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In-storage psychrophilic anaerobic digestion:

acclimated microbial kinetics
a a b a
Susan King , Pierre Courvoisier , Serge Guiot & Suzelle Barrington
a
Department of Bioresource Engineering , Macdonald Campus of McGill University , 21 111
Lakeshore, Ste Anne de Bellevue , QC , H9X 3V9 , Canada
b
Department of Environmental Bioengineering , Biotechnology Research Institute, National
Research Council of Canada , 6100 Royalmount, Montreal , QC , H4P 2R2 , Canada
Accepted author version posted online: 09 Dec 2011.Published online: 24 Jan 2012.

To cite this article: Susan King , Pierre Courvoisier , Serge Guiot & Suzelle Barrington (2012) In-storage
psychrophilic anaerobic digestion: acclimated microbial kinetics, Environmental Technology, 33:15, 1763-1772, DOI:
10.1080/09593330.2011.644867

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 Environmental Technology
Vol. 33, No. 15, August 2012, 17631772

In-storage psychrophilic anaerobic digestion: acclimated microbial kinetics

Susan Kinga , Pierre Courvoisiera , Serge Guiotb and Suzelle Barringtona
a Department
of Bioresource Engineering, Macdonald Campus of McGill University, 21 111 Lakeshore, Ste Anne de Bellevue QC H9X
3V9 Canada; b Department of Environmental Bioengineering, Biotechnology Research Institute, National Research Council of Canada,
6100 Royalmount, Montreal QC H4P 2R2 Canada
(Received 20 June 2011; nal version received 23 November 2011 )

In-storage psychrophilic anaerobic digestion develops by microbial acclimation in covered swine-manure storage tanks,
producing CH4 and stabilizing organic matter. To optimize the systems performance, the process kinetics must be understood.
The objective of this study was to evaluate kinetic parameters describing the major stages in the digestion process, and to
investigate the eect of temperature acclimation on these parameters. Specic activity tests were performed using manure
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inocula and ve substrates at three incubation temperatures. Extant substrate activities were determined analytically for
each case, and intrinsic kinetic parameters for glucose uptake were estimated by grid search tting to the Monod model.
The results demonstrate that this acclimated microbial community exhibits dierent kinetic parameters to those of the
mesophilic communities currently modelled in the literature, with increased activity at low temperatures, varying with
substrate and temperature. For glucose, the higher uptake is accompanied by lower microbial yield and half-saturation
constant. Decomposing these values suggests that active psychrophilic and mesophilic microbial populations co-exist within
the community. This work also conrms that a new method of assessing microbial substrate kinetics must be developed for
manure microbial communities, separating microbial mass from other suspended organics.
Keywords: psychrophilic anaerobic digestion; specic substrate uptake; manure treatment; microbial kinetics; swine manure

Introduction temperatures [7]. For example, after cultivation of granular

In-storage psychrophilic anaerobic digestion (ISPAD) is
a combined manure treatment and storage system devel-
oped for Canadian pork producers [1]. The process occurs
in manure storage tanks with air-tight covers when the
anaerobic microbial community in the manure has accli-
mated to the ambient operating conditions; manure solids
are reduced by 24%, and 63% of the potential methane is
released [2]. This performance could be optimized using
kinetic modelling, if the appropriate parameters are eval-
uated. Current modelling of manure decomposition, using
Monod kinetics and Arrhenius temperature correction fac-
tors, requires substrate uptake parameters for each microbial
population [3,4].
The substrate uptake kinetics of anaerobic microbial
communities may be assessed using specic activity assays
[5]. Substrates represent the major stages of anaerobic
digestion: glucose is the model substrate for acidogen-
esis; propionate and butyrate are used for acetogenesis;
acetate for aceticlastic methanogenesis; and H2 /CO2 for
hydrogenotrophic methanogenesis and homoacetogenesis
[6]. Microbial communities acclimated to psychrophilic
conditions exhibit increased substrate uptake at lower tem-
peratures, compared with non-acclimated communities,
but the increases are not uniform across substrates and
sludge for 306 days at 10 C, activities on acetate,
propionate and butyrate increased by factors of 3.65, 1.45
and 4.1, respectively at 10 C, and 2.44, 1.20 and 2.61
respectively at 30 C [8]. Similarly, after operating at 15 C
for 625 days, activities on acetate and butyrate increased
at both 15 and 37 C, whereas propionate activity remained
low, and H2 /CO2 activity increased continually throughout
the experimental period [9]. Propionate activity appears to
be the most sensitive to temperature change and the slow-
est to acclimate [10,11], while the highest acclimated VFA
activity is reported for butyrate [12].
When a mesophilic anaerobic microbial community
acclimates to psychrophilic conditions, such as those occur-
ring in ISPAD, for at least a year, the component popula-
tions generally exhibit maximum substrate uptake at 35 C,
which is taken to mean that they are still mesophilic [3,9].
At the same time, the greater increases in activity at low
temperatures indicate that these communities are psychro-
active [13]. Occasionally a fully psychrophilic microbial
population with a temperature optimum near 15 C is found
within the community [14,15]. However, psychrophilic and
mesophilic populations consuming a single substrate may
also coexist in a single community [16,17], in which case
the substrate uptake data may exhibit a bi-modal form with

