Solid line shows the model results, using the kinetic
coefficients in Table 2 and an initial substrate
concentration of 78,000 mg COD/L.
Department of Civil and Environmental Engineering at the
wat e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 4 4903University of Washington in Seattle.
r e f e r e n c e s
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to understand the state of the aceticlastic methanogenic
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for controlling operation and performance. These are:
1. It can indicate the degree of damage to a digester due to
a toxic shock or feed upset.
2. It can assess the recovery condition of a digester after an
3. It can be used to determine safe loading changes when
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risk for upset.
This research was funded by the National Science Foundation
grant number BES-0332118 and the King County Department
of Natural Resources and Parks, Advanced Wastewater
Anne Conklin, Jeffrey Zahller and Tom Chapman were
students at the University of Washington when the research
was conducted. Anne Conklin is now with Carollo Engineers
in Seattle,WA, Jeffrey Zahller is with HDR Inc. in Bellevue,WA
and TomChapman iswith Brown and Caldwell in Seattle,WA.
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wa t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 4 9 0 44904
Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacityIntroductionMethodsAcetate utilization capacity (Vmax,ac) activity testVplant,ac determination and acetate capacity numberWastewater treatment plant samplesLab-scale digester operationAnalytical methodsAcetotroph modeling
ResultsDeveloping the Vmax,ac testApplication of Vmax,ac test to monitoring digester upsetsInduced failure of a lab-scale digesterFull-scale digester upset
Application of Vmax,ac test to full- and pilot-scale digestersDiscussion