Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

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    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 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: (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.

    1. Introduction

    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 : Ltd. All rights reserveds in anaerobic digestion:

    D. Stenseld, J.F. Fergusond

    , USA


    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. Methods

    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

    measures Vmax.


    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).

    Vplant;acLCH4=Ldigester d

    VSfeed$Rfeed VSdigester$Rsludge

    ( 2 0 0 8 ) 4 8 9 5 4 9 0 4QV


    0:7 2

  • 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

    endogenous decay.


    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):

    DS S0

    K1SRT11 K1SRT1


    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)).

    Vmax;ac k"0:7Y



    i1 kdSRT1


    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

    Eq. (8):


    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).

    Vmax;ac k





    i1kdSRT1 0:7Y





    i1 kdSRT2

    375 (12)

    3. Results

    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

    100 mM.

    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)




    c (L



    ), A








    Acetate (m





    ), H

    2 (p




    Gas P


















    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



    4 (%





    CH4H2Gas Production








    0 2 4 6 8 10 12 14Time(days)








    12,000 Alk










    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)



    g R

    ate (kg



    latile A


    s as A

    cetic A






    c (L














    30 130 230 330 430

    Time from 3/1/02 (days)




    c an

    d V











    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

    feeding rate.

    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

    Source Temp.(C)

    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

    dACN SRT

    (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,.

  • 4. Discussion

    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

    Table 1.


    kg VSm3 d

    VS Load

    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

    Mesophilic Thermophilic

    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

    SRT (days)

    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

    SRT, days












    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












    c (L








    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


    5. Conclusions

    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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    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

    Technology Program.

    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

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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



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