Corresponding author. Email: Susan.King@mail.mcgill.ca

ISSN 0959-3330 print/ISSN 1479-487X online
2012 Taylor & Francis
http://dx.doi.org/10.1080/09593330.2011.644867
http://www.tandfonline.com
 1764 S. King et al.
mesophilic optimum, composed of two superposed uptake
curves [18].
Experimental substrate uptake data may be tted mathe-
matically to a model equation to estimate kinetic parameters
such as the Monod half-saturation constant, Ks [19,20].
The goodness of t is used to compare the suitability of
dierent relationship forms to the process represented by
the data [21]. Using this approach, a study of aceticlas-
tic methanogenesis data found that both the Monod and
Haldane models were accurate at 30 C, but the Haldane
model produced a better t between 6 and 22 C [22]. A
similar study concluded that the Haldane model was pre-
ferred at 22 C, while at 11 C both the Haldane and a
non-competitive model t the data equally well, proposing
that dierences could be attributed to the representation of
inhibition in each model [23]. However, a key diculty in
assessing and comparing kinetic parameters from substrate
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uptake data is the evaluation of the size of the active micro-
bial population involved [8]. In addition, the endogenous
production of substrate must be accounted for when eval-
uating the substrate uptake behaviour of manure microbial
communities [24].
The relationship between the maximum substrate
uptake kinetic parameter, qmax , and temperature is usually
described using the exponential Arrhenius equation [25].
Accordingly, in a study using granular sludge adapted to
10 C for 235 days, the temperature dependence of acetate
conversion was well described by an Arrhenius model;
however, propionate, butyrate and mixed volatile fatty
acid (VFA) activities were better described by a square-
root formulation [26]. Conversely, when digested sewage
sludge was adapted to 20 C for four months, the tempera-
ture dependence of the resulting methane production from
acetate was poorly described by both the Arrhenius and Hal-
dane equation forms; no better-tting model was proposed
in this study [23].
These results illustrate that acclimated anaerobic com-
munities may not always be treated as mesophilic, and that
parameters must be evaluated for each intermediate sub-
strate. In addition, the assumptions of Monod kinetics and
Arrhenius temperature dependence may not apply. As such,
the substrate uptake kinetics of an ISPAD community can-
not be estimated from known data, and must be dened in
the laboratory. Therefore the objective of this study was
to dene the kinetic parameters and temperature depen-
dence relationships of the main microbial populations in
an ISPAD microbial community. To accomplish this, swine
manure inocula from a three-year-old ISPAD installation
were evaluated using specic substrate activity tests. Assays
were performed at three temperatures, 8, 18 and 35 C,
using glucose, acetate, propionate, butyrate and H2 /CO2 as
substrates. Inocula from a similar uncovered storage tank,
and freshly produced manure were evaluated as controls.
Extant substrate uptake activity and optimal temperature
were dened for each population. Intrinsic activity was
estimated by curve tting to the Monod model.
Materials and methods
Manure inocula
In 2004, a full-scale ISPAD system was established in St.
Francois Xavier Quebec, Canada. The facility used a con-
crete tank, 30 m in diameter by 3.66 m deep, covered with
an air-tight membrane (GTI, Fredericton, NB, Canada). The
tank received swine manure on a regular basis. The contents
were removed for land-spreading twice yearly, except for a
depth of 0.30.6 m. Manure from this facility (Xf ) was used
to represent ISPAD in this study. The two controls, freshly
produced manure (Xm ) and one-year-old manure contained
in an uncovered storage tank (Xo ), were obtained from the
swine research facility of the McGill University Macdonald
Campus Experimental Centre, located in Montreal, Quebec,
Canada. Manures from the two operations were considered
comparable in terms of solids and nutrients, as they are pro-
duced by hogs fed a standard corn and soybean-based ration
[27]. Samples of each manure were collected in June 2007
as described previously [28].
Manure characterization
Sub-samples of all three manures (Xf , Xo and Xm ) were
analysed according to standard methods [29] to estab-
lish: solids, chemical oxygen demand (total and soluble)
and pH. The quantity of active microbial biomass in
each manure sample was estimated using the Luminultra
wastewater ATP kit (Luminultra, NB, Canada) and a lumi-
nometer (Sirius, model V3.2, Bethold Detection Systems,
TN, USA).
Specic substrate activity tests
Three sets of substrate activity tests (SAT) were performed
[30]: one using the microbial community contained in the
ISPAD manure as active biomass (Xf ), one using the com-
munity in the uncovered tank manure (Xo ) and the third
using the community in fresh manure (Xm ). Each set com-
prised three batches, one each at 8, 18 and 35 C. Each batch
included ve individual substrate assays: glucose, acetate,
propionate and butyrate were the liquid substrates, and
H2 /CO2 was the gaseous substrate used. All combinations
were run in triplicate.
For each batch, twelve 120 mL bottles (for liquid sub-
strates) and three 60 mL bottles (for gaseous substrate) were
prepared. Manure inoculum was added to each bottle, to
provide 5 g VSS L1 for liquids and 2 g VSS L1 for the
gaseous substrate. Phosphate buer was added, bringing
the volume of liquid in each bottle to 20 mL. The bot-
tles were sealed, ushed with N2 /CO2 gas (80%/20%)
and placed in a shaker (New Brunswick Scientic, Edi-
son, NJ, USA) operating at 100 rpm (400 rpm for gaseous
substrates), in a thermostatically controlled environment, in
the dark. The bottles were allowed three or four days under
these conditions for acclimation to the assay conditions.
 Environmental Technology 1765

Following the acclimation period, liquid substrate was Table 1. Initial periods for determination of extant substrate
injected through the cap of each 120 mL bottle, and the activity from SAT assays.
60 mL bottles were ushed and pressurized to 140 kPa with Initial period (hours)
H2 /CO2 . The rst sample was immediately taken: 0.5 mL
of liquid (for liquid substrate bottles) or 100 l of headspace
a,b
qmax Y a,c 35 C 18 C 8 C
gas (for H2 /CO2 bottles). Liquid samples were centrifuged Glucose 30 0.10 6 18 36
to remove solids. Sub-samples of supernatant were anal- Butyrate 20 0.06 14 45 91
ysed for glucose in the bottles with this substrate. For the Propionate 13 0.04 32 104 209
bottles fed acetate, butyrate or propionate, sub-samples of Acetate 8 0.05 42 136 272
H2 35 0.06 8 26 52
supernatant were diluted vefold for VFA analysis. For
the H2 /CO2 bottles, the headspace gas samples were anal- a Batstone et al. [3].
ysed immediately by gas chromatography. Sampling was b Maximum substrate uptake rate; COD COD1 d1 .
c Microbial yield; COD COD1 .
repeated at regular intervals during each assay period. At
the end of each assay, the bottle contents were analysed to
determine solids and pH. extant substrate uptake rate. This rate, divided by the VSS
concentration in the bottle, gave the extant activity, qext . In
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cases where the curve had a suitable form, it was tted to

Analytical methods
Glucose was measured using high-pressure liquid chro-
matography (Waters Chromatography Division, Milford,
MA, USA) equipped with an injector (model 717+), photo-
diode array detector (model 2996), pump (model 600) and
refractive index detector (model 2414). The column used for
the separation was an ICSep IC ION-300 column (Transge-
nomics, San Jose, CA, USA) of 300 mm 7.8 mm i.d. and
an ion guard GC-801 column (Transgenomics). The mobile
phase consisted of 0.035 N H2 SO4 at a pH of 4, owing at
a rate of 0.4 mL min1 . The measurements were conducted
using a wavelength of 210 nm. Analysis was carried out at
35 C.
Acetic, propionic and butyric acid concentrations were
measured using a gas chromatograph (Agilent, model 6890,
Wilmington, DE, USA) equipped with a ame ionization
detector of 0.2 L. The samples were fortied at a ratio of
1:1 (by volume) using an internal standard of iso-butyric
acid dissolved in 6% formic acid. These were directly
injected into a glass column of 1 mm 2 mm Carbopack C
(60 to 80 mesh) coated with 0.3% Carbowax 20 M and
0.1% H3 PO4 . The column was held at 130 C for four
minutes, and helium as carrier gas was injected at a rate
of 20 mL min1 . The injector and the detector were both
maintained at 200 C.
To quantify hydrogen consumption, biogas composition
(H2 , N2 + O2 , CH4 , CO2 ) was measured using a gas chro-
matograph (Hewlett Packard, 6890 Series, Wilmington,
DE, USA) equipped with a thermal conductivity detec-
tor and a 900 mm 3 mm 60/80 mesh Chromosorb 102
column (Supelco, Bellefonte, PA, USA).
Computations
All gas measurements were corrected to standard temper-
ature and pressure of 0 C and 101.3 kPa, according to the
ideal gas law. For each bottle in each assay, substrate con-
centration was plotted versus time. The maximum slope
of this curve within the initial period was dened as the
Monod equations, extracting intrinsic kinetic parameters,
Ks , Y and qmax .
Initial period denition
To avoid microbial growth during a specic activity assay,
sampling must be restricted to the rst few hours of the assay
[5,6,31]. Activity measured this way may be called extant,
while activity during the growth phase is termed intrinsic
[32,33]. To assess extant activities in this study, the initial
period was dened as equal to one doubling time for each
population, at the temperature of the assay.
The doubling time values for 35 C were determined
based on ADM1 model parameters [3]. The appropriate
values for 18 and 8 C were extrapolated by applying the
Q10 assumption to the maximum substrate uptake rate
qmax(T) = qmax(35 C) 1.072(T35) [34]. The doubling time
for each temperature (T) is then d(T) = ln(2)/(qmax(T) Y ).
The values of qmax(35 C) and Y and the calculated uptake
rates and doubling times for each population are presented
in Table 1.
Estimation of kinetic parameters
Kinetic parameters were estimated by tting the substrate
uptake data to the Monod-type equations for population
growth and substrate uptake [25]:
x = xi + Y (Si S)
dS/dt = (qmax S x)/(Ks + S)
where x is the concentration of active microbial biomass in
g L1 , Y is the yield of microbial biomass from the substrate
in g biomass (g substrate)1 , S is the concentration of sub-
strate in g L1 , qmax is the maximum substrate uptake rate in
g substrate (g biomass)1 d1 , and Ks is the half-saturation
constant in g substrate L1 . The subscript i indicates the
initial value of a parameter. The method of tting was devel-
oped using the means of the triplicate glucose consumption
 1766 S. King et al.

Table 2. Initial parameter estimates and boundary values for characterization data were collected in triplicate, on
glucose consumption at 35 C. sub-samples from the composite sample taken from each
Minimum Starting value Maximum tank. Signicance of dierences between these measured
values was evaluated by the Student NewmanKeuls
a,b
qmax 38.5 39.8 165.9 method in a simple analysis of variance based on a com-
Y a,c 0.008 0.075 0.128 pletely randomized design. The specic substrate activity
Ksa,d 0.022 0.469 0.591 assays used a randomized complete block design, consid-
xie 0.1 1.0 10.0
ering microbial community type (Xf , Xo and Xm ) as the
a Batstone et al. [3]. treatment factor, temperature (35, 18 and 8 C) as the block
b Maximum substrate uptake rate; g glucose (g biomass)1 d1 . factor, with substrate uptake rate (qext ) as the dependant
c Microbial yield; g biomass (g glucose)1 .
variable. Treatments were assigned randomly to experimen-
d Half-saturation constant; g glucose L1 .
tal units (bottles), and all treatmentblock combinations
e Biomass concentration; % VSS.
were completed in triplicate.

data for the uncovered tank biomass, Xo , which had a clearly Results and discussion
dened Monod-type form at each assay temperature. Once
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Characteristics of manure inocula

the method was perfected, it was applied with the same
algorithms, starting points, step sizes and assumptions to Table 3 lists the characteristics of the three manures used
the glucose consumption data sets for the fresh manure and as sources of active biomass in the specic activity assays.
the ISPAD tank communities. For each optimization, the The total solids concentrations illustrate dierences in water
starting and boundary values for the optimized parameters content: the ISPAD manure, Xf , and the uncovered tank
are presented in Table 2. manure, Xo , are respectively 19% and 52% less concen-
The ADM1 literature values of Ks , Y and qmax for glu- trated than the fresh manure, Xm . This is due to wash water
cose consumers were used with the 35 C data to nd a entering both storage tanks, as well as rain in the uncov-
starting value for xi . Values for all four variables were then ered installation. Evidence of anaerobic digestion in the
optimized using the grid search method, minimizing the ISPAD tank is seen in the volatile solids (VS), volatile dis-
sum of squares error (SSE) between the calculated value solved solids (VDS) and soluble chemical oxygen demand
and the experimental data. This method nds local minima (sCOD) contents of Xf . These are, respectively, 66%, 5%
but can be trapped in a local minimum and miss the global and 4% of total solids (TS), reduced from 72%, 20% and
minimum; however, because the tted values were expected 32% in fresh manure. Hydrolysis of VSS to VDS without
to be of the same order of magnitude as the ADM1 values, methanogenesis is evident in the uncovered tank, where VS
this was considered acceptable. The rst iteration step size and sCOD are equal to those of fresh manure in terms of
was 15% of the initial parameter value. For each subsequent percentage of TS, while VDS has increased to 32%. For
iteration, the step size was halved. Because the data con- all three manures, pH is within the normal range [35], and
tained a small number of points, the algorithm tended to the active microbial communities account for less than 2%
come up with extreme values that tted the data well, but
only reduced the SSE by negligible amounts. These values Table 3. Characteristics of experimental manures.
were also unrealistic in terms of the shape of the curve and
Fresh Uncovered ISPAD
the dierence from the starting values in the literature. To manure, Xm tank, Xo tank, Xf
control this behaviour, boundary values were added to the
algorithm. Boundary values for qmax , Y and Ks were selected TS (g L1 ) 48.0 (0.3) 22.7 (0.2) 38.7 (0.4)
from those used by the ADM1 model [3], while xi was kept VS (g L1 ) 34.3 (0.2) 16.5 (0.1) 25.4 (0.2)
FS (g L1 ) 13.7 (0.1) 6.3 (0.1) 13.3 (0.2)
within one order of magnitude from the starting value. The
VSS (g L1 ) 27.4 (1.2) 11.2 (0.6) 24.0 (0.2)
iterative process was then stopped when the change in SSE VDS (g L1 ) 7.0 (0.7) 5.3 (0.4) 1.4 (0.2)
from the previous iteration was less than 5% of the previous pH 6.9 (0.1) 7.3 (0.1) 7.5 (0.1)
SSE value. Total COD 2.4 (n.d.) 2.1 (0.1) 2.0 (0.1)
The optimized parameter values for the 35 C data were (g gVS1 )
then used as starting values for the 18 and 8 C data sets to Soluble COD 0.9 (n.d.) 0.7 (0.01) 0.1 (0.02)
(g gVS1 )
optimize Ks and qmax for each temperature, with the value ATP 12.0 10.7 16.7
of Y and the ratio of xi to VSS kept constant throughout. (g gVSS1 )
Active biomass 0.41.2 0.31.0 0.51.6
(% VSS)
Statistical analysis
Note: Values represent the average of three replicates; standard
All statistical analyses were performed using SAS deviations in brackets.
9.0 (2009, SAS Institute inc., Cary NC, USA). The FS = xed solids, n.d. = not determined.
 Environmental Technology 1767

Table 4. Form of substrate uptake response curve for each SAT

assay.

Fresh manure, Uncovered tank, ISPAD tank,

Glucose (mg L1)

Xm Xo Xf

Glucose
35 C E E E
18 C E E E
8 C L/E E E
Butyrate
35 C A/E L L
18 C A L L Figure 1. Exponential glucose uptake by uncovered tank
8 C A L L biomass, Xo . Data points represent the average of three replicates.
Error bars represent 1 standard deviation.
Propionate
35 C A/E L L/E
18 C A A E
8 C A A L
Acetate
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35 C A/E L/E L

Butyrate (mg L1)

18 C A A L
8 C A A L
H2 /CO2
35 C E E L
18 C E E L/E
8 C E L E

E = exponential, L = linear, A = accumulating.

of the VSS. The similar community size allows activities
for the three inocula, presented in terms of total VSS, to be
compared within the study.
Figure 2. Linear butyrate uptake by ISPAD tank biomass, Xf .
Data points represent the average of three replicates. Error bars
represent 1 standard deviation.

Substrate activity test (SAT) data sets

Propionate (mg L1)

Plots of the 45 SAT data sets displayed three distinct forms:

exponential, linear and accumulation. This categorization,
summarized in Table 4, dictated the kinetic parameters
that could be extracted from each set. Exponential curves,
such as those illustrated in Figure 1, allow the most com-
plete analysis. The initial slope represents qext ; in addition,
these curves may be accurately tted to the Monod equa-
tions, which estimate qmax and Ks . Linear data, illustrated
in Figure 2, denes qext , though qmax and Ks cannot be
evaluated. A combined linear-exponential response, such
as that seen in Figure 3, allows the same determinations as
the linear type, because the mild exponential portion does
not permit an acceptable t to the Monod equations. Accu-
mulation of a substrate is illustrated in Figure 4. Where
this is the complete response, no activity can be calcu-
lated, although some may be occurring concurrently with
the production of substrate from manure organic matter.
However, in cases where a linear or exponential pattern
followed initial accumulation, qext could be determined.
Extant substrate uptake activity
The extant substrate uptake activities, qext , for all substrate
biomass combinations are presented in Table 5. The ISPAD
Figure 3. Linearexponential propionate uptake by ISPAD tank
biomass, Xf . Data points represent the average of three replicates.
Error bars represent 1 standard deviation.
microbial community, Xf , is highly active on all substrates
tested and at all assay temperatures. This conrms its classi-
cation as a fully acclimated balanced anaerobic microbial
community [2,7]. The fresh manure community, Xm , which
shows measureable activity on glucose and H2 /CO2 only, is
not acclimated to anaerobic conditions. The community in
Xo is more active than this, consuming butyrate at all assay
temperatures, as well as acetate and propionate at 35 C.
Because butyrate activity is developed preferentially, fol-
lowed by acetate and then propionate activity later in the
acclimation process, the uncovered tank community may be
classied as semi-acclimated to anaerobic conditions [12].
 1768 S. King et al.

Table 6. Fitted kinetic parameters, biomass concentration and

error for glucose consumption.

Fresh manure, Uncovered tank, ISPAD tank,

Acetate (mg L1)

Xm Xo Xf
a
qmax
35 C 39.12 39.12 51.84
18 C 10.01 12.02 31.92
8 C 3.10 4.47 5.90
Yb
35 C 0.128 0.128 0.082
18 C 0.128 0.128 0.082
8 C 0.128 0.128 0.082
Figure 4. Accumulating and accumulating-linear acetate uptake Ksc
by fresh manure biomass, Xm . Data points represent the average 35 C 0.238 0.022 0.022
of three replicates. Error bars represent 1 standard deviation. 18 C 0.776 0.574 0.029
8 C 0.679 0.469 0.029
xid
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Table 5. Extant substrate activities, qext , from SAT assays. 35 C 7.16 5.26 7.41
18 C 7.16 5.26 7.41
Fresh manure, Uncovered tank, ISPAD tank, 8 C 7.16 5.26 7.41
Xm Xo Xf
e2e
Glucose 35 C 0.021 0.433 0.076
35 C 55 (68) 307 (243) 128 (100) 18 C 0.036 0.192 0.003
18 C 36 (23) 87 (12) 8 C 0.013 0.049 0.003
8 C 12 (1) 10 (3) 7 (7)
Fitting done using the average of three replicates.
Butyrate a Maximum substrate uptake rate; g glucose (g biomass)1 d1 .
35 C 18 (29) 204 (37) b Microbial yield; g biomass (g glucose)1 .
18 C 81 (1) 105 (23) c Half-saturation constant; g glucose L1 .
8 C 12 (8) 10 (2) d Biomass concentration; mg biomass L1 .
Propionate e Sum of squares error (goodness of t); (g glucose L1 )2
35 C 24 (35) 32 (10)
18 C 7 (9) acclimation process and be reduced in a fully acclimated
8 C 12 (5)
psychro-active community [7].
Acetate
35 C 83 (254) 316 (135)
18 C 28 (9)
8 C 47 (10)
Monod kinetic parameters
H2 /CO2 Since the Monod equations may only be tted to data sets
35 C 14 (7) 131 (225) with an exponential form, the glucose SAT data sets were
18 C 8 (1) 59 (24) analyzed in this way. Table 6 presents the resulting tted
8 C 52 (5) 1 (0.3) kinetic parameters qmax , Y and Ks , as well as the population
Note: Values represent the average of three replicates; standard size estimate, xi , for glucose consumption by each of the
deviations in brackets three studied microbial communities. The population sizes,
qext ; mg substrate (g VSS)1 d1 , = no measurable activity. representing 0.00.05% VSS, are much smaller than those
from the ATP analysis (Table 3), which is to be expected
since only a small number of the microbial species present
As reported by other researchers, the variations between are expected to be glucose consumers. However, because
communities, substrates and temperatures are not uni- the percentage VSS values obtained are similar for the three
form, and the values tend to support the mesophilic opti- communities, the resulting activity values may be compared
mum assumption, with maximum activities at 35 C. The on this basis.
H2 /CO2 activity of Xo , which increases with decreasing For Xo and Xm , the maximum substrate uptake rate,
temperature, is the exception. In this case, the test pro- qmax , at 35 C remains equal to the literature value pre-
cedure may have been a limiting factor, or it may be a sented in Table 2, at 39.12 g substrate (g biomass)1 d1 ,
further indication of acclimation occurring in the uncov- while Xf exhibits a 33% increase to 51.84 g substrate
ered tank. Whereas homoacetogens were reported to have (g biomass)1 d1 . This increase in the processing e-
a larger role in low-temperature digestion than in the ciency of the glucose-consuming population within Xf
mesophilic process [36], it has been proposed more recently illustrates acclimation to the substrate. This is further
that homoacetogen activity may be highest during the demonstrated at 18 and 8 C, where Xf shows increases
 Environmental Technology 1769

of 220% and 90%, respectively, in comparison with the

values calculated for Xm at these temperatures. These
greater increases at lower temperatures indicate acclima-
tion to the psychrophilic operating temperature of ISPAD,
as reported by other researchers [8,9]. In contrast, qmax for
the semi-acclimated Xo remains equal to that of Xm at 35 C,
showing modest increases of 20% and 44% at 18 and 8 C,
respectively.
The microbial yield, Y , exhibits a similar pattern,
with the Xf value 35% lower than that of Xo and Xm ,
which remain identical. This indicates a change in cellular
metabolic processing, as the glucose-consuming population
in Xf produces less microbial biomass from each gram of
substrate consumed, while concurrently consuming more
substrate. The excess consumed substrate, then, is converted
to intermediate products in the anaerobic digestion process.
This contributes eventually to the increase in methane pro-
Downloaded by [University of Guelph] at 02:24 14 November 2014

duction previously reported for the ISPAD biomass [2].
Because Y is expected to resist change for any given species
[37], this eect may represent a change in the predominance
of species making up the glucose-consuming population as
it acclimates to the ISPAD operating conditions. A follow-
up molecular biology investigation of the ISPAD microbial
community is planned, to explore this possibility.
The apparent half-saturation constant, Ks , indicates the
concentration at which the population is able to process the
substrate at half its maximum rate, which represents the
anity of the microbial population for the substrate. The
implication is that, at concentrations lower than Ks , process-
ing is slow and inecient and may result in accumulation.
Continuing the pattern seen with qmax and Y , the Ks val-
ues obtained for Xm remain similar to the original literature
values representing a standard mesophilic population. The
values of Ks representing Xf are 9096% lower, showing
that the glucose-consuming population in Xf is able to e-
ciently utilize substrate even at concentrations a full order
of magnitude lower than the standard mesophilic popula-
tion. This indicates that the ISPAD microbial community
in Xf is acclimated not only to temperature and substrate
but also to the low substrate concentrations which occur in
the lightly fed ISPAD tank. The uncovered tank commu-
nity in Xo , with a low Ks comparable to that of Xf at 35 C,
illustrates the beginning of the acclimation process, while
the higher values similar to Xm at 18 and 8 C indicate that
the community in Xo remains only semi-acclimated and
primarily mesophilic.
Temperature dependence
Figure 5 illustrates the relationship between the tted values
of qmax and temperature. These points describe an expo-
nential Arrhenius-type curve for Xo and Xm , as expected
for mesophilic populations. In contrast, for Xf the curve
is reversed. This suggests the coexistence of psychrophilic
and mesophilic populations [18]. Assuming that the qmax
values for Xm represent an average mesophilic population,
Figure 5. Maximum glucose uptake rates, qmax , for fresh manure
biomass, Xm , uncovered tank biomass, Xo , and ISPAD tank
biomass, Xf . Data points represent the average of three replicates.
Data are derived from curve tting. See Table 6 for error data for
the tting process.
these values were subtracted from the qmax values of Xf
to produce psychrophilic values. These are illustrated in
Figure 6, and represent the eect of the temperature accli-
mation process on the ISPAD microbial community. With
the clear peak near 18 C, these psychrophilic qmax values
for Xf illustrate the development of a true psychrophilic
population coexisting with the original mesophilic popu-
lation. This concept is corroborated by recent reports of
similar results [16,17]. Assessing qmax at other intermediate
temperatures could further dene the relationship.
Extant activity interpretation
Modest activity in the initial period of an SAT assay is
sometimes dismissed as a lag period, caused by microbial
acclimatization to the test conditions; however, these SAT
bottles were allowed three to four days for acclimatization
prior to the injection of specic substrates. This period did
not constitute starvation conditions, because of the pres-
ence of manure organic matter. Strong activity immediately
following substrate injection was evident in many bottles,
conrming that acclimatization had occurred. Therefore,
any lag due to acclimatization had occurred prior to sub-
strate injection, and activity during the initial period after
injection was considered to represent the original biomass.
Increasing activity following the initial period was assumed
to represent microbial growth due to substrate excess in the
bottle.
The extant activities reported in Table 5 exhibit a wide
variety of standard deviations. This was understood as
a compound error, because substrate concentration, time
elapsed and VSS concentration are all measured factors
 1770 S. King et al.
Downloaded by [University of Guelph] at 02:24 14 November 2014
Figure 6. Maximum glucose uptake rates, qmax , for possible
coexisting mesophilic and psychrophilic populations within the
ISPAD tank biomass, Xf . Data points represent the average of
three replicates. Data are derived from curve tting. See Table 6
for error data for the tting process.
exposed to experimental error, which combine to deter-
mine each specic substrate activity. Thus the errors, in
some cases, may be additive and, in others, may be subtrac-
tive, resulting in the wide variety of deviations reported.
Despite this variation, the data illustrate trends that have
been observed by other researchers, which strengthen their
validity. In fact, substrate uptake activities are known to be
highly variable, as many related parameters used by ADM1
are reported to have up to 300% variability [3].
Monod kinetics interpretation
The extant substrate uptake rate may, in cases where the
SAT response form was linear, be equivalent to the Monod
maximum uptake rate. However, further assays using dif-
ferent initial substrate concentrations would be required to
conrm this. Therefore, the Monod parameter was only
assessed for those assays demonstrating an exponential
uptake response. Likewise, assessment of the Monod half-
saturation constant, Ks , from SAT data requires that the rate
of substrate uptake varies during the assay. As a result, this
parameter could only be estimated for the data sets with
an exponential response pattern, as part of the curve-tting
procedure.
The SAT assay protocol is designed for specic
biomass-substrate ratios. The non-exponential responses
seen in this study may have been caused by variations in
this ratio, caused by both the presence of extremely small
anaerobic microbial populations in this biomass and the
presence of manure organic matter in the bottles, the degra-
dation of which may produce VFAs and mask concurrent
consumption by the appropriate population. It must be noted
that the assay results for the ISPAD biomass were more
accurate in many cases than those from the two control
samples, owing to the minimal concentration of dissolved
solids in the ISPAD-treated manure.
Both accumulation and minimal activity responses were
seen primarily in the data from assays using fresh manure.
Unlike the dairy manure used in other anaerobic digestion
studies, this seed community is not expected to harbour
a fully developed anaerobic population; pigs are non-
ruminants and have a dietary transit time of approximately
one day, which is less than the doubling time of some key
anaerobic populations [38]. Therefore these response types
are understood to be due to the exceptionally small popula-
tion, as well as the presence of other organic substrates in
the manure.
ISPAD kinetics
The kinetic impact of acclimation may be inferred by com-
paring the ISPAD qext values in Table 5 to the mesophilic
qmax values reported in Table 1. This can only be done
by ranking the activities in each case, owing to both the
population size and experimental uncertainties. Mesophilic
communities are most active on hydrogen, followed by glu-
cose, butyrate propionate and acetate in decreasing order
of magnitude. The ISPAD community, however, is most
active on acetate, followed by butyrate, hydrogen, glucose
and propionate, at 35 C; the rankings are led by butyrate
at 18 C, and glucose at 8 C, with variations in the fol-
lowing rankings as well. This comparison conrms that the
impact of acclimation on the kinetic parameters varies with
substrate and temperature.
The Monod kinetic parameters estimated for the ISPAD
glucose-consuming population reveal a higher qmax , lower
Y and lower Ks than a corresponding mesophilic popu-
lation, which illustrates acclimation to the psychrophilic
operating temperature as well as the low substrate concen-
trations found in the ISPAD tank. The tting process used
in the study is considered to be accurate because, for the two
control inocula, the algorithm selected parameters compa-
rable to the mesophilic literature data recommended by the
ADM1.
Decomposing the Monod uptake rates shows develop-
ment of coexisting mesophilic and psychrophilic glucose
consumers, which is an exciting development and conrma-
tion of the latest understanding of temperature acclimation.
Further study will be undertaken to see if this eect applies
to other substrates, and to rene estimates of the associated
parameters. In addition, this poses new questions on the
factors controlling the interaction of two such populations,
and the requirements for modelling such processes.
Follow-up
To evaluate the remaining ISPAD kinetic parameters, a
new manure-SAT protocol must be developed. The standard
 Environmental Technology
SAT assay requires an optimal ratio of substrate to biomass,
which cannot be obtained owing to the small proportion
of biomass in the manure and the confounding substrate
concentrations from the manure organic matter. This must
be overcome in developing the manure-SAT, which will
include measurement of the biomass concentration using
molecular biology methods and separation of the biomass
from all or part of the manure organic matter. A more elab-
orate series of control assays could be used to separate
production and consumption, or tracer studies could be used
[24].
The accurate kinetic parameters resulting from use of
the m-SAT could then be used in modelling ISPAD. How-
ever, because psychrophilic and mesophilic populations,
consuming a single substrate, coexist in ISPAD, current
models would have to be modied to include this condition.
One approach to modelling microbial diversity that could be
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appropriate, using multiple microbial populations for each
substrate, has recently been applied to the ADM1 [39].
Conclusions
The objective of this study was to measure the substrate
uptake activities of the main microbial populations in an
ISPAD microbial community and to dene from these
the kinetic parameters required for modelling the pro-
cess. Extant substrate uptake rates were determined for
the ISPAD biomass on ve substrates at three tempera-
tures. Half-saturation constants, maximum substrate uptake
rate and microbial yield were estimated by mathematical
curve-tting for glucose at all three temperatures. The study
concluded that:
1. The ISPAD biomass is described by kinetic param-
eters that are signicantly dierent from those of
an equivalent mesophilic community. These changes
vary with substrate and temperature.
2. For glucose consumption, the tted higher maxi-
mum uptake rate, lower microbial yield and lower
half-saturation constant illustrate acclimation to both
the psychrophilic operating temperature and low
substrate concentrations found in the ISPAD tank.
3. The glucose uptake rates can be decomposed to
show the development of coexisting psychrophilic
and mesophilic populations consuming glucose.
4. To accurately evaluate kinetic parameters of manure
microbial communities for mathematical modelling
requires development of a new substrate uptake
assay protocol, which evaluates the size of the micro-
bial community and removes the remaining manure
organic matter.
Acknowledgements
The authors wish to acknowledge the nancial contributions of the
Natural Science and Engineering Research Council of Canada,
1771
GTI of Fredericton, New Brunswick, Canada, and the Biotech-
nology Research Institute of the National Research Council of
Canada, as well as the personal contributions of Luis Ortega,
Helene Leblanc, Grant Clark and Dominic Frigon.
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